Biological characterisation of superficial bladder cancer by bivariate cytokeratin 7/DNA analysis, flow cytometric assessment of MIB- 1, and an immunohistochemical study.

A total of 238 cases of bladder carcinoma stages Ta, Tis, T1 were submitted prospectively to multiparameter flow cytometry and immunohistochemical study in order to determine the biological aggressiveness of the tumour. DNA index (DI), S-phase fraction (SPF) obtained by bivariate cytokeratin 7/DNA analyses, and the immunohistochemical evaluation of p53 and MIB-1 were studied in relation to the traditional prognostic factors in bladder cancer (stage and grade). the variance analysis results showed that DNA aneuploidy was significantly associated with high stage (p = 0.0001), high grade (p = 0.0001), high SPF value > or = 5.5% (p = 0.0001), MIB-1 positivity > or = 31% (p = 0.0001) and high expression of p53 (staining involving > 50% of cells, p = 0.0001). Even if there was no statistical significance the hypotetraploid class (1.70 < DI < 1.89) showed poor prognostic biomarkers more frequently than the other aneuploid classes. Out of 238 cases, 101 were also submitted to flow cytometric measurement of MIB-1 (fMIB-1) to study the correlation between cell proliferation and DNA content. Data obtained from fresh, 3:1 methanol/acetone fixed samples were compared with values obtained from both cell cycle analysis methods and routine application of the MIB-1 immunostaining in histological sections. fMIB-1 values were positively correlated with SPF values (r = 0.801, p < 0.01) and S+G2M fraction (percentage of cells in S and in G2M phases) (r = 0.763, p < 0.01) but no correlation with paraffin sections was found. A fMIB-1 value > 7% was strongly associated with aneuploidy (p = 0.0001). The determination of DNA content coupled with the study of the epithelial (cytokeratin 7) and proliferative (MIB-1) markers could be useful in providing important information on the biological behaviour of superficial bladder tumours.     

Cytologic grading of invasive breast carcinoma. Correlation with clinicopathologic variables and predictive value of nodal metastasis

OBJECTIVE: To estimate cytologic grade and correlate it with the other known prognostic factors, such as tumor differentiation, growth fraction, estrogen receptor status and nodal status. STUDY DESIGN: Fine needle aspirates from 104 invasive ductal carcinomas were stained by the Papanicolaou method and examined for necrosis, cellular size, nuclear/cytoplasmic ratio, nuclear pleomorphism, nucleoli, chromatin granularity and density of chromatin. We established a semiquantitative scoring system based on the above features and correlated cytologic findings with clinicopathologic variables. RESULTS: Histologic grade correlated positively with cytologic grade and negatively with estrogen receptor positivity. Moreover, high cytologic grade was associated with nodal metastasis and proliferative index labeling by MIB-1. CONCLUSION: This study showed that our grading system for breast cancer on fine needle aspiration cytology is feasible on a routine diagnostic basis. Cytologic grading can provide more information than usual on tumor biologic behavior.     

Value of the cribriformity factor in grading prostatic carcinoma.

Architectural and histometric features for the objective grading of prostate adenocarcinoma in histologic specimens were analyzed in five cases each of Gleason primary grades 2, 3A, 3B, 3C, 4A and 4B, selected as "typical" for the histopathologic images. Tissue sections from the selected cases were stained by the Feulgen method. Fifteen fields for each grade, for a total of 4,430 glands, were digitized by a video-based microphotometer at low resolution (pixel spacing of 2 microns). Outer and inner outlines of the glandular epithelium were traced manually using a mouse. For each field the number of glands, the gland area, the lumen area, the area of the glandular epithelium and the cribriformity factor were computed. The gland area and its variance proved to be useful indicators for lower-grade lesions, whereas the variance of cribriformity resulted in an excellent grading indicator in the Gleason 3-4 range when cribriform glands were present.     

Towards a quantitative grading of bladder tumors.

The histological inspection of tumor tissue for the purpose of reporting a tumor grade is a problem of significant clinical importance. The grading by a pathologist is only partly reproducible due to vaguely defined, subjective criteria. In this article we describe and evaluate a set of measurable features that quantitate the differences in tumor tissue. Different aspects of the reproducibility of the measurements under varying conditions of image selection, focus, and noise have been investigated. Three hundred thirty-three images were digitized from 111 bladder tissue sections (4 microns thick, Feulgen stained), using the ICAS microscope-camera platform. A segmentation routine was developed to segment the images into nuclei and background without any user interaction. Size, shape, optical density, and texture features were measured on and among the objects found by this segmentation routine using the image analysis package Acuity. The results of the measurements showed that there is a significant quantitative difference between grade 1 and grade 3 tumors. Grade 2 tumors can be described as "in between grade 1 and grade 3" and falling somewhere on an increasing scale between grades 1 and 3. Grade 2 tumors do not seem to represent a statistically distinct population. The procedure described here is shown to be quite reproducible in the presence of noise, reasonably reproducible in the event of a modest amount of defocusing (with grade 3 tumors exhibiting the most sensitivity), and less reproducible in the context of which fields-of-view are chosen for analysis.     

Automated measurement of MIB-1 positive area as an alternative to counting in follicular lymphoma

Manual counting of MIB-1 positive cells which has been suggested as an alternative to centroblast counting for the diagnostic grading of follicular lymphoma is a laborious task. In this study, the validity of automated measurement of the MIB-1 positive area is analyzed as an alternative approach. Archival MIB-1 stained tissue sections of 15 follicular lymphomas were assessed manually and automatically by three independent observers. Concordance correlation coefficients and coefficients of variation were calculated to study reproducibility and variability of both methods and to compare result from both methods. A good concordance was observed between the two methods. The reproducibility of the automated method was slightly better than the manual counting of positive nuclei. Measurement of MIB-1 positive surface area may be used as a simple and fast alternative to tedious manual counting of positive nuclei as a potential help in follicular lymphoma grading.     

Assessing agreement between multiple raters with missing rating information, applied to breast cancer tumour grading

Background

We consider the problem of assessing inter-rater agreement when there are missing data and a large number of raters. Previous studies have shown only ‘moderate’ agreement between pathologists in grading breast cancer tumour specimens. We analyse a large but incomplete data-set consisting of 24177 grades, on a discrete 1–3 scale, provided by 732 pathologists for 52 samples.

Methodology/Principal Findings

We review existing methods for analysing inter-rater agreement for multiple raters and demonstrate two further methods. Firstly, we examine a simple non-chance-corrected agreement score based on the observed proportion of agreements with the consensus for each sample, which makes no allowance for missing data. Secondly, treating grades as lying on a continuous scale representing tumour severity, we use a Bayesian latent trait method to model cumulative probabilities of assigning grade values as functions of the severity and clarity of the tumour and of rater-specific parameters representing boundaries between grades 1–2 and 2–3. We simulate from the fitted model to estimate, for each rater, the probability of agreement with the majority. Both methods suggest that there are differences between raters in terms of rating behaviour, most often caused by consistent over- or under-estimation of the grade boundaries, and also considerable variability in the distribution of grades assigned to many individual samples. The Bayesian model addresses the tendency of the agreement score to be biased upwards for raters who, by chance, see a relatively ‘easy’ set of samples.

Conclusions/Significance

Latent trait models can be adapted to provide novel information about the nature of inter-rater agreement when the number of raters is large and there are missing data. In this large study there is substantial variability between pathologists and uncertainty in the identity of the ‘true’ grade of many of the breast cancer tumours, a fact often ignored in clinical studies.     

Immunohistochemical analysis of MIB-1 proliferative activity in human endometrial cancer. Correlation with clinicopathological parameters, patient outcome, retinoblastoma immunoreactivity and K-ras codon 12 point mutations

To test the prognostic utility of MIB-1 in human endometrial neoplasias, the proliferative activities of fifty-two endometrial carcinomas obtained from Polish women were assessed. We also investigated the relationship between the MIB-1 Proliferative Index and the well-known clinicopathological features of cancer (clinical stage, histological type, histological grade, depth of myometrial invasion), patient's age, overall survival, retinoblastoma immunostaining and K-ras codon 12 point mutations. The mean MIB-1 Proliferation Index was 43.8%, with a median of 36.0%. Due to the great intratumour heterogeneity of the immunoreaction, the Index ranged from 0% to 98%. A significant relationship was noted between MIB-1 expression and histological grading (p=0.0004) and myometrial invasion of cancer (p=0.01). Multivariate Cox regression demonstrated that the clinical stage was the only independent prognostic factor during follow-up (p=0.025). There was a tendency towards a poorer outcome for women with a Proliferative Index of >31% than for patients whose Index was 31%; the difference, however, did not reach significance (p=0.25; log-rank test). Interestingly, uterine cancers lacking retinoblastoma protein expression had a mean MIB-1 Proliferation Index that was nearly twice as high as in those neoplasias that stained positively for retinoblastoma (70.33% and 42.14%, respectively; p=0.09; Mann-Whitney-U test). There were no significant differences between K-ras codon 12 point mutation-positive and -negative endometrial carcinomas regarding the proliferative activity of the cancer (mean Indexes 47.6% and 43.8%, respectively; p=0.66, Mann-Whitney-U test). Our data support the view that MIB-1 proliferative activity was significantly increased with a decrease of the histological grading and with the myometrial invasion of human endometrial cancer.     

Morphometry of bladder carcinoma: morphometry and grading complement each other

Subjective grading of bladder carcinoma is a good predictor of the clinical outcome in those patients whose tumours are grade 1 or grade 3. However, in grade 2 tumours, which account for 45% of cases, grading has little predictive value in an individual patient. We have complemented the use of subjective grading with measurement of nuclear area and used a calculation of the distribution of nuclear sizes as a predictor of the clinical course. When subjective grading was complemented by morphometry the outcome was correctly predicted in 55 of 58 cases and all cases with poor clinical outcome were identified.     

Cell proliferation in prostatic carcinoma: comparative analysis of Ki-67, MIB-1 and PCNA

Summary Antibodies to assess the proliferative index of tumours are being increasingly employed together with established markers for prognostic evaluation. This study set out to compare three cell proliferation markers, Ki-67, MIB-1 and PCNA, utilizing a semiquantitative method of assessment, in 20 human prostatic carcinomas. The streptavidin-biotin immunostaining system was used for the monoclonal antibodies MIB-1 and PCNA and an indirect immunoperoxidase assay for the monoclonal antibody Ki-67. Significant correlations were found between the expression of Ki-67 in frozen tissues and MIB-1 in formal saline-fixed wax-embedded tissues (p = 0.0003); between Ki-67 and PCNA expression in Bouin's-fixed tissues (p </ 0.0001); and MIB-1 (formalin-saline-fixed tissues) and PCNA (Bouin's-fixed tissues) (p </ 0.0001). A more intense nuclear staining pattern with less heterogeneity was observed for MIB-1 compared with PCNA, suggesting the antibody of choice, on formal saline-fixed tissues, is MIB-1, which closely correlated with Ki-67, a marker we have previously shown to be of prognostic value in prostatic carcinoma.     

Direct comparison of bromodeoxyuridine and Ki-67 labelling indices in human tumours

Direct comparison of bromodeoxyuridine (BrdUrd) and Ki-67 labelling indices was achieved by selecting similar areas from serial sections of human tumours. Fifteen patients were selected who had been administered BrdUrd in vivo and both proliferation markers were assessed by immunohistochemistry. The data show a good correlation between both BrdUrd LI and MIB-1 LI and T_pot (calculated using the flow cytometry derived duration of S phase) and MIB-1 LI. The contribution of BrdUrd LI to growth fraction varied as a function of proliferation characteristics. In tumours with a high LI, the number of DNA synthesizing cells represented half the growth fraction, whilst in tumours with lower LI's (<10%) the ratio of DNA precursor labelled cells as a function of growth fraction fell to between 10% and 20%. T_pot showed a linear correlation with MIB-1/BrdUrd ratio with a slope approaching unity. It was apparent that both intra- and interpatient variation in proliferation index was greater for BrdUrd labelling than for MIB-1 expression.     

Cytological grading of breast carcinoma—a feasible proposition?

Fine needle aspiration cytology (FNAC) of the breast is widely used in the diagnosis of breast carcinoma. In some centres this is sometimes the only diagnostic procedure performed prior to definitive treatment. A grading system based on cytology would be helpful in the selection of patients for appropriate therapy. The aim of this study, therefore, was to devise such a system for grading breast carcinoma based on cytological features alone. The features assessed were the degree of cell clustering, nuclear pleomorphism, nuclear diameter, the presence of multiple, easily visible nucleoli and necrosis. Cytological features were compared to the histological grade of the tumours following excision. Discriminant analysis showed that the features with the closest correlation with histological grade were nuclear diameter, nuclear pleomorphism and the presence of nucleoli. A scoring system based on these three parameters enabled the classification of tumours into high and low cytological grades which showed a close correlation with histological grade.     

Topometric analysis of diffuse astrocytomas

OBJECTIVE: To investigate differences between astrocytomas of WHO grade 2 and anaplastic astrocytomas of WHO grade 3 in terms of topometric variables characterizing individual tumor cell nuclei. STUDY DESIGN: Paraffin sections from surgical specimens from 25 astrocytomas (grade 2, n = 11; grade 3, n = 14) were analyzed by means of an image analysis system. At least 300 tumor cell nuclei were measured in the region with the highest Ki-67 proliferation index. Three different kinds of topometric variables were determined for each tumor cell nucleus: (1) several distances; (2) the variable Angle 2/1, the angle between the straight lines representing the distance to the nearest and second-nearest nucleus; and (3) the number of neighbors according to our mathematical definition. RESULTS: Most topometric variables showed distinct differences between the 2 tumor grades (multivariate analysis of variance), with 88% cases correctly reclassified by means of cross-validated discriminant analysis. The variables with the highest discriminatory power were the SD of Angle 2/1 and the ratio between the distance to the second-nearest and nearest tumor cell nucleus, with lower values for these variables in anaplastic astrocytomas. Even variables concerning neighborhood relationships showed significant differences. CONCLUSION: The results of this pilot study show that this first set of topometric variables is sufficient to detect differences between topologic characteristics of tumor cell nuclei in astrocytomas grade 2 and grade 3. Topometry seems to be an important tool for grading astrocytomas.     

Morphometry, densitometry and pattern analysis of plastic-embedded histologic material from urothelial cell carcinoma of the bladder.

An image analysis method of grading histologic sections of bladder carcinoma was tested. The method was new in four respects. First, for fixation of the biopsies a coagulant fixative was used. Second, 2-microns plastic sections were used to ensure the reproducibility of nuclear imaging. Third, a new stereologic approach was used for calculation of the nuclear volume and DNA content. Fourth, for the classification rule the morphometric, densitometric and texture features were used in concert. The IBAS 2000 instrument was used for the measurements. Texture analysis of the chromatin patterns was performed using Markovian texture features. Using discriminant analysis, of 22 parameters, 2 morphometric, 2 densitometric and 3 texture features were selected for the classification rule. With them, 89% of the bladder carcinomas were correctly classified into the three grades. All grade III tumors were classified correctly. Among the features tested, the densitometry of the DNA had the highest F values. All of the grade III tumors and 45% of the grade II tumor group had DNA histograms indicating aneuploidy. This study showed that plastic-embedded material is well suited to morphometry and densitometry and can be used for quantitative grading of bladder carcinoma.     

Apoptosis in breast cancer

OBJECTIVE: To evaluate apoptotic rates on fine needle aspiration (FNA) samples of infiltrating duct carcinoma of the breast and to determine whether cytologic grading improved with consideration of the apoptotic rate in comparison with histologic grading. STUDY DESIGN: We studied 35 women who underwent mastectomy following an FNA diagnosis of infiltrating ductal carcinoma. Concordance between cytologic and histologic grades was calculated. Next, cytologic grades were considered with the apoptotic rates and compared with the histologic grades. RESULTS: An overall concordance of 82.9% was noted between the cytologic and histologic grading systems, with maximum concordance in grade 1 and minimum in grade 3 breast cancers. A highly significant difference in the apoptotic rates, as calculated on cytology, existed between the three histologic grades, indicating a significant increase in apoptosis with rising histologic grade. Applying multiple regression analysis, a significant improvement of cytologic grade with consideration of the apoptotic rate was observed. CONCLUSION: Employing histologic grade as the yardstick, cytology was less sensitive for the purpose of grading breast ductal carcinoma. However, by considering the apoptotic rates, the sensitivity of cytologic grading significantly rose in relation to histologic grade. Larger studies are required to determine whether apoptosis can be incorporated into the existing cytologic grading systems to increase their sensitivity.     

Nucleolar organizing regions in renal cell carcinoma, renal oncocytoma and renal adenoma

Nucleolar organizing regions (NORs), as demonstrated by the silver-colloid staining technique, have been counted in 75 renal cell carcinomas (20 grade 1, 22 grade 2, 17 grade 3 and 16 sarcomatoid), eight renal oncocytomas and nine renal adenomas. Mean NOR counts were 3.27, 6.28, 9.24 and 8.12, respectively, for grades 1, 2, 3 and sarcomatoid tumours, 3.09 for renal oncocytomas and 2.63 for renal adenomas. Analysis of data using the unpaired Student's t-test showed significant difference between NOR counts of grade 1, 2 and 3/sarcomatoid renal cell carcinoma, and grades 2, 3 and sarcomatoid renal cell carcinomas when compared to renal oncocytomas and adenomas. The association between type and grade of tumour, NOR value and tumour proliferation is discussed.     

Prognostic significance of proliferative activity, DNA-ploidy, p53 and Ki-ras point mutations in colorectal liver metastases

Paired colorectal liver metastases (CLM) and normal tissue samples from a consecutive series of 36 patients were studied prospectively. MIB-1 expression was studied by immunohistochemistry on paraffin-embedded sections. DNA ploidy and S-phase fraction (SPF) measurements were performed by flow cytometry on frozen tissues. Mutations within the p53 (exons 5-8) and c- Ki-ras (codons 12 and 13) genes were detected by PCR single-strand conformation polymorphism analysis followed by sequencing. A high correlation was observed between the MIB-1 LI and SPF value (rho=0.81; P <0.01). Moreover, p53 gene mutations were associated with either high MIB-1 LI and high SPF. In univariate analysis, SPF and MIB-1 levels were related to risk of death. The association between overall survival and DNA-ploidy or p53 mutations did not reach statistical significance, but a slightly better survival was observed for patients either with DNA-diploid tumours or without mutations ( P =0.05 and P =0.06, respectively). SPF was shown by multivariate Cox model analysis to be an independent prognostic variable and thus it might be a useful prognostic factor in patients with CLM.     

Essay: Letters,numbers, symbols and words: how to communicate grades of evidence and recommendations

THE GRADE WORKING GROUP IS DEVELOPING and evaluating a common, sensible approach to grading quality of evidence and strength of recommendations in health care. In this article, we discuss the advantages and disadvantages of using letters, numbers, symbols or words to represent grades of evidence and recommendations. Using multiple strategies, we searched for comparative studies of alternative ways of representing ordered categories in any context. In addition, we contacted experts and reviewed theoretical work and qualitative research on how best to communicate grades of any kind quickly and clearly. We were unable to identify health care research that addressed, either directly or indirectly, the best way to present grades of evidence and recommendations. We found examples of symbols used by government, commercial and consumer organizations to communicate quality of evidence or strength of recommendations, but no comparative studies. Although a number of grading systems are used in health care and other fields, there is little or no evidence of how well various presentations are understood. Before promoting the use of specific symbols, numbers, letters or words, the extent to which the intended message is comprehended should be evaluated. Organizations such as the Canadian Task Force on Preventive Health Care¹ and more than 100 other groups² use various systems of codes to communicate grades of evidence and recommendations. The codes fall primarily into 3 categories: letters (e.g., A, B, C, etc.), numbers (e.g., I, II, III, etc.) and mixed letters and numbers (e.g., Ia, Ib, IIa, etc.).Health care practitioners, especially students, are often puzzled by the message a grade conveys. For example, the administration of oral anticoagulation in patients with atrial fibrillation and rheumatic mitral valve disease receives various grades of recommendation from different organizations: Class I based on level B evidence by the American Heart Association,³ grade C recommendation based on level IV evidence by SIGN² and grade 1C+ (where the 1 indicates the balance between benefit and harm and C+ the methodological quality of the underlying evidence) by the American College of Chest Physicians.⁴ Thus, the various grading systems may not be fulfilling their intended function: to communicate a clear message, quickly and concisely. Indeed, if the same code, used by different systems, represents different meanings, bewilderment and incomprehension may result.We formed the GRADE Working Group with the hope of reaching agreement on a common, sensible approach to grading quality of evidence and strength of recommendations. We consider here the advantages and disadvantages of using letters, numbers, symbols or words to represent different grades.     

Prognostic significance of proliferative activity, DNA-ploidy, p53 and Ki-ras point mutations in colorectal liver metastases

Abstract. Paired colorectal liver metastases (CLM) and normal tissue samples from a consecutive series of 36 patients were studied prospectively. MIB-1 expression was studied by immunohistochemistry on paraffin-embedded sections. DNA ploidy and S-phase fraction (SPF) measurements were performed by flow cytometry on frozen tissues. Mutations within the p53 (exons 5-8) and c-Ki-ras (codons 12 and 13) genes were detected by PCR single-strand conformation polymorphism analysis followed by sequencing. A high correlation was observed between the MIB-1 LI and SPF value (rho=0·81; P<0·01). Moreover, p53 gene mutations were associated with either high MIB-1 LI and high SPF. In univariate analysis, SPF and MIB-1 levels were related to risk of death. The association between overall survival and DNA-ploidy or p53 mutations did not reach statistical significance, but a slightly better survival was observed for patients either with DNA-diploid tumours or without mutations ( P =0·05 and P =0.06, respectively). SPF was shown by multivariate Cox model analysis to be an independent prognostic variable and thus it might be a useful prognostic factor in patients with CLM.