Mean Labeling Index Research Articles

Minichromosome maintenance‐7 and geminin are reliable prognostic markers in patients with oral squamous cell carcinoma: immunohistochemical study

Minichromosome maintenance (MCM) proteins act as the origin licensing machinery that regulates initiation of DNA replication. Geminin is a licensing repressor and prevents reinitiation of DNA replication by blocking reloading of MCM proteins at replication origins. Recent studies have proposed that MCM7 and geminin may be sensitive proliferative and prognostic markers of various malignant tumors. This study aimed to analyze the expression of MCM7 and geminin to clarify pathobiological significance in human oral squamous cell carcinomas (OSCCs). We performed immunohistochemical analysis on 10 specimens of normal oral epithelia, 50 lesions with dysplasia and 113 OSCCs. Labeling indices (LIs) for MCM7, geminin and Ki-67 were evaluated, comparing with clinicopathological profiles. The mean LIs for MCM7 were 29.2% for normal epithelia, 32.2% for dysplasias, and 51.1% for OSCCs; the value was significantly higher in the last than in the former two (P < 0.01). The mean LIs for geminin were 6.8% for normal epithelia, 9.2% for dysplasias, and 21.3% for OSCCs; the value was significantly higher in the OSCCs (P < 0.01). The MCM7 LIs were correlated with the histological grade of OSCCs, in which the highest LIs were noted in the poorly differentiated type (P < 0.01). The survival rate was significantly lower in patients with a higher MCM7 LI (>49.5%) than in those with a lower LI (P < 0.05) at stage III-IV. However, the survival rate in the patients with a higher geminin LI (>19.5%) was significantly higher than in those with a lower LI (P < 0.05) at stage IV.

MIB-1 labeling index correlated with magnetic resonance imaging detected tumor volume doubling time in pituitary adenoma

The purpose of this study was to evaluate whether MIB-1 labeling index (LI) could be used to predict growth velocity of residual pituitary adenomas after surgery. One hundred and sixty pituitary adenomas which had not received other treatment modality except for surgery were collected. Each of them had at least two post-operative magnetic resonance imaging (MRI) studies with an interval of at least 1 month apart. Tumor volume doubling time (TVDT) was calculated for those in which volume increased. Post-operative tumor progression was noted in 54 (33.8%) cases, of which 39 (72.2%) cases were non-functioning adenomas. The MIB-1 LIs of the functioning and non-functioning adenomas were not significantly different. The median TVDT of these 54 cases was 34.6 months. The mean and median MIB-1 LI were 2.7 and 1.1 respectively (range 0.4-20.6). The MIB-1 LI was significantly correlated with log(2)(TVDT) (r=-0.363, P=0.007); when LI was <0.8, the TVDT of 90.5% cases was >or= 2 years. Only one-third of the pituitary adenomas progressed after surgery, and their MIB-1 LIs were generally low. The MIB-1 LI was significantly correlated with the MRI detected TVDT of post-operative residual pituitary adenomas.

EGFR and Ki-67 expression in oral squamous cell carcinoma using tissue microarray technology

Our aim was to validate the use of tissue microarrays (TMA) in oral squamous cell carcinomas (OSCC) to analyse epidermal growth factor receptor (EGFR) and Ki-67 expression. We also analysed the relationship that the expression of these markers may have with clinical, pathological and survival variables. The study sample comprised 39 unselected patients diagnosed and treated for OSCC. We analysed Ki-67 and EGFR expression by immunohistochemistry on formalin-fixed, paraffin-embedded surgical specimens. Whole sections (WS) were compared with double 1.5 mm core-tissue microarrays. High EGFR expression was observed both on TMA (in 98% of the cases) and WS (in 100% of the cases) with substantial agreement kappa value (0.720). EGFR expression was not significantly associated with clinical, pathological and survival variables on TMA and WS. Ki-67 analysis showed a Spearman correlation of 0.741 with a Ki-67 mean labelling index of 45% in TMA and 56.8% in WS. We found a significant relationship between gender and Ki-67 labelling index on WS (P = 0.022) and TMA (P = 0.002). Clinical stage was the only parameter in multivariate analysis that had a significant predictive value. We demonstrate that dual 1.5 mm core TMA is a valid, rapid, economical and tissue-saving way to study OSCC biopsies and that it presents strong correlation with the WS. EGFR overexpression in OSCC suggests that these tumours may be a candidate for therapy investigation directed to EGFR.

Expression of Ki-67, PCNA, and p27kip1 in canine pituitary corticotroph adenomas

Pituitary-dependent hypercortisolism (PDH), which is caused by adrenocorticotropic hormone (ACTH)-secreting pituitary adenomas, is a common endocrinopathy in dogs. Dogs with non-enlarged pituitaries harboring a microadenoma have a better prognosis than those with enlarged pituitaries. The aim of this study was to investigate the expression of the proliferation markers Ki-67 and proliferating cell nuclear antigen (PCNA) and the cell-cycle inhibitor p27kip1 in corticotroph adenomas in enlarged and non-enlarged pituitaries. The expression of Ki-67, PCNA, and p27kip1 was analyzed by immunohistochemical staining of 17 pituitary adenoma samples harvested during pituitary surgery in dogs with PDH. The labeling index was calculated by counting the number of immunopositive cells per 1,000 cells. The mean (± standard deviation) labeling index for Ki-67 was 8.4% ± 14.2% for the group with enlarged pituitaries, and 8.8% ± 5.5% for the group with non-enlarged pituitaries; that for PCNA was 35.5% ± 12.2% and 37.0% ± 15.5%; and that for p27kip1 was 29.3% ± 22.6% and 42.5% ± 27.9%, respectively. No significant differences in Ki-67, PCNA, and p27kip1 labeling indices were found between enlarged and non-enlarged pituitaries. However, a trend toward significance was observed when comparing the expression of p27kip1 in enlarged pituitaries versus normal pituitary tissue. It is concluded that Ki-67 and PCNA are not useful as proliferative markers for studying the pathobiology of pituitary corticotroph adenomas in dogs.

Estudo citofotométrico da expressão dos marcadores tumorais Ki-67 e CD34 no adenocarcinoma de próstata

To quantify the percentage of immunostaining through the labeling index as well as the optical density of Ki-67 and CD34 in prostate adenocarcinoma and compare the results between markers. Markers Ki-67 and CD34 were studied using immunohistochemistry in 34 cases of prostate adenocarcinoma from radical prostatectomies performed at the Hospital Regional do Gama in Brasilia, Brazil from 2000 through 2005. Those markers were quantified using the SAMBA 4000 software - Automated Scanning Microscopic Analysis System - and the IMMUNO software in the analysis of the variables labeling index and optical density. Spearman's correlation coefficient was estimated in order to evaluate the association between the expression levels of the markers. For the comparison of lesion types, Student's paired t-test and the nonparametric Wilcoxon test were used. Of the 34 blocks referred for the study of the tumor markers, 15 were positive for Ki-67, 34 showed CD34 expression, and 14 were positive for both markers. The median value for the labeling index of CD34 was 72.72%; the minimum was 5.14% and the maximum, 88.81%. The median for the Ki-67 labeling index was 73.78%, while the minimum was 16.87%, and the maximum, 87.47%. The median value for the optical density of CD34 was 48.33, the minimum was 35.65 and the maximum, 85.86. For the optical density of Ki-67, the median was 40.03, while the minimum and maximum values were 21.53 and 52.43, respectively. The cytophotometric expression of Ki-67 had a mean labeling index of 64.04%, and the mean CD34 labeling index was 61.64%. The cytophotometric expression of the mean optical density of Ki-67 was 39.49, while for CD34 it was 53.69. There was a significant difference between Ki-67 and CD34 immunostaining with respect to optical density (P=0.025); no significant difference occurred regarding labeling index (P=0.470).

Immunohistochemical expression of geminin in colorectal cancer: Implication of prognostic significance

DNA should be duplicated precisely once per cell cycle to maintain genome integrity. After DNA replication, geminin binding to Cdt1 inhibits uploading of the minichromosome maintenance (MCM) complex as DNA helicase onto chromatin and prevents DNA re-replication in the same cell cycle. Expression of geminin indicates poor prognosis in some malignancies, such as breast and renal cell carcinoma. We evaluated the expression of geminin to clarify its pathobiological and prognostic significance in colorectal cancer, compared with expression of MCM7 and Ki-67. We performed Western blot analyses of 5 human colorectal cancer cell lines and immunohistochemistry on 191 surgically removed specimens of Dukes' B and C stage colorectal cancer. Double-labeling immunofluorescence was also carried out to identify co-expression of geminin, MCM7 and Ki-67. Geminin proteins were detected in all the 5 cell lines examined. Geminin, MCM7 and Ki-67 were co-expressed and cells that stained only for geminin were not detected. Mean labeling indices (LIs) for geminin, MCM7 and Ki-67 were 26.3, 58.2 and 40.8%, respectively. Patients with high geminin LIs had significantly unfavorable prognosis in stage II and III colorectal cancer (P=0.04). Patients with a tumor with a higher proliferating nature (i.e. high LIs for three markers) showed significantly unfavorable prognosis in multivariate Cox analysis. Our results indicate that assessment of geminin, MCM7 and Ki-67 may be useful for predicting prognosis in patients with colorectal cancer.

High frequency of RASSF1A and RARb2 gene promoter methylation in morphologically normal endometrium adjacent to endometrioid adenocarcinoma

To identify a DNA methylation signature of endometrioid carcinoma of the endometrium (EEC) in the early stages of endometrial carcinogenesis. Archival biopsy specimens of 39 EECs, 14 cases of atypical hyperplasia (AH), 11 histologically normal endometrial tissues adjacent to EECs and 24 normal control endometrial samples were retrieved. The cases were tested by quantitative methylation-specific polymerase chain reaction with primers hybridizing in the promoter regions of five genes frequently methylated in human cancer (RASSF1A, RARb2, P16, MGMT and GSTPi). Twenty-nine of 39 (74%) EECs and 7/14 (50%) AHs were methylated for the RASSF1A gene, whereas 17/39 (44%) EECs and 6/14 (43%) AHs were positive for the methylation of the RARb2 gene. No significant results were obtained for the other genes (P16, MGMT and GSTPi). Interestingly, 4/11 (36%) and 6/11 (55%) histologically normal endometrial tissues adjacent to EEC showed, respectively, RASSF1A and RARb2 gene methylation. Furthermore, these 11 specimens were microsatellite stable and showed similar proliferative, cell cycle and apoptotic mean labelling indices as the normal endometrial control tissues. Promoter region methylation of RASSF1A and RARb2 genes is an early event in endometrial carcinogenesis.

Aberrant expression of an “intestinal marker” Cdx2 in pyloric gland adenoma of the gallbladder

The aim of this study was to survey Cdx2 expression in pyloric gland adenoma (PGA) of the gallbladder. We reviewed 29 PGA cases, ten (34.4%) and seven (24.1%) of which showed intestinal metaplasia (IM) and squamous morule (SM), respectively. The immunostaining for Cdx2, beta-catenin, MUC5AC, MUC2, MUC6, and M-GGMC-1 was performed and scored (0 = negative, 1+ = <10%, 2+ = 10% to <30%, 3+ = 30% to <50%, 4+ = 50% to <70%, 5+ = 70-100%). Although its scores were relatively low (1+ or 2+), Cdx2 was frequently expressed in 27 cases (93.1%). Not only goblet and/or Paneth cells were positive but also non-IM cells in PGAs, as opposed to the lack of staining in the background mucosa. Cdx2 scores were not correlated with those of IM (p = 0.485) and MUC2 (p = 0.868). Of note, Cdx2 was positive in foci of SM in all seven cases, and there was a significant difference in Cdx2 scores between PGAs with and without SM. Furthermore, the p value of scores between Cdx2 and beta-catenin was 0.051, and both mean labeling indices (LIs) were correlated (r = 0.736). With Cdx2, higher morular LIs than glandular LIs were observed (p = 0.001). Finally, we concluded that aberrant Cdx2 expression in PGAs is closely associated with nuclear beta-catenin expression and SM in contrast with IM.

Quantitative analysis of mitotic Olig2 cells in adult human brain and gliomas: Implications for glioma histogenesis and biology

The capacity of adult human glial progenitor cells (AGPs), to proliferate and undergo multipotent differentiation, positions them as ideal candidate cells of origin for human gliomas. To investigate this potential role we identified AGPs as mitotically active Olig2 cells in nonneoplastic adult human brain and gliomas. We conservatively estimated that one in 5,000 human temporal lobe neocortical gray or subcortical white matter cells is mitotic. Extrapolating from a mean Olig2/Mib-1 labeling index (LI) of 52% and total cell number of 100 billion, we estimated the overall prevalence of mitotic Olig2 AGPs in nonneoplastic human brain parenchyma at 10 million. These data identify a large reservoir of Olig2 AGPs which could be potential targets for human gliomagenesis. The vast majority of mitotic cells in Grade II and Grade III gliomas of all histologic subtypes expressed Olig2 (mean LI 75%) but rarely S100B (LI 0.6%), identifying the Olig2 cell as a distinct contributor to the proliferating cell population of human gliomas of both oligodendroglial and astrocytic lineages. In the most malignant Grade IV glioma, or glioblastoma multiforme (GBM), the prevalence of Olig2/Mib-1 cells was significantly decreased (24.5%). The significantly lower Olig2/Mib-1 LI in GBMs suggests that a decrease in the prevalence of Olig2 cells to the total mitotic cell pool accompanies increasing malignancy. The novel framework provided by this quantitative and comparative analysis supports future studies to examine the histogenetic role of Olig2 AGPs in adult gliomas, their potential contribution to the tumor stroma and the molecular role of Olig2 in glioma pathogenesis.

Mullerian Adenosarcomas: An Immunophenotypic Analysis of 35 Cases

Mullerian adenosarcomas (MAs) are rare mixed mesenchymal and epithelial neoplasms that occur most commonly in the uterus. Although the epithelial component is typically benign, the mesenchymal component of most adenosarcomas morphologically resembles that observed in endometrial stromal tumors and is responsible for their clinical behavior. Thus, the differential diagnosis usually includes not only low-grade endometrial stromal tumors, but also adenofibroma, carcinosarcoma, and embryonal rhabdomyosarcoma especially in small samples. The objective of this study was to ascertain the immunophenotypic profile of the epithelial and mesenchymal components of MAs and delineate possible differences between conventional mesenchymal areas and areas of sarcomatous overgrowth. Representative sections from 35 MAs, 28 of them without sarcomatous overgrowth (MA-NSO) and 7 with sarcomatous overgrowth (MA-SO), were included in the study. Thirty tumors arose in the uterus, 4 were pelvic, and 1 originated in the colon. Adequate blocks were selected and immunostained for estrogen receptor (ER), progesterone receptor (PR), androgen receptor (AR), CD10, WT1, smooth muscle actin, desmin, AE1/3 cytokeratin, CD34, calretinin, inhibin, c-kit, and Ki-67. The mesenchymal component expressed ER in 21/27 MA-NSOs but in only 1/7 MA-SOs (65% overall). PR was expressed in 21/26 MA-NSOs and 4/7 MA-SOs (76% overall), whereas AR was positive in 10/27 MA-NSOs and 5/7 MA-SOs (35% overall). CD10 was expressed in 23/28 MA-NSOs but in only 2/7 MA-SOs (71% overall), and WT1 positivity was seen in 22/27 MA-NSOs and 6/7 MA-SOs (79% overall). Sixty-seven percent of MAs expressed smooth muscle actin, 32% desmin, including both examples of MA-SOs with rhabdomyoblastic differentiation, and 25% expressed AE1/3 cytokeratin. CD34 expression was found in 35% of the tumors, but it was almost always patchy in distribution and weak in intensity, as was calretinin expression, seen only in 12% of the cases. Expression of c-kit and inhibin in greater than 5% of the tumor cells was not encountered. The median and mean Ki-67 labeling indices were 10% and 12%, respectively (range, <5% to 40%). The median and mean Ki-67 indices were both 5% in MA-NSOs compared with 30% and 28%, respectively, in MA-SOs. The epithelial compartment demonstrated expression for ER (24/32), PR (23/31), and AE1/3 cytokeratin (33/33); rare cases expressed CD10 (4 cases) and AR (1 case). In summary, the immunophenotype of most MAs resembled that of endometrial stromal tumors (positive for ER, PR, WT1, and CD10, with variable expression of muscle markers, AR and cytokeratin). The proliferative rate in the stromal component was strongly related to the presence of sarcomatous overgrowth. ER, PR, and CD10 expression was lost in MA-SOs relative to conventional low-grade stromal areas of mullerian/mesodermal adenosarcomas, reflecting the "dedifferentiation" of this component.

Diversity of glial cell components in pilocytic astrocytoma

To characterize the cellular density and proliferative activity of GFAP-negative cells in pilocytic astrocytoma (PA), surgically excised tissues of PAs (n=37) and diffuse astrocytomas (DAs) (n=11) were examined morphologically and immunohistochemically using antibodies against GFAP, Olig2, Iba1 and Ki-67 (MIB-1). In PA, Olig2 immunoreactivity was significantly expressed in protoplasmic astrocytes in microcystic, loose areas and cells in oligodendroglioma-like areas. Iba1-positive, activated microglia/macrophages were also commonly observed in microcystic areas. In compact areas, a prominent reaction for GFAP was observed, but for Olig2 and Iba1 to a lesser degree. On semiquantitative analysis, the number of Olig2-positive cells was significantly higher in PAs (mean labeling index (LI) +/- standard deviation (SD): 46.8+/-15.4%) than in DAs (13.3+/-7.8%) (P<0.001). Many Iba1-positive, microglia/macrophages were observed in PAs (19.9+/-6.5%), similarly to DAs (20.9+/-9.9%). Re-immunostaining of PA demonstrated that most Ki-67-positive, proliferating cells expressed Olig2, whereas GFAP or Iba1 expression in Ki-67-positive cells was less frequent (14.7+/-13.7%, and 8.8+/-13.6%) in a double immunostaining study. Conversely, the percentage of Olig2-positive, proliferating cells in total Olig2-positive cells (7.2+/-3.9%) was higher than that of Iba1-positive, proliferating cells in total Iba1-positive cells (0.9+/-0.6%). In conclusion, the present study found that PA consisted of numerous GFAP-negative cells, including Olig2-positive cells with high proliferation. Semiquantitative analysis of Olig2 immunohistochemistry in microcystic areas might therefore be useful for the differential diagnosis of PA and DA.

The Diagnostic Value of Ki-67 and repp86 in Distinguishing Between Benign and Malignant Mesothelial Proliferations

The differentiation of benign mesothelial proliferations from malignant mesotheliomas may be difficult, especially when evaluating small specimens from pleural biopsies. To explore the potential value of 2 proliferative cell markers, Ki-67 and restrictedly expressed proliferation-associated protein 86 kDa (repp86), in distinguishing between malignant mesothelioma (MM) and benign reactive mesothelial hyperplasia (MH). Thirty-six cases of MM from 26 men and 10 women with a mean age of 62.9 years (range, 36-80 years) and 22 cases of benign reactive MH from 14 male and 8 female patients with a mean age of 51.5 years (range, 15- 88 years) were included in this study. The proliferative status of the lesions was assessed by immunohistochemistry using monoclonal antibodies to Ki-S2 (repp86) and Ki-S5 (Ki-67). The labeling indices were quantified. The mean labeling indexes for Ki-67 in MM and benign reactive MH were 24.6% (range, 1%-66%) and 6.23% (range, 0%-25%), respectively. The mean labeling indexes for repp86 in MM and benign reactive MH were 26.3% (range, 0%-50%) and 3.26% (range, 0%- 21%), respectively. The average proliferative cell count was significantly higher in MM compared with benign reactive MH (P < .001). Furthermore, both markers showed a significant correlation in their expression in MM and benign reactive MH (r = 77.5, P < .001). Sensitivities of 88% and 92% and specificities of 92% and 94% were obtained at a cutoff point of 9% for Ki-67 and repp86, respectively. Used in combination, Ki-67 and repp86 appear to be useful markers in differentiating MM from benign reactive MH.

The diagnostic value of Ki-67 and repp86 in distinguishing between benign and malignant mesothelial proliferations.

Abstract Context.—The differentiation of benign mesothelial proliferations from malignant mesotheliomas may be difficult, especially when evaluating small specimens from pleural biopsies. Objective.—To explore the potential value of 2 proliferative cell markers, Ki-67 and restrictedly expressed proliferation–associated protein 86 kDa (repp86), in distinguishing between malignant mesothelioma (MM) and benign reactive mesothelial hyperplasia (MH). Design.—Thirty-six cases of MM from 26 men and 10 women with a mean age of 62.9 years (range, 36–80 years) and 22 cases of benign reactive MH from 14 male and 8 female patients with a mean age of 51.5 years (range, 15– 88 years) were included in this study. The proliferative status of the lesions was assessed by immunohistochemistry using monoclonal antibodies to Ki-S2 (repp86) and Ki-S5 (Ki-67). The labeling indices were quantified. Results.—The mean labeling indexes for Ki-67 in MM and benign reactive MH were 24.6% (range, 1%–66%) and 6.23% (range, 0%–25%), resp...

Clinicopathologic significance and prognostic role of cyclin E and cyclin A expression in laryngeal epithelial lesions

Conclusions. The determination of cyclin A expression might be helpful in the identification of laryngeal squamous cell cancer (LSCC) patients with increased risk of metastases. The results suggest that cyclin A may be a more informative marker for cell proliferation than Ki-67. Abnormalities of cyclin E and cyclin A may play an important role in LSCC development and progression; however, the expression of cyclin E does not seem to have prognostic significance. Objective. The aim of the study was to elucidate a possible association between cyclin E and cyclin A expression and clinicopathologic factors and their potential role as prognostic markers for patients with laryngeal epithelial lesions. Materials and methods. Expression of cyclins E and A, and Ki-67 was examined immunohistochemically in a formalin-fixed, paraffin-embedded series of 46 LSCC; 23 epithelial dysplasias (ED); and 21 normal mucosae (NM). Results. The mean labeling indices (LIs) for cyclin E in LSCC, ED, and NM were 10.6%, 4.9%, and 0%, and for cyclin A 27.2%, 17.5%, and 7%, respectively. In LSCC, a statistically significant correlation was found between enhanced cyclin A expression and a higher incidence of locoregional lymph node metastasis (p<0.01). The enhanced expression of cyclin A was linked with cell proliferation in LSCC, ED, and NM. No association was observed between cyclin E and A and other clinicopathologic parameters or applied treatments. The prognostic significance of cyclin E, cyclin A, and Ki-67 in determining overall survival time showed no statistically significant differences.

A Comparative Analysis of Lichen Sclerosus of the Vulva and Lichen Sclerosus That Evolves to Vulvar Squamous Cell Carcinoma

Lichen sclerosus (LS) is a chronic inflammatory disease of unknown cause which is found most often in the vulvar region. Vulvar LS has been associated with squamous-cell carcinoma (SCC) of the vulva; estimates of the prospective risk of malignant transformation range from 3% to 5%. In this study, the proliferative index was determined using the cell proliferation marker Ki67 and expression of the tumor suppressors p16 and p53 was quantified in 8 women with histologically confirmed vulvar LS who, after 10 months to 9 years of follow-up, developed vulvar SCC. A comparison group consisted of 8 women with vulvar LS who did not develop cancer during follow-up for 9 years or longer. Immunoperoxidase staining utilized the monoclonal antibody MIBI. All patients described vulvar itching and/or burning. Mean ages were approximately 60 years for both groups of patients. Nuclear atypia was observed in epidermal keratinocytes in 2 of the 8 women with unchanged vulvar LS and in 5 of those who developed SCC, not a statistically significant difference. All cancers were of the keratinizing type with LS present in the nearby skin. Significantly higher MIBI labeling indices were found in cases evolving to vulvar SCC than in those that remained unchanged. The mean p53 labeling index was also significantly greater in evolving than in nonevolving cases of vulvar LS, but there was no such difference in pl6-positive staining between the 2 groups. Suprabasilar extension of p53 was observed in half of the evolving cases and in a single nonevolving case. All cases of evolving LS and none of those that did not become cancerous exhibited at least 2 of 3 characteristics: a MIBI labeling index (LI) exceeding 50%; a p53 LI exceeding 70%; suprabasilar extension of p53. Immunohistochemical staining for MIBI and p53 may help to identify those women with vulvar LS who are relatively likely to develop SCC. Periodic status checks or adjunctive biopsies would be appropriate for these patients.

Diagnostic and prognostic significance of cyclin A expression in low-grade astrocytomas: comparison with astrogliosis and high-grade tumours

Aim:Definitive distinction between low-grade astrocytoma and astrogliosis is a long-standing difficulty due to their similar histopathological characteristics. To clarify differences in biological significance, this study focused on various components of...

Relation between telomerase activity, hTERT and telomere length for intracranial tumours

Human linear chromosomes are capped by specialized DNA-protein structures called telomeres. The present study analysed the telomerase activity, hTERT protein and telomere length in meningiomas and gliomas in relation to their WHO grading. Fifty-three freshly dissected tumour biopsies were analysed for telomerase activity, hTERT protein expression and telomere length. Telomerase activity was examined in 41 of the 53 biopsies. Telomerase activity was detected in 3 of 35 (8.6%) screened meningiomas (1 benign, 1 atypical and 1 malignant meningioma). For hTERT expression, 56.4% of meningiomas were positive with a mean labelling index (hTERT LI) of 31.3% (SD=26.5) for the hTERT positive meningiomas. The mean telomere length for meningiomas was 6.983 kb (SD=1.969). For gliomas, no active telomerase was detected in 2 low-grade astrocytomas, whereas three of the four screened glioblastomas were positive for telomerase activity. The only hTERT protein positive astrocytoma had a mean labelling index of 9.0%. On the other hand, the hTERT LI for glioblastomas was 53.6% (SD=28.0). The two low-grade astrocytomas had a telomere length of 14.310 and 9.236 kb. The anaplastic astrocytoma had a telomere length of 4.903 kb and the glioblastomas 5.767 kb (SD=2.042). The normal meningeal and neuronal tissue is negative for telomerase activity and hTERT. The length was +/-10.000 kb. These results indicate that telomere shortening may be a critical step in pathogenesis of atypical and malignant meningiomas and gliomas. Critical telomere shortening in vitro was shown to activate telomerase.

Establishment of a novel orthotopic xenograft model of human gallbladder carcinoma

Gallbladder cancer is characterized by high morbidity and mortality. An appropriate human xenograft animal model could serve as a research tool to investigate new therapeutic strategies. To date, the few reports describing a xenograft animal model showed significant limitations. We improved a murine orthotopic human xenotransplantation model by implanting human gallbladder carcinoma cells directly into the lumen of the gallbladder. Mz-ChA-1 cells were orthotopically injected into the gallbladder of Severe Combined Immune Deficiency (SCID) beige mice inducing the growth of solid tumors. The natural course of the disease, tumor growth, and metastases were analyzed. The cytotoxic drug gemcitabine was tested in vitro and in vitro. All animals revealed solid tumors in the inoculated area with liver infiltration. The median tumor volume in the untreated group was significantly higher than in the gemcitabine-treated group. Immunohistochemical staining revealed expression of human cytokeratin 7 and cytokeratin 8. To analyze tumor cell proliferation, the tumors were stained for the antigen Ki-67, and labeling indices were calculated for both groups. Animals receiving gemcitabine treatment showed significantly lower mean labeling indices. In vitro investigation revealed a significant reduction of DNA synthesis. DNA fragmentation, as a measure of apoptosis, was elevated by roughly 20% within 24 h of treatment. With this, we successfully established an orthotopic xenotransplant animal model and investigated the in vitro and in vivo effects of gemcitabine in human xenografted Mz-ChA-1 gallbladder adenocarcinoma. This model resembles the clinical situation as closely as possible and offers a relevant option for the preclinical testing of new therapeutic strategies.

Expression patterns of cyclin E, cyclin A and CDC25 phosphatases in laryngeal carcinogenesis

The aim of the research was to evaluate the expression levels of cyclin E/A and CDC25A/B during laryngeal carcinogenesis. The expression was demonstrated using immunohistochemistry in 46 cases of laryngeal cancer (LSCC), 23 epithelial dysplasias (ED) and 21 samples of normal mucosa (NM). The mean labeling indices (LI) for cyclin E in LSCC, ED and NM were 10.6, 4.9 and 0%; for cyclin A 27.2, 17.5 and 7%; for CDC25A 73.9, 53 and 32% and for CDC25B 36.5, 25.9 and 0%, respectively. A gradual increase in cyclin A and CDC25A expression levels from mild through moderate and severe dysplasia to in situ carcinoma were noted. Cyclin A LI significantly increased also from NM through ED to LSCC. Cyclin A and CDC25A LI significantly increased from NM to ED. Overexpression of cyclin A and CDC25A was significantly associated with proliferation among ED. Linear interdependency was detected in ED between the expression of CDC25A and cyclin A. Cyclin E and CDC25B overexpression occurs as a late event in neoplastic transformation. The progressive expression of proteins supports the multistep model of laryngealcarcinogenesis. The results indicate a possible role of cyclin A as a marker reflecting cell proliferation. The enhanced immunoexpression of cyclin A and CDC25A suggests the potential for malignant formation in preneoplastic lesions.

Immunohistochemical Analysis of PCNA, Ki67 and p53 in Nasal Polyposis and Sinonasal Inverted Papillomas

Immunoreactivity of proliferating cell nuclear antigen (PCNA), Ki67 and p53 in inflammatory nasal polyp and inverted papilloma tissues was investigated. Immunohistochemistry was performed using a standard avidin-biotin-peroxidase method, and the immunoreactivity of PCNA, Ki67 and p53 was quantified by counting immunostained nuclei in at least 1000 epithelial cells. The mean labelling index (percentage of immunostained cells) for PCNA was 40.68 in the inverted papilloma group and 14.73 in the nasal polyp group, and for Ki67 was 15.43 in the inverted papilloma group and 2.64 in the nasal polyp group. Both of these differences between the inverted papilloma and nasal polyp groups were significant. Immunoreactivity for p53 was detected in five (35.7%) inverted papilloma patients and two (9.5%) nasal polyp patients. The increase in epithelial cell proliferation seemed to be greater in inverted papillomas than in inflammatory nasal polyps. Increased epithelial cell proliferation may be involved in the development of inverted papillomas.