DNA ploidy and immunomarking of bladder urothelial tumors before and after intravesical bacillus Calmette-Guérin treatment

OBJECTIVE: To investigate DNA ploidy and immunoexpression of Ki-67 and p53 as predictivefactors in cases of superficial urothelial cell carcinoma (UCC) treated with bacillus Calmette-Guérin (BCG). STUDY DESIGN: Samples were obtained from 66 patients with UCC (pTa grade 3 or high grade and pT1 independent of grade or with concomitant carcinoma in situ) before and after intravesical BCG treatment. DNA ploidy analysis (ploidy balance, degree of hyperploidy and aneuploidy, proliferation index) was done by static cytometry. Ki-67 and p53 were analyzed immunohistochemically in paraffin-embedded tissue, and their quantification was carried out using an image analysis system. RESULTS: During a mean follow-up of 63.8 months, 31 of the 66 patients developed recurrent tumors (46.9%). DNA ploidy analysis showed that ploidy balance as well as degree of hyperploidy and aneuploidy were not statistically different between recurrent and nonrecurrent tumors. Only proliferation index was statistically significant between recurrent and nonrecurrent tumors. No statistically significant difference was observed in the percentage of Ki-67- and p53-positive cells between primary tumors that recurred and those that did not. CONCLUSION: These findings suggest that only proliferation index has predictive value for recurrence and progression in UCC treated with BCG.     

DNA ploidy, Ki-67 and p53 as indicators of lymph node metastasis in early gastric carcinoma

OBJECTIVE: To perform flow cytometric and immunohistochemical analysis of early gastric carcinoma in order to investigate the clinical value of DNA ploidy, Ki-67 and p53 as indicators of lymph node metastasis. STUDY DESIGN: We studied 108 cases of surgically resected early gastric carcinoma, which comprised 21 cases showing positive lymph node metastasis (group A) and 87 cases showing negative lymph node metastasis (group B). RESULTS: Aneuploidy was seen in 16 cases (76.2%) in group A in contrast to 10 cases (11.5%) in group B (P = .0029). The mean value of the Ki-67 labeling rate in all cases was 21.9%, and 15 cases (79.0%) showed a Ki-67 labeling rate of more than 21.9% in group A, while they numbered 31 (37.4%) in group B (P = .0016). Cases with cell positive for p53 protein numbered 15 (75%) in group A, while they numbered 38 (46%) in group B (P = .0460). CONCLUSION: DNA aneuploidy, a high labeling rate of Ki-67 and overexpression of p53 are all useful indicators of lymph node metastasis in patients with early gastric carcinoma.     

Prospective study on prognostic significance of DNA ploidy and Ki-67 expression in colorectal cancer

The aim of the study was to correlate tumoral DNA ploidy and Ki-67 expression with therapy response, Overall Survival (OS), Disease Specific Survival (DSS) and Disease Free Survival (DFS). Three samples of colorectal cancer were collected from each patient. One sample of normal tissue was our internal control. DNA ploidy was evaluated by FACSCalibur cytometer and Ki-67 by immunohistochemistry. We studied 67 patients and we found aneuploidy in 65,7 percent of carcinoma with a Ki-67 median expression of 55 percent. After surgery and chemotherapy in 35 percent of the patients with aneuploid carcinoma and high proliferative activity (Ki-67 greater than 55 percent) there were no evidence of disease versus 100 percent of patients with DNA diploidy and low proliferative activity (Ki-67 less than 55 percent). Tumoral aneuploidy significantly correlated with lower OS, DSS and DFS (18 percent vs 86 percent at 30 months). Univariated analysis demonstrated a significant correlation between aneuploidy and develop disease progression (p=0,033, odd ratio=5.7), while the cut-off of 55 percent for Ki-67 expression did not correlate with OS, DSS and DFS. Preliminary results (the study is still in progress) seemed to suggest that DNA ploidy has a prognostic and predictive significance in colorectal carcinoma.     

Independent prognostic value of hormone receptor expression and S-phase fraction in advanced breast cancer

OBJECTIVE: TO assess the prognostic value of immunocytochemically assessed hormone receptor expression and DNA flow cytometry data in advanced breast cancer. STUDY DESIGN: This prospective study with long-term follow-up evaluated the above parameters in relation to overall survival in 392 patients with advanced breast cancer (stages IIB, n = 106; IIIA, n = 66; IIIB, n = 174; and IV, n = 46) using fine needle aspiration cytology. RESULTS: Estrogen and progesterone receptor positivity was detected in 65.1% and 46.1% of the tumors, respectively. DNA aneuploidy was present in 70.9% of the cases, and the median S-phase fraction (SPF) was 9.4%. There was a significant correlation of aneuploidy and high SPF with lack of hormone receptors. The median SPF and SPF tertiles (cutoff values, 6.5% and 12%), applied in the whole series, showed a significant correlation with survival, whereas if SPF was used according to ploidy status, no prognostic significance was found. No differences in relation to survival among different DNA aneuploidy subclasses were verified. In univariate analysis, clinical staging, hormone receptors, DNA ploidy and SPF showed a statistically significant correlation with the overall survival. In multivariate analysis only DNA ploidy did not maintain independent prognostic significance. CONCLUSION: Hormone receptor expression and flow cytometric SPF are independent prognostic factors in advanced breast cancer.     

Prognostic factors in prostate cancer

Prognostic factors in organ confined prostate cancer will reflect survival after surgical radical prostatectomy. Gleason score, tumour volume, surgical margins and Ki-67 index have the most significant prognosticators. Also the origins from the transitional zone, p53 status in cancer tissue, stage, and aneuploidy have shown prognostic significance. Progression-associated features include Gleason score, stage, and capsular invasion, but PSA is also highly significant. Progression can also be predicted with biological markers (E-cadherin, microvessel density, and aneuploidy) with high level of significance. Other prognostic features of clinical or PSA-associated progression include age, IGF-1, p27, and Ki-67. In patients who were treated with radiotherapy the survival was potentially predictable with age, race and p53, but available research on other markers is limited. The most significant published survival-associated prognosticators of prostate cancer with extension outside prostate are microvessel density and total blood PSA. However, survival can potentially be predicted by other markers like androgen receptor, and Ki-67-positive cell fraction. In advanced prostate cancer nuclear morphometry and Gleason score are the most highly significant progression-associated prognosticators. In conclusion, Gleason score, capsular invasion, blood PSA, stage, and aneuploidy are the best markers of progression in organ confined disease. Other biological markers are less important. In advanced disease Gleason score and nuclear morphometry can be used as predictors of progression. Compound prognostic factors based on combinations of single prognosticators, or on gene expression profiles (tested by DNA arrays) are promising, but clinically relevant data is still lacking.     

Prognostic evaluation of breast cancer in cytologic specimens

OBJECTIVE: To evaluate prognostic factors in breast cancer using cytologic samples and to determine the correlation between those factors and ploidy. STUDY DESIGN: Two hundred sixteen fine needle aspirates from patients with primary breast cancer were analyzed for expression of estrogen receptors (ERs), progesterone receptors (PRs), Ki-67 antigen, expression of p53 tumor suppressor gene and overexpression of c-erbB-2 using a standard immunochemical method. Not all subjects had all biomarker information because of the study design (c-erbB2 added later). The specimens were analyzed also for ploidy. We used the SAMBA 4000 image analysis system for quantification of the percent of cells stained positively by the different immunocytochemical stains andfor ploidy. RESULTS: A significant correlation wasfound between ER and PR and between Ki-67 and positive p53. Steroid receptor content was not significantly related to p53, Ki-67 or c-erbB2. No correlation was found between c-erbB2 and the other biomarkers. Ploidy had a significant correlation with all the biomarkers used. CONCLUSION: A reliable and rapid evaluation of markers for breast cancer can be achieved by measuring cells stained positively by immunocytochemical stains, as well as ploidy, by means of an image analysis system. ER, PR Ki-67, p53 and c-erbB2 had a significant correlation with ploidy and overall prognostic value in breast cancer.     

Overall survival in advanced breast cancer: relevance of progesterone receptor expression and DNA ploidy in fine-needle aspirates of 392 patients

Fine-needle aspiration cytology (FNAC) is essential for making a diagnosis in advanced breast cancer. The determination of hormone receptors in the material obtained is useful for predicting patient response to endocrine therapy, but the prognostic value of hormone receptor expression as well as the clinical utility of DNA flow cytometry are controversial. The aim of this prospective study with long-term follow-up (median: 81 months) was to evaluate these biomarkers in relation to overall survival in a series of 392 patients with advanced breast cancer (stage IIB, n=106; IIIA, n=66; IIIB, n=174; and IV, n=46) using FNAC. Estrogen and progesterone receptor expression was found in 65.1% and 46.1% of the tumors, respectively. Hormone receptors were not found to be associated with clinical staging. DNA aneuploidy was present in 70.9% of the cases and the median S-phase fraction (SPF) was 9.4%. There was a significant correlation of aneuploidy and high SPF with lack of hormone receptors. In univariate analysis, advanced disease stage, absence of hormone receptors, DNA aneuploidy and high SPF showed a statistically significant correlation with poor clinical outcome. In multivariate analysis, disease stage, progesterone receptors and DNA ploidy retained independent prognostic significance in relation to overall survival. These data indicate that progesterone receptor expression and DNA ploidy are independent prognostic factors in advanced breast cancer.     

Expression of p120, Ki-67 and PCNA as proliferation biomarkers in imprint smears of prostate carcinoma and their prognostic value

The cell proliferation markers p120, Ki-67 and proliferating cell nuclear antigen (PCNA) recognize nuclear antigens. The expression of these proteins by immunostaining methods was reported to be of value in determining the prognosis of patients with malignant diseases. In this study, we evaluated the prognostic significance of the expression of nuclear antigens p120, PCNA and Ki-67 in prostate cancer and compared the results with other prognostic factors. Imprint smear samples obtained from 70 patients immediately after radical prostatectomy for prostatic carcinoma were immunostained with monoclonal antibodies against p120, Ki-67 and PCNA. The immunostaining results were correlated with Gleason score, tumour differentiation, stage and prostatic specific antigen (PSA) levels. Our findings demonstrate that p120, Ki-67 and PCNA expression in prostatic carcinoma smears, correlated significantly with the degree of Gleason score (P < 0.001). When combining p120, Ki-67 and PCNA positivity with tumour differentiation there was a significant association among these parameters (P < 0.001). Overexpression of p120, Ki-67 and PCNA, was also associated with increased PSA serum levels (>4 ng/ml) (P < 0.001). The distribution of p120, Ki-67 and PCNA expression in prostate carcinomas was not statistically significant for Ki-67 (P = 0.69) and p120 (P = 0.22) but was significant for PCNA (P < 0.001) as far as the histological stage (T2a, T2b, T2c, T3a). P120, Ki-67 and PCNA expression had significant prognostic value for disease-free survival. Our results conclude that nuclear antigens p120, Ki-67 and PCNA appear to be additional markers in the field of prognosis of prostatic carcinoma.     

Human papillomaviruses and DNA ploidy in anal condylomata acuminata

Previous studies have emphasized the usefulness of DNA ploidy measurement and Human Papillomavirus (HPV) detection as prognostic markers in low grade cervical lesions. We addressed the eventual relationship between HPV type, DNA profile, and p53 tumor suppressor protein expression in anal condylomata acuminata to eventually determine parameters which may be considered as predictive risk factors for the development of cancer. DNA ploidy was assessed by image cytometry after Feulgen staining of contiguous serial sections of 45 anal condylomata acuminata without atypia containing HPV detected by in situ hybridization and Polymerase Chain Reaction (PCR). p53 expression was detected by immunohistochemistry. DNA aneuploidy was found in 53.3% of these lesions, 48.9% containing non oncogenic HPV types 6 and/or 11 and 4.4% harbouring HPV types 11 and 18. The DNA diploid lesions were all associated with non oncogenic HPV types 6 and/or 11 and one case also contained HPV type 33. There was no significant correlation between the detection of DNA aneuploidy and the presence of immuno-detected p53. DNA aneuploidy was not related to the presence of oncogenic HPV in anal condylomata acuminata. The DNA aneuploid profile frequently observed, especially in lesions associated with non oncogenic HPV types, is not yet well explained and cannot be considered as a prognostic factor. In contrast, a more intensive clinical follow-up should be proposed in patients with oncogenic HPV associated to DNA aneuploidy.     

Proportion of S-phase tumor cells measured by flow cytometry is an independent prognostic factor in meningioma tumors.

Meningiomas are tumors in arachnoid cells which represent up to one fifth of all intracranial tumors and up to a quarter of spinal neoplasias. Although meningiomas have classically been considered to be benign tumors, it has also been well-established that they show a heterogeneous clinical outcome. To the best of our knowledge no study has yet been performed in which the independent prognostic value of both DNA ploidy and cell cycle has been simultaneously assessed in a large series of meningioma tumors. The aim of the present study was to prospectively explore the prognostic value of DNA ploidy status and the proliferative rate of tumor cells in a series of 105 consecutive meningioma patients studied at diagnosis. Both the presence of DNA aneuploidy and the proportion of S-phase tumor cells were analyzed in all cases in fresh tumors obtained during diagnostic surgery. From the technical point of view, we followed the recommendations of the Consensus Conference on Flow Cytometry DNA Analysis held in October 1992. Our results show that meningioma tumors display a relatively low incidence of DNA aneuploidy (14%), and they usually show a low proliferative rate (mean percentage of S-phase cells of 1.3 +/- 0.3%). The presence of DNA aneuploidy was associated with a higher incidence of aggressive histopathologic subtypes (P = 0.045), a greater age (P = 0.009), location at the cerebral convexity (P = 0.004), and a greater proportion of S-phase cells (P = 0.005). In contrast, no significant association between the DNA ploidy status of meningioma patients and their disease-free survival was found (P = 0. 1). Regarding the proliferative activity of neoplastic cells, we found that a high proportion of S-phase cells (>1.8%) was associated with a significantly lower mean age (P = 0.007), aggressive histopathologic subtypes (P = 0.03), a higher incidence of DNA aneuploidy (P = 0.004), and a significantly shorter disease-free survival (P < 0.004). Multivariate analysis of prognostic factors showed that the proportion of S-phase tumor cells was the most powerful independent prognostic factor in meningioma patients (P = 0.02). In summary, we conclude that the proportion of S-phase tumor cells represents the individual parameter with the highest value for predicting disease-free survival in meningioma patients.     

DNA ploidy and S-phase fraction in carcinoma of the gallbladder related to histopathology, number of gallstones and survival.

Gallstones are a risk factor for the development of gallbladder cancer. We studied DNA ploidy and cell cycle composition by flow cytometry in archival specimens from 52 gall bladder carcinomas in relation to histopathological grade, tumour stage, gallstone number and survival. 69% of the gallbladder carcinomas showed aneuploidy. All tumours with single stones (N=11) were aneuploid while only 61% of tumours with multiple stones (N=41) were aneuploid (p=0.002). DNA aneuploidy was related to increase in T-category (p=0.01), grade (p=0.02), and nuclear pleomorphism (p=0.0005). The distribution of DNA ploidy shifted from tetraploid in low stage towards triploid positions in high stage tumours (p=0.02) combined with higher S-phase values in triploid tumours (p=0.05). S-phase fraction increased during development from normal tissue to dysplasia, cancer in situ and cancer in diploid cases (p=0.0002), and further at the change from diploid to aneuploid (p=0.004). At a median cancer specific survival time of four months patients with diploid tumours had a better survival than those with aneuploid tumours (p=0.02). In multivariate analysis of the tumour characteristic, only T-category and tumour grade were independent prognostic factors.The shift from diploid to aneuploid and the further shift of ploidy within aneuploid tumours are in agreement with the concept of a clonal development of gallbladder cancer. These changes are combined with a stepwise increase in the fraction of S-phase cells. Low frequency of symptoms in single stone patients may be the reason for detection of malignancy at a late stage of tumour development.     

Distinguishing Low-Risk Luminal A Breast Cancer Subtypes with Ki-67 and p53 Is More Predictive of Long-Term Survival

Overexpression of p53 is the most frequent genetic alteration in breast cancer. Recently, many studies have shown that the expression of mutant p53 differs for each subtype of breast cancer and is associated with different prognoses. In this study, we aimed to determine the suitable cut-off value to predict the clinical outcome of p53 overexpression and its usefulness as a prognostic factor in each subtype of breast cancer, especially in luminal A breast cancer. Approval was granted by the Institutional Review Board of Samsung Medical Center. We analyzed a total of 7,739 patients who were surgically treated for invasive breast cancer at Samsung Medical Center between Dec 1995 and Apr 2013. Luminal A subtype was defined as ER&PR + and HER2- and was further subclassified according to Ki-67 and p53 expression as follows: luminal A (Ki-67-,p53-), luminal A (Ki-67+, p53-), luminal A (Ki-67 -, p53+) and luminal A (Ki-67+, p53+). Low-risk luminal A subtype was defined as negative for both Ki-67 and p53 (luminal A [ki-67-, p53-]), and others subtypes were considered to be high-risk luminal A breast cancer. A cut-off value of 10% for p53 was a good predictor of clinical outcome in all patients and luminal A breast cancer patients. The prognostic role of p53 overexpression for OS and DFS was only significant in luminal A subtype. The combination of p53 and Ki-67 has been shown to have the best predictive power as calculated by the area under curve (AUC), especially for long-term overall survival. In this study, we have shown that overexpression of p53 and Ki-67 could be used to discriminate low-risk luminal A subtype in breast cancer. Therefore, using the combination of p53 and Ki-67 expression in discriminating low-risk luminal A breast cancer may improve the prognostic power and provide the greatest clinical utility.     

Flow cytometric analysis of DNA indices, expression of p53 and multidrug resistance genes in multiple myeloma patients

OBJECTIVE: To perform a quantitative analysis of DNA ploidy, S-phase fraction % (SPF%), p53 and multidrug resistance (MDR) gene expression as independent prognostic parameters and to compare these parameters with stage of the disease in multiple myeloma (MM) patients. STUDY DESIGN: Peripheral blood bone marrow samples were analyzed for DNA ploidy and SPF% using a FACScan flow cytometer (Becton Dickinson). Detection of p53 and MDR gene expression was done using immunocytochemistry. RESULTS: Aneuploidy was found in 5/48 (10.42%) total myeloma patients, all of whom revealed hyperdiploidy. High SPF% was noted in 18/37 (48.65%) newly diagnosed MM patients and 5/11 (45.45%) follow-up cases of myeloma. p53 Gene product was noted in 8/48 (16.66%) myeloma patients, 6 newly diagnosed and 2 on follow-up. MDR gene expression was detected in 4/27 (10.81%) newly diagnosed patients and in 1/11 (9.09%)follow-up patients. CONCLUSION: All myeloma patients with aneuploidy revealed hyperdiploidy. The majority of cases with high SPF% were at advanced stages, indicating the prognostic significance of SPF%. Although, there was no statistical significance of DNA ploidy, SPF%, p53 and MDR gene product expression, they are important prognostic parameters. Our results can provide baseline data for comparison with future studies since these parameters have not been reported earlier from the Indian subcontinent.     

Prognostic significance of DNA cytometry in carcinoma of the uterine cervix FIGO stage IB and II.

OBJECTIVE: To assess the prognostic value of DNA-image cytometry in cervical carcinoma of the uterus and its relation to other established prognostic factors. STUDY DESIGN: The study included 116 cases of cervical carcinoma FIGO stages IB and II which were treated with radical abdominal hysterectomy. The median follow-up was 55 months (range 1-162 months). DNA image cytometry was performed on cytologic specimens prepared by enzymatic cell separation from formalin-fixed, paraffin-embedded tissues. DNA stemline ploidy, DNA stemline aneuploidy, 5c exceeding rate, 9c exceeding rate, 2c deviation index, and DNA malignancy grade were computed. DNA-variables as well as various clinical and histological variables were related to survival rates. RESULTS: In multivariate statistical analysis DNA stemline ploidy using 2.2c as a cut-off value and FIGO stage showed to be statistically significant available presurgery predictors of survival, whereas the postsurgical parameters lymphonodal status, tumor size and parametrial involvement were significantly correlated with survival. The synopsis of all parameters in a multivariate Cox model indicated that - with declining relevance - the number of positive pelvic lymph nodes, DNA stemline ploidy using a cut-off level at a modal value of 2.2c, largest pelvic lymph node, 5c exceeding rate, and ratio of carcinoma area to cervix area, were of predictive value for survival. CONCLUSIONS: Our results suggest that prognostic information deducted from classical staging parameters is successfully complemented by DNA image cytometry which can be applied pretherapeutically.     

Prediction of aggressiveness of gastrointestinal stromal tumours based on immunostaining with bcl-2, Ki-67 and p53

OBJECTIVE: While the use of fine needle aspiration (FNA) in the diagnosis of gastrointestinal stromal tumours (GISTs) is well-established, it can be difficult to predict the prognosis of GIST based on morphology alone. The objective of the current study was to determine if expression of bcl-2, Ki-67 and p53 correlated with the outcome of GISTs based on cytological material. METHODS: Cell-blocks from 14 GISTs diagnosed by FNA were retrieved. Immunostaining was performed with antibodies against bcl-2, Ki-67 and p53. All cytological diagnoses were confirmed by positive immunostaining with c-kit and/or subsequent histological evaluation. Positivity for bcl-2, Ki-67 and p53 was defined as the presence of > or =10% cytoplasmic staining, > or =5% nuclear staining and > or =5% nuclear staining respectively. RESULTS: The 14 patients consisted of seven males and seven females with a mean age of 58 years. The average follow-up interval was 46 months. Six had a benign course and eight developed recurrences/metastases. Thirteen (93%) cases showed positive staining for bcl-2. Positive Ki-67 and p53 staining was noted in one (7%) and seven (50%) cases respectively. The difference in staining for p53 between aggressive and non-aggressive GISTs was statistically significant. No statistically significant difference was noted for bcl-2 staining or Ki-67 labelling index between the two groups. CONCLUSIONS: According to our observations, p53 immunostaining may be useful in predicting the outcome of GIST diagnosed by FNA; Ki-67 and bcl-2 are not useful as prognostic markers for GIST in FNA specimens.     

Mean weighted nuclear volumes in colonic adenocarcinomas. Evaluation of prognostic significance

OBJECTIVE: To investigate the correlation of stereologically estimated mean weighted nuclear volumes of tumor cell nuclei (MWNV) with other prognostic factors and survival in colonic adenocarcinomas. STUDY DESIGN: The study group consisted of 42 patients with colonic adenocarcinomas who were treated by a standardized protocol and had a mean follow-up of 89 months. The point sample intercepts method was used to estimate MWNV. In addition, immunohistochemical expression of p53 and Ki-67 was evaluated semiquantitatively. RESULTS: The calculated nuclear volumes correlated significantly with histologic grading and with the extent of tumor progression (organ confined/nonorgan confined). There was no correlation with p53 expression or proliferative activity determined by MIB-1 positivity. No correlation could be demonstrated with sex, age or clinical outcome of the patients. CONCLUSION: Assessment of MWNV in colonic adenocarcinomas does not provide additional information concerning the clinical course.     

Low levels of p27 in association with deregulated p53-pRb protein status enhance tumor proliferation and chromosomal instability in non-small cell lung carcinomas

BACKGROUND: Down-regulation or overexpression of the cyclin-dependent kinase inhibitor p27 have been observed in a range of malignancies, including lung cancer. To further elucidate the role of the molecule in tumor growth regulation, we evaluated p27 expression in a series of non-small cell lung carcinomas (NSCLCs), and examined its relation with histology, kinetic parameters, ploidy, and overall survival. We extended our investigation into the association of p27 levels with the presence of Ki-ras mutations, as well as with the expression status of p53 and pRb in tumor cells. MATERIAL AND METHODS: p27, p53, and pRb status were immunohistochemically evaluated in a total of 69 NSCLCs. In situ assays were employed to assess the kinetic parameters (Ki-67 immunohistochemistry for proliferation index, Tdt-mediated dUTP nick end labeling assay for apoptotic index). The ploidy status of the tumors was assessed after staining nuclei with the Feulgen procedure, and the presence of Ki-ras mutations was examined by restriction fragment length polymorphisms. All possible associations were assessed with a series of statistical methods. RESULTS: Immunoreactivity for p27 was observed in the entire series of specimens, with the mean percentage of positive cells being 33%. Adenocarcinomas (AdCs) exhibited higher p27 levels compared to squamous cell carcinomas (SqCCs) (p < 0.01). An inverse correlation was established between p27 expression and proliferation index (PI) (r = -0.834, p < 0.01) but not with apoptotic index (AI), whereas aneuploid tumors were characterized by lower p27 levels than diploid ones (p < 0.01). No difference in p27 immunostaining was observed with regard to the presence of Ki-ras mutations, whereas aberrant p53 and/or pRb expression patterns were associated with p27 underexpression (p < 0.01 for p53 status, p < 0.05 regarding pRb levels, and p < 0.01 for a combined deregulation of both proteins). Two or more alterations in the p27/p53/pRb protein network (i.e., p27 levels lower than the estimated mean value, overexpressed p53, and/or aberrant pRb) were associated with increased PI and aneuploidy (p < 0.001 and p < 0.01, respectively). A powerful trend was found between p27 expression and overall survival (p = 0.066). CONCLUSIONS: Our findings confirm the heterogeneity between AdCs and SqCCs, and are suggestive of an increased proliferative activity in NSCLCs underexpressing p27. Furthermore, our analysis supports the concept of p27 forming a functionally compact network with p53 and pRb, which is actively involved in the regulation of cellular proliferation and chromosomal stability.     

Immunohistochemical assay of p53, cyclin D1, c-erbB2, EGFr and Ki-67 proteins in HPV-positive and HPV-negative cervical cancers

The purpose of the present study was to analyse clinical correlation between HPV type 16 and 18 infection, expression of p53, cyclin D1, Ki-67, c-erbB2 and EGFr gene products in cervical cancer cells as well as their nuclear ploidy. The morphological parameters evaluated, such as differentiation of carcinomas, vascular invasion, ploidy and expression of oncogenic proteins, indicate the increased biological malignancy of HPV 16/18-positive carcinomas. The majority of them were poorly differentiated, revealed significantly higher frequence of vascular invasion (p<0.05), were more frequently aneuploid and showed overexpression of cyclin D1. The comparison of the data obtained with the mortality rate of the patients suggests that the overexpression of EGFr and moderate expression of Ki-67 seem to be unfavorable prognostic factors, regardless of the presence of HPV 16/18.     

Prognostic significance of DNA ploidy and proliferation rate in human astrocytic gliomas

DNA ploidy and the proliferative potential in 75 gliomas were investigated using bromodeoxyuridine labelling index (BrdUrd LI), S-phase fraction (SPF) and argyrophilic nucleolar organizer regions (AgNOR) technique. There were 53 highly malignant (AIII-AIV), and 22 low-grade (AI-AII) gliomas. One fragment of the tumour was fixed in Carnoy's solution for AgNOR test, while the other fragments were used for flow cytometric determination of the labelling index, SPF and DNA ploidy. For the BrdUrdLI, tumour samples from each patient were incubated in vitro for one hour at 37 degrees C with BrdUrd using the high pressure oxygen method. The tumours showed variability in the BrdUrdLI values, SPF and AgNOR counts/cell nucleus. The same percentage of DNA aneuploidy (55%) was found in high-grade as well as in low-grade gliomas. Univariate analysis showed that patients with grade I & II gliomas had significantly higher 3-year survival rate (p = 0.0193) than those with grade III and grade IV gliomas. Also patients with lower proliferation rate of tumours (BrdUrdLI < or =2.3% and AgNOR counts < or =2.6%/cell) had higher 3-year survival rate (p<0.03), which can be helpful in prognosis. Tumour ploidy or SPF had no influence on patients' survival (p = 0.7908). Cox multivariate analysis showed that only patients' age > 45 years and high tumour grade (III and IV) were significant unfavourable prognostic factors in terms of patients' survival.     

Further testing of morphometric grading in T(A,1) urothelial carcinomas of the urinary bladder and the additional value of p53 immunoquantitation

OBJECTIVE: To analyze the value for grading of a previously developed quantitative morphometric/cytometric multivariate grading model (consisting of the mean nuclear area of the 10 largest nuclei (MNA-10, mitotic activity index = MAI and Ki-67 area% = Ki-67) in two new independent test sets of urothelial carcinomas (UCs) of the urinary bladder and to evaluate the additional value of p53 area% (p53) in this model. STUDY DESIGN: Ki-67 immunoquantitation, morphometric MAI and MNA-10 assessments using a previously described, strict protocol and matching of the resulting morphometric grade with subjective grade in two test sets of 154 T(A,1) UCs of the bladder (consensus grade between two independent observers). Further testing of this morphometric grading model was performed in 57 cases that lacked initial interobserver agreement on grade. Single and multivariate analysis of all features (including p53) was performed. RESULTS: With the previously developed morphometric/cytometric grading model, 93% (grade 1 vs. 2) and 91% (grade 2 vs. 3) of the consensus cases were correctly classified. These percentages were very similar to previous results, suggesting that the model is robust. Of the 57 cases that lacked initial interobserver agreement on grade, 53/57 (93%) were classified unambiguously as grade 1, 2 or 3 with the quantitative morphometric/ cytometric grading model. In the exploratory analysis, p53 was significant but with more overlap than the other features had. In multivariate analysis p53 did not improve the overall classification result of the original morphometric/cytometric model. CONCLUSION: The value of MNA-10, MAI and Ki-67 for grading in T(A,1) urothelial carcinomas of the urinary bladder was confirmed. p53 Did not improve overall grading classification of this combination.