The high incidence of JC virus infection in urothelial carcinoma tissue in Taiwan

Human polyomaviruses, JC virus (JCV) and BK virus (BKV), usually remain latent in kidney and urothelial tissue after primary infection. Infection with human polyomavirus has still not been correlated conclusively with malignancy of kidney and urothelial tissue. The present study investigated further the possible relationship between JCV/BKV infection and urothelial carcinoma. Tissue samples were examined from 33 urothelial carcinomas and 5 renal cell carcinomas for JCV/BKV infection, using nested PCR with primers common to both JCV and BKV. The viral genotypes were further verified by endonuclease digestion and DNA sequencing following the PCR. In addition, immunohistochemistry and Western blotting were also performed to detect viral large tumor protein (LT) and the late capsid protein (VP1) in the tissue samples. The results from nested PCR showed that 90.1% (30/33) of the urothelial carcinomas samples and all of the renal cell carcinomas samples (5/5) were JCV DNA positive. Both archetypal and re-arranged JCV genotypes were detected. On the other hand, BKV DNA was detected in only one (3%) of the urothelial carcinoma tissue samples. The immunohistochemical results showed that 30% (10/33) of urothelial carcinoma tissues was stained positive for large tumor antigen (LT). However, the structural protein VP1 was not detectable in any of the tissue samples examined. The present study demonstrated that JCV is highly prevalent in urothelial carcinoma tissue as is the expression of large tumor antigen. Therefore, the findings support the hypothesis that JCV infection is associated with urothelial carcinoma.     

Detection of SV40 in colon cancer: A molecular case–control study from Northeast Italy

To explore the involvement of the simian polyomavirus SV40 in human colon cancer, a molecular case–control study was undertaken in patients and in their relatives living in an area where the spread of SV40 has already been documented. From 2006 to 2008, 94 colon cancer patients (age: 37–90 years) and 91 subjects (age: 32–70 years) relatives of each index case were enrolled. A blood sample and a specimen of cancer tissue or biopsy were collected, from each patient or control, respectively. Samples were analyzed twice for Polyomavirus (i.e., SV40, JCV, and BKV) by PCR and by quantitative real‐time PCR (RT‐qPCR) with reproducible results. No BKV/JCV was detected either in normal or pathological tissues. SV40 was not present in control subjects, either normal tissue or in biopsies from adenomas or polyps. All blood samples were negative. Conversely, six adenocarcinoma specimens were positive for SV40 sequences (overall prevalence 6.4%, P = 0.03 in comparison with controls). Nevertheless, the SV40‐associated colon cancer risk proved statistically not significant (OR = 3.91; P = 0.115) when adjusted for age. Quantitation of SV40 DNA performed by RT‐qPCR showed a low viral load ranging from 6.2 × 10¹ to 9 × 10³ copies per reaction. This molecular case–control survey showed, for the first time in fresh samples and by RT‐qPCR, that SV40 can be detected in colon cancer tissue. However, the finding was not statistically significant when compared with a well‐structured community control group. Thus, the role of SV40 and other polyomavirus in colon cancer genesis deserves further investigation. J. Med. Virol. 82: 1197–1200, 2010. © 2010 Wiley‐Liss, Inc.     

Detection of Merkel cell polyomavirus in Merkel cell carcinoma and Kaposi's sarcoma

Merkel cell carcinoma is a rare malignancy that sometimes occurs in the skin of elderly people. Recently, a new human polyomavirus, Merkel cell polyomavirus (MCPyV) was identified in Merkel cell carcinoma. In the present study, MCPyV‐DNA was detected in 6 of 11 (55%) cases of Merkel cell carcinoma by nested PCR and real‐time PCR. Histologically, MCPyV‐positive cases showed round and vesicular nuclei with a fine granular chromatin and small nucleoli, whereas MCPyV‐negative cases showed polygonal nuclei with diffusely distributed chromatin. Real‐time PCR analysis to detect the MCPyV gene revealed that viral copy numbers ranged 0.04–0.43 per cell in cases of Merkel cell carcinoma. MCPyV was also detected in 3 of 49 (6.1%) cases of Kaposi's sarcoma (KS), but not in 192 DNA samples of other diseases including 142 autopsy samples from 20 immunodeficient patients. The MCPyV copy number in KS was lower than that in Merkel cell carcinoma. PCR successfully amplified a full‐length MCPyV genome from a case of KS. Sequence analysis revealed that the MCPyV isolated from KS had 98% homology to the previously reported MCPyV genomes. These data suggest that the prevalence of MCPyV is low in Japan, and is at least partly associated with the pathogenesis of Merkel cell carcinoma. J. Med. Virol. 81:1951–1958, 2009. © 2009 Wiley‐Liss, Inc.     

Transitional cell carcinoma of the bladder: low incidence of human papillomavirus DNA detected by the polymerase chain reaction and in situ hybridization

Viral studies on mammalian urothelium have shown an association between the bovine papillomavirus and cancer of the bladder in cattle. However, the evidence for human papillomavirus (HPV) involvement in urinary bladder in man is less clear. The aim of this study was to investigate the association between HPV DNA and transitional cell carcinoma of the bladder, using the highly sensitive polymerase chain reaction (PCR) and non-isotopic DNA in situ hybridization on formalin-fixed paraffin-embedded tissues from 76 patients. An HPV type specific set of primers was localized on the E6-gene for HPV 16/18 DNA. The second and third set of primers were specific for HPV 6/11 DNA. A biotinylated DNA probe which recognizes HPV 6/11, 16/18, and 31/33/35 was used for in situ hybridization. Of the 76 cases investigated, PCR analysis showed positive signals in seven (9.2%) of cases–six for HPV 16 DNA, and one for HPV 16 DNA and HPV 6 DNA. Four (5.2%) were also reactive for HPV 16/18 DNA using in situ hybridization. Most transitional cell carcinomas (71.4%) associated with HPV DNA were of high pathological grade/stage. One case had koilocytosis. Our results suggest that HPV DNA in transitional cell carcinoma is probably a rare occurrence, although the finding of the high risk HPV 16 DNA may indicate a role for it in this tumour's aetiology.     

Evidence for aldosterone‐dependent growth of renal cell carcinoma

The aim if this study was to investigate the hypothesis that K‐RAS 4A is upregulated in a mineralocorticoid‐dependent manner in renal cell carcinoma and that this supports the proliferation and survival of some renal cancers. Expression of the K‐RAS in renal tumour tissues and cell lines was examined by real‐time PCR and Western blot and mineralocorticoid receptor, and its gatekeeper enzyme 11β‐hydroxysteroid dehydrogenase‐2 was examined by immunocytochemistry on a tissue microarray of 27 cases of renal cell carcinoma. Renal cancer cells lines 04A018 (RCC4 plus VHL) and 04A019 (RCC4 plus vector alone) were examined for the expression of K‐RAS4A and for the effect on K‐RAS expression of spironolactone blockade of the mineralocorticoid receptor. K‐RAS4A was suppressed by siRNA, and the effect on cell survival, proliferation and activation of the Akt and Raf signalling pathways was investigated in vitro. K‐RAS4A was expressed in RCC tissue and in the renal cancer cell lines but K‐RAS was downregulated by spironolactone and upregulated by aldosterone. Spironolactone treatment and K‐RAS suppression both led to a reduction in cell number in vitro. Both Akt and Raf pathways showed activation which was dependent on K‐RAS expression. K‐RAS expression in renal cell carcinoma is at least partially induced by aldosterone. Aldosterone supports the survival and proliferation of RCC cells by upregulation of K‐RAS acting through the Akt and Raf pathways.     

Mutations of the p53 gene in carcinomas of the urinary system

Deletion of p53, which is an anti-oncogene located on chromosome 17p, was reported to be present at a high incidence in tumor cells of colorectal carcinoma, as well as osteosarcoma of the familial cancer syndrome. Mutations of the p53 gene were investigated in 59 surgical specimens of primary carcinomas of the urinary system from 57 patients, using the polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) analysis. The PCR products were sequenced using the dideoxy chain termination method or the DNA sequencer. The tumors examined were 20 transitional cell carcinomas (TCC) and 39 renal cell carcinomas (RCC). Mutations of the p53 gene were detected in 20.0% (4/20) of TCC and were present in 16.7% (1/6) of the tumors invading the muscular layer. In two patients with simultaneous double bladder TCC, the mutations were found only in the larger tumors. In RCC, mutations were detected in 7.7% (3/39) of patients. No significant correlation between the presence of the mutation and the clinicopathologic parameters was found in RCC except that the three tumors with p53 gene mutations were clear cell carcinomas. These results suggest that p53 gene mutations play a possible role in both carcinogenesis and progression of TCC, but the p53 gene mutations may not be significant in development of RCC.     

An unusual case of non-disseminated bladder aspergillosis in a setting of transitional cell carcinoma

A bladder infection of Aspergillus with no evidence of dissemination is rare. We present a case of Aspergillus infection with transitional cell carcinoma of the urinary bladder without any evidence of systemic involvement. A 65-year-old male diabetic whose main complaints were intermittent painful haematuria and nocturia had undergone nephroureterectomy a year and a half back for transitional cell carcinoma of right renal pelvis. Cystoscopy revealed bladder mucosa having fixed broad tumour with encrustation and bleeding on touch at the right vesico-ureteric junction. The histopathologic diagnosis was a high-grade transitional carcinoma with Aspergillus infection. Fungal culture of urine obtained after bladder wash yielded Aspergillus fumigatus.     

Upregulated MT1-MMP/TIMP-2 axis in the TSU-Pr1-B1/B2 model of metastatic progression in transitional cell carcinoma of the bladder

Muscle invasive transitional cell carcinoma (TCC) of the bladder is associated with a high frequency of metastasis, resulting in poor prognosis for patients presenting with this disease. Models that capture and demonstrate step-wise enhancement of elements of the human metastatic cascade on a similar genetic background are useful research tools. We have utilized the transitional cell carcinoma cell line TSU-Pr1 to develop an in vivo experimental model of bladder TCC metastasis. TSU-Pr1 cells were inoculated into the left cardiac ventricle of SCID mice and the development of bone metastases was monitored using high resolution X-ray. Tumor tissue from a single bone lesion was excised and cultured in vitro to generate the TSU-Pr1-B1 subline. This cycle was repeated with the TSU-Pr1-B1 cells to generate the successive subline TSU-Pr1-B2. DNA profiling and karyotype analysis confirmed the genetic relationship of these three cell lines. In vitro, the growth rate of these cell lines was not significantly different. However, following intracardiac inoculation TSU-Pr1, TSU-Pr1-B1 and TSU-Pr1-B2 exhibited increasing metastatic potential with a concomitant decrease in time to the onset of radiologically detectable metastatic bone lesions. Significant elevations in the levels of mRNA expression of the matrix metalloproteases (MMPs) membrane type 1-MMP (MT1-MMP), MT2-MMP and MMP-9, and their inhibitor, tissue inhibitor of metalloprotease-2 (TIMP-2), across the progressively metastatic cell lines, were detected by quantitative PCR. Given the role of MT1-MMP and TIMP-2 in MMP-2 activation, and the upregulation of MMP-9, these data suggest an important role for matrix remodeling, particularly basement membrane, in this progression. The TSU-Pr1-B1/B2 model holds promise for further identification of important molecules.     

Polyomavirus (BK)-associated pleomorphic giant cell carcinoma of the urinary bladder: a case report

This report describes the morphological features of a pleomorphic giant cell carcinoma with focal trophoblastic differentiation of the urinary bladder in a male, 12 years post living related donor renal transplant. The voided urine cytology demonstrated rare decoy cells admixed with markedly atypical urothelial cell clusters, papillae and giant cells. Cystoprostatectomy demonstrated a nodular mass involving the trigone and right lateral-posterior wall, adjacent to the ureteral orifice. Hematoxylin-eosin stained sections showed two synchronous malignancies: (a) pleomorphic giant cell carcinoma with focal trophoblastic differentiation of the urinary bladder, metastatic to the omentum and (b) prostatic adenocarcinoma, Gleason score 3 + 4 = 7, involving the right prostate lobe. Strong diffuse expression of polyomavirus large T antigen was demonstrated in the primary and metastatic pleomorphic giant cell carcinoma, supporting a possible role for polyomavirus (BK) in the oncogenetic pathway. The prostatic adenocarcinoma was negative for polyomavirus large T antigen. Our findings of p63, CK7 and CK903 expression in pleomorphic giant cell carcinoma suggest that the tumor is of urothelial derivation. This is the first report describing the morphological features of urinary bladder pleomorphic giant cell carcinoma with trophoblastic differentiation, positive for polyomavirus large T antigen, arising in the background of BKV reactivation.     

The protein tyrosine phosphatase receptor type J is regulated by the pVHL–HIF axis in clear cell renal cell carcinoma

Mass spectrometry analysis of renal cancer cell lines recently suggested that the protein‐tyrosine phosphatase receptor type J (PTPRJ), an important regulator of tyrosine kinase receptors, is tightly linked to the von Hippel–Lindau protein (pVHL). Therefore, we aimed to characterize the biological relevance of PTPRJ for clear cell renal cell carcinoma (ccRCC). In pVHL‐negative ccRCC cell lines, both RNA and protein expression levels of PTPRJ were lower than those in the corresponding pVHL reconstituted cells. Quantitative RT‐PCR and western blot analysis of ccRCC with known VHL mutation status and normal matched tissues as well as RNA in situ hybridization on a tissue microarray (TMA) confirmed a decrease of PTPRJ expression in more than 80% of ccRCCs, but in only 12% of papillary RCCs. ccRCC patients with no or reduced PTPRJ mRNA expression had a less favourable outcome than those with a normal expression status (p = 0.05). Sequence analysis of 32 PTPRJ mRNA‐negative ccRCC samples showed five known polymorphisms but no mutations, implying other mechanisms leading to PTPRJ's down‐regulation. Selective silencing of HIF‐α by siRNA and reporter gene assays demonstrated that pVHL inactivation reduces PTPRJ expression through a HIF‐dependent mechanism, which is mainly driven by HIF‐2α stabilization. Our results suggest PTPRJ as a member of a pVHL‐controlled pathway whose suppression by HIF is critical for ccRCC development.     

Combined small and transitional cell carcinoma of the urinary bladder with CA19-9 production

It is well known that extrapulmonary small cell carcinoma, which exhibits morphological features similar to those observed in the lung, occurs in various organs. Clinically, most cases manifest aggressive biological behavior. A case of small cell carcinoma of the urinary bladder producing a high level of serum carbohydrate antigen (CA) 19-9, in which expression was confirmed in cancer cells of small as well as transitional cell carcinoma in the same tumor mass by immunostaining is reported. This paper documents combined small and transitional cell carcinoma of the urinary bladder with CA19-9 production, although it has already been reported that adenocarcinoma or transitional cell carcinoma in various organs frequently expresses CA19-9. Observations suggest that the histogenesis of some cases of combined small and transitional cell carcinoma in the urinary bladder may be the same, as both can produce CA19-9.     

Epigenetic inactivation of HOXA5 and MSH2 gene in clear cell renal cell carcinoma

The high‐throughput method using microarray is an easy and fast way to analyze the methylation status of hundreds of preselected genes and to screen them for signatures in methylation. The aim of our study is to detect hypermethylated genes and to analyze the association between methylation status and clinicopathological parameters of clear cell renal cell carcinoma. The genetic substrate included 62 cancer tissues and 62 matched adjacent normal kidney tissues. We adapted the GoldenGate genotyping assay to determine the methylation state of 1505 specific CpG sites in 807 genes. We identified two genes (HOXA5 and MSH2) with β‐value differences of more than 0.3 between cancer and normal tissues. The high methylation group in HOXA5 had high Fuhrman's nuclear grade (P= 0.041). Other data in HOXA5 and MSH2 were not significant with methylation status (P > 0.05). Survival curve of the high methylation group in HOXA5 was slightly lower than that of the low methylation group. However, the statistical significances of overall survival in HOXA5 and MSH2 were low (P > 0.05). We report the hypermethylation of two genes in clear cell renal cell carcinoma. The data we obtained could provide the basis for a diagnostic test pathological assessment, or prognosis in clear cell renal cell carcinoma.     

Genomic mutations of viral protein 1 and BK virus nephropathy in kidney transplant recipients

Genomic variability in the viral protein 1 region of BK polyomavirus (BKV) may change the ability of the virus to replicate. The significance of such changes was studied in clinical samples taken from kidney transplant patients with and without BKV nephropathy. A 94 base‐pair fragment of viral protein 1 was amplified from 68 urine, 28 blood, and 12 renal biopsy samples from eight patients with BKV nephropathy, and from 100 urine samples, 17 blood and three renal biopsy samples from 41 of 218 controls. The DNA was sequenced and the amino acid changes were predicted by the Expert Protein Analysis System program (ExPASy, Swiss Institute of Bioinformatics, Geneva, Switzerland). Single base‐pair mutations were detected more frequently in the samples from the BKV nephropathy patients than in the controls, and this was the only statistically significant finding of the study (P < 0.05), thus suggesting a greater genetic instability in BKV nephropathy associated strains. The amino acid changes were distributed at random in both BKV nephropathy patients and controls. However, one aspartic acid‐to‐asparagine substitution at residue 75 was detected in all samples of the one patient with BKV‐associated nephropathy, who developed disease progression confirmed by histology, and not in any of the other patient or control samples. Whether this specific amino acid change plays a role in disease deserves further study. J. Med. Virol. 81:1385–1393, 2009. © 2009 Wiley‐Liss, Inc.     

BK virus infection in human immunodeficiency virus-infected patients

The aim of this study is to evaluate the prevalence of BK virus (BKV) infection in HIV-positive patients receiving highly active antiretroviral therapy (HAART) in our hospital. The presence of BKV was analysed in urine and plasma samples from 78 non-selected HIV-infected patients. Clinical data were recorded using a pre-established protocol. We used a nested PCR to amplify a specific region of the BKV T-large antigen. Positive samples were quantified using real-time PCR. Mean CD4 count in HIV-infected patients was 472 cells/mm3 and median HIV viral load was <50 copies/mL. BKV viraemia was detected in only 1 HIV-positive patient, but 57.7% (45 out of 78) had BKV viruria, which was more common in patients with CD4 counts>500 cells/mm3 (74.3% vs 25.7%; p=0.007). Viruria was present in 21.7% of healthy controls (5 out of 23 samples, p=0.02). All viral loads were low (<100 copies/mL), and we could not find any association between BKV infection and renal or neurological manifestations. We provide an update on the prevalence of BKV in HIV-infected patients treated with HAART. BKV viruria was more common in HIV-infected patients; however, no role for BKV has been demonstrated in this population.     

Overexpression of PLCE1 in Kazakh esophageal squamous cell carcinoma: implications in cancer metastasis and aggressiveness

Three recent large‐scale genome‐wide association studies (GWAS) in Chinese Han populations have identified an esophageal squamous cell carcinoma (ESCC) susceptibility locus within phospholipase C epsilon 1 (PLCE1) gene, which encodes a phospholipase involved in intracellular signaling. The expressed PLCE1 in ESCC, however, are inconsistent. This study examined PLCE1 expression by immunohistochemistry (IHC) from 110 ethnic Kazakh ESCC patients and 50 from adjacent normal esophageal tissues (NETs). The expressed PLCE1 was localized in cytoplasm, especially in the peripheral layers of cancer cell nests, which was significantly higher in tumors than in NETs (p < 0.001). Increased expression of PLCE1 was correlated with advanced tumor‐node‐metastasis (TNM) stages (p = 0.015) and lymph node metastasis (p = 0.003) in patients with ESCC. Of the 110 patients, we examined 50 paired ESCC tissues and corresponding NETs by quantitative RT‐PCR (polymerase chain reaction) and the mean mRNA level of PLCE1 in ESCC was 1.85‐fold higher compared with those in corresponding NETs (p = 0.0012). Meanwhile, 4 of 5 ESCC cell lines also showed elevated expression of PLCE1 mRNA. Furthermore, elevated expression of PLCE1 mRNA in Kazakh ESCC was associated with its immunoreactivity (ρ = 0.297, p = 0.040), lymph node metastasis (p < 0.001), and advanced TNM stages of ESCC (p = 0.013). To our knowledge, this study demonstrates for the first time that PLCE1 overexpression correlates with lymph node metastasis and advanced TNM stages of Kazakh ESCC, implicating a role of PLCE1 in cancer metastasis and aggressiveness in ethnic Kazakh patients with ESCC. Furthermore, the current findings may warrant investigations into whether inhibiting PLCE1 could be a strategy for targeted anticancer therapy particularly for Kazakh ESCC.     

DNA from BK virus and JC virus and from KI, WU, and MC polyomaviruses as well as from simian virus 40 is not detected in non-UV-light-associated primary malignant melanomas of mucous membranes

The single most important causative factor for malignant melanomas of the skin is UV radiation. However, this is not true for melanomas on body surfaces sheltered from the sun; thus, it is important to seek new causative factors of melanoma genesis. Human papillomaviruses and gammaherpesviruses are associated with human skin cancer; for example, human papillomavirus types 5 and 8 are associated with epidermodysplasia verruciformis, and human herpesvirus 8 is associated with Kaposi's sarcoma. Recently, a newly described human polyomavirus, Merkel cell polyomavirus (MCPyV), has been associated with Merkel cell carcinoma, an unusual form of neurotropic skin cancer. Moreover, melanocytes are of neuroepithelial origin. This background impelled us to investigate if human polyomavirus DNA could play a role in the development of extracutaneous melanomas. Sixty-four extracutaneous melanomas were initially collected and dissected. Of these, 38 could be successfully used for further testing for the presence of the five human polyomaviruses known so far—BK virus (BKV), JC virus (JCV), KI polyomavirus (KIPyV), WU polyomavirus (WUPyV), and MCPyV—and of simian virus 40 (SV40). No polyomavirus DNA could be detected in any of the samples tested by use of a nested PCR detecting BKV, JCV, and SV40; a newly designed PCR detecting KIPyV and WUPyV; or a newly designed PCR for MCPyV. We conclude that since no human polyomavirus DNA was detected in primary malignant melanomas on non-sun-exposed body surfaces, these polyomaviruses presumably are not major factors for the development of extracutaneous melanomas.     

A case of urothelial carcinoma with triple variants featuring nested,plasmacytoid, and lipoid cell morphology

We present very rare variants of urothelial carcinoma featuring nested, plasmacytoid, and lipoid cell morphology in an 80‐year‐old female who was admitted to our hospital with mictritional pain and bilateral hydronephrosis. Abdominal computed tomography showed diffuse thickening of the urinary bladder, indicating probable invasion into the surrounding adipose tissue. Cytological tests on a urine specimen revealed medium‐sized cancer cell clusters with hyperchromatic nuclei and dense cytoplasm, small clusters of dyshesive cells with eccentric or crescent nuclei, and abundant or vacuolated cytoplasm mimicking plasma cells or lipoblasts. Histopathological findings of the bladder tumor revealed that the nested urothelial carcinoma variants were mainly located in the lamina propria, whereas the plasmacytoid and lipoid‐cell variants had deeply infiltrated into cells with a myxoid background. Immunohistochemically, the cancer cells were positive for cytokeratin, epithelial membrane antigen, but not for vimentin, S‐100, or hematopoietic markers of plasma cells such as CD79α and CD38. The patient received no radiosurgical therapies because of the advanced stage of her disease and died a few months after the initial diagnosis. To our knowledge, this is the first cytological and histological examination of urothelial carcinoma consisting of nested, plasmacytoid, and lipoid‐cell variants of the urinary bladder. Diagn. Cytopathol. 2009. © 2009 Wiley‐Liss, Inc.