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Ultrastructural changes in cell nuclei were studied in kidney biopsies from 18 patients suffering from Nephropathia epidemica. Three patients showed small nuclear particles in a large aggregate in a few cells of the distal tubule. The particles had a diameter of about 30 nm and their occurrence was not associated with intravenous glucose infusions. Large light nuclear bodies were numerous in the nuclei of both interstitial and tubular cells. Many showed strands of darker material about 35 nm in diameter at their center. Nuclear vesicles with varying amounts of membrane debris were seen in both tubular and interstitial cells. Occasional interstitial lymphocytes showed abnormal margination of chromatin. Of the changes observed the small nuclear particles are not found in kidney biopsies in other conditions and it is possible that they are aggregates of viral protein.

Flocculent densities appear in the mitochondrial matrix during ischemic damage. These densities were examined in kidneys after 2 hours of autolysis. The samples were embedded in water-miscible glycol methacrylate (GMA). Ultrathin sections on copper grids were digested with pronase, trypsin, ribonuclease T1, various phospholipases (A2, C, D), lipase, and desoxyribonuclease I. For statistical analysis the mean number (+/- SD) of densities per mitochondrion was determined in tubular epithelial cells. A statistically significant (p less than 0.001) decrease in the number of densities was apparent after treatment with pronase (75.5%) of the densities digested completely) and with trypsin (43.3% of the densities digested completely). Digestion with other enzymes did not significantly decrease the number of densities. This study provides quantitative support for the hypothesis that flocculent densities are composed of proteins, most probably mitochondrial membrane proteins or matrix proteins. The results do not support the hypothesis that RNA, neutral lipids, phospholipids or DNA are major constituents of these structures.

This is the first reported case in which neoplastic cells not of granulocytic or monocytic origin have been shown to contain lysozyme. A highly differentiated metastasizing adenocarcinoma of the jejunum is presented. Areas of the tumour contained cells resembling Paneth cells, that is, they contained cytoplasmic granules which 1) stained red with Masson's trichrome and 2) were shown to contain lysozyme by an immunoperoxidase technique. No argentaffin cells could be identified within the tumour. Staining of mucosubstance revealed large intestine-type sulfated glycoproteins in the tumour tissue. The presence of lysozyme-containing neoplastic Paneth cells suggests that the tumour: 1) originated from the mucosal crypts, and 2) had a high degree of cellular differentiation.

A report is given on a female patient, 32 years of age, with a history of slight joint distress, recurrent dependent oedema, hypokalaemia, hypergammaglobulinaemia and positive tests for rheumatoid factor. Electrolyte balance studies revealed latent renal tubular acidosis (RTA), impaired renal pH-regulation and impaired renal conservation of sodium and potassium. Systemic pH was found to be largely dependent on the dietary load of sodium chloride. Correction of the hypokalaemia resulted in frank systemic acidosis, whereas persistent hypokalaemia was associated with alkalosis. The situation thus differed from that found in primary RTA. Electron microscopic and light microscopic studies from a renal biopsy revealed simultaneous signs of tubular and interstitial nephritis and slight glomerular reaction. Immunoglobulin deposits were seen in the tubuli and the glomerular basement membrane by the immunofluorescence method.

Taurine content of 199 clinical muscle biopsies was determined and correlated to histometric data of 121 cases. Taurine concentration in muscles was markedly dependent on fiber type distribution, taurine being more abundant in the slow, oxidative type 1 fibers than in the type 2 fibers. Taurine concentration rose slightly with age and tended to be higher in denervations, muscular dystrophies and myotonias, but the differences from the control values were non-significant.

The aim of our study was to correlate apoptosis, assessed by means of lamin B degradation, with grade of tumour and immunohistochemical expression of p27Kip1 and retinoblastoma protein (pRB) in a series of 32 low-grade astrocytomas (LGA) and 43 high-grade astrocytomas (HGA). Determination and analysis of lamin B expression was achieved stereologically with the use of computerised interactive image analysis. The average stereological surface density of the lamin-positive nuclear surface of cells showing evidence of lamin degradation cells was 13.80 mm2/mm3 in LGA and 21.02 mm2/mm3 in HGA. The average range of immunopositivity of p27 and pRB expression was 2.85 and 2.10, respectively, in LGA and 1.80 and 0.88, respectively, in HGA. The rate of apoptosis indirectly correlated with the expression of both p27 and pRB but was not fully significant (Por = 0.09). Our findings suggest that there may be a loss of function of main cell-cycle regulators and increased uncontrolled cell death in addition to increased cell proliferation in high-grade astrocytomas. Moreover, we believe that the detection of lamin B degradation in astroglial tumours can be an alternative, precise and convenient assay for the detection of a defined set of apoptotic cells. However, before being applied more generally, comparative research should be done on different methods of quantifying apoptosis, with the intent of finding the difference between biologically different methods. Such studies would also be able to find the method associated with the strongest prognostic power.

In an effort to determine numerical thresholds for histological breast cancer grading, we evaluated the fraction of fields with tubular differentiation (FTD) in Nigerian breast cancers (n=300). Analyses were based on Kaplan-Meier survival curves, and univariate and multivariate analyses by Cox's regression. The mean (SD) value of FTD in Nigeria, 16.7 (19.3)%, was much lower than reported in European breast cancer. Decreasing FTD was associated with increasing histological grades (p0.0001) and clinical stage (p = 0.0190). The most significant cut-point for FTD in predicting outcome was 15.0%. In univariate analysis, FTD 15% was a significant prognosticator in the whole material, in larger than 5cm tumours, among postmenopausal and premenopausal patients, and LN+ patients. After multivariate analysis with mitotic count, FTD was an independent prognosticator among tumours larger than 5cm, but not in other groups. We conclude that the rational grading of breast cancers need be optimised according to diagnostic and therapeutic environment. We propose FTD 15% as a cut-point to grade breast cancer in Nigerian material.

The immunohistochemical detection of tumor marker CA 50 was studied in bladder cancer of WHO grades I–III. The material consisted of tumors in 83 patients and the mean clinical follow-up time was thirteen years (range 9.6–22 years). The fraction of CA 50-positive cells (FPtot) in microscopic image was scored 0–100 percent. Also the maximally staining region was selected, and the fraction of CA 50-positive cells in this region was scored 0–100 percent (FPmax). The average staining intensity of CA 50-positive cells was scored from 0 to 3 in the whole section (ASItot) and in the maximally staining area (ASImax). The inverse relation between histologic grade, FPtot (p = 0.0001), and ASItot (p = 0.006) was statistically significant. FPtot (p = 0. 039) and ASItot (p = 0.018) were also inversely related to clinical stage. Occurrence of metastasis during the follow-up was associated with low CA 50 positivity (FPtot, p = 0.003; ASItot, p = 0.002). The lower the staining intensity or the lower the fraction of CA 50-positive cells, the more aggressive was the tumor. In survival analysis, low FPtot (p = 0.002) and ASItot (p = 0.007) values were related to high risk of bladder cancer death. The results show that immunohistochemical staining of bladder tumor specimens with CA 50 can be used to predict bladder cancer aggressiveness and survival.

Two mitotic activity indices, volume fraction of neoplastic epithelium, nuclear area, nuclear perimeter, and shortest nuclear axis were estimated in 46 ovarian tumors by three observers in two independent laboratories. The mitotic activity index, the volume corrected mitotic index (M/V index) and subjective volume fraction estimates showed a very good correlation from the same fields by two observers in the same laboratory (r = .999, .995, .950). Repeat estimates from different fields by the same observer showed coefficient values of .949, .939, and .813, and estimates by two different observers within one laboratory values of .949, .936, and .789, respectively. The correlation between two independent observers in different laboratories was also good (r = .894, .834, .834, respectively). Grading based on morphometric measurements was uniformly performed in 90-100% of cases in an interfield interobserver intralaboratory situation, and in 79-97% (M/V index) or 76-93% (mitotic activity index) in an interlaboratory situation. Because the cases near the grade limits were more often differently graded than other cases, the methods allowed the pathologist to locate the probably incorrectly graded cases. Morphometry of ovarian tumors was shown to be easy, which makes morphometric malignancy grading systems potentially able to support diagnostic and therapeutic decisions in practice.

Carcinoma of the breast in children is an extremely rare disease, only 44 patients have been reported in the world literature. We report on a case of breast cancer in a 9-year-old girl, who was admitted to the hospital because of a four-month history of a painless mass in the right breast. Clinical examination and mammography resulted in a strong suspicion of malignancy. The histology corresponded to juvenile secretory carcinoma, the histological type without the characteristics of prepubertal cancer cases. Electron microscopy demonstrated intracellular large vacuoles and acini filled with secretory material. The mitotic index was low, the highest measured value was 4 mitotic figures per 10 high power fields corresponding to 2.78 mitotic figures per mm2 of epithelium in the microscope field. Flow cytometry showed that the neoplastic cells were diploid and had a low S-phase fraction. A simple mastectomy including axillary evacuation was performed. No lymph node or distant metastases were found. Postoperative radiation treatment or chemotherapy were not administered. Our patient has been in follow-up for six years without recurrence. In this case simple mastectomy with axillary evacuation seems to have given a cancer-free development into adulthood. Earlier reports have also shown a favourable outcome in these tumours.

The nuclear DNA content of archival paraffin-embedded bladder cancer samples (70 patients) of WHO grades I-III has been measured by flow cytometry. The female/male ratio was 15/55. The mean follow-up time was 13 years (range 9.6-22.0 years). 37 of 70 (53%) patients had DNA index 1.0 (diploid DNA content), and the remaining 33 (47%) patients had an aneuploid tumor. There was no significant difference in the age (mean +/- SD) of the patients having a diploid (66 +/- 9 years) or an aneuploid tumor (68 +/- 11 years) at the time of diagnosis. 47 deaths occurred during the follow-up period; 24 (51%) of these were due to bladder cancer (12 diploid, 12 aneuploid tumors). No significant difference was found after radical treatment during the disease-free interval (mean +/- SD) between diploid (48 +/- 45 months) and aneuploid (35.5 +/- 35 months) groups of patients. Recurrences during the follow-up period were equally common among aneuploid and diploid tumors. A statistically significant relation between histological grade and survival could be demonstrated, but DNA ploidy and S phase fraction had little prognostic value in this respect. There was no statistically significant difference in survival between aneuploid (30%) and diploid (35%) groups of tumors during the follow-up period. The study suggests that flow cytometric determination of nuclear DNA ploidy from paraffin-embedded samples in bladder tumors does not add to the prognostic power of subjective histological grading.

The potential of nuclear morphometry and volume-corrected mitotic index (M/V index) in grading cases of transitional cell carcinoma of the urinary bladder was studied. 30 cases of bladder cancer including all three WHO grades were evaluated. Four investigators selected independently the most atypical field from paraffin sections, and one investigator measured the nuclear areas from these fields using the IBAS 1&2 image analyzer system. Following the same sampling rule, four investigators counted the mitotic figures per area of neoplastic tissue in the microscopic image at an objective magnification of X40. The mean nuclear areas covered values from 28.1 to 139.0 microns 2 (mean +/- SD 57.2 +/- 18.9). The total variance of measurements was 359.1 and the mean variance between corresponding fields 110.2 (about 30% of the total variation). The efficiency was evaluated by estimating the fraction of falsely classified cases. Instrumental morphometry of nuclear area in a three-grade system gave an efficiency of 79% and of 90%, in a two-grade system. The M/V index varied from 0 to 54 (mean +/- SD 12.3 +/- 10.9). The total variance was 119.8 and the methodological variance 15.5 (about 13% of total variance). In a three-grade system this would correspond to an efficiency of about 75%; in a two-grade system the efficiency would be 88%. The results suggest that nuclear area and M/V index estimates constitute efficient grading systems in bladder carcinoma.

The immunohistochemical reactivity of tumour markers MCA and CA50 was determined in transitional cell carcinoma of the bladder of WHO grades I-III. The material consisted of paraffin-embedded biopsies from bladder tumors in 83 patients. Mean follow-up time was 13 years (range 9.6-22 years). Staining indexes for the whole section and for the area with maximal staining intensity were calculated (for MCA; IMCAtot, IMCAmax, for CA 50: ICA50tot, ICA50max). Also two combination indexes were created (Itot, Imax). The relations between histological grade on one hand, and IMCAtot (p less than 0.001), IMCAmax (p = 0.007), ICA50tot (p less than 0.001), and ICA50max (p less than 0.001) on the other were statistically significant. IMCAtot (p = 0.008) and ICA50max (p 0.040) had a statistically significant relation to clinical stage. IMCAtot (p = 0.004) and IMCAmax (p = 0.001) also predicted node involvement with statistical significance. Metastasizing behavior could be predicted with all four indexes with statistical significance (IMCAtot p = 0.021; IMCAmax, p = 0.001; ICA50tot, p = 0.021; ICA50max, p = 0.050), but best with IMCAmax. Bladder cancer survival was related to IMCAtot (p = 0.044) and to ICA50tot (p = 0.0009) with statistical significance. The higher the MCA positivity and the lower the CA50 positivity, the worse was the prognosis. The combination index (Itot) predicted survival (p = 0.0004) better than IMCAtot or ICA50tot separately. Itot and Imax did not offer any advantages over other indexes in predicting node involvement or metastasis. Grade II tumors were divided into two prognostic groups using Itot, but the difference between survival was not statistically significant.(ABSTRACT TRUNCATED AT 250 WORDS)

A retrospective follow-up (range 9.4-22 years, mean 13 years) study of 83 patients with grade I-III (WHO) bladder carcinomas was performed. Nuclear area (mean +/- SD 59.7 +/- 18.7 microns2) and the SD of nuclear area (mean +/- SD 19.7 +/- 13.4 microns2) were determined by using morphometric methods. The SD of nuclear area and histopathological grade exhibited a clearly significant relation, the relation between grade and nuclear area was weaker. The number of recurrences in the bladder and the recurrence-free period were not significantly related to histopathological grade, mean nuclear area or SD of nuclear area. The progress in nodal or metastatic stage could be predicted by histopathological grade, mean nuclear area and SD of the nuclear area. Prediction of crude survival, however, was not efficient. When only bladder cancer deaths were included in the analysis, histopathological grade (p less than 0.001), mean nuclear area (p = 0.011) and SD of the nuclear area (p = 0.001) showed a significant relation to survival. Grade II tumors could be divided into two prognostically different groups using nuclear area and SD of the nuclear area as classifiers. The results suggest that morphometric parameters are as good as histopathological grade in predicting long-term prognosis of bladder carcinomas, and better than the histopathological grade in predicting progress in nodal (N) or metastatic (M) stage.

The prognostic value of subjective histological and morphometric grading was studied in 75 primary ovarian carcinomas. Histological grading methods recommended by Czernobilsky and by Russell and the morphometric method of Baak and co-workers were compared in a two-observer system. The 5-year survival could be correctly predicted in about two-thirds of the patients with all three methods. When mitotic counting (volume corrected mitotic index, M/V-index) was compared with the above grading methods by using a receiver operating characteristic curve) the M/V-index was generally superior in its prognostic power regardless of the sensitivity/specificity level chosen. The morphometric grading method and the grading method based on the M/V index were also shown to be readily reproducible.

Kidney biopsy reports given during 2003 were collected from the authors' pathology database. A total of 111 biopsies were performed. Five tumor samples were not studied with electron microscopy (EM). Of the remaining 106 biopsies, 85 were studied with EM. EM was not performed in 10/24 transplant biopsies, or in 11/82 cases of suspected primary kidney disease. The role of EM was evaluated by grouping the samples in 3 categories: (1) EM was essential for diagnosis, (2) EM contributed to the interpretation and cleared uncertainties, and (3) EM had no influence on the diagnostic process. In transplant biopsies EM influenced the final diagnosis in 86% of cases (category 2). In biopsies performed for primary kidney disease EM was essential for diagnosis in 18.3% clearly contributed in 53.5%, and had no influence on the final diagnosis in 28.2% of cases. The study suggests that the importance of EM has not decreased during the last few years. Because only about 25% of the EM reports did not have any influence on the diagnostic process, it is recommended that kidney biopsy protocols should include EM in all biopsy cases, or at least tissue should be reserved for EM studies of all cases. Because of the influence of EM on the diagnostic process the need for EM in pathology training should be emphasized.

We compared the histology and patterns of occurrence of breast cancers in Nigeria (n = 297) and Finland (n = 285).The histology of invasive ductal carcinoma (IDC) was re-evaluated using similar criteria. The clinical data were extracted from medical records.The mean age at presentation was 42.7 (12.2) years in Nigeria vs. 58.7 (12.5) years in Finland. In both populations there was an association between reproductive factors and the occurrence of breast cancer. In Nigeria, 53.2% of cases belonged to stages 3 and 4 (vs. 6.7% in Finland). In Finland there were higher frequencies of lobular, tubular and mucinous types than in Nigeria. The Nigerian material had more medullary type (2.7% vs. 0.7% in Finland), extensive necrosis, nuclear atypia and pleomorphism, with coexisting pleomorphic ductal carcinoma in situ. At 2 years after treatment, the survival figures for Nigeria and Finland were 72.8% and 96.4%, respectively.The histology of Nigerian and Finnish cancers clearly differ. Nigerian cancers appear more advanced with higher grade. The atypical in situ component is clearly more common in Nigeria. Part of the differences can be explained by diagnostic and treatment delays associated with misguided socio-cultural beliefs, poor health care access and impaired immunity.

Differential analysis and cytological grouping of fine needle aspiration biopsy (FNAB) samples of the prostate are important in practice. We used image analytical DNA cytometry to achieve this and also studied the best method of interpretation of the histograms.Sixty-two FNAB samples of the prostate were stained with Feulgen stain and nuclear DNA histograms were produced by image cytometry. The most atypical cell groups were selected for measurements. Also, free epithelial cells between cell groups were studied. The cells presented in the histograms were grouped according to the nuclear DNA content and by application of different gates of observation for diploid status.Several DNA histogram features (histogram classification categories, benign and malignant histogram patterns, presence of5c-7c cells) showed significant relationships to differential diagnosis. Highly aneuploid (5c-7c) cells had the potential for distinguishing a progressive-type of prostate cancer.The fraction of tetraploid and aneuploid histograms increased from atypical but benign to definitely malignant samples. DNA histograms have potential in the differential diagnosis and evaluation of the progressive character of prostate cancer.

We tried to improve the evaluation of E-cadherin immunostaining in paraffin sections, to distinguish the less aggressive variants of ductal infiltrating breast cancer from other variants.The method graded the membrane staining and estimated the fraction of area of cancer tissue stained at the respective staining grade, resulting in an immunohistochemical staining index. At the cut-point 0.35 the index divided all 157 patients (P=0.0188), and 57 node-positive patients (P= 0.0006) into two groups of different survival. In multivariate analysis (all patients) E-cadherin immunoscore was inferior to mitotic index (SMI) (P=0.0002), but still significant (P=0.0031). Among node-positive patients E-cadherin was even more powerful and superior (P=0.0001) to the still significant SMI (P=0.0023), and E-cadherin immunostaining and the mitotic activity (SMI) combined did not need the support of other prognosticators in the Cox model.The study suggests that E-cadherin immunostaining can be used efficiently in finding patients with favourable outcome among node-positive patients.

Colon cancer is one of the most common malignancies in Finland. The purpose of the current study was to analyse the results of surgical treatment of right-sided colon cancers operated in the 1980s at Turku University Central Hospital. In addition, we compared the results to those reported from earlier decades.One hundred and fifty-three patients with primary proximal colon cancer were operated in 1981-1990. The results were analysed retrospectively from patient records.The crude five-year survival rate of the patients was 48%. The most crucial factor affecting survival was the stage of spreading of the tumour. Obstructive tumours had a poorer prognosis than non-obstructive ones.The results of surgical treatment of proximal colon cancer were satisfactory at Turku University Central Hospital and slightly better compared to earlier reports.

To find efficient morphometric nucleolar features for distinguishing different prostatic atypia groups in fine needle aspiration biopsies.A computerized interactive morphometry program was used to outline nucleoli of prostate cells. The samples were divided into three groups: group 1 (definitely benign [n = 10] and atypical but benign [n = 13]), group 2 (moderately atypical [n = 11] or highly suspicious [n = 13]) and group 3 (definitely malignant [n = 17]).The analysis revealed a difference in the number of nucleoli between definitely benign samples and other atypia groups but not between the latter. Nucleolar size features were best in distinguishing between atypia groups. The sample-associated mean size features were more powerful than cell group-associated size features.The sample-associated mean area, defined from cells selected by an experienced cytologist, if larger than 2 micron 2, was strongly associated with definitely malignant samples. The same was true for the largest nucleolar area if larger than 5.0 micron 2. Morphometric nucleolar size features appeared efficient in distinguishing between definitely malignant and other samples.

To study the potential of nuclear size and shape estimates in interpreting fine needle aspiration biopsy (FNAB) samples of the prostate.Morphometry was used to outline nuclei of prostate cells. Cell groups were selected by an experienced cytologist.The mean area of nuclei in the most atypical cell groups among definitely malignant samples (n = 17) varied from 26.3 to 93.3 micron 2 and in normal prostate cells (n = 10) from 15.6 to 33.7 micron 2. Perfect distinction of definitely benign and slightly atypical samples (n = 13) from definitely malignant samples was possible when the samples were characterized by the weighted means of the mean nuclear areas of the cell groups in the samples. The means of individual cell groups allowed correct distinction in only 84.8% of cell groups. Shape factors did not have any diagnostic value.Morphometric nuclear size estimates from ethanol-fixed FNAB samples of the prostate are of diagnostic value and can potentially be used as part of multivariate diagnostic models when selected by an experienced cytologist according to strict criteria. However, measurement should be done from several cell groups (at least three of the most-atypical cell groups) in each sample.

Evaluation of nuclear DNA staining intensity from histological breast cancer sections has not always been accepted, because of the difficulties in interpreting the histograms. One reason for this is the lack of evidence based interpretation guidelines.The DNA staining intensity of 140 breast cancer samples was measured with flow cytometry (FCM) and image cytometry (ICM). The methods were compared by using grading efficiency (GE).First, the ICM histograms were evaluated with a computer assisted image cytometry system using different cut off points for aneuploidy. The GE results varied from 67.9-76.4%. Subjective interpretation and evaluation according two previously published interpretation methods did not improve the GE. Secondly, we excluded histograms which showed clearly different cell clones in FCM and ICM. The GE of remaining histograms was 77.9%. Comparison of these histograms allowed formulation of interpretation guidelines which improved the GE to 85.3%.This study suggests that efficient interpretation guidelines of section-based DNA histograms can be created.

The purpose of the study is to evaluate histological measurement methods for quantitative assessment of the degree of tubular differentiation in breast cancer.We evaluated tubular differentiation in 20 cases of invasive breast cancer by four different assessment methods. Method 1 was the traditional subjective evaluation of the amount of malignant tubules in each sample. Method 2 evaluated the fraction of fields presenting tubular differentiation by registering the presence or absence of neoplastic tubular structures in each microscopic field. In method 3 the area fraction of malignant epithelial cells presenting tubular differentiation was assessed field-by-field and expressed as an average of the whole tumour area. Method 4 applied point counting for evaluating the fraction of malignant epithelial cells in tubular structures. By correlation and reproducibility analyses, method 1 was inferior to the other methods. Method 4 was accurate but too laborious and time-consuming for clinical use. Methods 2 and 3 were both efficient and reproducible and could be used interchangeably. With the time and effort used in the measurements taken into consideration method 2 was best applicable to clinical practice.Accurate evaluation of tubular differentiation in breast cancer is possible by defining the presence or absence of tubular differentiation in microscopic fields of a histological section. Assessment of the fraction of fields with tubular differentiation (FTD) is simple, unambiguous, objective and fast--even a large sample can be screened in less than 10 min. In our results, FTD has clear advantages over subjective or point counting-based evaluation methods of tubular differentiation.

To develop the necessary correction for the polymerase chain reaction-based oncogene amplification test applied to paraffin-embedded tissue sections.The theoretical basis for making the correction in test results and for determining the neoplastic cell fraction was clarified, and a method for microscopic sampling was developed. The cell count bias was corrected by applying an equation by Ebbeson and Tang and using two adjacent tissue sections of different thickness, cut from the block from which DNA was isolated.There was no need to determine the absolute thicknesses of the sections. Section thickness, as a relevant feature, was cancelled out in developing the formula for estimating the cell fraction. The correction was applied in practice, and when tested on 10 breast cancer samples, the correlation between amplification estimates from two observers was good (r = .96).This method is valuable for interpreting the test results based on paraffin sections and for developing simpler quantitating methods for special test applications. The study also showed that amplifications may be more common than expected on the basis of the crude results of this type of test.

To evaluate the formula of Ebbeson and Tang (FET) with respect to the disector (DS) principle.The DS principle has been proposed for avoiding cell count bias. DS is a slice of tissue, and from it those cells by area are counted in microscopy; the cells are not in contact with one of the surfaces of the slice. The resulting number divided by the thickness of the DS gives an accurate figure for cell number by volume. FET applies two sections of different thickness, usually cut adjacent. Cells seen in the sections are counted by area, the figures are subtracted from each other, and the difference is divided by the difference between the thicknesses of the sections. The result is cell number by volume: NV = (NA1 - NA2)/(t1 - t2).FET and the DS principle superficially appear different. However, from a geometric point of view they are based on the same principle. When the thickness of the thinner section of FET approaches zero, the situation is in all respects equal to the DS principle. The formula for DS can thus be written: NV = (NA1 - NA2)/t1.The result proves that in principle DS and FET are equivalent methods of counting cell numbers by volume in tissues. FET may be more easily applied in histopathology practice because visual comparison of the sections is not necessary. Section thickness, however, has to be measured from vertically embedded sections or with scanning laser confocal microscopy. FET shares the stereologically unbiased character of the DS principle and is independent of the size and shape of structures counted.

During the 20-year period from 1961 to 1980, 17 cases of extranodal head and neck non-Hodgkin's lymphoma in children under 15 years of age were diagnosed in Finland. Eight cases had tumours in tonsils or nasopharyngeal adenoids and 9 cases lymphomas of other sites of the head and neck. The age-adjusted annual incidence rate for all cases was 0.69 and for tumours of the pharyngeal lymphatic tissue 0.35 per 10(6) children. Seven of the 17 tumours were undifferentiated lymphomas (Burkitt's and non-Burkitt's types) and 5 lymphoblastic lymphomas. The overall 5-year disease-free survival rate was 37%. Five of the 9 patients with tonsillar or adenoid lymphomas were disease-free more than 5 years.

Three hundred sixty-four cases of invasive ductal breast cancer diagnosed during the years 1988 to 1991 were analyzed to determine quantitative thresholds for mitotic activity. Mitotic counts were calculated in each sample and expressed as standardized mitotic index (SMI) and mitotic activity index (MAI). Based on Kaplan-Meier curves, univariate and multivariate analysis of Cox's regression, and maximum efficiencies of ROC analysis, optimal thresholds were determined on the basis of survival and recurrence of disease. In our material, with a follow-up time of 5 years 9 months, we found two thresholds--a lower and a higher--for both SMI (17 mitoses/mm2 and 32 mitoses/mm2) and MAI (13 mitoses/10 HPF and 35 mitoses/10 HPF). The thresholds were the same in the whole material and in subgroups divided according to the patients' age and axillary lymph node status at the time of diagnosis, and tumor size. The thresholds clearly separated patients with favorable, intermediate, and unfavourable outcome of disease. In our material, the risk of breast cancer death associated with the determined thresholds (ranging from 4.7 to 3.8) clearly exceeded those of menopausal status, axillary lymph node status and tumor size. The risk of breast cancer death associated with the determined thresholds was still emphasized in the groups of premenopausal and axillary lymph node-negative patients, and with tumor size less than 2 cm in diameter (risk ratios, 11.8, 6.0, and 6.7, respectively). The results suggest that the presented quantitative thresholds could be applied in grading of invasive ductal breast cancer.

We compared the performance of multivariate models based on mitotic activity index, lymph node status, and tumor size in the prognostication of breast cancer.Cox and discriminant models for survival were created for two patient groups: a) 120 breast cancer patients, and b) 86 patients with ductal infiltrating carcinoma. The models were compared with the model of Baak et al (1985).The models distinguished between dying and surviving patients with an efficiency of 70.9-77.9% in mutual tests. With a single cutoff the model of Baak et al was less efficient (50.8-65.8%). If a region of uncertainty was allowed between two cutpoints, the efficiencies below and above the cutpoints increased. When the uncertain region included a third of the patients, the efficiency varied between 73.8 and 84.7%.Multivariate models seem to need a region of uncertainty between two discriminating cutpoints. These models resulted in the correct prediction of prognosis in about 75% and more of patients. With different materials the models differed in efficiency. With a region of uncertainty the model of Baak et al performed well with completely independent material.

The G1/S checkpoint of the cell cycle is regulated by p16, p53 and RB tumor suppressor genes. Loss of expression of the p16INK4 tumor suppressor protein, the product of the CDKN2 gene, has been associated with a wide variety of human malignancies. Mutations, loss of heterozygosity and deletions of the CDKN2 locus have been reported in sporadic and familial cutaneous malignant melanomas (CMM). To investigate the role of the alterations of p16 expression in melanoma, we evaluated by immunohistochemistry the p16 expression and cell proliferation in 79 primary CMM and 10 benign melanocytic nevi (BMN). Forty-six melanomas (58%) and all BMN were found to be p16 positive; 33 melanomas (42%) were considered p16 negative. The extent of invasion according to Clark was significantly higher in p16-negative tumors than in p16-positive tumors. Cell proliferation as expressed by the proportion of positive cells in Ki-67 immunostaining was found to be significantly higher in p16-negative tumors than in p16-positive tumors, although there was no significant difference in the mitotic index between p16-positive and p16-negative tumors. In p16-positive tumors, the number of Ki-67-positive cells correlated with the mitotic index; in p16-negative tumors, there was no correlation between these parameters. Our data suggest that loss of p16 expression is more common in advanced melanomas, and that G1/S checkpoint regulation is disrupted in p16-negative melanomas. Our results show that loss of p16 expression is a common event in primary melanomas, which further substantiates the role of p16 as a major tumor suppressor.

Two medical laboratory histotechnologists were trained on mitotic counting for 6 months and 1 year. The training included detailed theoretical education on mitotic counts and microscopy sessions with a motivated pathologist. Most of the training time, however, was used for mitotic counts from a large collection of breast cancer samples. The progress in performance was followed by allowing the technicians to count 20 breast cancer samples several times during the training. The mean value of the mitotic counts of technician 1 varied dramatically during the early phases of training but then stabilized near the levels of technician 2, whose mean values were more constant throughout the training period. Both observers showed changes in the standard deviation and coefficient of variation in the early phases of training but later stabilized at the same level. The proportion of the counting results of the technicians within the 95% confidence limits determined by the performance of already trained, experienced technicians varied considerably at first, but then stabilized at values 0.7 and above. The study showed that training improved the performance in mitotic counting. We suggest that the training of mitotic counts provides the means to achieve economical and reproducible estimates of tumor cell proliferation.

In this study, we found an unexpected association (crude odds ratio = 2.8; 95% confidence interval = 0.9-8.4) between definite work-related exposure to asbestos and carcinoma of the urinary bladder in a small group of patients (n = 28) initially recruited as referents for an epidemiological feasibility study on the occupational causes of lung cancer. We extended the study by using molecular methods to examine mutations in the p53 tumor suppressor gene in the same cases of bladder cancers. The same number of archival samples of transitional cell carcinoma, mainly of grade 3, were added to the analysis. We failed to show any association between occupational exposure to asbestos and p53 mutations among bladder cancer patients. We observed an increasing occurrence of p53 mutations in nonsmokers (5 of 17, 29%), former smokers (8 of 21, 38%), and current smokers (9 of 16, 56%) in that order; however, this was not statistically significant. The most prevalent type of mutation was G:C to A:T transition. Tumor grade was not associated with the frequency of mutations, but the higher stage (T3-T4) tumors appeared to have mutations more frequently than did the less invasive tumors (T1-T2).

The reproducibility of quantitative image analytic assessment of agarose gel separated PCR products was tested in a computerised core facility image analysis unit serving several research groups. Routine type use of the instrumentation showed considerable variation (measurements of individuals bands: CV about 20%; ratio measurements: CV about 6.5%). Careful concentration on the methodology improved the result (CV about 9% and 4.5%, respectively). Focusing into the methodology clearly showed that the baseline correction was necessary. The magnification of the image and the width of the measurement window were important variation sources. If these were kept constant, the reproducibility improved remarkably (CVs of 1.69% and 2.90%, respectively). Further analysis showed that the results did not change during the warm up period of the instrumentation. Measurements based on integrated optical density or related variables were well correlated, but did not show linear relation with peak height estimates. The most reproducible results were achieved when the thresholds were placed at the point of 45 degrees inclination of the image density curve of the band, or at the half height points of the curve.

We studied nuclear morphometry of human breast cancer with special emphasis on two sources of variation: freezing prior to fixation and selection of measured objects on the basis of different sampling rules. Samples of 147 histologically verified invasive breast cancer cases were examined with a computer-based image overlay drawing system. Thirty-eight of the 147 samples of tissue frozen before embedding in paraffin were analyzed separately. Among the latter we found shrinkage of 35% and 46% (depending on the sampling rule) of the nuclear profile area as compared with samples not frozen before the standard tissue processing. These findings confirm that nuclear morphometry results from frozen and unfrozen tissue are not comparable. Frozen tissue later embedded in paraffin should not be used with prognostication models based on traditionally fixed tissue. In morphometry we applied two sampling rules that differed in the criteria used for selecting nuclei for measurement. We registered a significant difference in nuclear size and in the variation of nuclear size between the two sampling methods. Of the morphometric features studied, nuclear area was affected most. Finally, we examined the two sampling rules in light of the established prognosticators in breast cancer: tumor size, axillary lymph node status, and the Multivariate Prognostic Index (MPI). The two sampling rules resulted in different distributions of morphometric results in the prognostic groups. Our findings emphasize the significance of the sources of variation in nuclear morphometry. They also stress the need for well-standardized morphometric methods in predicting the outcome of breast cancer.

We asked whether reinnervating axons are Schwann cell mitogens in vivo as they are in vitro. Left sciatic nerves of 50 Wistar rats were transected. In one-half of the animals, axonal reinnervation from the proximal to the distal stump was allowed to take place, while in the other half, sutures were placed on the transected nerve ends to prevent reinnervation. Samples were collected from 3 days up to 8 weeks after the transection proximally and distally from the point of transection. PCNA-immunostaining was performed on paraffin sections to determine the number of proliferating cells. Axonal reinnervation was followed by Bielschowsky staining and Schwann cell number was determined by counting S-100-immunopositive cells from paraffin sections. In the distal stump Schwann cell proliferation was similar in both experimental groups. There was no statistical evidence of S-100 negative cell proliferation during the study. Proximally to the site of transection the number of small initial axonal sprouts and also the number of Schwann cells increased if the nerve stump had been sutured. In conclusion, although axons may be mitogenic for Schwann cells, axonal reinnervation into the distal stump of the transected peripheral nerve does not influence the proliferation of Schwann cells to a greater extent than other potential effects associated with nerve transection.

The reproducibility in quantitation of proliferation activity, determined using the monoclonal antibody 19A2 to proliferating cell nuclear antigen (PCNA), was tested in visual and computer-assisted analyses of brain tumor material. The PCNA labeling index was scored using count (PCNA-LI, visual and computer analyses) and area (PCNA-LIa, computer analysis) estimates of immunopositivity. The quality of immunostaining was the most important reason for variation in the assessment results. Other significant variation sources in the assessment were experience in selecting microscopic fields and distinguishing immunopositive nuclei from immunonegative ones. Computer-assisted analysis improved the reproducibility of quantitation between different observers (visual rPCNA-LI = 0.624 versus computer assisted rPCNA-LI = 0.904). Also, the use of PCNA-LIa improved the intraobserver and interobserver reproducibility in different stainings (observer 1:rPCNA-LI = 0.857 versus rPCNA-LIa = 0.874; observers 1 and 2: rPCNA-LI = 0.904 versus rPCNA-LIa = 0.927; observers 3 and 4: rPCNA-LI = 0.848 versus rPCNA-LIa = 0.906). PCNA-LIa by computerized image analysis improves accuracy in the evaluation of the granularly expressed PCNA level. Furthermore, the effect of tumor heterogeneity on the assessment results can be diminished with the computerized method because large tissue areas can be analyzed faster.

Evaluation of the treatment response to hormonal therapy in experimental mammary carcinoma models usually consists of recording the changes in tumour volume or in number of tumours. We performed quantitative histology on dimethylbenz[a]anthracene(DMBA) induced rat mammary carcinoma treated with the antioestrogen toremifene. The inhibition of growth of the treated regressing tumours was not only associated with inhibition of mitosis and reduction of the neoplastic epithelium, but toremifene seemed to reduce all main tissue components. The percentual area fractions of epithelium and stroma were equal in the untreated and the treated tumours. The estimated absolute volumes of epithelium, stroma and glandular luminae as well as the mitotic index were significantly reduced during toremifene treatment in those tumours which were responding by regression to the treatment. The findings indicate growth inhibiting effect on both epithelium and stroma or on interactions between epithelium and stroma. Adding quantitative histology to the gross response analysis in experimental animal models is feasible and may elucidate the mechanisms of action of a novel hormonal treatment.

The antiestrogen toremifene treatment results in regression or stabilization of dimethylbenz(a)anthracene(DMBA)-induced rat mammary tumors by reducing the mitotic activity and possibly by modifying the abundant spontaneous apoptosis seen in these tumors. A comparative study on the modes of cell loss was performed in untreated DMBA tumors and in tumors treated with toremifene and ovariectomy. Ovariectomy results in massive apoptotic cell death throughout the tumors with apoptotic cells exfoliating into the glandular lumina. Apoptosis accounts for most of the cell death also in the untreated tumors. The distinctly different morphology of the apoptotic cells in the untreated tumors (condensed apoptosis) and tumors treated with ovariectomy (blebbing apoptosis) may reflect the different type or schedule of the apoptotic process. Both morphologies of apoptotic cells were seen in the toremifene-treated tumors. Blebbing apoptosis is not easily examined in paraffin-embedded material but is best detectable in plastic sections and by electron microscopy.