MicroRNA-126 inhibits invasion in bladder cancer via regulation of ADAM9.

BACKGROUND: The miRNA deregulation is commonly observed in human malignancies, where they act as tumour suppressors or oncogenes. Despite the association of several miRNAs with bladder cancer, little is known about the miRNAs that contribute to bladder cancer progression from non-muscle invasive (NMI) to muscle-invasive (MI) disease. METHODS: We first profiled the expression of miRNAs and mRNAs in a cohort of urothelial carcinomas and further characterised the role of miR-126 in invasion, as it emerged as the most downregulated miRNA between MI and NMI tumours. RESULTS: We found that restoration of miR-126 levels attenuated the invasive potential of bladder cancer cells. Mechanistically, we identified the role of miR-126 in invasion through its ability to target ADAM9. Notably, a significant inverse correlation between miR-126 and ADAM9 expression was observed, where ADAM9 was upregulated in MI bladder cancer cells. While knockdown of ADAM9 attenuated the invasiveness of cells with low miR-126 levels, experimental upregulation of ADAM9 recapitulated the invasive phenotype. Furthermore, ADAM9 expression assessed by immunohistochemistry significantly correlated with poor prognosis in patients with urothelial carcinoma. CONCLUSIONS: In this study we describe the role of miR-126 in bladder cancer progression, identifying miR-126 and ADAM9 as potential clinical biomarkers of disease aggressiveness.

Pten is essential for embryonic development and tumour suppression.

The PTEN gene encodes a dual-specificity phosphatase mutated in a variety of human cancers. PTEN germline mutations are found in three related human autosomal dominant disorders, Cowden disease (CD), Lhermitte-Duclos disease (LDD) and Bannayan-Zonana syndrome (BZS), characterized by tumour susceptibility and developmental defects. To examine the role of PTEN in ontogenesis and tumour suppression, we disrupted mouse Pten by homologous recombination. Pten inactivation resulted in early embryonic lethality. Pten-/- ES cells formed aberrant embryoid bodies and displayed an altered ability to differentiate into endodermal, ectodermal and mesodermal derivatives. Pten+/- mice and chimaeric mice derived from Pten+/- ES cells showed hyperplastic-dysplastic changes in the prostate, skin and colon, which are characteristic of CD, LDD and BZS. They also spontaneously developed germ cell, gonadostromal, thyroid and colon tumours. In addition, Pten inactivation enhanced the ability of ES cells to generate tumours in nude and syngeneic mice, due to increased anchorage-independent growth and aberrant differentiation. These results support the notion that PTEN haploinsufficiency plays a causal role in CD, LDD and BZS pathogenesis, and demonstrate that Pten is a tumour suppressor essential for embryonic development.

Pten and p27KIP1 cooperate in prostate cancer tumor suppression in the mouse.

The genetic bases underlying prostate tumorigenesis are poorly understood. Inactivation of the tumor-suppressor gene PTEN and lack of p27(KIP1) expression have been detected in most advanced prostate cancers. But mice deficient for Cdkn1b (encoding p27(Kip1)) do not develop prostate cancer. PTEN activity leads to the induction of p27(KIP1) expression, which in turn can negatively regulate the transition through the cell cycle. Thus, the inactivation of p27(KIP1) may be epistatic to PTEN in the control of the cell cycle. Here we show that the concomitant inactivation of one Pten allele and one or both Cdkn1b alleles accelerates spontaneous neoplastic transformation and incidence of tumors of various histological origins. Cell proliferation, but not cell survival, is increased in Pten(+/-)/Cdkn1b(-/-) mice. Moreover, Pten(+/-)/Cdkn1b(-/-) mice develop prostate carcinoma at complete penetrance within three months from birth. These cancers recapitulate the natural history and pathological features of human prostate cancer. Our findings reveal the crucial relevance of the combined tumor-suppressive activity of Pten and p27(Kip1) through the control of cell-cycle progression.

Cell surface antigens of human melanocytes and melanoma. Expression of adenosine deaminase binding protein is extinguished with melanocyte transformation.

It has been proposed that the pathogenesis of melanoma proceeds through multiple stages, ranging from benign proliferation of melanocytic cells to acquisition of the capacity to invade tissues and metastasize. During investigations of cell surface antigens expressed by melanocytes and melanoma, we identified an antigen system that was expressed by cultured normal melanocytes but not by melanoma cell lines. mAbs against this antigen detected a 120-kD cell surface glycoprotein on melanocytes. This molecule had been identified previously as the binding protein for adenosine deaminase (ADAbp). ADAbp was expressed by 51 melanocyte cell lines derived from normal fetal, newborn, and adult skin and adult choroid, but not by 102 melanoma cell lines derived from primary and metastatic lesions. Studies with radiolabeled bovine adenosine deaminase, confirmed that melanocytes expressed binding sites for adenosine deaminase, but no binding sites were detected on cultured melanoma cells. Further studies showed that ADAbp+ melanocytes became ADAbp- upon malignant transformation in vitro. Immunohistochemical studies on a panel of frozen tissues demonstrated reactivity of anti-ADAbp mAbs with epidermal melanocytes and benign junctional nevi, but not with potentially premalignant dysplastic nevi or primary/metastatic melanoma lesions. These studies demonstrate that ADAbp expression is lost with malignant transformation of melanocytes, presumably at an early stage in the transformation process.

Phenotypic heterogeneity of melanoma. Relation to the differentiation program of melanoma cells.

Phenotypic heterogeneity is a characteristic feature of tumor lesions in patients with melanoma. Variability can be observed in cell morphology, pigmentation, and antigen expression. To test whether phenotypic heterogeneity could be the result of events regulated during cell differentiation, we evaluated the expression of a panel of differentiation traits on melanoma cells. Metastatic melanoma lesions from two patients, designated FD and AP, were examined histologically and found to contain mixed populations of cells. Established melanoma cell lines derived from each of these lesions were subcloned at early passage in culture (passages 7 and 8) to create a panel of clones derived from each tumor. There was heterogeneity in the expression of differentiation-related traits in clones, corresponding to distinct phenotypes observed within the original tumors. Clones from patient FD corresponded to early to intermediate stages of melanocyte differentiation, and clones from patient AP ranged from intermediate to late stages. The influence of cholera toxin and PMA on differentiation of parental cultures and subclone was studied. Results of induction studies demonstrated a number of features of differentiation of melanoma cells: regulation of differentiation traits is coordinated as a program of traits expressed sequentially at specific stages; early traits, such as the epidermal growth factor receptor and the melanoma chondroitin sulfate proteoglycan antigen, are downregulated as melanoma cells differentiate, whereas late markers, including melanin, tyrosinase activity, and antigens expressed in mature melanosomes, are upregulated; Ia (class II major histocompatibility) antigens are characteristically expressed on melanomas corresponding to early or intermediate stages of differentiation and are regulated as part of the differentiation program; minimal changes in stage of differentiation were observed during induction of parental cultures with either cholera toxin or PMA, whereas definite shifts in differentiation could be induced in selected cloned subpopulations. We conclude that melanoma cells are not frozen at a specific stage of differentiation, but rather are capable of differentiating when exposed to appropriate signals. Diversity in the differentiation state of melanoma cells can account for much of the phenotypic heterogeneity observed in melanoma lesions.

Role of promyelocytic leukemia (PML) protein in tumor suppression.

The promyelocytic leukemia (PML) gene encodes a putative tumor suppressor gene involved in the control of apoptosis, which is fused to the retinoic acid receptor alpha (RARalpha) gene in the vast majority of acute promyelocytic leukemia (APL) patients as a consequence of chromosomal translocations. The PMLRARalpha oncoprotein is thought to antagonize the function of PML through its ability to heterodimerize with and delocalize PML from the nuclear body. In APL, this may be facilitated by the reduction to heterozygosity of the normal PML allele. To determine whether PML acts as a tumor suppressor in vivo and what the consequences of deregulated programmed cell death in leukemia and epithelial cancer pathogenesis are, we crossed PML(-/-) mice with human cathepsin G (hCG)-PMLRARalpha or mammary tumor virus (MMTV)/neu transgenic mice (TM), models of leukemia and breast cancer, respectively. The progressive reduction of the dose of PML resulted in a dramatic increase in the incidence of leukemia, and in an acceleration of leukemia onset in PMLRARalpha TM. By contrast, PML inactivation did not affect neu-induced tumorigenesis. In hemopoietic cells from PMLRARalpha TM, PML inactivation resulted in impaired response to differentiating agents such as RA and vitamin D3 as well as in a marked survival advantage upon proapoptotic stimuli. These results demonstrate that: (a) PML acts in vivo as a tumor suppressor by rendering the cells resistant to proapoptotic and differentiating stimuli; (b) PML haploinsufficiency and the functional impairment of PML by PMLRARalpha are critical events in APL pathogenesis; and (c) aberrant control of programmed cell death plays a differential role in solid tumor and leukemia pathogenesis.

Ku70 is required for DNA repair but not for T cell antigen receptor gene recombination In vivo.

Ku is a complex of two proteins, Ku70 and Ku80, and functions as a heterodimer to bind DNA double-strand breaks (DSB) and activate DNA-dependent protein kinase. The role of the Ku70 subunit in DNA DSB repair, hypersensitivity to ionizing radiation, and V(D)J recombination was examined in mice that lack Ku70 (Ku70(-/-)). Like Ku80(-/-) mice, Ku70(-/-) mice showed a profound deficiency in DNA DSB repair and were proportional dwarfs. Surprisingly, in contrast to Ku80(-/-) mice in which both T and B lymphocyte development were arrested at an early stage, lack of Ku70 was compatible with T cell receptor gene recombination and the development of mature CD4+CD8- and CD4-CD8+ T cells. Our data shows, for the first time, that Ku70 plays an essential role in DNA DSB repair, but is not required for TCR V(D)J recombination. These results suggest that distinct but overlapping repair pathways may mediate DNA DSB repair and V(D)J recombination.

Lipopolysaccharide induces disseminated endothelial apoptosis requiring ceramide generation.

The endotoxic shock syndrome is characterized by systemic inflammation, multiple organ damage, circulatory collapse and death. Systemic release of tumor necrosis factor (TNF)-alpha and other cytokines purportedly mediates this process. However, the primary tissue target remains unidentified. The present studies provide evidence that endotoxic shock results from disseminated endothelial apoptosis. Injection of lipopolysaccharide (LPS), and its putative effector TNF-alpha, into C57BL/6 mice induced apoptosis in endothelium of intestine, lung, fat and thymus after 6 h, preceding nonendothelial tissue damage. LPS or TNF-alpha injection was followed within 1 h by tissue generation of the pro-apoptotic lipid ceramide. TNF-binding protein, which protects against LPS-induced death, blocked LPS-induced ceramide generation and endothelial apoptosis, suggesting systemic TNF is required for both responses. Acid sphingomyelinase knockout mice displayed a normal increase in serum TNF-alpha in response to LPS, yet were protected against endothelial apoptosis and animal death, defining a role for ceramide in mediating the endotoxic response. Furthermore, intravenous injection of basic fibroblast growth factor, which acts as an intravascular survival factor for endothelial cells, blocked LPS-induced ceramide elevation, endothelial apoptosis and animal death, but did not affect LPS-induced elevation of serum TNF-alpha. These investigations demonstrate that LPS induces a disseminated form of endothelial apoptosis, mediated sequentially by TNF and ceramide generation, and suggest that this cascade is mandatory for evolution of the endotoxic syndrome.

Personalized approach to prostate cancer prognosis.

Personalized medicine in the management of patients with prostate cancer is limited to the integration of patient attributes such as age, genetic risk and comorbidities with specific clinical-pathologic variables including serum prostate specific antigen (PSA), imaging and features from the diagnostic prostate needle biopsy or prostatectomy specimen including tumor differentiation (i.e. Gleason), volume and extent of disease (i.e. tumor length and / or percentage, number of positive cores at diagnosis or pathologic stage post surgery including margin status). Although the development of various clinical statistical instruments such as nomograms have provided a mechanism to interrogate such variables, most urologists rely on basic prognostic features of stage, grade and PSA along with clinical judgment to define and understand individual risk and predict health outcomes. Furthermore, unlike other tumor types such as breast cancer, there are no routine ancillary diagnostic studies performed on the prostate needle biopsy or prostatectomy specimen to support and refine the treatment decision process for the individual patient. In this review we will provide a summary of the current practice of predictive statistical modeling in prostate cancer and explore how technical advances in functional histology have played a role in the development and incorporation of a systems based platform for providing a patient-specific risk profile useful for clinical decision making.

Role of PML in cell growth and the retinoic acid pathway.

The PML gene is fused to the retinoic acid receptor alpha (RARalpha) gene in chromosomal translocations associated with acute promyelocytic leukemia (APL). Ablation of murine PML protein by homologous recombination revealed that PML regulates hemopoietic differentiation and controls cell growth and tumorigenesis. PML function was essential for the tumor-growth-suppressive activity of retinoic acid (RA) and for its ability to induce terminal myeloid differentiation of precursor cells. PML was needed for the RA-dependent transactivation of the p21WAF1/CIP1 gene, which regulates cell cycle progression and cellular differentiation. These results indicate that PML is a critical component of the RA pathway and that disruption of its activity by the PML-RARalpha fusion protein may be important in APL pathogenesis.

Impaired Fas response and autoimmunity in Pten+/- mice.

Inactivating mutations in the PTEN tumor suppressor gene, encoding a phosphatase, occur in three related human autosomal dominant disorders characterized by tumor susceptibility. Here it is shown that Pten heterozygous (Pten+/-) mutants develop a lethal polyclonal autoimmune disorder with features reminiscent of those observed in Fas-deficient mutants. Fas-mediated apoptosis was impaired in Pten+/- mice, and T lymphocytes from these mice show reduced activation-induced cell death and increased proliferation upon activation. Phosphatidylinositol (PI) 3-kinase inhibitors restored Fas responsiveness in Pten+/- cells. These results indicate that Pten is an essential mediator of the Fas response and a repressor of autoimmunity and thus implicate the PI 3-kinase/Akt pathway in Fas-mediated apoptosis.

Endothelial apoptosis as the primary lesion initiating intestinal radiation damage in mice.

Gastrointestinal (GI) tract damage by chemotherapy or radiation limits their efficacy in cancer treatment. Radiation has been postulated to target epithelial stem cells within the crypts of Lieberkühn to initiate the lethal GI syndrome. Here, we show in mouse models that microvascular endothelial apoptosis is the primary lesion leading to stem cell dysfunction. Radiation-induced crypt damage, organ failure, and death from the GI syndrome were prevented when endothelial apoptosis was inhibited pharmacologically by intravenous basic fibroblast growth factor (bFGF) or genetically by deletion of the acid sphingomyelinase gene. Endothelial, but not crypt, cells express FGF receptor transcripts, suggesting that the endothelial lesion occurs before crypt stem cell damage in the evolution of the GI syndrome. This study provides a basis for new approaches to prevent radiation damage to the bowel.

Delivery of replication-competent retrovirus expressing Escherichia coli purine nucleoside phosphorylase increases the metabolism of the prodrug, fludarabine phosphate and suppresses the growth of bladder tumor xenografts.

We have developed unique replication-competent retroviral (RCR) vectors based on murine leukemia virus that provide improved efficiency of viral delivery, allow for long-term transgene expression and demonstrate an intrinsic selectivity for transduction of rapidly dividing tumor cells. The purpose of this study was to evaluate the in vivo transduction efficiency and the therapeutic efficacy of the RCR vector mediated delivery of Escherichia coli purine nucleoside phosphorylase (PNP) in combination with fludarabine phosphate for bladder cancer. We constructed vectors containing green fluorescent protein (GFP) gene (ACE)-GFP) or PNP gene (ACE-PNP). KU-19-19 bladder tumors exhibited 28.3+/-16.1, 46.6+/-5.8 and 93.7+/-7.8% of GFP expression on 14, 18 and 26 days after intratumoral injection of ACE-GFP, respectively. GFP expression could not be observed in normal tissues surrounding the injected tumors. No detectable polymerase chain reaction products of GFP gene could be observed in any distant organs. Intratumoral injection of ACE-PNP, followed by systemically administered fludarabine phosphate, significantly inhibited the growth of pre-established KU-19-19 tumors. Our results indicate that RCR vectors are a potentially efficient gene delivery method and that the RCR vector mediated PNP gene transfer and fludarabine phosphate treatment might be a novel and potentially therapeutic modality for bladder cancer.

Altered expression of the retinoblastoma gene product: prognostic indicator in bladder cancer.

BACKGROUND: It has been reported that 50%-70% of patients with bladder cancer experience recurrence after initial successful treatment and about 10%-20% of these patients die of the disease. Despite precise pathologic staging and grading, we are unable to predict clinical outcome in all patients. The retinoblastoma-susceptibility (RB) gene, a prototype of tumor suppressor genes, has recently been associated with development and/or progression of bladder cancer, as well as sarcoma and small-cell lung cancer. In transitional cell carcinomas of the bladder, we have observed altered expression of the Rb gene product--a nuclear phosphoprotein thought to function as a cell cycle regulator. PURPOSE: The aim of this study was to investigate the hypothesis that altered patterns of Rb expression correlate with prognosis in bladder cancer. METHODS: Expression of the RB gene was evaluated in specimens from 48 primary bladder tumors obtained by cystectomy or transurethral resection. Rb protein expression was correlated with disease outcome in these patients. Rb expression was examined by immunohistochemistry, using the mouse monoclonal antibody Rb-PMG3-245 on frozen tissue sections. Computerized image analysis was used to quantify the level of Rb protein in individual tumor cells. RESULTS: The overall 5-year disease-free survival was 66%, with a median follow-up of 42 months. Normal levels of Rb protein expression were found in 34 patients (Rb-positive group). A spectrum of altered patterns of expression from undetectable levels to heterogeneous expression, however, was observed in 14 patients (altered Rb group). Of the 38 patients with muscle-invasive tumors, 13 were categorized as having altered expression of Rb protein. Only one of 10 patients with superficial carcinomas had altered expression of Rb protein. The 5-year survival was significantly decreased in patients with altered Rb protein compared with the survival in patients with positive Rb expression (P less than .001). CONCLUSIONS: The results suggest that tumors exhibiting decreased expression of the RB gene-coded product (Rb protein) had a more aggressive biological behavior than those that expressed the Rb protein in the majority of their tumor cells. IMPLICATIONS: This study demonstrates that altered patterns of Rb protein expression may be an important prognostic variable in patients presenting with invasive bladder cancer.

Association of p27Kip1 levels with recurrence and survival in patients with stage C prostate carcinoma.

BACKGROUND: There are few biologic determinants that are prognostic for patients with localized prostate cancer. We examined whether cellular levels of the cyclin-kinase inhibitor p27Kip1 (also known as p27) in prostate tumors could be used to predict progression of this disease. METHODS: Levels of p27 in tumor cell nuclei were assessed by immunohistochemical analysis of tissue sections from the primary tumors of 96 patients with stage C prostate carcinoma who had been treated by radical prostatectomy. Tumors were classified into one of the following three groups on the basis of the percentage of tumor cells showing nuclear p27 reactivity: low (0%-10%), moderate (11%-50%), and high (>50%). The Mantel-Haenszel test, Kaplan-Meier analysis, and the logrank test were used to calculate the probability that nuclear p27 levels were associated with tumor grade and substage, with a serum prostate-specific antigen (PSA) recurrence (defined as the finding of a detectable level [0.4 ng/mL or greater] of serum PSA following radical prostatectomy), with the recurrence of clinically evident disease, and with survival. All reported P values are two-sided. RESULTS: Luminal cells and basal cells of normal prostate glands showed high levels of nuclear p27 immunoreactivity in all tissue sections examined. Fifty-three tumors showed high p27 reactivity, 31 showed moderate reactivity, and 12 showed low or no detectable reactivity. Decreased levels of p27 were associated with tumor grade (P = .004). Tumor levels of p27 were not associated with preoperative prostate-specific antigen levels (P = .360) or with tumor substage (P = .320). However, decreased p27 reactivity was significantly associated with an increased probability of recurrence (P = .004) and decreased survival (P = .010). The median recurrence-free interval for patients with tumors showing high, moderate, or low p27 reactivity was 13.7 years, 8.4 years, and 4.7 years, respectively. Median survival times were more than 14 years, more than 13.5 years, and 8.1 years for patients in the high, moderate, and low p27 reactivity groups, respectively. CONCLUSION: Levels of nuclear p27 immunoreactivity in the primary tumor can be used to predict recurrence and survival among patients with localized prostate cancer.

Altered patterns of MDM2 and TP53 expression in human bladder cancer.

BACKGROUND: The TP53 gene maps to the short arm of chromosome 17 (17p13.1) and encodes for a nuclear phosphoprotein of 53 kd (p53) involved in cell cycle control. The MDM2 gene is located on the long arm of chromosome 12 (12q13-14), and it encodes for a nuclear protein (Mdm2) of 90 kd of molecular mass. Genetic alterations in the TP53 gene have been reported as frequent events in bladder cancer and are associated with disease progression. The MDM2 gene has been shown to be amplified and overexpressed in sarcomas; however, these changes have not yet been analyzed in neoplastic lesions of the urinary bladder. PURPOSE: We undertook the present study in order to determine the frequency of MDM2 and TP53 abnormalities in bladder tumors, as well as to examine the clinical relevance of identifying their altered patterns of expression in patients affected with bladder cancer. METHODS: We analyzed a cohort of 87 patients affected by bladder tumors. Altered patterns of expression of Mdm2 proteins were determined using an immunohistochemical assay with monoclonal antibody 2A10, and MDM2 gene amplifications were studied by Southern blotting. Mutant p53 proteins were identified using monoclonal antibody PAb1801. The presence of intragenic mutations in the TP53 gene were assessed utilizing single-strand conformation polymorphism and further characterized by sequencing. Associations were assessed statistically by the two-tailed Fisher's exact test. RESULTS: Twenty-six of 87 cases had abnormally high levels of Mdm2 proteins; however, only one case showed an MDM2 amplification. Thirty-six of 87 cases displayed p53 nuclear overexpression. Sixteen cases had abnormally high levels of both Mdm2 and p53 proteins. There was a strong statistical association between Mdm2 and p53 overexpression (Fisher's exact test: P = .018). Moreover, there was a striking association between Mdm2 overexpression and low-stage, low-grade bladder tumors (Fisher's exact test: P = .0005). CONCLUSIONS: The results suggest that aberrant Mdm2 and p53 phenotypes are frequent events in bladder cancer and may be involved in tumorigenesis or tumor progression in urothelial neoplasias. IMPLICATIONS: This study is the first to report altered patterns of MDM2 expression in human bladder tumors and demonstrates that aberrant Mdm2 and p53 phenotypes may be important diagnostic and prognostic markers in patients affected by bladder cancer.

Deletion of the p16 and p15 genes in human bladder tumors.

BACKGROUND: Two genes, p16 (also known as CDKN2, INK4A, or MTS1) and p15 (also described as INK4B or MTS2), are found in tandem at chromosome 9p21. These genes are designated as candidate tumor suppressor genes because they encode proteins that function as negative cell cycle regulators. (The encoded polypeptides inactivate specific cyclin-protein kinase complexes that are required for progression through the cell cycle.) Molecular genetic studies have revealed that deletion of the p16 and p15 genes occurs frequently in cancer cell lines and in certain malignant neoplasms. PURPOSE: We evaluated the frequency of p16 and p15 gene alterations in a well-characterized cohort of human transitional cell bladder cancers, and we explored potential associations between alterations in these genes and tumor stage and/or grade. METHODS: Tumor tissue and normal tissue from 110 patients with transitional cell carcinoma of the urinary bladder were examined. The status of the p16 and p15 genes in these tissues was determined by Southern blotting and hybridization with gene-specific probes, by coupled polymerase chain reaction and single-strand conformation polymorphism analysis (PCR-SSCP), and by sequencing DNA fragments produced during PCR. Associations between alterations in the genes and tumor stage and/or grade were evaluated using the two-tailed Fisher's exact test. RESULTS: Homozygous deletion (both alleles lost) of the p16 and the p15 genes was observed in 11 and nine bladder tumors, respectively. Eight of the 11 tumors exhibiting complete loss of the p16 gene also displayed homozygous deletion of the p15 gene. Exclusive loss of either gene was detected in only three tumors. Hemizygous deletion (one allele lost, also referred to as loss of heterozygosity [LOH] of the p16 and/or p15 genes was observed in eight tumors. Rearrangement of the two genes was indicated in three additional tumors. No point mutations were identified in either gene. The overall frequency of alteration in this cohort of bladder tumors was approximately 18% for each gene (in 20 [18.3%, 95% confidence interval (CI) = 11.1%-25.6%] of 109 informative tumors for the p16 gene and in 18 [18%, 95% CI = 10.5%-25.5%] of 100 informative tumors for the p15 gene). A statistically significant association between p16 gene alteration and bladder tumors of low stage (P < .01) and grade (P < .01) was observed; a significant association between p15 gene alteration and tumors of low stage (P < .01) was also detected. CONCLUSIONS: Alteration of the p16 and p15 genes, especially coincident homozygous deletion, appears to be a common event in bladder cancer.

Nuclear overexpression of p53 protein in transitional cell bladder carcinoma: a marker for disease progression.

BACKGROUND: Approximately one third of the patients with superficially infiltrative transitional cell (T1-TNM pathological staging system) bladder carcinoma who are treated with transurethral resection alone have disease progression. Despite precise pathologic staging and grading, clinical outcome in these patients is not predictable. Recent reports reveal that mutations of the p53 tumor suppressor gene (also known as TP53) occur commonly in bladder cancers. PURPOSE: The aim of this study was to investigate the hypothesis that altered patterns of expression of the protein product(s) of the mutated p53 tumor suppressor gene are associated with tumor progression in patients with T1 bladder cancer. METHODS: We examined deparaffinized tumor tissue specimens from transurethral resection in 43 patients with T1 bladder cancer who had not received adjuvant therapy. Nuclear overexpression of p53 protein was detected by immunohistochemical analysis using the mouse monoclonal antibody PAb1801, which stains both wild-type and mutant p53 proteins. The data were then correlated with the following conventional prognostic variables: age, sex, histologic presence of associated carcinoma in situ, and vascular invasion of tumor. Disease progression rates per 100 person-years were calculated. RESULTS: Median follow-up was 119 months. None of the urothelial and stromal cells from normal bladder specimens showed nuclear overexpression of p53 protein, but patients with T1 bladder tumors could be stratified into two groups with different patterns of staining for p53 protein. Eighteen patients (42%) had no more than 20% tumor cells with positive nuclear staining (group A), while the remaining 25 patients (58%) had 20% or more tumor cells with nuclear immunoreactivity (group B). Patients in group B had a significantly lower progression-free interval (P < .001). Disease progression rates were 20.5% per year for group B and 2.5% for group A. CONCLUSION: These results suggest that T1 bladder cancers exhibiting nuclear overexpression of p53 protein have a higher probability of disease progression. This study also suggests that p53 overexpression is an important prognostic factor in these patients and may be useful in selecting appropriate therapy. IMPLICATIONS: Large prospective studies are needed to confirm these results and to evaluate nuclear overexpression of p53 protein as a prognostic marker in bladder cancer.

Prognostic significance of transcription factor E2F-1 in bladder cancer: genotypic and phenotypic characterization.

BACKGROUND: We sought to identify and characterize potential alterations in E2F-1, a transcription factor that binds to the retinoblastoma protein (pRB), in bladder neoplasms and to elucidate a possible role for E2F-1 as an oncogene or a tumor suppressor gene. METHODS: Tumor samples from 133 evaluable patients with bladder cancer were analyzed for E2F-1 gene mutations by use of polymerase chain reaction-single-strand conformational polymorphism (PCR-SSCP) analysis and DNA sequencing. In addition, tumors were studied for E2F-1 and pRB protein expression by use of immunohistochemistry. Results from the above analyses were correlated with clinicopathologic parameters and outcome. All P values are two-sided. RESULTS: A polymorphism, consisting of a nucleotide change at amino acid codon 393 in exon 7 (GGC-->AGC [Gly-->Ser]), was identified in seven of 133 case patients, being present in both tumor and corresponding normal tissues. No bandshifts were identified in the nuclear-localization or DNA-binding domains on PCR-SSCP analysis. On immunohistochemical analysis, E2F-1 nuclear reactivity was observed in less than 5% of the cells from 53 tumors and in 5%-75% of the cells from the remaining 80 tumors. The pattern of E2F-1 protein expression was not altered in relation to the identified polymorphism. pRB nuclear reactivity greater than 20% (of tumor cells stained) was present in 66% of the samples. E2F-1 nuclear reactivity correlated inversely with the percentage of cells showing pRB reactivity (Kendall tau(b) = -0.18; P = .019). On multivariate analysis, patients with lower E2F-1 reactivity had statistically significantly increased risks of progression to metastases (P = .001) and death (P = .02). CONCLUSIONS: E2F-1 alterations occur at the phenotypic level, rather than at the genotypic level, in bladder cancer. The adverse outcome for patients whose tumors exhibit low E2F-1 nuclear expression suggests a possible tumor suppressor role for E2F-1 in bladder cancer.

Alterations of INK4A and INK4B genes in adult soft tissue sarcomas: effect on survival.

BACKGROUND: The INK4A and INK4B genes map to chromosome 9p21, with the INK4A gene encoding two protein products, p16 and pl9ARF. Alterations of the INK4A and INK4B genes occur frequently in certain primary malignant neoplasms. This study was undertaken to evaluate the frequency of INK4A and INK4B gene alterations in a cohort of adult soft tissue sarcomas. METHODS: The status of the INK4A and INK4B genes was determined in 46 soft tissue sarcomas by use of the following methods: Southern blotting, polymerase chain reaction (PCR), single-strand conformation polymorphism analysis, comparative multiplex PCR, and a methylation assay focusing on the p16 promoter. Associations between alterations of the INK4A and INK4B genes and clinicopathologic variables, as well as with p53 and pRB (retinoblastoma protein) status, were evaluated by use of the two-tailed Fisher's exact test. Disease-specific survival was evaluated by use of the Kaplan-Meier method and the logrank test. Proportional hazards analysis was used to obtain estimates of relative risks. All P values are two-sided. RESULTS: Homozygous and hemizygous deletions, but no point mutations, were observed in these two genes. The overall frequency of gene alteration (deletion or rearrangement) was approximately 15% for the INK4A and INK4B genes, with changes restricted to high-grade sarcomas. Statistically significant associations were observed between INK4A/INK4B deletions (P = .036) or alterations (P = .005) and poor survival. Alteration of the INK4A and INK4B genes was the only statistically significant predictor for poor survival when controlling for tumor grade and size (P = .03). CONCLUSION/IMPLICATIONS: Coincident homozygous deletion of the INK4A and INK4B genes occurs frequently in adult soft tissue sarcomas. Loss of p16 and pl9ARF function in primary tumors, although not equivalent to alterations in p53 and pRB function, appears to be associated with cancers that have an aggressive biologic behavior.