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

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

Apoptosis in human gastric mucosa, chronic gastritis, dysplasia and carcinoma: analysis by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling

We examined the existence and distribution of apoptotic cells in human gastric mucosa, chronic gastritis, adenomatous dysplasias and carcinomas in 15 surgically removed stomachs in which dysplasia and carcinoma were found simultaneously. Serial sections were cut for immunohistochemistry for p53 oncoprotein and Ki-67 antigen, and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labelling (TUNEL). TUNEL signal-positive apoptotic cells were rare in normal mucosa, while a few apoptotic cells were noted in gastritic mucosa and intestinal metaplasia, intermingled with Ki-67 antigen-positive cells forming a generative cell zone. This suggests the cell-cycle-dependent apoptosis of gastric mucosa. The frequency of apoptotic cells per crypt was higher in incomplete than in complete metaplasia, implying greater underlying DNA damage in the former. TUNEL indices (TI; percentage of TUNEL-positive cells in tumour cells) were slightly higher in adenomatous dysplasias (4.9±2.1) than in carcinoma (3.9±1.1), but there was no no statistical difference. Ki-67 indices (KI; percentage of Ki-67 antigen-positive cells in tumour cells) were significantly (P<0.05) higher in carcinomas than in dysplasias. Thus, gastric adenomatous dysplasias were characterized by relatively higher TI and lower KI, which might reflect a more static growth potential. The expression of p53 oncoprotein in cancer cells is thought to be an apoptosis-suppressing event, although its precise role remains to be elucidated. Overall, these results indicate that apoptosis plays a crucial part in the morphogenesis of gastritic mucosa including intestinal metaplasia, and that the process is correlated both with tumourigenesis and with proliferative activity.     

Differential expression of fatty acid synthase (FAS) and ErbB2 in nonmalignant and malignant oral keratinocytes

The aim of this study was to investigate fatty acid synthase (FAS) and ErbB2 expression in nonmalignant oral epithelium and oral or head and neck squamous cell carcinomas (OSCC/HNSCC). Morphologically normal, hyperkeratotic, and dysplastic oral epithelium as well as well-differentiated and poorly differentiated OSCC were immunohistochemically evaluated for FAS, ErbB2, and Ki-67. These proteins were also analyzed in a tissue microarray with 55 HNSCC. SCC-9 cells were used to study FAS and ErbB2 during differentiation. FAS expression was higher in hyperkeratosis, dysplasias, and OSCC than in normal epithelium. Well-differentiated OSCC/HNSCC were more positive for FAS than the poorly differentiated tumors. ErbB2 was observed at the surface of nonmalignant and well-differentiated OSCC/HNSCC keratinocytes and in the cytoplasm of poorly differentiated cells. Ki-67 index was progressively higher from normal oral epithelium to OSCC, inversely correlated with cell surface ErbB2, and positively correlated with intracytoplasmic ErbB2. Finally, SCC-9 cell cultures were enriched in membrane ErbB2-positive cells after differentiation by anchorage deprivation. In conclusion, FAS is overexpressed in OSCC/HNSCC and hyperkeratotic oral epithelium and ErbB2 is found at the cell surface of differentiating keratinocytes and in the cytoplasm of poorly differentiated tumor cells. Ki-67 index is higher in epithelial dysplasias and OSCC than in morphologically normal oral epithelium.     

Detection of laminin receptor mRNA in human cancer cell lines and colorectal tissues by in situ hybridization.

The 67-kd high-affinity laminin receptor (67 LR) is a gene product whose expression appears to be associated with the invasive and metastatic phenotype of a variety of human cancer cells. Northern blot hybridization has been routinely used to quantify the level of 67 LR mRNA from total cellular RNA extracts of homogenized tissue specimens or in vitro grown cell populations. This technique is useful to assess the average expression of the 67 LR mRNA of a particular sample but does not provide information about expression in specific cell types nor about heterogeneity of expression from cell to cell. In this study, we analyzed the expression of 67 LR mRNA in four human cancer cell lines with varying degrees of expression of 67 LR protein (renal cancer A-704, breast carcinoma MCF-7/4 and MCF-7/7, and pancreatic cancer Panc-1) using in situ hybridization performed with 67 LR riboprobes. Total cellular RNA was simultaneously extracted from the cell lines and hybridized on Northern blots with a 67 LR cDNA probe to assess the validity of the mRNA detection by in situ hybridization. Sixty-seven LR mRNA expression was higher in Panc-1 and MCF-7/4 cells than in MCF-7/7 and renal carcinoma A-704. There was a direct correlation (R2 = 0.88) between the in situ hybridization analysis and the mRNA levels detected by Northern blot analysis. The in situ hybridization method showed a heterogeneous expression of the 67 LR mRNA in the four cell lines with different subpopulations of cells showing a range from negative to high levels of the message. Sixteen freshly frozen human colorectal tissues (seven adenocarcinomas, five matched normal mucosae, and four adenomas) were also analyzed by in situ hybridization. The 67 LR mRNA was localized in normal and neoplastic epithelial cells. Adenocarcinoma cells showed a 1.6- to 5-fold higher expression (P < 0.02 according to the Wilcoxon-Mann-Whitney test) than did epithelial colonic cells from normal mucosae or adenomas. The signal tended to be stronger in poorly differentiated carcinomas and carcinomas with metastases than in moderately differentiated and nonmetastatic tumors. We conclude that the high expression of 67 LR mRNA in colorectal tumors is due to an increased production by tumor cells. Furthermore, in situ hybridization is an effective method to detect the expression of LR mRNA in cultured cell lines as well as in frozen tissue sections.     

Divergent expression of L-type amino acid transporter 1 during uterine cervical carcinogenesis

L-type amino acid transporter 1 (LAT1) is a Na?-independent neutral amino acid transporter that has an essential role in cell proliferation. Although LAT1 expression is observed in various tumor cell lines and immunohistochemical expression of LAT1 has been investigated in carcinomas of various organs, LAT1 expression in uterine cervical neoplasm has not been reported. Therefore, in the present study, we immunohistochemically analyzed LAT1 expression along with the well-known markers of cervical carcinogenesis Ki-67 and p16 in normal uterine cervical mucosa (49 specimens) as well as cervical intraepithelial neoplasia (17 mild or moderate dysplasias and 19 severe dysplasias or carcinomas in situ) and invasive carcinomas (17 squamous cell carcinomas and 9 adenocarcinomas). LAT1 expression was limited to the basal layer of normal squamous epithelium, and it was significantly decreased in cervical intraepithelial neoplasia (P < .001), generally paralleled by increased expression of Ki-67 and p16. Interestingly, in invasive squamous cell carcinoma, LAT1 expression again increased especially at the invasive fronts (P < .001), whereas Ki-67 and p16 expressions were almost unchanged relative to noninvasive neoplasia. Although virtually no LAT1 expression was demonstrated in normal uterine cervical glands, LAT1 expression was observed in some adenocarcinomas (P < .001). The present study suggests that LAT1 expression decreases because of human papillomavirus infection as reflected by p16 overexpression in cervical intraepithelial neoplasia, whereas LAT1 expression in invasive carcinoma is associated with acquired malignant potential.     

Clinicopathological significance of WT1 expression in ovarian cancer: a possible accelerator of tumor progression in serous adenocarcinoma

Recently, oncogenic potential of the WT1 gene has been proposed in some human solid tumors and leukemias. Although previous studies have shown the frequent expression of the WT1 protein in ovarian serous adenocarcinomas (OSAs), its clinicopathologic significance is still unclear. We immunohistochemically examined the expression status of WT1 in 119 OSAs and analyzed the correlation of the intensity of WT1 immunoreactivity with the level of WT1 mRNA expression by quantitative real-time polymerase chain reaction, clinicopathologic variables, expression of p53, Bcl-2, and Ki-67 labeling index (LI). Of 119 OSAs, nuclear WT1 immunoreactivity was positive in 99 (83%), of which 44 (44%) and 55 (56%) exhibited high and low WT1 immunoreactivities, respectively. The quantitative WT1 mRNA levels were significantly correlated with the intensity of WT1 immunoreactivity (P < 0.05). In comparison with WT1-negative OSAs, the WT1-positive OSAs showed a higher grade (P = 0.007), advanced stage (P = 0.018), and higher Ki-67 LI (P < 0.001). Additionally, high WT1 immunoreactivity was correlated with a higher grade (P = 0.003), Ki-67 LI (P = 0.012), Bcl-2 expression (P = 0.003), and poorer patient outcome (5-year survival, 36.5 vs 63.8%, P = 0.008 by log-rank test). The WT1 protein may be an accelerator of the progression of OSA.     

Expression and clinical significance of the Kv3.4 potassium channel subunit in the development and progression of head and neck squamous cell carcinomas

The concept of ion channels as membrane therapeutic targets and diagnostic/prognostic biomarkers has attracted growing attention. We therefore investigated the expression pattern and clinical significance of the Kv3.4 potassium channel subunit during the development and progression of head and neck squamous cell carcinomas (HNSCCs). KCNC4 mRNA levels were determined by real‐time RT‐PCR in both HNSCC tissue specimens and derived cell lines. Kv3.4 protein expression was evaluated by immunohistochemistry in paraffin‐embedded tissue specimens from 84 patients with laryngeal/pharyngeal squamous cell carcinomas and 67 patients with laryngeal dysplasias. Molecular alterations were correlated with clinicopathological parameters and patient outcome. Increased KCNC4 mRNA levels were found in 15 (54%) of 28 tumours, compared to the corresponding normal epithelia and varied mRNA levels were detected in 12 HNSCC‐derived cell lines analysed. Increased Kv3.4 protein expression was observed in 34 (40%) of 84 carcinomas and also at early stages of HNSCC tumourigenesis. Thus, 35 (52%) of 67 laryngeal lesions displayed Kv3.4‐positive staining in the dysplastic areas, whereas both stromal cells and normal adjacent epithelia exhibited negligible expression. No significant correlations were found between Kv3.4‐positive expression in HNSCC and clinical data; however, Kv3.4 expression tended to diminish in advanced‐stage tumours. Interestingly, patients carrying Kv3.4‐positive dysplasias experienced a significantly higher laryngeal cancer incidence than did those with negative lesions (p = 0.0209). In addition, functional studies using HNSCC cells revealed that inhibition of Kv3.4 expression by siRNA leads to the inhibition of cell proliferation via selective cell cycle arrest at the G2/M phase without affecting apoptosis. Collectively, these data demonstrate for the first time that Kv3.4 expression is frequently increased during HNSCC tumourigenesis and correlated significantly with a higher cancer risk. Our findings support a role for Kv3.4 in malignant transformation and provide original evidence for the potential clinical utility of Kv3.4 expression as a biomarker for cancer risk assessment. Copyright © 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

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.     

Apoptosis regulation differs between ulcerative colitis-associated and sporadic colonic tumors. Association with survivin and bcl-2

To clarify kinetics in ulcerative colitis (UC)-associated lesions, cell proliferation, apoptosis, and expression of apoptosis-inhibitory proteins were studied. Ki-67 labeling and survivin and bcl-2 expression were examined immunohistochemically in 22 low-grade dysplasias (LGDs), 25 high-grade dysplasias (HGDs), and 13 adenocarcinomas associated with UC, and for comparison in 21 sporadic adenomas with LGD, 22 sporadic adenomas with HGD, and 21 invasive adenocarcinomas. Apoptosis was studied with nick-end labeling and immunohistochemical analysis of single-stranded DNA. In UC-associated LGDs, Ki-67--positive cells were more frequent in the lower than the upper half of the crypt, related to bcl-2 expression, while in sporadic adenomas such cells were more common in the upper half. No difference in apoptosis was found between UC-associated LGDs and sporadic adenomas with LGD or between UC-associated HGDs and sporadic adenomas with HGD. However, UC-associated carcinomas exhibited a lower apoptotic count than their sporadic invasive counterparts. This seemed related to higher survivin expression without a significant difference between the 2 types of invasive lesions regarding bcl-2 levels. Apoptosis is less frequent in UC-associated than in sporadic invasive colon carcinomas, this being linked to elevated survivin expression. The control of apoptosis may be different in the 2 types of tumorigenesis.     

Expression of cyclin kinase inhibitor p27(Kip1) in skin tumours of dogs

Skin tumours (n=148) of epidermal or hair follicle origin were examined immunohistochemically to determine the expression of p27(Kip1)(p27), a cyclin-dependent kinase inhibitor (CDKI), and of Ki-67. In normal skin, a large number of basal cells of the epidermis and hair follicles were positive for Ki-67 and many suprabasal epithelial cells were positive for p27. Most of the hair matrix cells were positive for Ki-67 but negative for p27. Hair papillae were strongly positive for p27. Squamous cell carcinomas had a p27 positive index (PI) significantly lower than that of trichoepitheliomas (P<0.005), basal cell tumours (P<0.05) and intracutaneous cornifying epitheliomas (P<0.001). In contrast, Ki-67 PIs of squamous cell carcinomas and pilomatrixomas were significantly higher than those of trichoepitheliomas, basal cell tumours and intracutaneous cornifying epitheliomas (P<0.01 to P<0.001). No significant difference was observed between the Ki-67 PI values of squamous cell carcinomas and pilomatrixomas. The results suggested that p27 is capable of suppressing cell proliferation in the differentiation of normal canine skin. In spite of being a benign neoplasm, pilomatrixomas had a low p27 expression; this may be a reflection of the proliferative potential of the hair matrix. The expression of p27 may be a useful marker for the analysis of cell kinetics.     

Expression of the cyclin dependent kinase inhibitor p21WAF1/CIP1 in oesophageal squamous cell carcinomas

 To elucidate the role of CDK inhibitor p21^WAF1/CIP1 in human oesophageal squamous cell carcinomas, we examined its expression immunohistochemically using surgically resected tissues from 25 patients, and have analyzed the relationship with alteration of p53 gene (F-SSCP analysis), proliferative activity (Ki-67 labelling index), frequency of apoptosis (in situ DNA nick end labelling), and degree of differentiation. P21 expression was observed in 11 cases (44%) with a percentage of positive cells ranging between 1% and 10%. Of the 25 cases, 4 cases showed >5% of positive cells. As for the relationship with p53 gene, all 7 p53-mutation positive cases were negative for p21 expression, whereas 11 out of 18 mutation negative cases showed positive for p21 expression. As for the relationship with degree of tumour differentiation, 6 out of 8 well differentiated type cases showed positive for p21 expression. By contrast, all 8 cases of poorly differentiated type were negative for p21 expression. Frequency of apoptotic cells was significantly higher in p21 positive cases than negative cases although Ki-67 labelling index was almost the same regardless of the expression of p21. P21 expressing cells were distributed mainly in the middle layers of the invading nests, especially around the keratinization, which was almost similar to the distribution of apoptotic cells. Our results suggest that expression of p21 in human oesophageal squamous cell carcinomas is induced by a p53-dependent pathway and affects apoptosis and differentiation of carcinoma cells. Received: 9 October 1996 / Accepted: 23 December 1996     

Immunohistochemical Analysis of the Cell Cycle-Associated Antigens Ki-67 and Retinoblastoma Protein in Parathyroid Carcinomas and Adenomas

The morphologic distinction between parathyroid carcinoma and adenoma can be a difficult diagnostic problem. We analyzed nuclear immunoreactivity for the cell cycle-associated antigen Ki-67 with monoclonal antibody (MAb) MIB-1 and for retinoblastoma (RB) protein with two polyclonal antisera in 24 parathyroid carcinomas and 35 adenomas, which were formalin fixed and paraffin embedded to determine if these antibodies could assist in distinguishing between carcinomas and adenomas. In addition, 10 cases of parathyroid hyperplasia and 5 cases of normal parathyroids were examined as control tissues. The Ki-67 labeling index was significantly higher in parathyroid carcinomas compared to adenomas (7.1 ± 1.0% vs 2.4 ± 0.2%,p<0.001). No patient with a parathyroid adenoma, parathyroid hyperplasia, or normal parathyroid gland had a Ki-67 labeling index >5.3%. Analysis of the primary tumors from patients with recurrent carcinomas and from those with nonrecurrent carcinomas showed a higher mean Ki-67 labeling index (7.8 ± 1.5% vs 5.2 ± 1.1%), in the former group, although these differences were not statistically significant. The RB protein immunoreactivity was not useful in distinguishing between parathyroid carcinomas and adenomas in paraffin-tissue sections. These results indicate that nuclear immunoreactivity for the cell cycle-associated antigen Ki-67 may be another useful method to assist in distinguishing parathyroid carcinomas from adenomas.     

Differential expression of human telomerase catalytic subunit mRNA by In situ hybridization in pheochromocytomas

In pheochromocytomas, it is very difficult to predict malignant potential by conventional histology or immunohistochemical and molecular markers. We investigated the expression of human telomerase catalytic component (hTERT) mRNA, hTERT protein, Ki-67 antigen, and p27^kip1 in pheochromocytomas (27 benign, 7 suspected malignant, and 7 malignant), and evaluated the possibility of expressions of these proteins, and hTERT mRNA serve as diagnostic markers for predicting the biological behavior of these tumors. All tumors showed the classical histology and typical immunohistochemical pattern. By in situ hybridization, hTERT mRNA was expressed in 5/7 malignant tumors (defined as the presence of metastasis and/or extensive local invasion) as compared with 3/27 benign tumors. We examined the hTERT by immunohistochemistry to confirm the mRNA. hTERT mRNA expression was correlated with hTERT protein expression. All benign tumors exhibited no immunopositivity or <1% of cells stained for Ki-67 antigen. Six out of seven malignant tumors have shown either hTERT mRNA expression or Ki-67 immunoreactivity While no statistical difference in p27^kip1 expressions was observed among benign, malignant, and suspected malignant tumors, there was a statistical difference between the normal adrenal medulla samples and tumors (p<0.001). Thus, hTERT mRNA detection by in situ hybridization, hTERT expression, and Ki-67 antigen expression are all useful tools for differentiating malignant from benign pheochromocytomas.     

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.     

Immunohistochemical analysis of cell kinetic parameters in colonic adenocarcinomas, adenomas, and normal mucosa

The monoclonal antibody Ki-67 identifies a nuclear antigen that is expressed in proliferating cells in G1, G2, S, and M phases of the cell cycle. An immunoperoxidase method and this antibody were used to identify proliferating cells in sections of colorectal tissues--normal colon (n = 10), colorectal polyps (n = 20), and adenocarcinoma (n = 28). Colorectal adenomas showed a uniform distribution of positive nuclear staining throughout the sections, including the cells of the adenoma surface, while staining in the normal mucosa was confined to the middle third and lower third of the crypts. Areas of polyps with numerous Ki-67-positive epithelial cells invariably showed immature or dysplastic histology and, conversely, glands that lacked such histologic features had low Ki-67 staining frequency or were negative. In adenomas, nuclei located toward the luminal surface of glands were more likely to be Ki-67-positive than those located basally in the cells. The mean Ki-67 score (a measure of positive staining nuclei) for adenomas was 45.5 compared to a mean score of 66.3 for adenocarcinomas in the carcinomas studied (P less than .001). Ki-67 score did not correlate with histologic grade or Duke's stage. Ki-67 staining can be used to characterize the proliferative characteristics of normal colonic mucosa, adenomas, and carcinomas.     

Loss of Retinoic Acid Receptor-β Expression Is an Early Event during Esophageal Carcinogenesis

We recently observed that growth inhibition of esophageal cancer cells by retinoic acid (RA) was associated with both constitutive expression and RA-induced up-regulation of RA receptor beta (RAR-beta). Cell lines that did not express RAR-beta were also resistant to RA. To explore the expression of RAR-beta mRNA in vivo, we analyzed esophageal tissue specimens from 16 normal mucosae, 30 dysplastic lesions, and 157 esophageal tumors by in situ hybridization. RAR-beta was detected in 88% (14/16) of normal esophageal tissues and in 96% (96/100) of distant normal esophageal mucosa from cancer specimens. In contrast, RAR-beta was expressed in only 57% (17/30) of dysplastic lesions and in 54% (84/157) of carcinomas. Among esophageal carcinomas RAR-beta mRNA was expressed in 62% (26/42) of well-differentiated, 54% (27/50) of moderately differentiated, and only 29% (4/14) of poorly differentiated SCCs. Our data suggest that the loss of RAR-beta expression is an early event associated with esophageal carcinogenesis and the status of squamous differentiation.     

Merkel cell carcinomas: expression of S-phase kinase-associated protein 2 (Skp2), p27, and proliferation markers

Merkel cell carcinomas are rare and aggressive tumors about which the expression of cell cycle regulatory proteins are not well known. We evaluated the clinicopathologic features of Merkel cell carcinomas and examined the expression of the cell cycle regulatory markers p27 and S-phase kinase-associated protein 2 (Skp2) and the proliferation marks Ki-67 and DNA topoisomerase II alpha (topo II alpha) in a group of these tumors. Thirty-nine cases of Merkel cell carcinoma were studied, 19 from the Mayo Clinic, Rochester, MN, and 20 from the University of Torino, Torino, Italy. Although the University of Torino patients tended to be slightly older at time of surgery compared to the Mayo Clinic patients, no clinical, pathologic, or immunohistochemical feature was statistically significantly different between the two groups. Of the 39 patients, 20 were male and 19 were female. The age at surgery averaged 72 yr. Formalin-fixed paraffin-embedded archival tissues from the 39 Merkel cell carcinomas were analyzed by immunohistochemistry for p27, Skp2, Ki-67, and topo II alpha with the avidin-biotin peroxidase system. The distribution of immunoreactivity was analyzed by quantifying the percentage of positive nuclei, which was expressed as the labeling index. There was a statistically significant inverse relationsnip between p27 and Skp2 (p=0.005). Most tumors with increased levels of Skp2 were associated with reduced p27, and tumors with high levels of p27 expression were associated with reduced levels of Skp2. These results suggest that Skp2 regulates p27 expressions in Merkel cell carcinomas. Tumors showing increased Skp2 expression were not always correlated with increased proliferation as evaluated by Ki-67 and topo II alpha, suggesting that Skp2 may be involved in Merkel cell tumorigenesis, but that other factors may also influence cell proliferation in these tumors.