Paricalcitol aggravates perivascular fibrosis in rats with renal insufficiency and low calcitriol.

Cardiovascular complications are a major problem in chronic renal failure. We examined the effects of plasma calcium, phosphate, parathyroid hormone (PTH), and calcitriol on cardiac morphology in 5/6 nephrectomized rats. Fifteen weeks after nephrectomy rats were given a control diet, high-calcium or -phosphorus diet, or given paricalcitol treatment for 12 weeks. Sham-operated rats were on a control diet. Blood pressure, plasma phosphate, and PTH were increased, while the creatinine clearance was reduced in remnant kidney rats. Phosphate and PTH were further elevated by the high-phosphate diet but suppressed by the high-calcium diet, while paricalcitol reduced PTH without influencing phosphate or calcium. The high-calcium diet increased, while the high-phosphate diet reduced plasma calcium. Plasma calcitriol was significantly reduced in other remnant kidney groups, but further decreased after paricalcitol. Cardiac perivascular fibrosis and connective tissue growth factor were significantly increased in the remnant kidney groups, and further increased in paricalcitol-treated rats. Hence, regardless of the calcium, phosphate, or PTH levels, cardiac perivascular fibrosis and connective tissue growth factor increase in rats with renal insufficiency in association with low calcitriol. Possible explanations are that aggravated perivascular fibrosis after paricalcitol in renal insufficiency may be due to further suppression of calcitriol, or to a direct effect of the vitamin D analog.

Severity of tubulointerstitial inflammation and prognosis in immunoglobulin A nephropathy.

Many risk factors for progression in immunoglobulin A nephropathy (IgAN) have been found. We focused on renal leukocyte infiltrations and cytokines in IgAN. The subjects were 204 IgAN patients. Renal histopathological changes were semiquantitatively graded. Expression of tubulointerstitial Leukocyte common antigen (LCA), CD3, CD68, interleukin (IL)-1beta, and IL-10 was evaluated by immunohistochemistry. These parameters were correlated with progression of IgAN. The significance of these correlations was tested by a multivariate analysis. Glomerulosclerosis, tubular atrophy, interstitial inflammation, and hyaline arteriolosclerosis correlated with progression in all patients and also in patients with initially normal serum creatinine. Tubulointerstitial LCA, CD3, CD68, and IL-1beta expression correlated with progression. CD3 had the strongest correlation. In the multivariate analysis, tubulointerstitial CD3, hypertriglyceridemia, elevated serum creatinine concentration, and interstitial fibrosis were independently associated with progressive disease in all patients, and tubulointerstitial CD3 expression and hyaline arteriolosclerosis in patients with initially normal serum creatinine. We found parameters reflecting tubulointerstitial inflammation to predict deterioration of renal function in IgAN. This was also seen in patients whose serum creatinine was normal at the time of renal biopsy. Our findings show that, an immunohistochemical evaluation of tubulointerstitial inflammation seems to be a useful tool in determining the prognosis in IgAN.

Grading of diffusely infiltrating astrocytomas by quantitative histopathology, cell proliferation and image cytometric DNA analysis. Comparison of 133 tumours in the context of the WHO 1979 and WHO 1993 grading schemes.

The aim of the study was to evaluate the applicability of quantitative histopathology as an aid for grading diffusely infiltrating astrocytomas. Primary astrocytomas were analysed for parameters (mean nuclear size, mitosis count, area fraction of endothelial cells and tumour necrosis, area fraction of nuclei, and Ki-67 (MIB-1) labelling index), which are closely related to the World Health Organization (WHO) 1979 and WHO 1993 grading criteria. All estimates correlated with the WHO histopathological grade and patient outcome. According to the receiver-operating characteristics curve, the presence of tumour necrosis and mitosis count (cut-off at 3 mitoses/mm2 of neoplastic tissue) showed the best sensitivity and specificity in separating patients with different survival. The multivariate survival analyses confirmed this result. A decision-tree model was constructed based on these two variables: twig I with less than 3 mitoses/mm2, twig II with equal or more than 3 mitoses/mm2 but no necrosis, and twig III with tumour necrosis. This model was found to be more strongly associated with survival than the WHO 1979 or WHO 1993 grading schemes. Low-malignancy astrocytomas (WHO grade II or twig I tumours) could be further divided into two prognostic categories by the image cytometric DNA analysis. The results put an emphasis on astrocytoma grading on mitosis counts (grade II vs. III) and tumour necrosis (grade III vs. IV). To standardize the sampling for mitosis counting, it is suggested that a parallel Ki-67 immunostaining be used for the identification of the most proliferative areas.

Cyclin D1 expression in astrocytomas is associated with cell proliferation activity and patient prognosis.

An important positive regulator of the cell cycle, cyclin D1, is often amplified and overexpressed in malignancies. Cyclin D1 aberrations were analysed in grade II-IV astrocytomas by fluorescence in situ hybridization (FISH), mRNA in situ hybridization and immunohistochemistry. Proliferation activity was determined by Ki-67(MIB-1) immunolabelling and mitotic counting. High cyclin D1 expression was observed in grade IV astrocytomas (grades II-III versus grade IV; mRNA expression: p<0.001; immunoexpression: p=0.013), and correlated with poor patient survival (p<0.001, n=46). Upregulated cyclin D1 expression was also closely associated with poor patient prognosis in grade II-III astrocytomas (p<0.001, n=30). Cyclin D1 gene was not found to be amplified (n=7). Cell proliferation activity was significantly increased in tumours exhibiting high cyclin D1 mRNA levels (Ki-67(MIB-1): p<0.001; mitotic count: p<0.001) and high cyclin D1 protein expression (Ki-67(MIB-1): p=0.002; mitotic count: p=0.012). These results indicate that increased production of cyclin D1 is closely associated with high cell proliferation activity and aggressive behaviour in diffusely infiltrating astrocytomas.

Age-related appearance of amyloid P component in human renal glomeruli.

Amyloid P component (AP) is a matrix glycoprotein of adult renal glomeruli. To establish whether the deposition of AP in glomeruli is an age-related phenomenon, this study used indirect immunofluorescence (IF) to investigate 34 renal biopsy specimens and 11 renal autopsy specimens. The biopsy specimens were taken from 9 patients (age range from 2 to 38 years) with normal glomerular morphology and from 25 patients (age range from 4 to 56 years) with various renal diseases. All autopsy specimens (age range form 2 months to 28 years) showed normal glomerular morphology. AP was not detected in glomeruli before age 6. By age 14, the IF intensity reached the level of the adult specimens, in which a strong fluorescence was seen along the basement membranes and within the mesangial matrix. In renal diseases, glomerular AP also appeared after age 6, although varying in its location and intensity. In conclusion, our results indicate that the appearance of AP in glomeruli is an age-related phenomenon, the pattern of which varies in renal diseases.

Reduced E-cadherin expression is associated with invasiveness and unfavorable prognosis in breast cancer.

E-cadherin (E-cad) is a calcium-dependent, epithelial cell adhesion molecule whose reduced or lost expression has been associated with tumor dedifferentiation and increased metastatic potential in human carcinomas. The authors studied immunohistochemically E-cad expression in frozen sections of 362 breast carcinomas using a monoclonal antibody (HECD-1). The immunohistochemical detection of reduced E-cad expression was confirmed by mRNA in situ hybridization with two different oligonucleotide probes. THe proportion of tumors with reduced or lost E-cad expression increased significantly from pure intraductal carcinomas (20%, 4 of 20) through invasive ductal (IDCs; 52%, 124 of 239) to recurrent carcinomas (64%, 18 of 28; chi square test for trend, P = .004). Invasive lobular carcinomas (ILCs) and IDCs differed from each other in their E-cad expression. None of the ILCs (n=55) retained normal E-cad expression in contrast to 48% (115 of 239) of the IDCs. In 259 primary IDCs, reduced E-cad expression was associated with high histologic grade (chi square test for trend, P < .001), negative estrogen receptor status (ER; Fisher's exact test; P = .042), and marginally with axillary node involvement (Fisher's exact test, P = .063). In a subset of 109 primary IDC patients whose clinical follow-up was available (median follow-up 51 months), reduced E-cad expression was associated with shortened disease-free survival (DFS; Mantel-Cox test, P = .027). In Cox's multivariate regression analysis, progesterone receptor status (P = .018) and E-cad expression (P = .072) were selected as independent predictors of DFS. Our findings provide clinical evidence that loss of normal E-cad expression is an indicator of increased invasiveness and dedifferentiation in breast carcinoma. E-cad is a potentially important prognostic factor in primary IDCs.

Comparison of different immunohistochemical methods in the assessment of angiogenesis: lack of prognostic value in a group of 77 selected node-negative breast carcinomas.

There is evidence that tumor angiogenesis, as detected by immunohistochemical staining of endothelium, is of prognostic significance in breast cancer. However, little attention has been paid to possible differences between antibodies or to quantitation of the stained microvessels. We compared three endothelial cell antibodies [anti-human von Willebrand factor (anti-VWF, also termed factor VIII), anti-CD31, and anti-CD34] in archival paraffin-embedded specimens. Anti-CD34 and anti-VWF showed better staining performances than anti-CD31, although the staining results with different antibodies were comparable. Two different methods of microvessel quantitation (the highest microvessel count and percentage microvessel area) were evaluated and also showed significant correlation. From a retrospective database (n = 1000), 77 axillary node-negative invasive ductal breast carcinomas were selected on the basis of clinical outcome to maximize the prognostic power of the sample set (37 died due to a metastatic breast carcinoma, 40 showed no recurrence during 8-yr follow-up). Microvessel quantitations were related to flow cytometric DNA ploidy, c-erb-B-2 overexpression, and estrogen receptor status of the tumor. Surprisingly, neither highest microvessel counts nor microvessel area measurements quantitated with anti-CD34 or anti-VWF immunohistochemistry were able to discriminate between favorable and unfavorable outcome patients. Thus, our results suggest that further evidence is still needed on tumor angiogenesis immunohistochemistry before it can be adopted as a prognostic marker in routine, clinical practice.

Prognostication of astrocytoma patient survival by Ki-67 (MIB-1), PCNA, and S-phase fraction using archival paraffin-embedded samples.

The prognostic power of three proliferation estimation methods, Ki-67 (MIB-1) and PCNA immunohistochemistry, and flow cytometry (S-phase and S + G2/M fractions, respectively), were evaluated in 50 cases of astrocytoma. Each proliferation index showed a strong association with the grade of malignancy (grades I-IV). The MIB-1 labelling index (LI) provided additional information, as it could be used for the discrimination of grade II and grade III astrocytomas (P = 0.0357). All three proliferation estimation methods also had strong prognostic potential (MIB-1 LI: P < 0.0001; PCNA Li: P < 0.0001; S-phase: P = 0.0004; S + G2/M: P = 0.0124). According to the receiver operating characteristics (ROC) curve, the MIB-1 LI showed generally the best sensitivity and specificity in placing the patients correctly into groups of survivors and non-survivors, which was further confirmed in the multivariate analysis. Only 4 per cent of the patients having high MIB-1 scores (> 15.3 per cent) were alive after 2-years' follow-up. In contrast, 72 per cent of patients with tumours of low proliferation activity survived. It appears that Ki-67 (MIB-1) immunolabelling using archival paraffin-embedded samples is of value in predicting prognosis in astrocytic tumours.

Comparison of three quantitation methods for PCNA immunostaining: applicability and relation to survival in 83 astrocytic neoplasms.

Recent studies on astrocytic tumours demonstrated a close association between patient prognosis and neoplastic proliferation estimated by such methods as Ki-67 and bromodeoxyuridine labelling. Novel monoclonal PCNA antibodies and special antigen-retrieval techniques have the advantage of working on routinely fixed and embedded specimens and thus make the estimation of proliferation simpler. In addition to PCNA-positive cell count expressed in percentages (PCNA-LI), we estimated the number of PCNA-immunopositive cells count expressed in percentages (PCNA-LI), we estimated the number of PCNA-immunopositive cells of 83 astrocytomas in two ways: (1) per mm2 of neoplastic tissue (uncorrected PCNA index); and (2) per mm2 of total neoplastic nuclear area (corrected PCNA index). Both of these methods were reproducible and showed a good correlation with PCNA-LI and malignancy grade (I-IV). With quantitation methods 1 and 2, the proliferative status of about 2000 cells could be estimated in about 7-10 min, whereas the PCNA count by PCNA-LI of 200 cells took approximately the same time. The proliferation indices obtained by all three quantitation methods were highly significantly related to patient prognosis. The corrected PCNA index, having a close association with the neoplastic cellularity, even divided the glioblastoma group (grade IV) into two significantly different prognostic groups in which 56 and 17 per cent of the patients were alive after 1-year follow-up. The combination of PCNA immunohistochemistry and morphometry seems to give important prognostic information about astrocytomas independent of the histopathological grade.

Intratumor variation in cell proliferation in breast carcinoma as determined by antiproliferating cell nuclear antigen monoclonal antibody and automated image analysis.

Immunohistochemical detection of the proliferating cell nuclear antigen (PCNA) represents a potentially useful tool for the study of tumor proliferative activity. To study the intratumor heterogeneity of tumor growth, 88 breast carcinomas were immunostained with the anti-PCNA antibody 19F4 and analyzed with the CAS 200 image analysis system (Cell Analysis System, Inc., Lombard, IL). For each sample, 12 fields from both the central and the peripheral areas of the tumor were measured. The proportion of PCNA-positive nuclear area in the whole tumor (PCNAt score) varied from 0.7% to 45.2% (median, 14.4%). There was considerable intratumor heterogeneity in the staining for PCNA. In 79% of the specimens, the PCNA score was higher in peripheral areas than in the center of the tumor, the average difference being +3.4% (range, -9.2- +15.1%; P < 0.0001, Student's t-test). The S-phase fraction, determined by DNA flow cytometry of the same tumors, varied from 2.0% to 32.6% (median, 10.0%). The PCNA score showed a significant correlation with the S-phase fraction (r = 0.469, P < 0.001). Most divergent results were those with high PCNA scores and low S-phase fraction; possible explanations for this are discussed. The PCNA score also was related to the histologic grade of the tumors (P = 0.03, analysis of variance). In conclusion, proliferation indices obtained from different areas of a tumor can differ significantly because of intratumor heterogeneity in growth fractions. The PCNA immunostaining correlates with well-known prognostic factors (S-phase fraction and histologic tumor grade) in breast carcinoma.

IgA-IgM nephropathy. A subgroup of primary mesangial glomerulonephritis.

The renal biopsy material of Tampere University Central Hospital comprises 1992 renal biopsy specimens, accessioned during the years 1978-1989. Among these, there were three cases of mesangial glomerulonephritis with a peculiar type of immunofluorescent reactivity. Striking mesangial deposits of both IgA and IgM were found in glomeruli, whereas C3 deposits were absent or present in slight amounts. The light microscopic findings ranged from mild mesangial glomerulonephritis to more advanced forms of sclerosing glomerulopathy. Electron microscopic examination disclosed an increase of mesangial matrix, together with mesangial and paramesangial electron-dense deposits. Two of the patients had microscopic hematuria associated with proteinuria, and one had isolated proteinuria. The authors propose that this group of cases may represent a new subgroup of primary mesangial glomerulonephritis that has not been described previously. They differ immunohistologically from both IgA nephropathy and IgM nephropathy, and therefore could be designated as IgA-IgM nephropathy.