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Immunohistochemical study (IHC) is a critical tool in the clinical diagnosis of breast cancer. One common assessment is the expression level of the HER2 receptor in breast cancer tissue samples with the aim of stratifying patients for applicability of the therapeutic antibody Herceptin. In this study, we aimed to investigate whether a novel assay, real-time IHC combined with Interaction Map analysis, offers the possibility of objective assessment of HER2 expression. Interaction Map presents real-time interaction data as a collection of peaks on a surface, and it was performed on 20 patient tissue samples previously scored for HER2 expression. The result shows that the relative weight of the peaks in the maps contains novel information that could discriminate between high and low HER2 expression in an operator-independent manner (P

Generation of monospecific antibodies (msAbs) (multiepitope) through affinity purification of polyclonal antisera is a plausible strategy for high-throughput production of affinity reagents toward large sets of proteins. These antibodies are generated using readily accessible gene sequence information from publicly available databases. The resulting antibodies have the potential to be used in a variety of assays, probing differentially presented and altered proteins with high sensitivity and specificity. In the present study, 48 msAbs were compared with corresponding commercial analogs. Immunohistochemical staining properties were evaluated on tissue microarrays, representing various normal human tissues from 144 different individuals. MsAbs showed similar immunostaining patterns as compared with corresponding commercial analogs in 44 out of totally 48 (92%) antibody pairs analyzed. Although only few antibody pairs showed major discrepancies, minor dissimilarities were frequently seen. Our results suggest that msAbs are reliable and valuable tools in antibody-based proteomics, enabling analysis of protein expression patterns in cells and tissues. High-throughput strategies employing such antibodies provide a consistent approach in the exploration of the human proteome.

We studied 363 patients with stage Ta bladder tumors during long-term followup who were classified according to the 1998 WHO and International Society of Urological Pathology consensus classifications. We determine whether various immunohistochemical and molecular markers could predict tumor progression.A total of 680 patients in western Sweden with a first diagnosis of bladder carcinoma in 1987 and 1988 were registered and followed for at least 5 years. There were 363 (53%) tumors that were papillary stage pTa. The tumors were classified as papillary urothelial neoplasm of low malignant potential in 95 patients, low grade papillary urothelial carcinoma in 160 and high grade carcinoma in 108. Of the patients in the latter group 95 were subdivided into WHO grade 2 and 13 into WHO grade 3. Tissue from the primary tumors that progressed in stage during followup was further analyzed with immunohistochemical methods (p21, p53, Ki67 and pRb), DNA ploidy and mitotic frequency. The results were compared with those in matched controls (nonprogressors).Recurrence developed in 35% of patients with papillary urothelial neoplasm of low malignant potential compared to 71% with low grade urothelial carcinoma and 73% with high grade carcinoma (p0.0001). No papillary urothelial neoplasm of low malignant potential progressed in stage. Disease progressed in 4% of patients with low grade compared to 23% with high grade carcinoma (p0.0001). Of the patients with WHO grade 3 disease progressed in 45% compared to grade 2 in 20% (p0.0011). At first diagnosis p53 score was significantly higher (p0.0022) among patients with WHO grade 2 carcinoma which later progressed compared to that in matched controls but there was no significant difference regarding the other markers. In contrast to grade 2 most grade 3 carcinoma was aneuploid, had high mitosis frequency, high p53 and Ki67 scores as well as loss of retinoblastoma gene expression.The 1988 WHO and International Society of Urological Pathology consensus classifications divide noninvasive papillary bladder tumors into 3 subgroups with different clinical behavior, which seems to be an advantage compared with the 1973 WHO classification. A disadvantage is that the high grade carcinoma group contains 2 subgroups with different progression rates and immunohistochemical marker profiles, corresponding to the 1999 WHO grades 2 and 3. Grade 2 tumors in patients that progressed in stage years later seem to have different immunohistochemical and molecular marker profiles compared to those in matched controls.

It has been observed that immunoreactivity in paraffin sections decreased during storage. In this study, stored paraffin sections from both biopsy material and cultured cells were assessed for changes in immunoreactivity, using color-based image analysis to quantitate extent and intensity of the stainings. For seven of the 11 antibodies studied, storage at 20 degrees C for 16 weeks reduced the extent of immunostaining compared with that of freshly cut sections. Furthermore, increased storage temperatures resulted in a progressive loss of immunoreactivity. After 2 weeks of storage, at both 4 degrees C and 20 degrees C, p53 protein- and MIB1-antigen expression was significantly reduced regarding extent and intensity. The extent of the immunoreactivity reduced more for p53 protein than for MIB1 antigen, but the intensity did not. Boric acid was used for antigen retrieval on sections stored for 12 weeks at 20 degrees C. For both p53 protein and MIB1 antigen, this resulted in an extent and intensity of immunostaining equal to or higher than (MIB1) that obtained in freshly cut sections, using citrate buffer. Staining of cultured cells confirmed the results from biopsy material on the influence of storage temperature. Fixation time only marginally influenced the storage-related decrease in immunoreactivity. In conclusion, storage of paraffin sections leads to a varying degree of decreased immunoreactivity for several antibodies. The degree is at least partly dependent on storage time and temperature but not fixation time. However, this may be compensated for by optimizing the antigen retrieval protocol.

In vitro tumor models could support the process of development of new cytotoxic drugs and selection of suitable drugs for the individual patient. We investigated whether the testing of tumor cells from patients with kidney or urinary bladder carcinoma by fluorometric microculture cytotoxicity assay (FMCA) could provide clinically relevant data for these tumor types.A total of 45 tumor samples from patients with kidney or urinary bladder carcinoma were compared with 247 samples of other tumor types with respect to sensitivity to 8 standard and 6 investigational cytotoxic drugs in the FMCA, a 72 hour assay based on the concept of total cell kill. In bladder carcinomas, sensitivity to standard drugs was correlated to various tumor characteristics.The technical success rate for kidney and bladder carcinomas was high; approximately 90% of the samples could be analyzed successfully. Kidney carcinomas were highly resistant to standard drugs and bladder carcinomas essentially as sensitive as carcinomas of the breast and ovary but with a steeper dose-response relationship. In bladder carcinoma there was no clear relationship between tumor stage, grade, ploidy, mitoses or p53 expression and drug sensitivity. Except for suramin, kidney carcinomas were poorly sensitive to the investigational drugs CdA, gemcitabine, paclitaxel, vinorelbine and topotecan. In bladder carcinomas paclitaxel, gemcitabine and suramin showed promising activity.The FMCA seems suitable for cytotoxic drug sensitivity testing of urinary tract carcinomas. This technique may have a role in new drug development in these tumor types.

Purpose: We report the 5-year followup of a randomized comparison of mitomycin C and bacillus Calmette-Guerin (BCG) in patients with superficial bladder carcinoma. Recurrence, progression and survival rates, crossover results, prognostic factors and long-term side effects were analyzed.Materials and Methods: A total of 261 patients were enrolled in the study, and the inclusion criteria were primary Tis, dysplasia G2, T1 G3 and multiple recurrent Ta/T1 G1-2 disease. Intravesical instillations of 40 mg. mitomycin C and 120 mg. Pasteur BCG, Danish strain 1331, were given for 2 years.Results: After a median followup of 64 months 101 of the 250 evaluable patients (42%) were disease-free. A significant difference was noted in disease-free survival with BCG (p = 0.04), which was most pronounced for stage Tis disease. No difference in tumor progression, or crude or corrected survival was found between the 2 arms. Crossover treatment was successful in 39% of patients with second line BCG and 19% with secon...

Bladder cancers frequently exhibit an increased number of epidermal growth factor receptors (EGFR) in comparison to normal urothelium. The EGFR could potentially be a target for toxic conjugates. The aim of our study was to compare the expression of EGFR in metastases with concurrent or primary tumour in the urinary bladder using immunohistochemical techniques and a monoclonal antibody. Tumour material from 20 patients was investigated. The majority (13/20) of the metastases were homogeneously stained and showed a moderate to strong membranous staining for EGFR. The expression of EGFR in primary bladder tumours and metastases was similar. There was no indication that tumour tissue exposed to chemotherapy or radiation had a decreased number of EGFR. Targeting of the EGFR thus seems potentially applicable to metastatic disease.