PORES OF THE INTERNAL LIMITING MEMBRANE: A Common Finding in Vitreomaculopathies.

PURPOSE: To describe presence and distribution of pores of the inner limiting membrane (ILM) in eyes with vitreomaculopathies. METHODS: Inner limiting membrane specimens were harvested from 117 eyes of 117 patients during vitrectomy with membrane peeling from eyes with vitreomacular traction syndrome, idiopathic and secondary epiretinal gliosis, and idiopathic full-thickness macular hole. All specimens were processed as flat-mounts for immunocytochemistry and examined by phase-contrast, interference, and fluorescence microscopy. Demographic and clinical data were correlated. RESULTS: Inner limiting membrane pores were found in all vitreomaculopathies. They were identified in 47 (40.2%) of 117 eyes being most evident with antilaminin. In eyes with full-thickness macular hole >400 µ m, pores were seen in more than half of all eyes. They occur as numerous and uniformly distributed defects of the flat-mounted ILM with a mean diameter of 9.5 ± 2.4 µ m. Edges of ILM pores are round with an irregular contour and no specific cellular pattern. Pores were distinguished from retinal vessel thinning and iatrogenic artefacts. CONCLUSION: Contrary to previous reports, ILM pores are a common finding in vitreomaculopathies easily visible with antilaminin staining. Further studies are needed to clarify whether their presence correlates with differences in disease progression or imaging before and after vitrectomy with ILM peeling.

Matrix metalloproteinase 12 is an independent prognostic factor predicting postoperative relapse of conventional renal cell carcinoma - a short report.

PURPOSE: Approximately 15% of clinically localised conventional renal cell carcinomas (cRCC) develop metastases within 5 years of follow-up. Sarcomatous cRCC is a highly malignant cancer of the kidney. The aim of our study was to identify biomarkers for estimating the postoperative progression of cRCCs. METHODS: Global microarray-based gene expression analysis of RCCs with and without sarcomatous changes revealed that a high MMP12 expression was associated with a sarcomatous histology. Additionally, we analysed MMP12 expression using a multi-tissue array comprising 736 cRCC patients without metastasis at the time of surgery. The median follow-up time was 66 ± 29 months. RESULTS: Immunohistochemistry revealed MMP12 expression in 187 of 736 cRCCs with good follow-up data. Subsequent Kaplan-Meier analysis revealed that patients with MMP12 positive tumours exhibited a significantly shorter tumour-free survival (p < 0.001). In multivariate Cox regression analysis a weak to strong MMP12 expression indicated a 2.4-2.8 times higher risk of postoperative tumour relapse (p < 0.001; p < 0.003, respectively). CONCLUSIONS: MMP12 may serve as a biomarker to estimate postoperative cRCC relapse and as a possible target for penfluridol therapy.

Expression of TXNIP is associated with angiogenesis and postoperative relapse of conventional renal cell carcinoma.

One of the common mediator of tumour progression is the oxidative stress induced by inflammatory tumour microenvironment (TME). Activated fibroblasts, local and immune cells produce reactive oxygen species (ROS) supporting tumour cell proliferation and pave the way for metastatic tumour growth. TXNIP regulates ROS generation by inhibiting the antioxidative function of thioredoxin (TXN). The shift of TXNIP/TXN balance towards overexpression of TXNIP is associated with proliferation of endothelial cells during tumor angiogenesis. The oxidative stress activates the hypoxia inducible factor-1 (HIF-1), which plays an important role in the biology of conventional RCC (cRCC). Under oxydative stress TXNIP interacts with NLRP3 inflammasome leading to maturation and secretion of inflammatory cytokine IL1ß. To establish the role of TXNIP and downstream genes HIF1α and IL1ß in the biology of cRCC, we have applied immunohistochemistry to multi-tissue arrays containing tumours of 691 patients without detectable metastases at the time of operation. We found that cRCC displaying a fine organised capillary network with nuclear translocation of TXNIP and expressing IL1ß have a good prognosis. In contrary, we showed a significant correlation between cytoplasmic TXNIP expression, inefficient vascularisation by unorganized and tortuous vessels causing tumour cell necrosis and postoperative tumour relapse of cRCC.

Ureteric Bud-derivatives in Wilms Tumor and Nephrogenic Rest.

BACKGROUND/AIM: Recent studies suggest that not only the nephrogenic blastema but also the ureteric bud is involved in oncogenesis of Wilms' tumor (WT). However, the occurrence of ureteric bud (UB) derivatives in nephrogenic rest is not yet known. The aim of our study was to find UB derivatives in WT. MATERIALS AND METHODS: Keratin 17 (KRT17) is expressed exclusively in UB in foetal kidneys. In this study KRT17 immunohistochemistry was used to detect UB-derivatives in 21 triphasic, 2 stromal and 3 epithelial predominant WTs and 9 nephrogenic rests. RESULTS: We have detected KRT17 positive tubular structures resembling UB in 3 of 9 nephrogenic rests and 15 of 26 WTs. CONCLUSION: Not only the metanephric blastema but also the UB is involved in the histogenesis of nephrogenic rest and WT.

The Role of Genetic Analysis in Correct Diagnosis of Eosinophilic Variant of Chromophobe Renal Cell Carcinoma.

BACKGROUND/AIM: It has been suggested that eosinophilic variant of chromophobe renal cell carcinoma (chRCC) with low chromosome number or lack of genomic alteration has an excellent prognosis in comparison to classic chRCC. The aim of our study was to analyse the phenotypical variations of 77 chRCCs, including 7 eosinophilic ones, each diagnosed unequivocally by genetic means. MATERIALS AND METHODS: DNA isolated from chRCCs was subjected to array comparative genomic hybridisation (CGH) for establishing the chromosome alteration. Original histological slides were evaluated for cellular phenotype and growth pattern and compared to the genetic alterations. RESULTS: Loss of the entire chromosome 1, 2, 6, 10, 13, 17 and 21 occurred in 95%, 94%, 86% 90% 82% 90% and 66% of the cases, respectively. The number of chromosome alterations in eosinophilic forms of chRCC corresponded to those found in classic chRCC with pale-reticular cytoplasm or mixed cellular characteristics. Three of seven eosinophilic variants with loss of 4, 10 and 11 chromosomes showed metastasis at the time of diagnosis whereas only 3 metastatic tumors were noticed among the 70 classic chRCC. We did not find discriminating difference in number of chromosome alteration between classic and eosinophilic forms of chRCC. CONCLUSION: Eosinophilic chRCC has a more aggressive biology than the classic form. To avoid diagnostic pitfall of eosinophilic renal cell tumors with uncertain diagnosis, a genetic analysis should be carried out.

Impaired Vitamin D Signaling Is Associated With Frequent Development of Renal Cell Tumor in End-stage Kidney Disease.

BACKGROUND/AIM: End-stage kidney disease is characterized by chronic inflammation and frequent development of cancer. The level of circulating vitamin D is generally low in patients with end-stage renal disease (ESRD). Experimental studies have implicated the role of dysfunctional vitamin D metabolism in tumorigenesis. PATIENTS AND METHODS: We analyzed the expression of vitamin D receptor (VDR), cytochrome P450 family 27 subfamily B member 1 (CYP27B1) and cytochrome P450 family 24 subfamily A member 1 (CYP24A1), the key genes involved in vitamin D signaling, in kidneys from patients with ESRD, tissue microarrays containing ESRD-associated renal cell tumors, as well as in their precursor lesions by immunohistochemistry. RESULTS: Kidneys from patients with ESRD showed strong structural rearrangement with only few tubules and epithelial cell groups embedded in fibrotic-inflammatory stroma. Only an estimated 1-3% of the epithelial cells showed positive staining with antibodies to VDR, CYP27B1 and CYP24A1, which contrasted with the 100%, 40-50% and 40-50% of positively stained cells, respectively, found in normal kidneys. Down-regulation of the vitamin D signaling proteins was found in patients with renal cancer, with the exception of tumors and their precursors occurring exclusively in ESRD. CONCLUSION: The significantly reduced activity of CYP27B1 in kidney from patients with ESRD explains the low level of circulating vitamin D. We suggest that the lack of anti-tumorigenic effect of vitamin D is a crucial factor in the frequent development of unique types of renal cell cancer in in patients with ESRD.

Cytoplasmic Expression of AXL Is Associated With High Risk of Postoperative Relapse of Conventional Renal Cell Carcinoma.

BACKGROUND/AIM: Despite early detection by widespread use of abdominal imaging more than 40% of patients with conventional renal cell carcinoma (RCC) will die due to metastatic disease. Small kinase inhibitors for AXL receptor tyrosine kinase may delay the progression of metastatic cRCC. PATIENTS AND METHODS: We analysed AXL expression by immunohistochemistry on tissue multi arrays of 691 conventional RCC without metastasis at the time of nephrectomy. RESULTS: The Kaplan-Meier survival analysis indicated a poor disease-specific survival rates for patients with tumour showing cytoplasmic AXL staining, whereas expression on the cell membrane is associated with excellent disease outcome. Multivariate Cox regression analysis identified cytoplasmic AXL expression as an independent prognostic factor indicating a five-times higher risk of postoperative tumour progression (RR=5.048; 95% CI=2.391-10.657; p<0.001). CONCLUSION: Detecting cytoplasmic expression of AXL can be used to define a subset of conventional RCC with high risk of progression, thus identifying patients for more aggressive surveillance and adjuvant AXL inhibitor treatment as early as possible.

Expression of RARRES1 and AGBL2 and progression of conventional renal cell carcinoma.

BACKGROUND: Approximately 15% of clinically localised conventional renal cell carcinoma (RCC) will develop metastasis within 5 years of follow-up. The aim of this study was to identify biomarkers predicting the postoperative tumour relapse. METHODS: Tissue microarrays of conventional RCC from a cohort of 691 patients without metastasis at the time of operation were analysed by immunohistochemistry for the expression of carboxypeptase inhibitor RARRES1 and its substrate carboxypeptidase AGBL2. Univariate and multivariate Cox regression models were addressed to postoperative tumour relapse and the metastasis-free survival time was estimated by Kaplan-Meier analysis. RESULTS: In multivariate analysis, the lack of staining or cytoplasmic staining of RARRES1 was a significant risk factor indicating five times higher risk of cancer relapse. Combining its co-expression with AGBL2, we found that RARRES1 cytoplasmic/negative and AGBL2-positive/negative staining is a significant risk factor for tumour progression indicating 11-15 times higher risk of cancer relapse, whereas the membranous RARRES1 expression, especially its co-expression with AGBL2, associated with excellent disease outcome. CONCLUSIONS: RARRES1 and AGBL2 expression defines groups of patients at low and high risk of tumour progression and may direct an active surveillance to detect metastasis as early as possible and to apply adjuvant therapy.

M2 Macrophage Marker Chitinase 3-Like 2 (CHI3L2) Associates With Progression of Conventional Renal Cell Carcinoma.

BACKGROUND/AIM: In spite of early detection, appoximately 15% of the small renal cell carcinomas (RCC) will develop metastasis within 5 years follow-up. The aim of this study was to identify new biomarkers to estimate the postoperative relapse of the most common conventional RCC. PATIENTS AND METHODS: Tissue multi arrays of conventional RCC without metastasis at the time of operation from a cohort of 634 patients were analysed by immunohistochemistry for expression of the chitinase 3-like protein 2 (CHI3L2). Cancer specific survival of patients was estimated with Kaplan-Meier analysis, univariate and multivariate Cox regression models. RESULTS: Kaplan-Meier analysis estimated a shorter cancer-free survival for patients with CHI3L2 positive tumors. In multivariate analysis, the CHI3L2 positivity associated with a 3.5 times higher risk for tumor relapse (p<0.001). CONCLUSION: Expression of CHI3L2 in tumor cells of conventional RCC define a group of patients at high risk for postoperative progression.

Embryonal Origin of Metanephric Adenoma and its Differential Diagnosis.

BACKGROUND/AIM: The association of Wilms' tumor (WT), papillary renal cell tumor (PRCT) and mucinous tubular and spindle cell carcinoma (MTSCC) with embryonal rests has already been documented, but the cellular origin of metanephric adenoma (MA) is not yet known. The aim of this study was to understand their developmental evolution and find diagnostic markers. MATERIALS AND METHODS: CD57, KRT7, AMACR, SCEL, WT1 and CDH17 expression was analysed by immunohistochemistry in the four types of tumors and the associated pre-neoplastic lesions. RESULTS: Immunohistochemistry was able to differentiate WT, MA, MTSCC and PRCT. A phenotypic correlation between MA and perilobar nephrogenic rest associated with WT was identified. CONCLUSION: Perilobar nephrogenic rest and MA arise from differentiation arrested cells of the proximal domain of the S-shape body. We propose that WT1, MA, MTSCC and PRCT derive from different forms of maturation arrested embryonal rests.

Recalling Cohnheim's Theory: Papillary Renal Cell Tumor as a Model of Tumorigenesis from Impaired Embryonal Differentiation to Malignant Tumors in Adults.

We have suggested that papillary renal cell tumor (PRCT) of the kidney arises from nephrogenic rest-like lesions. To approve our hypothesis, we worked up 14 kidneys bearing papillary and 14 ones with conventional renal cell carcinoma (CRCC) histologically and found 42 papillary lesions in average per kidney bearing PRCT. PRCTs are characterized by loss of the Y chromosome and trisomy of chromosomes 7 and 17. The MET and HNF1B are localized to chromosome 7q31 and 17q21 and are frequently amplified in PRCT. We have analyzed the expression of the mutant MET in hereditary PRCTs and precursor lesions and found duplication and expression of the mutated allele. Because both genes are involved in early stage of nephron development, we have analyzed the expression of MET and HNF1B by immunohistochemistry in fetal kidneys, precursor lesions and PRCTs. We detected strong expression of MET and HNF1B in distal compartment of S-shaped body of fetal kidneys and in nephrogenic rest-like precursor lesions. Our finding suggests an association between expression of MET and HNF1B in precursor lesions and development of PRCT. We propose a model involving chromosomal clonal evolution and corresponding gene expression for development of PRCTs from embryonic rests due to impaired differentiation. Our model suggests that PRCT have a natural history distinct from that of most common CRCC.

Embryonal Origin of MTSCC of Kidney May Explain its Morphological Heterogeneity: Diagnostic Impact of Genetic Analysis.

BACKGROUND/AIM: Previous genetic and morphologic characterisation of mucinous tubular and spindle cell carcinoma (MTSCC) have yielded controversial results. The aim of this study was to explain the phenotypic heterogeneity of MTSCC diagnosed by genetic means. MATERIALS AND METHODS: We analyzed 7 MTSCC by array CGH and microsatellite allelotyping and by histology for morphological variation. We worked-up two entire kidneys with MTSCC to find microscopic alterations. RESULTS: We confirmed the diagnosis of MTSCC by detecting copy number changes at chromosomes 1, 4, 6, 8, 13, 14, 15, 18 and 22. We detected 13 small, microscopic precursor lesions in the two kidneys and found similar histological structures in precursor lesions and MTSCC. CONCLUSION: MTSCC develops from embryonal rest-like lesions of impaired differentiation which may explain its morphological variations. Until diagnosis of a "malignant" MTSCC" is not confirmed by genetic means, it should not be called carcinoma.

Cytoplasmic expression of ß-catenin is an independent predictor of progression of conventional renal cell carcinoma: a simple immunostaining score.

AIMS: The aims of this study were to investigate the potential of ß-catenin as a biomarker for predicting cancer-specific survival, and to find a reproducible mode of evaluation of immunohistochemistry. METHODS AND RESULTS: ß-Catenin expression was analysed by immunohistochemistry in a cohort of 488 patients with conventional renal cell carcinoma (RCC) operated on between 2000 and 2010. The association between ß-catenin expression and cancer-specific survival was assessed with univariate and multivariate Cox regression models in relation to conventional clinical pathological prognostic factors, and by Kaplan-Meier survival analysis with the log rank test. The univariate Cox regression model revealed an association of cytoplasmic ß-catenin positivity and pathological variables with cancer-specific death. The multivariate Cox regression model analysis of tumours without metastatic disease at the first presentation identified the T-classification (P < 0.001) and cytoplasmic ß-catenin positivity as risk factors for postoperative tumour progression. Specifically, cytoplasmic ß-catenin expression was an independent factor indicating an unfavourable prognosis, with a four-fold higher risk of cancer-specific death (relative risk 4.017; 95% confidence interval 2.489-6.482; P < 0.001). The median survival time for patients with tumours showing cytoplasmic accumulation of ß-catenin was 48 months, whereas the overall survival time was 166 months. CONCLUSIONS: Cytoplasmic ß-catenin expression is an independent prognostic factor for conventional RCC, and may help to identify patients with a high risk of cancer-specific death and to direct optimized active surveillance or adjuvant therapy.

Absence of Canonical WNT Signaling in Adult Renal Cell Tumors of Embryonal Origin.

BACKGROUND/AIM: The canonical ß-catenin pathway is involved in the development of Wilms' tumor, but its role in adult renal cell tumors (RCT) of embryonal origin is not yet known. MATERIALS AND METHODS: We sequenced the catenin beta 1 (CTNNB1) gene in papillary RCTs, applied the TOPflash/FOPflash reporter plasmid system on cell lines, and examined the ß-catenin protein expression by immunohistochemistry. RESULTS: The absence of mutations in CTNNB1 and low TOPflash/FOPflash ratio in tumor cell lines indicated the absence of active Wingless-type MMTV integration site family (WNT) signaling in RCTs. The weakly cytoplasmic tending towards membranous expression of ß-catenin in RCT is analogous to cellular differentiation in the embryonal kidney rather than tumorigenic activation of WNT signaling. CONCLUSION: The localization of ß-catenin in papillary RCT, metanephric adenoma and mucinous tubular and spindle-cell carcinoma corresponds to that of emerging tubules of kidney at distinct stage of maturation, indicating their embryonal origin.

Examination of PACAP-Like Immunoreactivity in Urogenital Tumor Samples.

Numerous studies investigated the localization of pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptors in different tumors and described the effects of analogs on tumor growth to show its potential role in oncogenesis. Recently, our research group has found significantly lower levels of PACAP27-like immunorreactivity (LI) and PACAP38-LI in different human samples of primary small cell lung cancer and colon cancer compared to normal healthy tissues. There are only few human studies showing the presence of PACAP and its receptors in urogenital tumors; therefore, the aim of the present study was to compare PACAP-LI in different healthy and pathological human samples from urogenital organs (kidney, urinary bladder, prostate, testis) with radioimmunoassay (RIA) method. Similar to our earlier observations, the PACAP27-LI was significantly lower compared to PACAP38-LI in all samples. We did not find significant alterations in PACAP-LI between healthy and tumoral samples from the urinary bladder and testis. On the other hand, we found significantly lower PACAP38-LI level in kidney tumors compared with healthy tissue samples, and we showed higher PACAP27-LI in prostatic cancer compared to samples from benign prostatic hyperplasia. These data indicate that PACAP levels of different tissue samples are altered under pathological conditions suggesting a potential role of PACAP in the development of different urogenital tumors.

Sciellin is a marker for papillary renal cell tumours.

There are no adequate immunohistochemical markers for papillary renal cell tumours. The aim of this study was to establish a gene expression profile of papillary renal cell tumours using an expression microarray approach. Through hierarchical clustering and significant analysis of microarrays, we have selected the best 13 genes and analysed their expression by real-time polymerase chain reaction (RT-PCR). Of these genes, we selected SCEL as potential marker of interest. Immunohistochemical staining of tissue microarrays containing all major types of kidney cancers revealed positive staining for sciellin in 87 of 114 papillary renal cell tumours and in 13 of 19 precursor lesions. No other renal tumour types were positive for sciellin. Our study indicates that although not all tumours express sciellin, its expression may help to confirm the diagnosis papillary renal cell tumour.

Effects of angiotensin II and AIII microinjections into the zona incerta after intra- and extracellular fluid loss.

Our recent results showed that angiotensin II or III (AII, AIII) microinjected into the zona incerta (ZI) significantly increased water intake. The most effective doses of AII and AIII were also defined. The two neuropeptides had their effects differently on drinking via different receptors. AII bound to AT(1) that was blocked by AT(1) receptor antagonist Losartan and the effect of AIII was eliminated by prior application of AT(2) receptor antagonist PD 123319. After different hydrational challenges, the effects of AII and AIII in the ZI have never been experimented, however. In the present experiments, the previously defined effective doses of AII (100 ng) or AIII (200 ng) were microinjected into the ZI after different types of challenges: (1). lowered thirst motivation when animals ingested approximately 40% of their daily fluid need during the consequent 60-min-daily-drinking period before the injection, (2). 48-h water deprivation, (3). intracellular dehydration and (4). extracellular dehydration. In all of the cases, incertally injected AII increased the animals' water ingestion. While Losartan could block these effects, PD 123319 was ineffective. Experiments were repeated by AIII, but in none of the cases differences were experienced between the groups. The finding that following hydrational challenges water intake increased only after AII injections and it could be blocked only by Losartan suggests that AII and AT(1) receptor play a pivotal role in the ZI in maintaining the body water balance.