Molecular profiles and urinary biomarkers of upper tract urothelial carcinomas associated with aristolochic acid exposure.

Recurrent upper tract urothelial carcinomas (UTUCs) arise in the context of nephropathy linked to exposure to the herbal carcinogen aristolochic acid (AA). Here we delineated the molecular programs underlying UTUC tumorigenesis in patients from endemic aristolochic acid nephropathy (AAN) regions in Southern Europe. We applied an integrative multiomics analysis of UTUCs, corresponding unaffected tissues and of patient urines. Quantitative microRNA (miRNA) and messenger ribonucleic acid (mRNA) expression profiling, immunohistochemical analysis by tissue microarrays and exome and transcriptome sequencing were performed in UTUC and nontumor tissues. Urinary miRNAs of cases undergoing surgery were profiled before and after tumor resection. Ribonucleic acid (RNA) and protein levels were analyzed using appropriate statistical tests and trend assessment. Dedicated bioinformatic tools were used for analysis of pathways, mutational signatures and result visualization. The results delineate UTUC-specific miRNA:mRNA networks comprising 89 miRNAs associated with 1,862 target mRNAs, involving deregulation of cell cycle, deoxyribonucleic acid (DNA) damage response, DNA repair, bladder cancer, oncogenes, tumor suppressors, chromatin structure regulators and developmental signaling pathways. Key UTUC-specific transcripts were confirmed at the protein level. Exome and transcriptome sequencing of UTUCs revealed AA-specific mutational signature SBS22, with 68% to 76% AA-specific, deleterious mutations propagated at the transcript level, a possible basis for neoantigen formation and immunotherapy targeting. We next identified a signature of UTUC-specific miRNAs consistently more abundant in the patients' urine prior to tumor resection, thereby defining biomarkers of tumor presence. The complex gene regulation programs of AAN-associated UTUC tumors involve regulatory miRNAs prospectively applicable to noninvasive urine-based screening of AAN patients for cancer presence and recurrence.

Collecting duct carcinoma and endemic nephropathy - case reportS and literature review.

Although collecting duct carcinoma is a subtype of renal cell carcinoma, several studies implicate association with urothelial carcinoma. The coexistence of collecting duct carcinoma and another renal neoplasm is rare. Endemic nephropathy is a renal disease causing chronic renal failure. It is highly associated with urothelial neoplasm and occurs in endemic villages in Bosnia, Croatia, Bulgaria, Romania and Serbia. Recent studies have confirmed the important role of exposure to aristolochic acid as an etiologic factor. We present three cases of collecting duct carcinoma with literature overview. In one case, we describe collecting duct carcinoma with metachronous urothelial carcinoma of the pyelon and urinary bladder in an endemic nephropathy patient. To our knowledge, this is the first case report describing this coexistence. Certain similarities between collecting duct carcinoma and urothelial carcinoma were found, e.g., higher incidence in female compared to male, higher mean age, and multifocal and multicentric occurrence of the tumor. Our observations support the hypothesis that collecting duct carcinoma and urothelial carcinoma could be connected.

Low-Coverage Exome Sequencing Screen in Formalin-Fixed Paraffin-Embedded Tumors Reveals Evidence of Exposure to Carcinogenic Aristolochic Acid.

BACKGROUND: Dietary exposure to cytotoxic and carcinogenic aristolochic acid (AA) causes severe nephropathy typically associated with urologic cancers. Monitoring of AA exposure uses biomarkers such as aristolactam-DNA adducts, detected by mass spectrometry in the kidney cortex, or the somatic A>T transversion pattern characteristic of exposure to AA, as revealed by previous DNA-sequencing studies using fresh-frozen tumors. METHODS: Here, we report a low-coverage whole-exome sequencing method (LC-WES) optimized for multisample detection of the AA mutational signature, and demonstrate its utility in 17 formalin-fixed paraffin-embedded urothelial tumors obtained from 15 patients with endemic nephropathy, an environmental form of AA nephropathy. RESULTS: LC-WES identified the AA signature, alongside signatures of age and APOBEC enzyme activity, in 15 samples sequenced at the average per-base coverage of approximately 10×. Analysis at 3 to 9× coverage revealed the signature in 91% of the positive samples. The exome-wide distribution of the predominant A>T transversions exhibited a stochastic pattern, whereas 83 cancer driver genes were enriched for recurrent nonsynonymous A>T mutations. In two patients, pairs of tumors from different parts of the urinary tract, including the bladder, harbored overlapping mutation patterns, suggesting tumor dissemination via cell seeding. CONCLUSIONS: LC-WES analysis of archived tumor tissues is a reliable method applicable to investigations of both the exposure to AA and its biologic effects in human carcinomas. IMPACT: By detecting cancers associated with AA exposure in high-risk populations, LC-WES can support future molecular epidemiology studies and provide evidence-base for relevant preventive measures.

Renal cell carcinomas of chronic kidney disease patients harbor the mutational signature of carcinogenic aristolochic acid.

Aristolochic acid (AA) is a potent dietary cytotoxin and carcinogen, and an established etiological agent underlying severe human nephropathies and associated upper urinary tract urothelial cancers, collectively designated aristolochic acid nephropathy (AAN). Its genome-wide mutational signature, marked by predominant A:T > T:A transversions occurring in the 5'-CpApG-3' trinucleotide context and enriched on the nontranscribed gene strand, has been identified in human upper urinary tract urothelial carcinomas from East Asian patients and in experimental systems. Here we report a whole-exome sequencing screen performed on DNA from formalin-fixed, paraffin-embedded renal cell carcinomas (RCC) arising in chronic renal disease patients from a Balkan endemic nephropathy (EN) region. In the EN regions, the disease results from the consumption of bread made from wheat contaminated by seeds of Aristolochia clematitis, an AA-containing plant. In five of eight (62.5%) tested RCC tumor specimens, we observed the characteristic global mutational signature consistent with the mutagenic effects of AA. This signature was absent in the control RCC samples obtained from patients from a nonendemic, metropolitan region. By identifying a new tumor type associated with the AA-driven genome-wide mutagenic process in the context of renal disease, our results suggest new epidemiological and public health implications for the RCC incidence worldwide, particularly for the high-risk regions with unregulated use of AA-containing traditional herbal medicines.

Value of cytology in small cell lung carcinoma diagnostic--single-center study.

Small cell carcinoma of the lung (SCLC) together with the large cell neuroendocrine carcinoma (LCNEC), typical carcinoid (TC), and atypical carcinoid (AC) make a group of morphologically identifiable neuroendocrine tumors. The differential diagnosis of SCLC includes, first of all, other neuroendocrine tumors, and primary or metastatic non-small cell carcinomas. Although the criteria for the morphologic separation from other tumors of the lung are defined, in everyday practice it can be a problem, both in cytology and with histological samples. Accurate and early differentiation of the SCLC is important because it exhibits aggressive behavior, rapid growth, early spread to distant sites, but also exquisite sensitivity to chemotherapy and radiation. The study included 127 patients who underwent bronchoscopic examination or percutaneous transthoracic fine-needle aspiration (PTTFNA) during the period from early 2003 to 2007 in University Hospital Center Osijek whose cytological diagnosis was SCLC. The value of cytological diagnosis was determined by comparing it with histological findings obtained from a biopsy sample during bronchoscopy or on a resection specimen in 50 patients. In the remaining 77 patients, histological verification of cytological diagnosis was not made and the patients were treated based on cytological diagnosis of small cell carcinoma. In 76% of cases (38/50) cytological diagnosis of small cell lung carcinoma was also confirmed histologically. In 8% of cases (4/50) adenocarcinoma was histologically confirmed, in 10% (5/50) of the cases the squamous carcinoma was confirmed, and there was one case of urothelial carcinoma, one case of sarcoma and one undifferentiated carcinoma. Cytological diagnosis of SCLC was made in all cases in a brush smear while the catheter aspirate was positive in only 32 cases (25.8%). Median survival in the group of patients with histologically confirmed small cell cancer was 238 days, for women 250 days, and for men 237 days. Cumulative survival was 63.2% for 6 months, 26.3% for 12 months, 13.2% for 18 months and 7.9% for two years. In conclusion, cytology is a reliable and relatively non-invasive method for patients. Our results confirm that there is a good correlation between cytology and histology diagnoses, especially when it comes to malignant lesions. In determining the type of tumor cytology must be supported with additional methods, especially in cases when it is not possible to take samples for histological verification.

Endemic (Balkan) nephropathy is aristolochic acid nephropathy.

Endemic nephropathy is a syndrome that comprises two entities: chronic interstitial nephropathy and urothelial cell cancers predominantly of the upper urinary tract. The etiological agent for the disease is aristolochic acid, a compound found in the plants of Aristolochia spp. The development of urothelial cancers is characterized by the formation of aristolactam DNA adducts leading to mutations, predominantly A: T->T: A transversions. In order to comprehensively understand the gene regulation programs in upper urothelial cancers we performed integrated miRNA and mRNA expression profiling of paired tumours and unaffected urothelium samples. The obtained data will help us to understand the carcinogenesis caused by aristolochic acid and might be the source for the design of a diagnostic biomarker.

Correlation of vascular endothelial growth factor and hypoxia-inducible factor-1α expression with pathological renal artery changes in patients with renal cell carcinoma.

OBJECTIVE: The aim of this study was to correlate the expression of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1α) with pathological renal artery changes in patients with renal cell carcinoma (RCC). A further aim was to correlate intratumoral microvessel density (MVD) with VEGF and HIF-1α expression and prognostic factors for RCC, including tumour necrosis. MATERIAL AND METHODS: Formalin-fixed and paraffin-embedded tissue blocks from 150 patients with RCC and 50 patients with non-tumorous kidney diseases were analysed. The control group consisted of specimens from both renal arteries obtained from 25 decedents at routine autopsy (50 cases in total). Immunohistochemistry was performed using primary antibodies to VEGF, HIF-1α and CD31. RESULTS: Pathological renal artery changes were more common in patients with RCC and non-tumorous kidney diseases than in the control group. MVD was higher in the RCCs of patients with pathological renal artery changes. Tumours with higher HIF-1α expression had higher MVD; however, VEGF expression was not associated with MVD. A significant association was also found between MVD and the extent of tumour necrosis, in that less necrotic tumours had higher MVD. No association between renal artery changes and VEGF and HIF-1α expression was established. CONCLUSION: Considering the results of this study, the evaluation of renal artery changes in forthcoming research on RCC would be helpful for several reasons: to estimate their incidence in a larger number of patients, to clarify their connection with RCC and to reveal their relationship with MVD in RCC.

Consensus statement on screening, diagnosis, classification and treatment of endemic (Balkan) nephropathy.

Currently used diagnostic criteria in different endemic (Balkan) nephropathy (EN) centers involve different combinations of parameters, various cut-off values and many of them are not in agreement with proposed international guidelines. Leaders of EN centers began to address these problems at scientific meetings, and this paper is the outgrowth of those discussions. The main aim is to provide recommendations for clinical work on current knowledge and expertise. This document is developed for use by general physicians, nephrologists, urologist, public health experts and epidemiologist, and it is hoped that it will be adopted by responsible institutions in countries harboring EN. National medical providers should cover costs of screening and diagnostic procedures and treatment of EN patients with or without upper urothelial cancers.

Human formalin-fixed paraffin-embedded tissues: an untapped specimen for biomonitoring of carcinogen DNA adducts by mass spectrometry.

DNA adducts represent internal dosimeters to measure exposure to environmental and endogenous genotoxicants. Unfortunately, in molecular epidemiologic studies, measurements of DNA adducts often are precluded by the unavailability of fresh tissue. In contrast, formalin-fixed paraffin embedded (FFPE) tissues frequently are accessible for biomarker discovery. We report here that DNA adducts of aristolochic acids (AAs) can be measured in FFPE tissues at a level of sensitivity comparable to freshly frozen tissue. AAs are nephrotoxic and carcinogenic compounds found in Aristolochia herbaceous plants, many of which have been used worldwide for medicinal purposes. AAs are implicated in the etiology of aristolochic acid nephropathy and upper urinary tract carcinoma. 8-Methoxy-6-nitrophenanthro-[3,4-d]-1,3-dioxole-5-carboxylic acid (AA-I) is a component of Aristolochia herbs and a potent human urothelial carcinogen. AA-I reacts with DNA to form the aristolactam (AL-I)-DNA adduct 7-(deoxyadenosin-N(6)-yl) aristolactam I (dA-AL-I). We established a method to quantitatively retrieve dA-AL-I from FFPE tissue. Adducts were measured, using ultraperformance liquid chromatography/mass spectrometry, in liver and kidney tissues of mice exposed to AA-I, at doses ranging from 0.001 to 1 mg/kg body weight. dA-AL-I was then measured in 10-µm thick tissue-sections of FFPE kidney from patients with upper urinary tract cancers; the values were comparable to those observed in fresh frozen samples. The limit of quantification of dA-AL-I was 3 adducts per 10(9) DNA bases per 2.5 µg of DNA. The ability to retrospectively analyze FFPE tissues for DNA adducts may provide clues to the origin of human cancers for which an environmental cause is suspected.

Aristolactam-DNA adducts are a biomarker of environmental exposure to aristolochic acid.

Endemic (Balkan) nephropathy is a chronic tubulointerstitial disease frequently accompanied by urothelial cell carcinomas of the upper urinary tract. This disorder has recently been linked to exposure to aristolochic acid, a powerful nephrotoxin and human carcinogen. Following metabolic activation, aristolochic acid reacts with genomic DNA to form aristolactam-DNA adducts that generate a unique TP53 mutational spectrum in the urothelium. The aristolactam-DNA adducts are concentrated in the renal cortex, thus serving as biomarkers of internal exposure to aristolochic acid. Here, we present molecular epidemiologic evidence relating carcinomas of the upper urinary tract to dietary exposure to aristolochic acid. DNA was extracted from the renal cortex and urothelial tumor tissue of 67 patients that underwent nephroureterectomy for carcinomas of the upper urinary tract and resided in regions of known endemic nephropathy. Ten patients from nonendemic regions with carcinomas of the upper urinary tract served as controls. Aristolactam-DNA adducts were quantified by (32)P-postlabeling, the adduct was confirmed by mass spectrometry, and TP53 mutations in tumor tissues were identified by chip sequencing. Adducts were present in 70% of the endemic cohort and in 94% of patients with specific A:T to T:A mutations in TP53. In contrast, neither aristolactam-DNA adducts nor specific mutations were detected in tissues of patients residing in nonendemic regions. Thus, in genetically susceptible individuals, dietary exposure to aristolochic acid is causally related to endemic nephropathy and carcinomas of the upper urinary tract.

Higher apoptotic cell rate in Balkan endemic nephropathy--stereologic analysis.

The aim of this study was to correlate apoptotic cell rate from different nephron segments between control group and groups of patients with Balkan endemic nephropathy (BEN). Kidney specimens of20 patients with clinically and epidemiologically confirmed BEN were compared with biopsy material of 10 patients (group I, non BEN) without glomerular or tubulointerstitial disease. Out of 20 patients with BEN, 10 suffered and died from BEN (group II, BEN) and 10 patients (group III, BEN/CV) suffered from BEN but died from cardiovascular disease. Patient age ranged from 40 to 50 years. The apoptotic cell rate was measured in proximal and distal tubules and in collecting ducts using the 40X objective with a calibrated eyepiece multipurpose M 42 test system according to Weibel. Comparison of all three nephron segments yielded statistically significant differences in volume density of apoptotic cells in proximal tubules and in collecting ducts among all three patient groups (non BEN vs. BEN, non BEN vs. BEN/CV and BEN vs. BEN/CV, P<0.001 all). Statistically significant difference in apoptotic cell rate was also found in distal tubules between non BEN and BEN groups and non BEN and BEN/CV groups, but not between BEN and BEN/CV groups. Our results showed a statistically significant increase of apoptotic cells in all three nephron segments in patients with BEN (BEN and BEN/CV) compared to control group. The highest number of apoptotic cells was found in distal tubules in the groups of patients with BEN and BEN with coexisting cardiovascular disease, suggesting that these cells might be most frequently and most severely injured in patients with BEN.

TP53 Mutational signature for aristolochic acid: an environmental carcinogen.

This study was designed to establish the TP53 mutational spectrum of aristolochic acid (AA), examined in the context of endemic (Balkan) nephropathy, an environmental disease associated with transitional cell (urothelial) carcinomas of the upper urinary tract (UUC). Tumor tissue was obtained from residents of regions in Bosnia, Croatia and Serbia where endemic nephropathy has been prevalent for over 50 years. Fifty-nine TP53 mutations were detected in 42 of the 97 tumors analyzed. Mutational spectra were dominated by A:T to T:A transversions with the mutated adenines located almost exclusively on the nontranscribed strand. This marked strand bias is attributed to selective processing of aristolactam-dA adducts by transcription-coupled nucleotide excision repair. Hotspots for A:T to T:A mutations include codons 131 and 179 and the 5'-AG acceptor splice site of intron 6. The unique TP53 mutational signature for AA identified in this study can be used to explore the hypothesis that botanical products containing this human carcinogen and nephrotoxin are responsible, in part, for the high prevalence of UUC and chronic renal disease in countries where Aristolochia herbal remedies traditionally have been used for medicinal purposes.

Comparison of occurrence of upper urinary tract carcinomas in the region with endemic villages and non-endemic nephropathy region in Croatia.

Balkan endemic nephropathy (BEN) is a chronic tubulointerstitial renal disease of a still unknown etiology, associated with an increased frequency of urothelial carcinoma, particularly of the upper urinary tract (UUT). The aim of the study was to compare the occurrence of UUT carcinomas between Brodsko-Posavska Region (BPR) which is the region with endemic villages and the non-endemic region of Zagreb (ZG) in two six-year periods with a 20 year period separating the two, pointing out a possible difference in occurrence regarding war in Croatia (1991-1995). Comparing BPR and ZG regions we found a more then 5 times higher frequency of UUT carcinomas in BPR in the first period and more than 4.5 times higher frequency in the second period. Women in BPR were more frequently affected with UUT carcinomas.

Morphometric analysis of renal arteries in patients with renal cell carcinoma.

The aim of this study was to analyze morphometric parameters of renal arteries (longest diameter and tunica media thickness) in patients with renal cell carcinoma (RCC), to look into their relationship to tumor necrosis and to compare them with morphometric parameters recorded in a control group. We analyzed archival cases of RCC diagnosed in 2003 that also contained routinely sampled specimens of distal segments of renal artery. The control group consisted of specimens from both renal arteries obtained from 16 patients at routine autopsy during 2004-2005. Autopsy, as well as further histological analysis, did not disclose any malignant disease in the control group. Morphometric analysis of diameter and thickness of the renal artery tunica media was performed using Issa 3.1 software (Vamstek 2002, Zagreb, Croatia). The comparison of tunica media thickness showed that renal arteries from RCC cases were significantly thicker compared to distal parts of renal arteries in the control group (p=0.0002). Although renal artery samples from cases with necrotic tumor areas were thicker than those without tumor necrosis, the difference was not statistically significant. It is concluded that significantly thicker tunica media characterizes renal arteries in the group of patients with RCC when compared with the control group.

An unusual pattern of pseudoepitheliomatous hyperplasia associated with cutaneous primary melanoma: report of two cases with analysis of p53 and bcl-2 immunoreactivity.

Pseudoepitheliomatous hyperplasia (PEH) is a benign, reactive epithelial proliferation. PEH is characterized by hyperplasia of the epidermis or adnexal epithelium into irregular squamous strands that extend deep down into the subjacent dermis. PEH occurs in response to underlying infections, inflammatory or neoplastic conditions. The presence of PEH overlying cutaneous melanoma is rare. The clinical and histological features of PEH can closely mimic squamous cell carcinoma and could be misinterpreted. We report two cases of cutaneous primary melanoma associated with PEH and discuss differential diagnoses and potential role of p53 and bcl-2 in the pathogenesis of PEH.

The correlation between the tumor necrosis and renal artery changes in renal cell carcinoma.

Necrosis, cysts, hemorrhage, and calcification represent common findings in renal cell carcinoma. Different lesions, including arteriosclerosis or fibromuscular dysplasia, or both, may involve the main renal artery. This study analyzed the relationship between the presence and extent of necrosis in renal cell carcinoma with renal artery changes in a consecutive series of 112 patients (71 men, 41 women) with mean renal cell carcinoma of 7.7 cm (range, 2 to 20 cm). Necrosis was seen macroscopically and confirmed microscopically in 88 cases (78.6%), with 64 tumors having less than 50% and 24 more than 50% necrosis. Fibromuscular dysplasia was found in 41 patients (36.6%; 17 men, 24 women) and atherosclerotic changes in 21 patients (18.8%; 18 men, 3 women). The results suggest that necrosis of renal cell carcinoma was significantly more common in women with associated fibromuscular dysplasia (especially type I) and men with atherosclerotic changes of renal artery.

Synchronous anorectal melanoma.

Anorectal melanoma is a very rare tumor with poor prognosis. Rectal bleeding is the most frequent symptom and surgical treatment ranges from local excision to radical abdominoperineal resection. We report a case of a 75-years-old male patient who presented with a history of recurrent rectal bleeding, and whose histopathological diagnosis was melanoma. Macroscopically, we found two distinct tumors in anorectal region, 0.5 cm and 1.5 cm from dentate line. The first one was pedunculated, on a thin stalk, measuring 1 cm in greatest diameter, and the second one was sessile and nodular measuring up to 2.8 cm in largest diameter. Microscopic examination and immunohistochemical analysis of both tumors confirmed the diagnosis of melanoma. This case represents multiple synchronous primary melanoma of the anorectal region, with a possibility that one of the lesions is primary melanoma and the second one is a satellite lesion.

Local recurrence of primary non-ampullary adenocarcinoma of duodenum after surgical treatment--a case report and a literature review.

Worldwide there is no general attitude on optimal surgical procedure in treatment of primary non-ampullary adenocarcinoma of duodenum, especially for early stage of duodenal cancer. Some authors prefer local excision and segmental resection while others rather perform duodenopancreatic resection, even in the case of early stage of duodenal cancer with aim to avoid tumor recurrence. In this paper we present a rare clinical course of a 60-year-old male patient with an endoscopically and pathohistologically proven early stage duodenal cancer that was treated by wide local excision. Three years after operation, control endoscopy showed "flat" polyp in the duodenum and radical duodenopancreatic resection was performed. Pathohistological examination of resected specimen showed cancer that had spread throughout the duodenal wall with metastases in the regional lymph nodes. According to our findings and literature review we gave some direction concerning the optimal diagnostic and surgical procedure for this rare tumor.