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Patricia Harnden

Publications and source records attributed to Patricia Harnden.

At least 19 recordsLinked to original sources
PubMed2007

The prognostic significance of perineural invasion in prostatic cancer biopsies: a systematic review.

Men with clinically localized prostate cancer are faced with a wide range of treatment options, and only Gleason grading is universally used as a histopathological prognostic factor for this disease. The significance of perineural invasion in diagnostic biopsies is controversial. Opinion about whether or not it should influence treatment decisions is currently almost equally divided. To address this, the authors performed a systematic review of studies that examine the association between perineural invasion and prostate cancer recurrence. MEDLINE, Embase, and the Web of Knowledge were searched from January 1990 to December 2005. Outcomes analyzed were the development of biochemical or clinical recurrence. Twenty-one articles on the association of perineural invasion in biopsies and prostate cancer recurrence after radical prostatectomy (n = 10) or radiotherapy (n = 11) were found but none on its significance in the context of watchful waiting. Structured data extraction was performed to allow comparisons between articles and to identify sources of heterogeneity to explain discrepancies in results. The considerable variation in study design, execution, and reporting precluded meta-analysis and quantitative risk estimation, but the weight of evidence suggested that perineural invasion in biopsies was a significant prognostic indicator, particularly in specific patient groups defined by presenting serum prostate-specific antigen levels and biopsy Gleason scores. Immediate treatment rather than watchful waiting may be more appropriate for patients with localized prostatic cancer and perineural invasion. However, the data are limited, and well-designed studies that use predefined stringent protocols are required to provide robust estimates of risk to aid in treatment planning.

Adenocarcinoma↗
PubMed2006

Urinary biomarker profiling in transitional cell carcinoma.

Urinary biomarkers or profiles that allow noninvasive detection of recurrent transitional cell carcinoma (TCC) of the bladder are urgently needed. We obtained duplicate proteomic (SELDI) profiles from 227 subjects (118 TCC, 77 healthy controls and 32 controls with benign urological conditions) and used linear mixed effects models to identify peaks that are differentially expressed between TCC and controls and within TCC subgroups. A Random Forest classifier was trained on 130 profiles to develop an algorithm to predict the presence of TCC in a randomly selected initial test set (n = 54) and an independent validation set (n = 43) several months later. Twenty two peaks were differentially expressed between all TCC and controls (p < 10(-7)). However potential confounding effects of age, sex and analytical run were identified. In an age-matched sub-set, 23 peaks were differentially expressed between TCC and combined benign and healthy controls at the 0.005 significance level. Using the Random Forest classifier, TCC was predicted with 71.7% sensitivity and 62.5% specificity in the initial set and with 78.3% sensitivity and 65.0% specificity in the validation set after 6 months, compared with controls. Several peaks of importance were also identified in the linear mixed effects model. We conclude that SELDI profiling of urine samples can identify patients with TCC with comparable sensitivities and specificities to current tumor marker tests. This is the first time that reproducibility has been demonstrated on an independent test set analyzed several months later. Identification of the relevant peaks may facilitate multiplex marker assay development for detection of recurrent disease.

Adult↗
PubMed2006

Genetic and epigenetic analysis of von Hippel-Lindau (VHL) gene alterations and relationship with clinical variables in sporadic renal cancer.

Genetic and epigenetic changes in the von Hippel-Lindau (VHL) tumor suppressor gene are common in sporadic conventional renal cell carcinoma (cRCC). Further insight into the clinical significance of these changes may lead to increased biological understanding and identification of subgroups of patients differing prognostically or who may benefit from specific targeted treatments. We have comprehensively examined the VHL status in tissue samples from 115 patients undergoing nephrectomy, including 96 with sporadic cRCC. In patients with cRCC, loss of heterozygosity was found in 78.4%, mutation in 71%, and promoter methylation in 20.4% of samples. Multiplex ligation-dependent probe amplification identified intragenic copy number changes in several samples including two which were otherwise thought to be VHL-noninvolved. Overall, evidence of biallelic inactivation was found in 74.2% of patients with cRCC. Many of the mutations were novel and approximately two-thirds were potentially truncating. Examination of these and other published findings confirmed mutation hotspots affecting codons 117 and 164, and revealed a common region of mutation in codons 60 to 78. Gender-specific differences in methylation and mutation were seen, although not quite achieving statistical significance (P = 0.068 and 0.11), and a possible association between methylation and polymorphism was identified. No significant differences were seen between VHL subgroups with regard to clinicopathologic features including stage, grade, tumor size, cancer-free and overall survival, with the exception of a significant association between loss of heterozygosity and grade, although a possible trend for survival differences based on mutation location was apparent.

Adult↗
PubMed2006

Proteomic analysis of primary cell lines identifies protein changes present in renal cell carcinoma.

New markers/targets for renal cell carcinoma (RCC) are needed to enable earlier detection and monitoring of disease and therapeutic targeting. To identify such molecules, normal and RCC-derived primary cell lines have been used as a simplified model system for studying changes that accompany tumorigenesis. Short-term cultures allow enrichment of relevant cell types from tissue samples, which is balanced against the potential for in vitro changes. Examination of 3 proteins with altered expression in RCC tissue showed the maintenance of normal-tumour differences in culture, although some changes were apparent, including alteration in the isoform of aldolase. Comparative analysis of primary cell lines by 2-DE found 43 proteins up-regulated and 29 down-regulated in at least three out of five tumour cell lines. Many of the observed changes have been previously reported in RCC, including up-regulation of several glycolytic enzymes, vimentin and heat shock protein 27, validating the approach. Additionally, several novel changes in protein expression were found, including up-regulation of several proteins involved in actin cytoskeleton organisation such as radixin and moesin, two members of the septin family, and the actin bundling protein, fascin. Validation studies using Western blotting and immunohistochemistry indicate that several of these molecules may be useful as markers for RCC.

Blotting, Western↗
PubMed2006

Proteomic identification of a role for the von Hippel Lindau tumour suppressor in changes in the expression of mitochondrial proteins and septin 2 in renal cell carcinoma.

The von Hippel Lindau (VHL) tumour suppressor gene, VHL, plays a central role in development of sporadic conventional renal cell carcinomas (RCCs). Studying VHL function may, therefore, increase understanding of the pathogenesis of RCC and identify markers/therapeutic targets. Comparison of 2-DE protein profiles of VHL-defective RCC cells (UMRC2) transfected with control vector or wild-type VHL showed differences in 30 proteins, including several novel changes. One of the findings confirmed by Western blotting was up-regulation of the mitochondrial protein ubiquinol cytochrome c reductase complex core protein 2 following VHL transfection, a change that was also observed in two other cell line backgrounds. A marked decrease in expression of this and several other mitochondrial proteins was demonstrated in RCC tissues and using VHL-transfectants, several were shown to exhibit VHL-dependent regulation. Thus, VHL may contribute to the decreased mitochondrial function seen in RCC. A form of septin 2 down-regulated following VHL transfection was also identified. Septin 2 was up-regulated in 12/16 RCCs, while alteration of the form present was also observed in 1/3 tumours analysed. Thus, increased expression of septin 2 is a common event in RCC and protein modification may also alter septin 2 function in a subset of tumours.

Blotting, Western↗
PubMed2005

APE1 and XRCC1 protein expression levels predict cancer-specific survival following radical radiotherapy in bladder cancer.

INTRODUCTION: Radiotherapy offers the potential of bladder preservation in muscle-invasive bladder cancer, but only a proportion of tumors respond, and there are no accurate predictive methods. The ability of tumor cells to repair DNA damage induced by ionizing radiation influences radiosensitivity. We therefore investigated the prognostic value of the DNA repair proteins APE1 and XRCC1 in patients with muscle-invasive bladder cancer treated by radical radiotherapy. MATERIALS AND METHODS: The tumors of 90 patients with muscle-invasive transitional cell carcinoma and known clinical outcomes were immunostained with APE1 and XRCC1 antibodies. Levels of protein expression were assessed as a percentage of tumor cells with positive nuclear staining (1,000 cells per tumor). RESULTS: The median percentage of nuclear staining for APE1 was 98.7% (range, 42.2-100%) and for XRCC1 was 96.5% (range, 0.6-99.6%). High expression levels of APE1 or XRCC1 (> or = 95% positivity) were associated with improved patient cancer-specific survival (log-rank, P = 0.02 and 0.006, respectively). In a multivariate Cox regression model, APE1 and XRCC1 expression and hydronephrosis were the only independent predictors of patient survival. CONCLUSIONS: Expression levels of both APE1 and XRCC1 proteins were strongly associated with patient outcome following radiotherapy, separating patients with good outcome from the 50% with poor outcome (82% and 44%, 3-year cause-specific survival, respectively). If prospectively validated, this simple test could be incorporated into clinical practice to select patients likely to respond to radiotherapy and consider alternative forms of therapy for those unlikely to respond.

Adult↗
PubMed2005

Comprehensive analysis of CDKN2A status in microdissected urothelial cell carcinoma reveals potential haploinsufficiency, a high frequency of homozygous co-deletion and associations with clinical phenotype.

PURPOSE: There are significant differences in reported frequencies, modes of inactivation, and clinical significance of CDKN2A in urothelial cell carcinoma (UCC). We aimed to address these issues by investigating all possible modes of inactivation and clinicopathologic variables in a single tumor panel. EXPERIMENTAL DESIGN: Fifty microdissected UCCs were examined. CDKN2A gene dosage (quantitative real-time PCR), allelic status (microsatellite analysis), hypermethylation (methylation-specific PCR), mutation status (denaturing high-performance liquid chromatography and sequencing), protein expression (immunohistochemistry), and clinicopathologic variables (stage, grade, and disease recurrence during follow-up) were assessed. RESULTS: Exon 2 was underrepresented in 20 of 46 (43%) and exon 1beta in 21 of 46 (46%) of cases. Underrepresentation of exon 2 was accompanied by loss of heterozygosity (LOH) of 9p in 6 of 18 (30%) and of exon 1beta in 11 of 19 assessable cases (58%). Overall, LOH of 9p was identified in 15/41 (37%). Homozygous deletion of exons 2 and 1beta was detected in 16 of 46 (35%) and 10 of 46 tumors (22%), respectively. Co-deletion was most common, but exon 2-specific homozygous deletion was also detected. In tumors without homozygous deletion, p16 promoter hypermethylation was detected in 1 of 18 (6%). Hypermethylation of the p14ARF promoter or mutations in CDKN2A were not observed. Homozygous deletion of exon 2 or LOH on 9p were associated with invasion. Homozygous deletion of exon 2 or exon 1beta was associated with recurrent disease. CONCLUSIONS: These results confirm CDKN2A as a clinically relevant target for inactivation in UCC and show that the true frequency of alteration is only revealed by comprehensive analysis. Our results suggest that CDKN2A may be haploinsufficient in human cancer.

Chromosomes, Human, Pair 9↗
PubMed2005

Intrinsic chemotherapy resistance to the tubulin-binding antimitotic agents in renal cell carcinoma.

Renal cancer is one of the most chemoresistant tumor types. Using a panel of 10 established renal cancer cell lines that have not been subjected to prior drug selection, the range of functional resistance phenotypes to the tubulin-binding agents paclitaxel, vinblastine, vincristine and patupilone (epothilone B, EPO906) was determined, together with expression of P-glycoprotein (PgP), multidrug resistance associated protein-2 (MRP2) and major vault protein (MVP) proteins. The IC(50) values for vincristine correlated positively with PgP expression (r = 0.73; p = 0.031), with values for paclitaxel and vinblastine just failing to reach significance. A significant positive correlation was observed for sensitivity to paclitaxel and MRP2 expression only (r = 0.8; p = 0.013). MVP expression did not correlate with sensitivity to any of the drugs examined. All cell lines exhibited much greater sensitivity to patupilone, demonstrating for the first time the potential use of patupilone in this cancer. In tissue samples from chemotherapy-naive renal cell carcinoma (RCC) patients, marked downregulation or absence of PgP in many tumor cells with expression levels more similar to sensitive cell lines rather than the resistant lines was seen. Similarly, MRP2 was absent or only weakly present in tumor cells, whereas MVP was very strongly upregulated in most tumor samples. This study illustrating discrepancies between results exclusively based on studies in cell lines and findings in vivo suggests that the role of PgP and MRP2 in intrinsic resistance in RCC in vivo may be less than expected from the in vitro findings and supports a potential role for MVP on the basis of in vivo expression studies.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2005

Resistance to the tubulin-binding agents in renal cell carcinoma: no mutations in the class I beta-tubulin gene but changes in tubulin isotype protein expression.

PURPOSE: The primary purpose of this study was to determine whether mutations of the class I beta-tubulin gene may be implicated in the inherent resistance to tubulin-binding agents (TBA) in renal cancer, with a small number of samples and cell lines also being examined for class I and III beta-tubulin isotype protein expression. EXPERIMENTAL DESIGN: DNA was extracted from 90 renal tumors and the class I beta-tubulin gene analyzed for mutations. For each sample, eight PCRs were used to cover the complete coding sequence with intronic primers ensuring highly homologous pseudogenes were not coamplified. Additionally, expression levels of class I and III beta-tubulin isotypes in 17 matched normal and malignant renal samples and a panel of renal cell carcinoma cell lines with differing intrinsic resistance to the TBAs was examined by Western blotting. RESULTS: Four polymorphic sequence changes of the class I beta-tubulin gene were identified with no mutations. Class I protein expression levels were higher in tumor tissue versus normal tissue, whereas class III expression showed no consistent change. In renal cancer cell lines, a significant correlation between class III isotype expression and vinblastine sensitivity was observed. CONCLUSIONS: These results do not support a role for mutations in the class I beta-tubulin gene in the intrinsic resistance of renal cancer to TBAs. Class III isotype expression may be implicated in resistance in vitro but in vivo, changes in class I isotype expression in renal cell carcinoma tissue may support a role in resistance to the TBAs and warrants further investigation.

Adult↗
PubMed2004

Mutation analysis of the 8p candidate tumour suppressor genes DBC2 (RHOBTB2) and LZTS1 in bladder cancer.

Genomic deletions of the short arm of chromosome 8 are common in many human cancers and are frequently associated with a more aggressive tumour phenotype. One of the regions of loss of heterozygosity (LOH) on 8p22 identified in bladder cancer contains two genes, LZTS1 (FEZ1) and DBC2 (RHOBTB2) that have been shown to be mutated at low frequency in other cancers. We screened a panel of bladder tumours and bladder tumour-derived cell lines for mutations in these genes. Forty two percent of the tumours were found to have LOH in the 8p22 region and many of the cell lines have known loss of 8p. Several known polymorphisms and novel polymorphisms were detected. One possible mutation of LZTS1 (G374S) was found in a cell line. The functional significance of this is unknown but the novel serine residue created may represent a novel phosphorylation site. In DBC2, we found a single somatic mutation in a tumour (E349D) that lies in a highly conserved region of the protein. mRNA levels for both genes were reduced in the majority of bladder cancer cell lines. We conclude that neither LZTS1 nor DBC2 is commonly mutated in bladder cancer. However, neither can yet be excluded as the target of 8p22 LOH. The finding of a somatic mutation of DBC2 in a tumour sample and the down-regulation of both gene transcripts in bladder tumour cell lines may indicate that an alternative mechanism of inactivation of the second allele, for example promoter hypermethylation, is more common than mutation and this must now be examined.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Cytokine-modified Mycobacterium smegmatis as a novel anticancer immunotherapy.

Intravesical administration of live M. bovis BCG organisms for carcinoma in situ of the urinary bladder is the most successful immunotherapy for solid malignancy. Nevertheless BCG-therapy is associated with significant toxicity and is ineffective in 30-40% of cases. Recently it has been proposed that cytokine-modified mycobacteria may give greater efficacy. As any immunotherapy involving administration of live BCG organisms (wild-type or recombinant) is likely to have associated toxicity (notably in the immunocompromised), we examined the anti-tumour potential of the closely related nonpathogenic organism, Mycobacterium smegmatis, and a TNFalpha gene-modified recombinant M. smegmatis. When wild-type M. smegmatis were delivered to immunocompetent C57Bl/6 mice bearing the transplantable MB49 bladder tumour, efficacy comparable to live BCG was observed with 10-20% long-term survival. However, this effect was lost in both Nude and Beige mice, lacking functional T and NK cells, respectively. Recombinant M. smegmatis secreting TNFalpha, however, gave a 70% durable tumour-free survival. Lymphocytes from draining lymph-nodes and spleens of these mice exhibited pronounced IFNgamma production to mycobacterial-antigen and tumour-lysate, indicating a bias towards cell-mediated immunity. This was further supported by histopathological examination of the tumour site, which revealed significantly increased numbers of CD3+ lymphocytes in animals receiving the recombinant vaccine, but not in those receiving wild-type bacteria. Importantly, tumour rejection following M. smegmatis/TNFalpha was independent of T lymphocytes, as athymic Nude mice efficiently eradicated MB49 tumours. In contrast, the therapeutic efficacy of M. smegmatis/TNFalpha was reduced in animals deficient in NK cytolytic function, suggesting a role for NK-cells in initial tumour destruction. Furthermore the absence of NK-function in Beige mice did not prevent the establishment of tumour-protective memory. No toxicity was observed with wild-type or recombinant M. smegmatis in immunocompetent, T-deficient or NK-deficient models. We demonstrate for the first time that recombinant mycobacteria expressing mammalian cytokines have markedly increased anti-tumour properties. The lack of toxicity suggests that M. smegmatis is a "safe" vehicle for use in immunocompromised patients.

Animals↗
PubMed2004

DNA double strand break repair in human bladder cancer is error prone and involves microhomology-associated end-joining.

In human cells DNA double strand breaks (DSBs) can be repaired by the non-homologous end-joining (NHEJ) pathway. In a background of NHEJ deficiency, DSBs with mismatched ends can be joined by an error-prone mechanism involving joining between regions of nucleotide microhomology. The majority of joins formed from a DSB with partially incompatible 3' overhangs by cell-free extracts from human glioblastoma (MO59K) and urothelial (NHU) cell lines were accurate and produced by the overlap/fill-in of mismatched termini by NHEJ. However, repair of DSBs by extracts using tissue from four high-grade bladder carcinomas resulted in no accurate join formation. Junctions were formed by the non-random deletion of terminal nucleotides and showed a preference for annealing at a microhomology of 8 nt buried within the DNA substrate; this process was not dependent on functional Ku70, DNA-PK or XRCC4. Junctions were repaired in the same manner in MO59K extracts in which accurate NHEJ was inactivated by inhibition of Ku70 or DNA-PK(cs). These data indicate that bladder tumour extracts are unable to perform accurate NHEJ such that error-prone joining predominates. Therefore, in high-grade tumours mismatched DSBs are repaired by a highly mutagenic, microhomology-mediated, alternative end-joining pathway, a process that may contribute to genomic instability observed in bladder cancer.

Antigens, Nuclear↗
PubMed2003

Sonic hedgehog regulates prostatic growth and epithelial differentiation.

The Sonic hedgehog (SHH)-signalling pathway mediates epithelial-mesenchymal interactions in several tissues during development and disease, and we have investigated its role in rat ventral prostate (VP) development. We have demonstrated that Shh and Ptc expression correlates with growth and development of the prostate and that their expression is not regulated by androgens in the VP. Prostatic budding was induced in response to testosterone in Shh null mouse urogenital sinus (UGS) explants grown in vitro and in rat UGS explants cultured with cyclopamine, suggesting that SHH-signalling is not critical for prostatic induction. SHH-signalling was disrupted at later stages of VP development (in vitro), resulting in a reduction in organ size, an increase in ductal tip number, and reduced proliferation of ductal tip epithelia. The addition of recombinant SHH to VPs grown in vitro caused a decrease in ductal tip number and expansion of the mesenchyme. In the presence of testosterone, inhibition of SHH-signalling accelerated the canalisation of prostatic epithelial ducts and resulted in ducts that showed morphological similarities to cribiform prostatic intraepithelial neoplasia (PIN). The epithelia of these ducts also demonstrated precocious and aberrant differentiation, when examined by immunohistochemistry for p63 and cytokeratin 14. In conclusion, we show that SHH-signalling is not essential for prostatic induction, but is important for prostatic growth, branching, and proliferation, and that androgen-stimulated growth in the absence of signalling from the SHH pathway results in aberrant epithelial differentiation.

Androgens↗
PubMed2003

Uroplakin gene expression in normal human tissues and locally advanced bladder cancer.

The uroplakins are widely regarded as urothelium-specific markers of terminal urothelial cytodifferentiation. This study investigated the expression of the four uroplakin genes, UPIa, UPIb, UPII and UPIII, in a wide range of normal human tissues to determine tissue specificity and in advanced transitional cell carcinoma (TCC) to examine gene expression in primary and metastatic disease. In the urinary tract, all four uroplakins were expressed by urothelium and UPIII was also expressed by prostatic glandular epithelium. UPIa and UPII appeared to be urothelium-specific, but UPIb was detected in several non-urothelial tissues, including the respiratory tract, where it was associated with squamous metaplasia of tracheal and bronchial epithelia. The ten cases of primary TCC and corresponding lymph node metastases demonstrated that each uroplakin gene could be expressed at the mRNA level. No single uroplakin gene was expressed in all primary tumours or metastases, but 80% of the primary tumours and 70% of the lymph node metastases expressed at least one uroplakin gene. UPIII mRNA was often expressed in the absence of UPIII protein. These results confirm that in human tissues the expression of UPIa and UPII genes is highly specific to urothelium and suggest that the tight differentiation-restricted expression of uroplakin genes in normal urothelium is lost following malignant transformation.

Carcinoma, Transitional Cell↗
PubMed2003

Proteomic changes in renal cancer and co-ordinate demonstration of both the glycolytic and mitochondrial aspects of the Warburg effect.

Renal cell carcinoma (RCC) is the tenth most common cancer although the incidence is increasing. The main clinical problems stem from the relatively late presentation of many patients due to the often asymptomatic nature of the illness, and the relative insensitivity of metastatic disease to conventional chemotherapy and radiotherapy. Despite increasing knowledge of some of the genetic changes underlying sporadic renal cancer such as those involving the Von Hippel Lindau (VHL) gene, many of the underlying pathophysiological changes are ill-defined and there remains a need for the identification of disease markers for use in diagnosis and prognosis or as potential therapeutic targets. This study has used a proteomic approach, based on two-dimensional gel electrophoresis and mass spectrometry, to compare the protein profiles of conventional RCC tissue with patient-matched normal kidney cortex. Sequencing of 32 protein spots with significantly increased expression in RCC samples (>/= 4/6 patients) and 41 proteins whose levels decreased (6/6 patients) confirmed several previously known RCC-associated changes such as increases in Mn-superoxide dismutase, lactate dehydrogenase-A, aldolase A and C, pyruvate kinase M2, and thymidine phosphorylase. Additionally, several previously unknown changes were identified, including increased expression of three members of the annexin family and increased levels of the actin depolymerisation factor cofilin. The Warburg effect was also demonstrated with the identification of increases in proteins involved in the majority of steps in the glycolytic pathway and decreases in the gluconeogenic reactions, together with a parallel decrease in several mitochondrial enzymes. A number of the alterations seen were further confirmed in additional samples by immunohistochemistry, Western blotting, and laser capture microdissection.

Blotting, Western↗
PubMed2003

Serological and proteomic evaluation of antibody responses in the identification of tumor antigens in renal cell carcinoma.

Renal cell carcinoma (RCC) is relatively resistant to conventional chemotherapy and radiotherapy. However, reports of spontaneous regression along with promising results in clinical trials suggest that immunotherapuetic strategies may be of clinical benefit. Few RCC related antigens have been identified to date, and the technical difficulty and time constraints of current antigen identification techniques preclude the screening of large numbers of patients. A comparatively rapid strategy has been used to identify components of tumors that elicit an antibody response in the patient - the serological and proteomic evaluation of antibody responses (SPEAR) approach. This combines two-dimensional polyarylamide gel electrophoresis of tumor and normal kidney samples with immunoblotting using autologous patient sera and protein identification by mass spectrometry. Using the SPEAR approach to screen RCC patients for naturally occurring antitumor antibody responses, a number of candidate immunogens have been identified in patients with high-grade disease and their relative expression levels in tumor tissue compared to normal tissue have been studied. These proteins include annexins I and IV, thymidine phosphorylase (TP), carbonic anhydrase I, Mn-superoxide dismutase and major vault protein (MVP). Downstream analysis of the tissue expression of some of these proteins shows that MVP is up-regulated in 2/4 of RCC tumors but is also expressed in normal kidney whereas TP is up-regulated in 100% (11/11) of RCC cases examined with no or minimal expression in normal kidney, indicating a potential use as a therapeutic target.

Annexins↗
PubMed2003

Identification of genes up-regulated in urothelial tumors: the 67-kd laminin receptor and tumor-associated trypsin inhibitor.

Studies investigating changes in gene expression in urothelial carcinoma have generally compared tumors of different stages and grades but comparisons between low-grade, noninvasive tumors and normal urothelium are needed to identify genes involved in early tumor development. We isolated the urothelium from a low-grade tumor and corresponding normal mucosa by laser capture microdissection on frozen sections. The RNA extracted was amplified to generate suppressive subtractive cDNA libraries. Random sequencing of cDNA clones identified approximately 100 unique species. Of these 83% were known genes, 15% had homology to genes with an unknown function in humans, and 2% did not show homology to any published gene sequence. Two of the known genes, the 67-kd laminin receptor (67LR) and tumor-associated trypsin inhibitor (TATI), had previously been associated with metastatic progression in many tumor types, although 67LR has not been investigated in urothelial tumors. Immunolabeling of the original tissue with antibodies against these two genes confirmed overexpression, validating our strategy: 67LR was not expressed in the normal urothelium but was present in the tumor, whereas TATI expression was confined to umbrella cells in the normal urothelium, but extended to all cell layers in the tumor. We investigated both markers further in a separate series of tumors of different stages and grades. TATI was more consistently overexpressed than 67LR in all tumor grades and stages. Levels of secreted TATI were significantly higher in urine samples from patients with tumors compared to controls. Our strategy, combining laser capture microdissection and cDNA library construction, has identified genes that may be involved in the early phases of urothelial tumor development rather than with disease progression, highlighting the importance of comparing tumor with normal rather than just tumors of different stages and grades.

Carcinoma↗