Bcl-2 immunostaining: a way to finding unresponsive postmenopausal N+ breast cancer patients.

BACKGROUND: Bcl-2 staining positivity has shown limited prognostic value. However, we have decided to study this issue in the era of mammography screening, and adjuvant treatment protocols. METHODS: Paraffin sections of 414 breast cancers were stained for bcl-2 and staining intensity graded. Association of bcl-2 with established prognosticators was analysed with chi 2 test and odds ratios in 2 x 2 tables. Kaplan-Mayer analysis and Cox's regression were used to evaluate the prognostic value of bcl-2 and other prognosticators. RESULTS: Bcl-2 immunostaining was associated with tumor size, lymph node status, histological type, multivariate prognostic index, standardized mitotic index, Ki-67 fraction, DNA-index, proportion of cells with DNA above 5c, estrogen receptor status, and histological grade. ER status showed the best association with bcl-2 positivity (odds ratio 11.3, 95% CI 5.6-22.7). In the whole group of patients bcl-2 positivity was not an independent prognosticator. However, among N+ patients bcl-2 staining was significant, and among postmenopausal N+ patients bcl-2 immunostaining was a stronger independent prognosticator than tumor size. CONCLUSIONS: The prognosis of N+ breast cancers can potentially be evaluated with bcl-2 positivity, in association with tumor size, and mitotic activity. Among postmenopausal N+ patients, most of whom have received anti-estrogen therapy, bcl-2 positivity is an independent prognosticator. Also, the close association of bcl-2 positivity with ER status supports the view that bcl-2 negativity reveals a patient group which might benefit from additional treatment in association with anti-estrogen therapy.

Cathepsin H expression distinguishes oncocytomas from renal cell carcinomas.

BACKGROUND: Because the differential diagnosis of oncocytoma and renal cell carcinoma lacks specificity, new methods supporting correct diagnostic decisions are welcome. MATERIAL AND METHODS: Sixteen cases of renal oncocytoma, and 16 sex-, age-, and stage- matched controls of renal cell carcinoma (T1-2N0M0) were studied. The minimum follow up exceeded ten years. There were no deaths due to neoplasm among oncocytomas, but 4 patient died with metastatic disease among cancer patients. Immunohistochemical staining for cathepsin H was quantified by 3 histoscores. The histoscores evaluated: 1) even cytoplasmic staining of neoplastic cells, 2) granular staining, or 3) total staining. RESULTS: 100% distinction was possible with the even cytoplasmic staining score. Total staining distinguished 87.5%, and granular staining 25% of neoplasms. CAM 5.2 cytokeratin, or vimentin distinguished 84.4% or 56.6% of these tumors, respectively. CONCLUSIONS: Cathepsin H histoscore on even cytoplasmic immunostaining is an excellent method for the distinction of oncocytomas and renal cell carcinomas.

Nuclear volume and breast cancer prognosis.

BACKGROUND: In breast cancer, nuclear volume estimates can be expected to be better prognosticators than nuclear profile areas because biological variation is wider in volume estimates than in area measurements. MATERIALS AND METHODS: 191 invasive breast cancer samples were available for nuclear volume measurements. To estimate the volume weighted mean nuclear volume, point-sampled linear intercepts were used on micrographs. The nuclear profile area was measured from 148 cases matching volume measurements and run by the Prodit morphometry program. Measurements were compared as prognosticators after a follow-up of 5 years. A computerized method on a randomly selected large number of nuclei was also applied in 17 cases. Bcl-2 immunostaining was compared with nuclear measurements. RESULTS: The correlation of volume and area measurements was statistically significant, but the correlation coefficients were low. The nuclear area showed a significant difference in survival at the 75 percentile cut-point but the volume-weighted mean nuclear volume did not allow distinction of different prognostic groups. Computerized volume measurements based on a large number of nuclei and measurements based on the simpler method did not show statistically significant correlation. Bcl-2 staining did not show any correlation with volume or area measurements. CONCLUSIONS: Although the prognostic value of nuclear area was shown in our study, the volume-weighted mean nuclear volume did not show prognostic significance. Improvement of the methodology which could decrease method variation and increase reproducibility of measurements is urgent for verification of the prognostic value of nuclear volume measurements. Bcl-2 immunostaining and nuclear area measurements were independent prognostic variables.

Use of the mitotic counts for the prognosis and grading of breast cancer. Method evaluation study.

Reproducibility of the volume fraction-corrected mitotic index (M/VV index) was studied in 144 unselected breast cancer specimens. The influence on decision making of variation in determining the index was also analysed. In the complete series of specimens the correlation between two observers, one subjectively estimating the epithelial fraction of tumor epithelium and the other using point-counting (10 x 10 ocular grid), was good (Pearson's r = 0.82, p < 0.001, 95% CI 0.70-0.92). A subset of 30 specimens was used to evaluate the grading efficiency (GE) of the M/VV index method. The mean grading efficiency as estimated from this subset varied between 90% and 93%. The average minimum GE value was 82.8% (SD = 3.4%). The findings suggest that when the M/VV index method is used, the grading is correct on average in 90% or more of the cases, but dependent on the cutoff point. The over-all grading efficiency of the M/VV index method was comparable to that obtained from published S-phase fraction data on breast cancer specimens from three independed laboratories. We conclude that the M/VV index in breast cancer analysis is a sufficiently reproducible method in mitosis counting, and that it can be used with subjective or point count estimation of the area fraction of neoplastic epithelium.

Standardized mitotic counts in breast cancer. Evaluation of the method.

Twenty-one pathologists and technicians participated in a study evaluating the variation present in mitotic counts for prognostication of breast cancer. The participants counted the mitotic figures in 20 breast cancer samples from ten high power fields (mitotic activity index, MAI, giving the results in mitotic figures per 10 fields) and also made a correction for field size and area fraction of the neoplastic epithelium to get the standardized mitotic index (volume fraction corrected mitotic index, or M/VV index, giving the result in mitotic figures per square mm of neoplastic epithelium). The difference in variation between the two methods was not big, but the standardized mitotic index (SMI) showed consistently smaller variation among all participants and different subgroups. Experienced pathologists had the highest variation in mitotic counts, and specially trained technicians, the lowest. The efficiency of the mitotic counts in grading (the grading efficiency) was used to evaluate the mitotic counts. In groups without special training for mitotic counts the mean grading efficiency was lower (experienced and training pathologists both on average had the potential to grade 88% of the cases correctly) than in the group specially trained for the purpose (trained technicians had the potential to grade 95% of the cases correctly). Among the specially trained technicians, the grading efficiency was of the same magnitude as the grading efficiency achieved in determining the S-Phase fraction of cells from paraffin embedded breast cancers by flow cytometry in different laboratories. The results suggest that special training is helpful in making mitotic counts more reproducible, and that in trained hands, the mitotic counts give results comparable to more sophisticated methods of determining proliferative activity in breast cancer.

Target length and primer concentration affect the gain of c-erbB2 and p53 amplicons.

Although quantitative polymerase chain reaction (PCR) using internal standards may perform reproducibly, the calibration of the normal level is problematic. Three test sequences (171, 213, and 260 base pairs [bp]) from the c-erbB2 oncogene were separately coamplified with a 133 bp control sequence from the single copy gene p53 in differential PCR. Sequence length differences between the oncogene and control sequences influenced the ratio estimates, obviously because of less efficient synthesis of the longer sequence. Increase in primer concentration of the longer oncogene sequences adjusted this imbalance, and the expected ratio of 1.0 could be reached. The primer or target sequence also influenced the ratio estimate, since the 171 bp oncogene sequence was as efficiently synthesized as the 133 bp control sequence but with lower primer concentration. The 260 bp oncogene sequence produced the most stable results, probably because of clear band separation in polyacrylamide gel electrophoresis. A ratio estimate of 1.0 was produced by oncogene and control gene primer concentrations of 3.5 pmol/microl and 0.6 pmol/microl, respectively. Calibrated quantitative PCR methodology is applicable to many areas and offers an excellent tool for screening allele deletions, supernumerary alleles, or chromosomes associated with familial diseases or disease syndromes.

Section thickness and mitotic counts in ovarian mucinous carcinoma. Methodological study with scanning confocal microscopy.

To study the effect of section thickness on mitotic counts, paraffin sections of 28 cases of mucinous ovarian carcinomas were analyzed. Mitotic counts, estimated with the number of mitoses per mm2 of neoplastic epithelium (M/Vv index) and per mm3 from optical sections and through the whole section thickness were done. Section thickness was measured with scanning confocal microscopy from each specimen of 3 series of sections with different nominal thicknesses (5, 8, and 10 microns). Section thickness varied considerably within each series of sections. The sections of the 5-microns group tended to be thicker and the sections of the 10-microns group thinner than expected on the basis of their nominal values. As expected, mitotic counts through the section gave higher M/Vv index values than counts done using optical sections. M/Vv values obtained using optical sections increased with increasing nominal section thickness. The study suggests that, when the section thickness cannot be measured, it is advisable to do mitosis counting by using optical sections in lightly stained tissue sections in the thickness range of 8-15 microns.

Some reflections on the history, and presence of quantitative pathology.

The quantitative approach has always been present in pathology, but this approach is now practiced more often and in more numerous fields than before. Today pathology is applying DNA cytometry, morphometry, stereology, and quantitative immunohistochemistry, all less useful or useless without the quantitative approach. The present fast development of computerized instrumentation has much helped in the spread and application of quantitative principles.

Organized quantitative pathology. Short review of the activities of the Committee for Diagnostic Quantitative Pathology from 1981 to the foundation of the International Society of Diagnostic Quantitative Pathology in 1994.

The Committee for Diagnostic Quantitative Pathology (CDQP), known originally as the Committee for Diagnostic Morphometry, ceased to exist under the original title and was converted to the International Society for Diagnostic Quantitative Pathology (ISDQP) in Amsterdam, September 14, 1994. The history of this society started in 1981 in a conference <> held at Koli, Finland. Since the original meeting the group of quantitative pathologists organized yearly gatherings: Symposia on Diagnostic Quantitative Pathology (earlier known as Symposia on Morphometry in Morphological Diagnosis) every other year, and meetings in association with the European Society of Pathology Congresses in the intervening years. In 1981, the symposium had 23 participants, in 1994, the International Society for Diagnostic Quantitative Pathology had over 300 members from six continents. During the short period of its existence the society has witnessed a steadily growing trend in educational courses on quantitative pathology. The general policy of the Committee, now Society, has willingly supported all activities which can be expected to lead to valuable results in quantitative microscopy and associated fields either through development of education, methodology or practical applications. By arranging a course of Diagnostic Quantitative Pathology the society participates in the activities of the European School of Pathology in Torino. The next symposia of the Society will be arranged in Heidelberg, October 1995 and in Sendai, Japan, October/November 1996.

Primary malignant melanoma of the skin. Relationships of nuclear DNA content, nuclear morphometric variables, Clark level and tumor thickness.

OBJECTIVE: To investigate how nuclear morphometric variables, tumor thickness (measured according to Breslow), invasion depth (classified according to Clark), nuclear DNA content and type of DNA histogram are associated with each other in primary malignant melanomas of the skin. STUDY DESIGN: Image analysis DNA cytometry and nuclear morphometry were performed on 85 primary skin melanomas. The relationships of size, sphericity and DNA content of melanoma cell nuclei; melanoma thickness; and Clark level were analyzed in detail. The effect of melanin bleaching on DNA cytometry results was studied. RESULTS: Melanoma thickness correlated with nuclear size in aneuploid, but not diploid, melanomas. The prevalence of aneuploidy did not increase with tumor thickness. In aneuploid melanomas the proportion of cells with higher-than-diploid and higher-than-tetraploid DNA content increased with tumor size. CONCLUSION: Aneuploidy is as common in thin as in thick melanomas. Genetic instability in aneuploid melanomas correlates with melanoma thickness. This correlation in aneuploid melanomas partially explains the correlation between nuclear size and melanoma thickness. In diploid melanomas no correlation was observed between nuclear size and melanoma thickness. DNA cytometry is a valuable tool for studies on the background of phenotypic changes in skin melanomas.

Cell proliferation in dimethylbenz(A)anthracene(DMBA)-induced rat mammary carcinoma treated with antiestrogen toremifene.

Cell proliferation during antiestrogen toremifene treatment was studied using the DMBA-induced rat mammary carcinoma model. The volume corrected mitotic index (M/V INDEX) and the S-phase fraction (SPF) determined by flow cytometry (FCM) were used as proliferation markers. Two series of rats (A and B) treated with two dose levels of toremifene were used. The two series of tumors appeared to have different growth properties. In series A the tumors were rapidly growing with high proliferation rate. In this series, toremifene (3 mg/kg for 4 weeks) reduced significantly the mean MV/INDEX, but the slight reduction of the mean SPF was not significant. In series B the tumors grew slowly and had low levels of proliferation markers. One-third of the tumors were spontaneously stable in the untreated group. Higher dose of toremifene was used in this series (12 mg/kg for 4 weeks), and the number of regressing or stable tumors was 58% compared with 31% in series A. Taking into consideration the high number of spontaneously stable tumors in series B, it may be concluded that about one-third of the tumors regressed or remained stable due to toremifene treatment in both series. The reduction of the M/V INDEX was significant only when the regressing treated tumors were compared with the growing controls. The reduction of the SPF was not significant. We think that the M/V INDEX is a more appropriate method to measure cell proliferation than is the SPF in this tumor model, where the tumors are heterogenous and, e.g., spontaneous apoptosis is known to be frequent.

Bleaching of melanin before image cytometry of the DNA content of pigmented skin tumors.

OBJECTIVE: To study the effect of bleaching of melanin with KMnO4 on the results of DNA image cytometry in pigmented skin tumors. STUDY DESIGN: Image cytometry of nuclear DNA content was performed on sections from 14 melanocytic skin tumors stained with Feulgen stain both with and without prior bleaching with KMnO4. RESULTS: The nuclear staining intensity of Feulgen stain was lower in the bleached sections, but this did not significantly affect the evaluation of ploidy. Heavy pigmentation caused some false peaks in the histograms (4 of 28 measurements made on unbleached slides). CONCLUSION: Bleaching of sections with KMnO4 can be useful when heavy melanin pigmentation would make DNA measurements impossible or difficult in image analysis cytometry. Bleaching is not advisable when only lightly pigmented tumors are analyzed if nuclei obscured by any pigment granules are to be avoided. In large series containing both bleached and nonbleached specimens, statistical analysis of these groups should be separated.

P90 exceeding rate as a prognostic factor in primary malignant melanoma of the skin. A DNA image cytometric study.

OBJECTIVE: To study the prognostic value of DNA image cytometry in primary skin melanomas. STUDY DESIGN: DNA image cytometry was performed on 62 stage I, Clark level II-V, primary skin melanomas. The DNA histograms were classified into three categories (diploid, nondiploid and aneuploid) according to the percentages of cells with higher-than-diploid and higher-than-twice-the-diploid DNA content (the P90 and 2P90 exceeding rates [ERs]). The prognostic value of P90ER, 2P90ER, type of DNA histogram, melanoma thickness, Clark level, and patient age and sex were analyzed for disease-specific survival with Cox's stepwise proportional hazards model. RESULTS: Aneuploid DNA histograms were as common in thin as in thick melanomas. Melanoma thickness and P90ER had prognostic value in univariate analysis, but in the multivariate analysis only P90ER had independent and significant prognostic value. CONCLUSION: Aneuploidy is a common feature of malignant melanoma, and it is as common in thin as in thick melanomas. P90ER has more prognostic value than the type of DNA histogram. The prognostic value of P90ER as compared with melanoma thickness should be studied further.

Subjective breast cancer grading. Analyses of reproducibility after application of Bayesian belief networks.

OBJECTIVE: To examine the influence of Bayesian belief networks (BBNs) on the reproducibility of subjective breast cancer grading. STUDY DESIGN: Twenty samples were analyzed for intraobserver and 128 samples for interobserver reproducibility using the Bloom-Richardson and Helpap grading systems. The expression of diagnostic features was evaluated subjectively, and for each a decision it was determined to what extent it represented one of the different outcomes. Evidence was then entered, for each diagnostic feature, into four different BBNs, recently described for breast cancer grading, in the form of a relative likelihood ratio vector. RESULTS: With all cases considered, the use of decision support based on the Bloom-Richardson and Helpap grading systems did not improve intraobserver reproducibility. This was found to be 68% and 80% in subjective gradings, respectively, and 60% and 70% in the BBN-supported method. Interobserver reproducibility was not improved (58% and 70% in subjective gradings and 51-59% based on assessment with decision support). However, when only cases associated with high beliefs were considered, both intraobserver reproducibility (agreement rose from 68% to 93%) and interobserver reproducibility (agreement rose from 60% to 87%) of BBN-supported gradings exceeded the results of subjective assessments. CONCLUSION: The results showed that the observers did not reach the same diagnosis (or grade) and that their observational assessment of histologic features lacked agreement. Since BBNs reflected only the data entered, poor agreement existed in the contribution to the final diagnostic belief by the different features and, ultimately, in belief in the final decision.

Nuclear morphometry, immunohistochemical staining with Ki-67 antibody and mitotic index in the assessment of proliferative activity and prognosis of primary malignant melanomas of the skin.

Nuclear morphometry, immunohistochemical staining with Ki-67 antibody and mitotic index were studied in primary cutaneous malignant melanomas. The number of Ki-67 positive cells/ 200 tumor cells did not correlate with any nuclear morphometrical parameters, and it only approached but did not reach significant correlation with melanoma thickness according to Breslow. The nuclear area, short axis and long axis correlated with melanoma thickness, but the nuclear axis ratio (which reflects the sphericity of nuclei) and melanoma thickness did not show significant correlation. Mitotic index was higher in thick melanomas and in melanomas with high Ki-67 positivity, large nuclear area, long nuclear short axis, and small nuclear axis ratio. In Cox's stepwise proportional hazard model, melanoma thickness and the nuclear axis ratio were significant independent prognostic factors for patient survival, while the nuclear area, short axis and long axis, gender, age, Clark level, mitotic index and Ki-67 positivity lacked significant independent prognostic value. The results suggest that the proliferative activity of tumor cells does not alone explain the great importance of tumor thickness as prognosticator in melanoma. The thickness of melanoma measured according to Breslow and the nuclear axis ratio are more efficient prognosticators in melanoma than parameters associated with proliferation.

Nuclear morphometry in differential diagnosis of renal oncocytoma and renal cell carcinoma.

PURPOSE: Morphometric distinction of renal oncocytoma and renal cell carcinoma was attempted. MATERIALS AND METHODS: Nuclear morphometric measurements were done from sections of 16 histologically diagnosed renal oncocytomas and renal cell carcinoma controls (grade 1 or 2, stage T1 to 2N0M0). RESULTS: Oncocytomas could be distinguished from carcinomas on the basis of nuclear morphometry in more than 80% of the cases. Especially, the shape descriptors (form factors) were significantly different between oncocytomas and carcinomas. CONCLUSIONS: Morphometrically determined nuclear shape descriptors can be used to support diagnostic decisions in problematic cases.

Management of uncertainty in breast cancer grading with Bayesian belief networks.

OBJECTIVE: To examine the potential of different constructs of Bayesian belief networks (BBN) to manage uncertainty in breast cancer grading. STUDY DESIGN: We developed four networks, two based on Bloom-Richardson's and two on Helpap's grading systems. The function of the networks was based either on an expert's experience or frequency counts derived from subjective grading of a large number of samples. The four BBNs were tested on 20 specimens, and the resulting final beliefs were compared with the subjective gradings. RESULTS: The BBNs showed agreement with the subjective gradings in 60-85% of cases. Different constructs of BBNs, however, differed in their performance. The mean beliefs in frequency-based networks were slightly higher than in experience-based networks. In addition, as compared with the Bloom-Richardson-based networks, the Helpap-based BBNs resulted in higher maximum beliefs but produced a larger fraction of discrepancies with the subjectively graded cases. Depending on the type of network, 65-90% of the BBN grades were associated with high beliefs. CONCLUSION: The results suggest that for reliable results, grading systems with more than three or four variables may be necessary. When based on relevant information, BBNs seem to have the potential to become a valuable method of assisting the pathologist in breast cancer grading.

Immunohistochemical labelling for prostate-specific antigen in breast carcinomas.

Immunohistochemical detection of prostate-specific antigen (PSA) is an aid in determining the prostatic origin of metastatic cells. However, small amounts of PSA have also been found in non-prostatic tissues and tumors, for example in some breast carcinomas, by highly sensitive immunofluorometric methods, but also by immunohistochemistry. Our aim was to evaluate the prevalence and prognostic value of histologically confirmed PSA immunoreactivity in breast carcinoma. Sections of formalin-fixed, paraffin-embedded samples from 171 breast carcinomas were immunostained for PSA. The staining results were compared with the mitotic activity, tumor size, histological grade, steroid receptors and follow-up data. For analysis the material was divided into subgroups according to the patients' age (pre- and postmenopausal). PSA was found by immunohistochemistry in 54 (32%) breast carcinomas. In survival analysis of the whole patient material PSA positivity did not show prognostic value. Among premenopausal patients concomitant estrogen receptor and PSA-negativity proved to be associated with high risk of breast cancer death (RR 6.2), also after adjustment for tumor size, histological grade, and axillary lymph node status. Among postmenopausal patients PSA positivity was associated with progesterone receptor positivity and high differentiation but not with age, nodal status, or mitotic activity. PSA can be detected by immunohistochemistry in a considerable number of breast carcinomas. PSA immunoreactivity alone does not seem to have any value as general prognosticator of breast carcinoma patients. However, concomitant absence of PSA and estrogen receptors was an indicator of unfavourable prognosis among premenopausal patients.

Prognostic studies in breast cancer. Multivariate combination of nodal status, proliferation index, tumor size, and DNA ploidy.

We studied histological samples and clinical data from 111 breast carcinoma patients originally treated in 1975-1977 who did not have distant metastases at the time of diagnosis. A multivariate survival analysis using the Cox model selected the studied variables in the following order according to their prognostic association (breast cancer deaths, or deaths from any cause): axillary lymph node status, tumor size, mitotic index, and DNA ploidy status. The association of the different variables to the prognosis in respect to breast cancer deaths was evaluated 1-10 years after treatment by stepwise logistic regression. Lymph node status, tumor size, mitotic index, and DNA ploidy all showed significant relation to the prognosis but this association varied considerably with time of observation.

Uptake and metabolism of hydroxymatairesinol in relation to its anticarcinogenicity in DMBA-induced rat mammary carcinoma model.

The chemopreventive effects of hydroxymatairesinol (HMR), a lignan extracted from Norway spruce (Picea abies), on the development of mammary carcinoma induced by 7,12-dimethylbenz[a]anthracene (DMBA) was studied in rats. HMR administered via diet in an average daily dose of 4.7 mg/kg body wt starting before DMBA induction reduced tumor volume and tumor growth, but no significant reduction in tumor multiplicity (number of tumors/rat) was observed. The predominant histological type in the control group was type B (well-differentiated adenocarcinoma, 78%). The proportion of type B tumors decreased to 35% in the HMR group, while the type A (poorly differentiated) and type C (atrophic) tumor proportions increased. Anticarcinogenic effects of dietary HMR (4.7 mg/kg) were also evident when the administration started after DMBA induction and was seen as growth inhibition of established tumors. Dietary HMR supplementation significantly increased serum and urinary enterolactone and HMR concentrations but had no significant effect on the uterine weight, suggesting that HMR or its major metabolite enterolactone did not have an antiestrogenic effect. Further studies are warranted to further clarify and verify HMR action and the associated mechanisms in mammary tumorigenesis.