A comparison of the efficacy and tolerability of solifenacin succinate and extended release tolterodine at treating overactive bladder syndrome: results of the STAR trial.

OBJECTIVE: To compare two new generation antimuscarinics at their recommended doses for treatment of overactive bladder syndrome (OAB). METHODS: A prospective, double blind, double-dummy, two-arm, parallel-group, 12-week study was conducted to compare the efficacy and safety of solifenacin 5 or 10 mg and tolterodine extended release (ER) 4 mg once daily in OAB patients. After 4 weeks of treatment patients had the option to request a dose increase but were dummied throughout as approved product labelling only allowed an increase for those on solifenacin. RESULTS: Solifenacin, with a flexible dosing regimen, showed greater efficacy to tolterodine in decreasing urgency episodes, incontinence, urge incontinence and pad usage and increasing the volume voided per micturition. More solifenacin treated patients became continent and reported improvements in perception of bladder condition assessments. The majority of side effects were mild to moderate in nature, and discontinuations were comparable and low in both groups. CONCLUSIONS: Solifenacin, with a flexible dosing regimen, was found to be superior to tolterodine ER with respect to the majority of the efficacy variables.

Elevated serum creatine kinase activity in a patient with acute pancreatitis.

A 62-year-old man presented with a five-day history of a 'flu-like' illness, epigastric pain and a state of increasing confusion. His serum values for amylase and glucose were grossly elevated, as was the creatine kinase (CK) activity, being 23 times above the upper limit of normal. CK-MB was less than 5% of his total CK activity. There was no past history of diabetes or recent history of intramuscular injections or injury. A diagnosis of acute pancreatitis complicated by hyperosmolar non-ketotic (HONK) diabetic pre-coma was made. The patient was treated with intravenous fluids, insulin and subcutaneous heparin. Normal values for serum amylase and CK activity were recorded with convalescence. This case indicates a possible association of a rise in total CK activity with acute pancreatitis complicated by HONK diabetic pre-coma. This observation was made in the absence of clinically evident muscle pathology.

PCR Analysis of CD44 Variants in Tumors.

The CD44 molecule is a transmembrane glycoprotein, it is an acidic, sul fated protein that contains multiple phosphoserine residues and several intrachain disulfide bonds. It is essentially composed of a 37-kDa protein core highly glycosylated by N- and O-linked sugars to form an 85-90-kDa product and is sometimes additionally linked to chondroitin sulfate side chains to produce a 180-220 kDa form on sodium dodecyl sulfate (SDS) gels (1,2).

Telomerase activity and its inhibition in benign and malignant breast lesions.

Many types of human tumours and immortal cell lines have been demonstrated to exhibit telomerase activity with the recently formulated telomeric repeat amplification protocol (TRAP assay). However, a small proportion of undoubted tumour samples give a negative result and it has been postulated that, on occasion, the assay can be blocked by inhibitory factors in the cell or tissue extracts. To resolve this issue, a modified TRAP assay has been used to re-examine 45 previously negative breast tissue specimens. Phenol--chloroform extraction of the sample after the telomerase extension reaction revealed the presence of polymerase chain reaction (PCR) inhibitory factors in tissue from 6 of 14 (43 per cent) breast biopsies of fibrocystic disease (FCD), 6 of 12 (50 per cent) fibroadenomas (FAs), none of five carcinomas in situ, and 1 of 13 (8 per cent) invasive carcinoma (CA) tissue specimens. These results demonstrated that the enzyme telomerase can be active in some benign lesions as well as in carcinomas of the breast. Specimens which still remained negative for telomerase in the above experiment were next assayed for the presence of biologically relevant inhibitors of the enzyme by mixing the extracts with confirmed positive samples. Extracts from 12 of 17 carcinoma specimens (all of five carcinomas in situ and 7 of 12 invasive carcinomas showed dose-dependent inhibitory activity against telomere extension, whereas no inhibition was observed in those of three of eight FCD and 2 of seven FAs. These results indicate that telomerase activity may be regulated by a balance between inhibitory factors and an activated enzyme.

Rapid analysis of distinctive CD44 RNA splicing preferences that characterize colonic tumors.

In normal tissues, the steady-state level of CD44 mRNA is low, and the variety of alternatively spliced transcripts produced by this complex gene is limited. Conversely, increased and disorderly expression of this gene has been observed in a number of types of cancer. This study analyzed the order in which the CD44 variant exons are spliced together in gastrointestinal tumor cell lines and in 20 colonic carcinomas and matched normal mucosa. We used a PCR-based assay to analyze specific exon junctions at the boundary of the standard and variant regions of the CD44 gene transcripts. This revealed characteristically different splicing preferences in colonic tumor and normal tissues. The junction of exon 5 to exon 8 appeared to be the most prevalent in normal mucosa, whereas the presence of junctions between exon 5 and either exon 7, 9, or 11 were increased markedly in tumor samples. These observations demonstrate that the unusual variety of CD44 transcripts in cancer cells results from the fidelity of alternative splicing mechanisms being compromised and are potentially useful as tumor cell markers in diagnostic assays.

Telomerase activity in bladder carcinoma and its implication for noninvasive diagnosis by detection of exfoliated cancer cells in urine.

BACKGROUND: Telomerase is an enzyme that can reconstitute the ends (telomeres) of chromosomes after cell division and thus circumvent the cumulative damage that occurs in normal adult somatic cells during successive mitotic cycles. Recently, it has been proposed that this enzyme should, therefore, be detectable in immortal malignant cells but not in their normal counterparts, which stop dividing and senesce. Accordingly, telomerase activity has been reported in many types of malignant tumors, including those of the gastrointestinal tract, breast, and lung but little information was available regarding its status in bladder carcinoma or in exfoliated cancer cells. METHODS: In the current study, telomerase activity was examined by a polymerase chain reaction-based assay designated TRAP (telomeric repeat amplification protocol) in tissue samples from 56 bladder carcinomas, 17 nonneoplastic bladder lesions, and 2 dysplastic lesions of the urinary tract. The feasibility of identifying cancer patients by the detection of telomerase activity in exfoliated cancer cells in the urine was also investigated. Such activity was assayed in centrifuged urine cell pellets from 26 bladder carcinoma patients and from 83 patients with no evidence of malignant disease. RESULTS: Evidence of telomerase was detected in solid tissue specimens from 48 of the 56 bladder carcinomas (86%) regardless of tumor stage or differentiation, whereas it was not found in any normal bladder tissue specimen. However, it was present in the dysplastic bladder lesions as well as in nearly all Stage I well differentiated carcinomas, suggesting that its activation occurs for the early stages of carcinogenesis and could perhaps be a useful marker for the detection of early primary or recurrent bladder tumors. Telomerase activity was detected with various signal intensities in urine specimens from 16 of the 26 patients with bladder carcinoma (62% sensitivity), whereas only 3 of 83 nonmalignant urine samples showed any activity (96.4% specificity); this was very weak. CONCLUSIONS: These results suggest that telomerase could be a good diagnostic marker for the early noninvasive identification of patients with bladder carcinoma by facilitating the detection of exfoliated immortal cancer cells in their urine.

Correlation between serum gamma-glutamyl transferase activity and the platelet count.

The serum gamma-glutamyl transferase (GGT) activity correlated significantly (P < 0.008) with the platelet count in 55 patients with thrombocytosis. All patients were selected so as to have normal liver function tests(including serum GGT). This correlation was present in both genders and there was no significant correlation with the other liver function tests. These findings are additional evidence that platelets may contribute to the total serum GGT activity.

Accumulation of Immature Intron-containing CD44 Gene Transcripts in Breast Cancer Tissues.

Background: Disorderly expression of the CD44 gene is a characteristic feature of many common types of human malignancy. The explanation for the diversity, abundancy, and abnormally large size of the resulting transcripts is under investigation. Methods and Results: This study used reverse transcription-polymerase chain reaction (RT-PCR)/Southern blot hybridization to examine the expression of the CD44 gene in fresh tissue samples from 21 breast cancers and 11 matched non-neoplastic breast tissue specimens from the same patients. Using probes for several exons and for a noncoding region (intron 9) of the gene, it was found that the previously described elevated levels of CD44 transcripts in malignant tumors of this organ include many unusual, alternatively spliced isotypes as well as immature forms that retain this intron and probably several others. All of the exons that were tested were involved in the disorderly overexpression of this gene observed in all of the cancer tissues, and we were able to detect retention of the intron 9 sequence in 14 (67%) of the 21 tumor samples. In contrast, it was possible to detect signals in only 3 (27%) of the 11 samples from nonmalignant breast tissue with this probe, and these were extremely faint. Conclusions: These findings imply that there is a profound disorder in the regulation of production, splicing, and processing of CD44 pre-mRNA in breast cancer tissues comparable to that described in tumors of many other organs. The clinical implication of this information is that analysis of tissue samples containing borderline or suspected premalignant lesions for the presence of these molecular abnormalities, which appear to be characteristic of neoplasia, may in due course help assessment of individual patient prognosis and optimization of treatment.

Semiquantitative Detection of Abnormal CD44 Transcripts in Colon Carcinomas by Reverse Transcription-Polymerase Chain Reaction Enzyme-linked Immunosorbant Assay (RT-PCR ELISA).

Background: Abnormal expression of CD44 variant RNA has been detected in a variety of human tumors and has been shown to be a potential diagnostic marker. To date, such analysis requires time-consuming gel electrophoresis, blotting, and autoradiographic procedures, and this approach may not be suitable for routine laboratory examinations. We have developed a rapid and semiquantitative reverse transcription-polymerase chain reaction enzyme-linked immunosorbent assay (RT-PCR ELISA) method and used it to analyze CD44 expression in colon carcinoma tissues and exfoliated cancer cells in colon luminal washings. Methods and Results: RNA was extracted from sample cells and tissues and converted to cDNA. PCR amplification products, labeled by incorporation of digoxigenin-11-dUTP, were hybridized with biotinylated probes complementary to CD44 exon 12 or to exons in the standard portion (CD44s) of the gene. Hybridized DNA complexes were immobilized on streptavidin-coated microtiter plates, and the bound PCR products were detected with a peroxidase-conjugated antibody to digoxigenin. CD44-derived PCR products were quantified by absorbance of a chromogenic reaction. Elevated expression of CD44 variant exon 12 was detected initially by Southern blot analysis in all of the 9 colon carcinoma tissues, while weak expression was observed in only 3 of 9 normal mucosas. This tumor-related differential expression was confirmed by the newly developed PCR-ELISA method. Elevated expression of CD44 exon 12 was also detected in exfoliated colonic epithelial cells from 10 of 13 carcinoma cases but not in exfoliated cells from 4 patients with inflammatory bowel disease. Conclusions: Raised expression of CD44 variant exon transcripts can be detected reliably in colonic tumor tissue and in exfoliated colonic cancer cells by a semiquantitative RT-PCR ELISA method. This was shown to be as sensitive as conventional RT-PCR using chemiluminescent detection. Therefore, CD44-based RT-PCR ELISA could facilitate detection of neoplasia in clinical specimens including colon washings and naturally micturated urine.

Progressive loss of CD44 gene expression in invasive bladder cancer.

This study investigated CD44 gene expression at both the RNA and protein level in well differentiated superficial and in deeply invasive bladder carcinomas. Proteins were studied by immunohistochemistry using antibodies against standard (CD44s) and variant (CD44v) isoforms. mRNA was analyzed by reverse transcriptase polymerase chain reaction/Southern blotting and in situ hybridization. Immunostaining with antibodies against CD44s and CD44v2, -5 and -6 (exons 7, 10, and 11, respectively) showed that carcinoma cells in all papillary tumors expressed strong signals throughout the epithelium but especially in the basal layer, which abuts on the stroma. However, invasive tumors, which are believed to originate mainly from flat urothelial tumors or, less frequently, from papillary carcinomas, progressively lost CD44 proteins as they penetrated deeper and became less differentiated. This change was paralleled at the mRNA level, by the gradual loss of expression of CD44s and CD44v transcripts in deeply invasive tumors until they were virtually undetectable. Conversely, papillary tumors contained multiple higher molecular weight transcripts, suggesting that the loss of CD44 proteins in the more aggressive tumors is due to a disturbance in transcription. This concept was confirmed by in situ hybridization studies with a probe showing that substantial variant CD44 mRNA is located in the urothelium in papillary carcinomas but is absent from deeply invasive carcinomas. These results indicate that initially there is a striking increase in CD44 gene expression in early bladder carcinomas, relative to normal urothelium, but that this diminishes as the tumors acquire a more aggressive phenotype.

Telomerase activity in human breast cancer and benign breast lesions: diagnostic applications in clinical specimens, including fine needle aspirates.

We analysed telomerase activity in normal, benign and malignant breast tissues and in fine needle aspirates by a PCR-based assay. The tissue samples we used in this assay consisted of 20 cryostat sections, 10 microns thick, from each breast biopsy. This method was used to obtain effective extraction from small samples and to confirm the histological identity of the specimen by microscopical examination of serial sections. Fifty-two of 71 breast carcinomas were positive for telomerase activity, and the intensity of this was strong in most cases, whereas all 6 samples of normal breast tissue and 17 of fibrocystic disease were negative and only 1 of 15 fibroadenomas was positive. Invasive ductal carcinomas were more frequently positive than invasive lobular carcinomas. There was no correlation of telomerase activity with tumour size or the occurrence of lymph node metastasis. Evaluation of our assay system showed that a signal of telomerase activity was detectable in extracts from single cryostat sections (< 1 mm2) of a cancer specimen and from as few as 4 cells of a human breast cancer cell line. On the basis of the above data, we applied this assay to fine needle aspirates of breast lesions. Ten of 15 aspirates which had been cytopathologically diagnosed as cancer were strongly positive, while 26 of 29 benign aspirates were totally negative and the remaining 3 showed only borderline activity. In 3 cases, the telomerase result could have helped establish a diagnosis when the cytological observations were inconclusive. Our results indicate that this sensitive assay could become a useful new modality for supplementing microscopic cytopathology in the detection of cancer cells in small tissue biopsies and fine needle aspirates.

Distribution of CD44 messenger RNA in archival paraffin wax embedded tumours and normal tissues viewed by in situ hybridisation.

Aims-We have previously demonstrated the abnormal localisation of expression of the CD44 gene in carcinoma cells in cryostat sections of fresh frozen tumour tissues, using radioactive in situ hybridisation (RISH). In order to facilitate further analysis of the expression of this gene in a wider range of neoplastic and non-neoplastic conditions, we have developed a technique which can visualise its low copy number transcripts in archival paraffin wax embedded specimens.Methods-(35)S labelled riboprobes complementary to transcripts from the standard (CD44s) and variant (CD44v) regions of the gene were used on paraffin wax embedded sections of tumours and corresponding normal tissues of the colon, breast and uterine cervix.Results-Elevated levels of signals for CD44s and CD44v transcripts were observed in carcinoma cells relative to their non-neoplastic counterparts in all tissues examined.Conclusion-This method permits easy access to material which can be selected for suitability, handled at room temperature without degradation and relied upon to show good histological detail. Comparison of the results with those on frozen tissues showed similar distributions of signals. Furthermore, the resolution and morphological detail was improved in paraffin wax sections.

Detection of exfoliated carcinoma cells in colonic luminal washings by identification of deranged patterns of expression of the CD44 gene.

AIMS: To investigate whether colonic cancer cells exfoliated into the lumen of the organ can be detected by identification of their abnormal CD44 gene products. METHODS: Exfoliated cells were obtained by centrifugation of saline wash-outs of 27 surgically resected colon specimens obtained from 15 patients with carcinoma, seven with ulcerative colitis and five with Crohn's disease. After extracting cellular mRNA, amplification by the reverse transcription-polymerase chain reaction (RT-PCR) technique and analysis by Southern blot hybridisation was carried out to examine the levels and patterns of transcription of exons 11(v6), and 12(v7) and intron 9 of the CD44 gene. The transcription of these CD44 components was also examined by RT-PCR of snap-frozen solid tissue specimens from 11 of the above patients with colorectal carcinoma, seven with ulcerative colitis and five with Crohn's disease. RESULTS: Abnormal expression of exons 11(v6) and 12(v7) was detected in exfoliated cells from 11 (73%) of 15 patients with carcinoma, but not in any patients with inflammatory bowel disease (IBD). The retention of intron 9 in CD44 mRNA transcripts was detected in washings from four (27%) carcinoma specimens but not in washings from non-malignant specimens. It was confirmed that in solid tissue samples from the same carcinomas there was abnormal over-expression of numerous alternatively spliced CD44 species containing transcripts of exons 11 and 12 and retention of intron 9. Low level expression of these exons was detected in tissue from inflammatory lesions from five of seven patients with ulcerative colitis and four of five with Crohn's disease. The retention of intron 9 was not seen in normal mucosa nor IBD. CONCLUSION: Abnormal expression of the variant exons and of intron 9 of the CD44 gene in tumour cells exfoliated into the colonic lumen may be helpful markers for the early, non-invasive, diagnosis of colorectal cancer.