Percentage Of Positive Nuclei Research Articles

Abstract P2-06-01: Ki67 Proliferative Index as an Invaluable Biomarker in Hormone Receptor (HR)-Positive Breast Cancer: Ki67 Labelling Index Can Reflect the Differences between Luminal A and B Subtypes Better Than HER2 Expression

Abstract Background: Differentiation by multigene signatures with excellent performance of hormone receptor (HR)-positive BC largely related to their proliferation genes. Despite questions about its usefulness, there is increasing evidence that Ki67 is a valuable prognostic marker. Standardization of Ki67 pathological assessment is the main problem to interpret reported trials. The aims of the study are to evaluate the role Ki67 as prognostic marker to predict relapse in HR-positive BC patients (luminal A and B) in adjuvant setting using prospective patients’ cohort. In addition, cut-off value and significant level of Ki67 were investigated comparing with other biomarkers including HER2 in luminal BCs. Method: We retrospectively analyzed the clinicopathologic characteristics of 1,070 postoperative breast cancer patients including Ki67 and clinical outcomes in terms of relapse free survival (RFS) between 2004 and 2007 at the Samsung Medical Center. Ki67 labelling index was measured in quantitative and semiquantitative method, independently. The percentage of positive nuclei stained for Ki67 was calculated each section based on the approximately 1,000 carcinoma cell nuclei. In addition, Ki67 was graded on a scale from 0 to 4, where 0 = staining of 0-4% of tumor cells, 1 = staining of 5-25% of tumor cells, 2 = 26-50% of tumor cells, 3 = staining of 51-75% of tumor cells, and 4 = staining of more than 76% of tumor cells. ROC curve was drawn to evaluate the usefulness of Ki67 index to get AUC then, find out the proper cut-off value of Ki67 to predict relapse. Multivariate analyses with Cox-regression model were performed. Results: Among 1,564 patients who received curative surgery for invasive breast cancer from January 2004 to June 2007, 1,070 patients with HR-positive were included in this analysis excluding 494 with HER2-enriched or triple negative breast cancer patients. Median follow-up duration was 56.9 months (range 36-77 months). Median age was 46 years (range 22-83 years). Ki67 threshold >19.5%, corresponding to a sensitivity 78.3%, a specificity 51.6% was chosen as cut-off value for relapse in adjuvant patients’ cohort. The AUC was 0.689 (P<0.0001 by Mann-Whitney U test). Overall relapse rate was 5.6%. In univariate analysis by log-rank test for relapse, ER negativity (p=0.010), HER2 positivity (p=0.017), histologic (p=0.001), and nuclear grade (p=0.012), lymphovascular invasion (P<0.0001), TNM stage (0.001), and Ki67 (P<0.0001) were identified risk factors to predict relapse. However, ER negativity, Ki67, and stage were identified as independent risk factors for relapse (Hazard Ratio (HR) 2.7, p=0.031 for ER negativity, HR 3.4, p <0.0001 for Ki67, HR 1.6, p=0.017 for stage) in Cox-regression multivariate analysis. Conclusion: Ki67 could strongly predict clinical outcomes for patients with luminal. 19.5% may be useful cut-off value of Ki67 labelling index. Ki67 may be a better biomarker to predict clinical outcomes than HER2 expression in luminal BCs. ER-/PR+ subset probably has different biology with other luminal BCs. Prospective clinical trials to choose therapeutic option using Ki67 are warranted. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P2-06-01.

Assessment of malignant potential of oral submucous fibrosis through evaluation of p63, E-cadherin and CD105 expression

BackgroundThe assessment of malignant potential of oral submucous fibrosis grades vis-à-vis their progression towards malignancy is associated with expression of possible multiple molecular markers.AimsTo analyse p63, E-cadherin and CD105 expression...

An Immunohistochemical Comparison of P53 and Bcl-2 as Apoptotic and MIB1 as Proliferative Markers in Low-Grade and High-Grade Ovarian Serous Carcinomas

Traditionally, ovarian serous carcinomas (OSCs) have been graded using a 3-tiered system, as well differentiated, moderately differentiated, and poorly differentiated. Recently, a 2-tiered system has been proposed depending on the nuclear atypia and mitotic count. The dualistic pathway of ovarian serous carcinogenesis with accumulating molecular genetic evidence forms the basis for this grading system. To investigate the expression of apoptotic proteins, p53 and bcl-2, and MIB1 index in low-grade and high-grade OSCs. Eighteen cases of low-grade OSCs and 28 cases of high-grade OSCs were stained immunohistochemically with antibodies against p53, bcl-2, and MIB1. For p53 and bcl-2, staining was evaluated on a semiquantitative scale depending on the number of cells showing positivity. For MIB1, the percentage of positive nuclei was calculated. Of 28 cases of high-grade OSCs, 15 (53.6%) showed 5+ staining with p53 compared with 3 (16.7%) of 18 cases of low-grade OSCs. Of 28 cases of high-grade OSCs, 10 (35.7%) showed 5+ staining with bcl-2 compared with 2 (11.1%) of 18 cases of low-grade OSCs. The mean MIB1 index was 42.1% in high-grade OSCs (range, 10%-90%) in contrast to the 19.4% (range, 10%-40%) in low-grade OSCs. The differences in apoptotic protein expression and proliferative index between the low-grade and high-grade OSCs were statistically significant (P < 0.05). The expression of apoptotic markers is higher in high-grade OSCs, which also have a higher proliferative activity compared with those in low-grade OSCs, which supports the dualistic pathway of ovarian serous carcinogenesis.

Can p53, Ki-67 and bcl-2 predict biochemical failure after radical prostatectomy?

Background and ObjectiveTo analyze p53, Ki-67 and bcl-2 expressions immunohistochemically and their predictive role in biochemical recurrence after radical prostatectomy.Materials and MethodsSeventy one patients who had undergone radical prostatectomy between 1992 and 2001 were randomly selected. Tissue microarrays were constructed from their radical prostatectomy specimens. They contained four cores from neoplastic and additional four cores from corresponding non-neoplastic regions. Gleason score ranged from 6-9, and pathological stage ranged from T2N0Mx to T3BN1. Staining for bcl-2 was scored visually taking percent negative, weak, moderate and strong positivity into consideration. Strong immunoreactivity was considered positive for p53. Ki-67 index was measured as the percentage of positive nuclei among tumor cells. Statistical analysis was performed to explore correlations between staining patterns and clinicopathological prognostic parameters.ResultsThe follow-up period extended from 13 to 112 months with a mean 60 (48 ± 23, 2) months. Of all, 38.02% had no evidence of disease, 52.1% were alive with disease and 9.8% were died during follow-up. The expression of p53, Ki-67 and bcl-2 in tumors were 39%, 76% and 5% respectively. While the secretory layer showed negative or weak bcl-2 staining in most cases, expression in basal cells was often stronger. Statistical analysis revealed differences in staining between normal and carcinoma for all three markers. There was no correlation between staining patterns and time to biochemical relapse. On the other hand, cases with higher Gleason sum showed the tendency for over expression of p53, Ki-67 and bcl-2 although the differences were not statistically different. Multivariate analysis revealed CMS group and seminal vesicle invasion as the independent predictors of PSA failure (log rank P = 0.0039 and P = 0.001, respectively).ConclusionThe proteins bcl-2, p53 and Ki-67 were expressed at a different rate in normal and neoplastic prostate tissue. Bcl-2 was mainly expressed by basal cells in normal glands. p53 and Ki-67 expression were increased in most prostate carcinomas. However, overall expression levels did not correlate with biochemical recurrence in this study.

CK2β Is Expressed in Endometrial Carcinoma and Has a Role in Apoptosis Resistance and Cell Proliferation

Protein kinase CK2 (CK2) is a serine/threonine kinase that participates in important cellular processes. We have recently demonstrated that CK2 plays a role in resistance to TRAIL/Fas-induced apoptosis in endometrial carcinoma (EC) by regulating FLIP. Here, we assessed the immunohistochemical expression of CK2beta in EC and checked its role in cell proliferation and anchorage-independent cell growth. CK2beta immunostaining was assessed in two tissue microarrays, one constructed from paraffin-embedded blocks of 95 ECs and another from 70 samples of normal endometrium. CK2beta expression was correlated with histological type; grade and stage; cell proliferation (Ki-67) and apoptotic index; immunostaining for cyclin D1, PTEN, AKT, beta-catenin, and FLIP. Moreover, the Ishikawa EC cell line was subjected to down-regulation of CK2 by shRNA. CK2beta expression was frequent in EC (nuclear, 100%; cytoplasmic, 87.5%). The staining was more intense in EC than in normal endometrium (P = 0.000), and statistically correlated with AKT, PTEN, beta-catenin, and FLIP. In EC, CK2beta expression correlated with cell proliferation. Knock-down of CK2beta blocked colony formation of EC in soft agar, and also resulted in decreased expression of cyclin D1 and ERK phosphorylation. The results confirm that CK2beta is widely expressed in EC, and suggest a role in cell proliferation and anchorage-independent cell growth.

Expression pattern of p63 in oral epithelial lesions and submucous fibrosis associated with betel-quid chewing in Sri Lanka

Betel-quid chewing, which is closely related to the high incidence of oral cancer, is prevalent in Sri Lanka. p63 has a remarkable structural similarity to p53, suggesting an aberrant expression in oral cancer. Using anti-p63 antibody and immunohistochemistry, the present study investigated the expression pattern of p63 in oral epithelial lesions, including different types of squamous cell carcinoma (SCC), different grades of epithelial dysplasia, and submucosal fibrosis associated with betel-quid chewing. Nuclear immunoreactivity for p63 was detected in all the cases, including normal oral epithelium and epithelial lesions. In normal oral epithelium, nuclear positivity for p63 was observed in some of the basal cell layers and focally in the parabasal layer. Nuclear positivity increased in the epithelial lesions. The percentage of positive nuclei in the epithelial lesions was significantly higher than in normal epithelium (P < 0.01) and was also significantly higher in oral submucosal fibrosis than in epithelial dysplasia (P < 0.05). The results indicate that the overexpression of p63 in oral precancerous lesions and SCC in betel-quid chewers in Sri Lanka may be a useful marker for oral precancerous lesions.

Phase II breast cancer chemoprevention study with celecoxib in women at increased risk for breast cancer

1010 Background: Short-term chemoprevention trials offer a convenient model to screen chemopreventive agents and identify endpoint biomarkers. One of the potential agents is celecoxib (C), which has antiproliferative and apoptosis inducing properties. In this prospective study, our primary aim was to evaluate changes in proliferation induced by C in breast tissue of high risk women. Here, we report changes in estrogen receptor (ER) proliferation index. Methods: 42 eligible high risk women were enrolled into the study, underwent fine needle aspiration (FNA) and started celecoxib treatment at 400 mg BID. Median age: 51.9 years. Risk factors: Gail risk &gt; 1.67% (n=13), lobular carcinoma insitu (n=13), atypical hyperplasia (n=11), previous history of breast cancer (n=5). For ER and Ki-67 testing, thin preparations slides were incubated with primary mouse monoclonal antibody 6F11 against the ER and clone MIB-1, respectively. Appropriate negative and positive controls were included. At least 100 epithelial cells were evaluated per slide. Immunoreactivity for each marker was scored as the percentage of positive nuclei. We assessed the difference in ER and Ki-67 levels before and after treatment using a Wilcoxon signed rank test. Results: The average pre-treatment ER expression in FNA samples was 35.9% and Ki-67 was 2.4%. 19 (45%) showed hyperplasia or atypical hyperplasia. 39 patients underwent also post-treatment FNAs. The pre-and post treatment ER expression in this group was 35.7% (range 0–100%) and 27.4% (range: 0–100%), respectively. The difference in ER levels was statistically significant (p = 0.04). Twenty-six patients had Ki-67 levels measured both before and after treatment. The median difference in Ki-67 levels was 0 (range 0- 5). This change was not statistically significant (p = 0.63). Conclusions: We have completed accrual to a prospective short-term chemoprevention trial with celecoxib. We have found a significant downregulation of ER expression with 6 months celecoxib. Since ER expression is a marker of proliferation, this finding confirms celecoxibs antiproliferative properties. Currently, we have not observed a change in Ki-67; this could be partly due to the small number of samples and the fact that Ki-67 is low in normal epithelium. [Table: see text]

A Strategy for Defining Biologically Relevant Levels of p53 Protein Expression in Clinical Samples with Reference to Endometrial Neoplasia

Numerous studies have investigated p53 immunohistochemistry as a diagnostic, prognostic or predictive marker in various neoplasms. However, the literature is confused and contradictory paying little attention to pivotal aspects of p53 biology nor basic immunohistochemical principles. Here we highlight the effect of varying antibody concentration on p53 immunohistochemical expression. Six uterine endometrioid carcinomas, one uterine serous carcinoma (USC) and a proliferative endometrium were stained with antip53 antibody D07 at varying dilutions. Cases were scored on a scale of 0-6 depending on the percentage of positive nuclei. Greater than 95% of epithelial cells in the USC were positive at all dilutions. The proliferative endometrium exhibited positive staining of >95% of epithelial cells at low dilutions but at high dilutions most epithelial cells were negative. The proportion of positive tumor nuclei in the endometrioid carcinomas varied markedly with antibody concentration. We illustrate that the signal obtained from p53 immunohistochemistry is dependent on antibody concentration. This has diverse implications. First, the p53 labeling index is unreliable without attention to such issues. Second, comparisons between studies are not valid since different antibody concentrations (and other variables) are used. Thirdly, standardization between laboratories is necessary if clinical utility is to be attached to markers including p53. We propose a strategy for determining the optimal p53 antibody concentration.

High expression of skp2 correlates with poor prognosis in endometrial endometrioid adenocarcinoma

Skp2 interacts with the degradation of cyclin-dependent kinase inhibitor p27. This study aimed to investigate the correlation of skp2 expression with the expression of p27 and other cell cycle regulators, and clinicopathological parameters in endometrial endometrioid adenocarcinoma. Tissue samples of 136 endometrioid adenocarcinomas, in addition to 20 endometrial hyperplasias and 20 normal endometria, were immunohistochemically stained for skp2. The expression was represented as a labeling index (LI), which indicates the percentage of positive nuclei. Skp2 staining was localized in the nuclei of the glandular cells of the proliferative phase endometrium, and endometrial hyperplasia and carcinoma cells. Skp2 expression was increased significantly in those of higher histological grade. The high level of skp2 expression was significantly correlated with the presence of lymph node metastasis and lymph-vascular space involvement. The LI of skp2 in endometrial carcinoma was significantly correlated with that of p27, Ki-67, cdk2, cyclin A, cyclin D1, cyclin E, p53 and PTEN. The high level of skp2 expression (LI> or =20%) was significantly correlated with the patients' poor survival. The skp2 level might have increased due to p27 accumulation and may be a good indicator of proliferative activity and poor prognosis.

An Immunohistochemical Comparison Between Low-Grade and High-Grade Ovarian Serous Carcinomas

Ovarian serous carcinoma (OSC) is the most common ovarian epithelial malignancy. Recently, a dualistic pathway of ovarian serous carcinogenesis has been proposed based on morphologic observations and molecular genetic analysis. In this scheme, low-grade OSC arises in a stepwise fashion from a benign serous cystadenoma through a usual serous borderline tumor through a micropapillary variant of serous borderline tumor. In contrast, the more common high-grade OSC arises de novo from the ovarian surface epithelium or the epithelium of cortical inclusion cysts with an as yet unrecognized precursor lesion. Although the division of OSC into low- and high-grade variants is gaining greater acceptance, and although there is accumulating molecular genetic evidence for this, there is little published information regarding a comparison of protein expression between these two types of OSC. In this study, we have investigated the immunohistochemical expression of a wide range of proteins in cases of low-grade (n = 22) and high-grade (n = 47) OSC. Antibodies used were p53, MIB1, BCL2, WT1, HER-2/neu, C-KIT, osteopontin, and survivin. For all antibodies, except MIB1, cases were scored as 0 (negative or occasional positive cells), 1+ (<10% cells positive), 2+ (10%-25% cells positive), 3+ (26%-50% cells positive), 4+ (51%-75% cells positive) or 5+ (>75% cells positive). For MIB1, the percentage of positive nuclei was calculated. There was a statistically significant higher expression of p53, MIB1, BCL2, HER-2/neu, and C-KIT in high-grade compared with low-grade OSC (P < 0.05). Thirty of 47 (64%) cases of high-grade OSC exhibited 5+ staining with p53 compared with 4 of 22 (18%) low-grade neoplasms. Twelve of 47 (26%) high-grade OSCs exhibited 5+ staining with BCL2 compared with 1 of 22 (5%) low-grade OSCs. The mean MIB1 proliferative index in high-grade OSCs was 55.4% compared with 23.0% in low-grade OSCs. Virtually all cases of both low-grade and high-grade OSCs exhibited diffuse nuclear positivity with WT1 and diffuse cytoplasmic positivity with survivin. Osteopontin expression was variable with no significant difference in expression between low-grade and high-grade OSC. Although expression of both HER-2/neu and C-KIT was significantly higher in high-grade compared with low-grade OSC, only rare cases exhibited strong positivity with these antibodies, which could be of therapeutic value in individual cases, although this would require additional molecular investigations. The significant differences in protein expression between low-grade and high-grade OSC provides further support for a different underlying pathogenesis. In particular, the differences in p53 immunoreactivity are in keeping with the observation that p53 gene mutation is more common in high-grade than low-grade OSC.

Modulation of peroxisome proliferator-activated receptors (PPARs) by PPARα- and PPARγ-specific ligands and by 17β-estradiol in isolated zebrafish hepatocytes

Peroxisome proliferation is a phenomenon occurring when responsive animals are exposed to certain compounds so-called peroxisome proliferators and is regulated through a nuclear receptor named peroxisome proliferator-activated receptor (PPAR). PPAR family members exhibit a strong binding affinity for both saturated and unsaturated fatty acids. Activators of PPARα include a variety of endogenously present fatty acids, leukotrienes and hydroxyeicosatetraenoic acids (HETEs) and clinically used drugs, such as fibrates. PPARβ activators include fatty acids, prostaglandin A 2 (PGA 2) and prostacyclin (PGI 2). PPARγ is the most selective receptor and, among others, 15-deoxy-Δ 12,14 prostaglandin J 2 (PGJ 2) has been described to be a PPARγ-specific ligand. The aim of the present study was to determine if known PPARα and PPARγ ligands were able to alter the expression of these subtypes in an in vitro model of zebrafish primary hepatocyte culture. With this purpose, a PPARα specific ligand (8S-HETE), a PPARγ specific ligand (PGJ 2) and a peroxisome proliferator of the fibrate class (clofibrate) were selected. In addition, the female hormone 17β-estradiol was also used as it is known to interact with PPARs. After cell exposure for 24 h, cells were immunohistochemically stained for both PPARs and immunolabeling was quantified as percentage of positive nuclei and cells. Levels of expression of PPARs were also measured by image analysis as grey level per cell. Expression was induced for both PPARα and PPARγ by clofibrate (at 0.5 mM for PPARα and at 1 and 2 mM for PPARγ), by HETE (1 μM), and by PGJ 2 (0.3 and 1 μM for PPARα and 0.3 μM for PPARγ). Expression of PPARγ was also induced at 10 μM by 17β-estradiol. The percentage of PPARα positive nuclei increased significantly at 1 μM HETE and the percentage of PPARγ positive cells decreased at 10 μM 17β-estradiol. As a conclusion, clofibrate, HETE and PGJ 2 are able to induce expression of both PPARα and PPARγ in zebrafish primary hepatocyte cultures. Further studies are needed to identify how the expression of different PPAR subtypes is regulated and to elucidate the implication of PPAR subtypes in zebrafish cell functions.

Baseline proliferation markers and cytopathologic findings in breast epithelium of women at increased risk for breast cancer

9694 Background: The aim of phase II chemoprevention (CP) trials is to screen potential chemopreventive agents and to identify surrogate endpoint biomarkers that can be used in large prevention studies. Tamoxifen is approved for the risk reduction of breast cancer and was shown to reduce proliferation in breast epithelium. Therefore, proliferation markers are one of the potential markers, which can be used in phase II CP studies. We are currently conducting 2 prospective clinical prevention studies evaluating changes in proliferation and apoptosis induced by celecoxib (C) or anastrazole (A) in breast tissue of high risk women. Here, we report baseline cytopathologic findings, expression of ER and Ki-67. Methods: High risk women were enrolled and underwent baseline fine needle aspiration (FNA) and started C (400 mg BID) or A (1 mg QD). ER and Ki-67 in FNA was assessed using immunohostochemistry. Immunoreactivity was scored as the percentage of positive nuclei; ≥ 10% was considered positive for ER. Ki-67% w...

Desdiferenciação do câncer da próstata após terapia antiandrogênica

Neoadjuvant androgen deprivation in prostate cancer induces tumor volume regression but does not improve outcome of the patient. A possible explanation for this phenomenon could be an increase of the residual tumor aggressiveness brought about by antiandrogen therapy. The purpose of the present study was to evaluate the frequency of tumor dedifferentiation following androgen blockade in prostate cancer and to determine if the remaining tumor shows signs of increased aggressiveness. Thirty patients bearing locally advanced prostate cancer (stages T2c - T3) were submitted to neoadjuvant anti-androgenic therapy during four months followed by radical prostatectomy. Gleason scores from biopsy and surgical specimens were compared. Furthermore, the cell proliferation index was evaluated by immunohistochemistry assay for PCNA, tests with strong nuclear staining were considered positive. The percentage of positive nuclei, counted in 500 cells, was determined in several categories of the Gleason score from surgical specimens. In 11(37%) surgical specimens the Gleason score was equal or lower than that found in the biopsy and in 19 (63%) the total score was higher in the surgical specimens (p<0.05). The median of PCNA expression was 4.5%, 10%, 12% and 14% in Gleason scores 2-4, 5-6,7 and 8-10, respectively (p>0.05). The median of cell proliferation indexes was 9% for glandular or specimen confined tumors and was 17% for extraprostatic tumors (p<0.05). The lower Gleason score was found in almost 2/3 of patients submitted to antiandrogen therapy. However, the cell proliferation index measured by PCNA was the same for tumors with lower or higher Gleason scores. It seems that cell dedifferentiation seen after neoadjuvant androgen deprivation represents a mere morphologic phenomenon and not a real increase in tumor aggressiveness.

Expression of tumor suppressor gene product p14ARF in endometrioid adenocarcinoma of the uterine corpus.

p14 activates p53 by inhibiting MDM2 expression and arrests the cell cycle in G1 and G2/M. Abnormal p14 expression has been reported in various human cancers. This study investigated p14 expression in endometrioid adenocarcinoma of the uterine corpus in an attempt to clarify its correlation with other cell cycle-regulators and clinicopathologic parameters. The specimen studied consisted of 124 endometrioid adenocarcinomas, 20 normal endometria, and 20 endometrial hyperplasias. Immunohistochemical staining of formalin-fixed and paraffin-embedded tissues was performed using a Catalyzed Signal Amplification System. Cells with >5% positive staining were classified as positive for p14. A staining score of 1 was adopted when the percentage of positive nuclei was <5%, a score of 2 when it was 5 to 50%, and a score of 3 when it was >50%. In normal endometrium, the frequency of positive staining in the proliferative phase and secretory phase was 50% (4/8) and 58.3% (7/12), with staining scores of 1.8+/-0.9 and 1.6+/-0.5, respectively. The frequency of staining in simple hyperplasia (SH), complex hyperplasia (CH), and complex atypical hyperplasia (CAH) was 88.9% (8/9), 25% (1/4), and 42.9% (3/7), respectively; the staining scores were 1.9+/-0.3, 1.3+/-0.5, and 1.4+/-0.5, respectively. Among endometrioid adenocarcinomas, the frequency of staining of well-differentiated (G1), moderately differentiated (G2), and poorly differentiated (G3) adenocarcinomas was 69% (49/71), 64% (16/25), and 42.9% (12/28) respectively, with staining scores of 2.1+/-0.8, 2+/-0.9, and 1.8+/-1, respectively. Thus expression levels of p14 were higher in G1 tumors than in normal endometria or endometrial hyperplasias, and the frequency of its staining in endometrioid carcinomas was inversely correlated with histologic grade. The staining score for endometrioid adenocarcinomas also was inversely correlated with the labeling index (LI) of Ki-67, but not with that of cyclin A, cyclin D1, cyclin E, cdk2, p27, p53, or other clinicopathologic parameters. In conclusion, p14 expression correlated with histologic grade and Ki-67, but not other prognostic factors in endometrioid adenocarcinoma. Long-term follow-up studies are needed to analyze the significance of p14 expression in these tumors.

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 markers 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 relationship 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 expression 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.

Effects of EGF on expression of phosphorylated p44/42 MAPK in rat small intestine after ischemia-reperfusion injury

AIM:To investigate the effects of EGF on the characteristics of phosphrylated p44/42 MAPK expression and its biological significance in EGF-induced gut repair after ischemia-reperfusion (I/R) injury. METHODS:A total of 80 Wistar rats were randomly divided into four groups, namely EGF treated group (E), normal saline control (R), ischemia group (I) and sham operated control (C). In group E and R, the rats were treated with intravenous EGF 100 µg/kg/rat or normal saline respectively after 45 minutes of superior mesenteric artery occlusion. Blood samples were collected at 2, 6, 12 and 24 hours after reperfusion and plasma D-lactate were determined. Tissue samples from intestine were also taken for histological analysis and immunohistochemical analysis of phospho-p44/42 MAPK. RESULTS:The changes of histological structure and D-lactate indicated that the intestinal barrier was damaged after intestinal I/R injury, while EGF treatment significantly improved the outcome. In group C and I positive signals of phospho-p44/ 42 MAPK were mainly located in the cytoplasm of the intestinal villi and crypts, while in group I positive cells increased significantly (P <0.05). In group R, positive signals were found in almost all the cells and the percentage of positive nuclei increased with the time of reperfusion, reaching its peak after 12h of reperfusion. In group E, the percentages were higher than those in group R and the peak of nuclear expression was earlier. CONCLUSION:EGF administration improves the outcome of I/R induced intestinal damage. After I/R the expression and nuclear translocation of phspho-p44/42 MAPK increases with the time of reperfusion, suggesting its role in intestinal reconstitution. EGF treatment induces its early expression and translocation into the nucleus, suggesting the significance of p44/42 MAPK signaling pathway in EGF-induced gut repair. Li P, Xin F, Fu XB, Yang YH, Guo BC. Effects of EGF on expression of phosphorylated p44/42 MAPK in rat small intestine after ischemiareperfusion injury. Shijie Huaren Xiaohua Zazhi 2003;11(5):578-582

Immunohistochemical expression of cyclin E in endometrial adenocarcinoma (endometrioid type) and its clinicopathological significance.

Cyclin E is known as a G1-S phase regulatory protein and its abnormal expression has been implicated in cellular proliferation. This study aimed to investigate the correlation of cyclin E expression with tumorigenesis of the endometrium, proliferative activity, and clinicopathological features of endometrial adenocarcinoma. Immunohistochemical staining for cyclin E in addition to cyclin-dependent kinase 2 (cdk2), Ki67, p27, and p53 was performed by the labeled streptavidin-biotin method on formalin-fixed, paraffin-embedded tissues of normal endometria (20 cases), endometrial hyperplasias (20 cases), and endometrial adenocarcinomas (endometrioid type) (127 cases). Positive staining was expressed as a labeling index (LI) based on percentages of positive nuclei in tumor cells. Immunohistochemistry showed that the nuclei of the cells were positive for cyclin E. Both proliferative and secretory endometria, and endometrial hyperplasia regardless of type were negligible for cyclin E expression. The expression in normal endometrium and hyperplasia was significantly less than that in endometrial adenocarcinomas (P<0.0001). LIs of cyclin E in well-differentiated, moderately differentiated, and poorly differentiated endometrial adenocarcinomas were 31.5+/-33.3%, 37.8+/-31.9%, and 51.1+/-30.8%, respectively. Cyclin E expression increased significantly more in histological grades. The LI of cyclin E in carcinoma was positively correlated with that of cdk2, Ki67, and p53 but not with p27. The cyclin E expression was correlated with myometrial invasion and lymph-vascular space involvement, but not with FIGO stage, lymph node metastasis, coexisting endometrial hyperplasia, estrogen receptor, progesterone receptor, and menopause. Cyclin E as a complex with cdk2 is associated with carcinogenesis and disease progression in endometrial adenocarcinoma, and might be a prognostic indicator of endometrial adenocarcinoma.

HnRNP B1 expression in benign and malignant lung disease.

Evidence is accumulating to suggest that hnRNP B1 expression may be a useful tool in the early diagnosis of lung cancer. This study examined the immunohistochemical expression of hnRNP B1 in archived sections of resected lung cancers and compared the patterns of expression with those seen in similar archived sections of non-neoplastic lung. Particular attention was paid to the expression of hnRNP B1 in the benign bronchial cells in both cases, to establish if overexpression of this protein in respiratory epithelial cells is specific for malignancy. Nineteen cases of different types of non-small cell carcinoma were examined (eight squamous cell, six adenocarcinomas, two carcinosarcomas, two undifferentiated large cell carcinomas, and one mucoepidermoid carcinoma) and compared with sections from 16 open lung biopsies (three cases of cryptogenic fibrosing alveolitis, two cases of sarcoidosis, two cases of organizing pneumonia, and one case each of tuberculosis, extrinsic allergic alveolitis, non-specific interstitial pneumonitis, pneumocystis pneumonia, aspergilloma, respiratory bronchiolitis-interstitial lung disease, mineral dust disease, Sjögren's syndrome and systemic sclerosis vascular variant). All the tumours showed positive staining, with the vast majority, 16/19 (84%), showing strong diffuse nuclear staining. The background cells of these cases showed positive staining in alveolar macrophages, lymph node germinal centres, bronchial mucous glands, and bronchial epithelial cells. No significant difference was seen in the percentage of positive bronchial epithelial cells in bronchi adjacent to the tumour compared with the resection margins. In the benign lung cases, positive bronchial epithelial cells were seen in a small percentage, 3/16 (18%), of cases, but the majority of cases showed no or very focal staining. The levels of expression between benign epithelial cells of malignant cases, compared with benign, showed a significant difference when the staining was assessed in percentage of positive nuclei (p = 0.001, Fisher's exact test). The results confirm that hnRNP B1 is widely expressed in a range of lung carcinomas; that expression is seen in benign bronchial epithelial cells and inflammatory cells; and that expression in background bronchial epithelial cells appears to be higher in malignant than in benign lung disease. It is feasible that this biomarker may be of use in the detection of early lung cancer, provided that levels of expression can be accurately quantified.

Immunohistochemical study of cell cycle regulatory proteins in intraductal breast carcinomas—a preliminary study

The aim of this study was to assess the levels of cell cycle regulatory proteins p21waf1 (p21), p53, Cyclin A, Cyclin D1 and Ki-67 to see whether they correlated with recurrence-free survival (RFS). From 1982 to 1996, 50 patients aged less than 51 years underwent lumpectomy followed by radiotherapy for a pure ductal carcinoma in situ (DCIS). For each case, the following immunohistochemical stains were carried out: Ki-67, Cyclin A, Cyclin D1, p53 and p21waf1 (p21). The percentage of positive nuclei was assessed. Multiple combinations of these factors were performed; in particular, we called the sum of Ki-67 and Cyclin A a global proliferation factor (GPF). Correlations with classical clinicopathological data were assessed. After a multivariate analysis, only GPF, Van Nuys Prognostic Index (VNPI) grade and mitotic index were independent predictive factors of recurrence in the whole population. In the population with close surgical margins, when the GPF level was less than the 25th percentile or more than the 75th percentile recurrence was low. In this preliminary study, GPF seems to be of interest to help in the decision process in the post-surgical management of the patient.

Prognostic significance of p21WAF1/CIP1 expression in tongue squamous cell carcinomas.

To investigate the prognostic significance of p21(WAF1/CIP1) expression and its relationship with p53 accumulation and other apoptotic markers such as Bax, Bcl-2, and apoptotic index in relation to disease-specific survival in oral tongue squamous cell carcinoma (SCC). A 10-year retrospective clinical study. Information about clinical findings, treatment, and follow-up has been recorded prospectively. Diagnostic, formalin-fixed, paraffin-embedded sections taken from 80 randomly selected patients before treatment for T1 to T4 oral tongue SCC were stained immunohistochemically with p21(WAF1/CIP1). The percentages of positive nuclei that stained positive for tumor were determined. The significance of prognostic parameters was tested by log-rank and Kaplan-Meier methods for the univariate analysis. The Cox proportional hazards regression model was used for multivariate analysis. Expression of p21(WAF1/CIP1) correlated inversely with T classification and clinical stage (P =.02 and P =.006, respectively) but not with N classification, tumor differentiation, or apoptosis-related variables such as p53 accumulation, apoptotic index, and Bax and Bcl-2 expression. Patients with tumors expressing high p21(WAF1/CIP1) values had increased disease-specific survival time (P =.03). The correlation between p21(WAF1/CIP1) expression and disease-specific survival was even more significant when restricted to p53-negative tumors (P =.002). A 2-parameter combination between p21(WAF1/CIP1) expression and Bax expression or p21(WAF1/CIP1) expression and p53 accumulation, respectively, revealed an enhanced prognostic potential (P<.001) when compared with single parameters. The expression of p21(WAF1/CIP1), particularly in combination with p53 accumulation or Bax expression, has prognostic value in oral tongue SCC.