Application of Genomic Sequencing to Refine Patient Stratification for Adjuvant Therapy in Renal Cell Carcinoma.

PURPOSE: Patients with resected localized clear-cell renal cell carcinoma (ccRCC) remain at variable risk of recurrence. Incorporation of biomarkers may refine risk prediction and inform adjuvant treatment decisions. We explored the role of tumor genomics in this setting, leveraging the largest cohort to date of localized ccRCC tissues subjected to targeted gene sequencing. EXPERIMENTAL DESIGN: The somatic mutation status of 12 genes was determined in 943 ccRCC cases from a multinational cohort of patients, and associations to outcomes were examined in a Discovery (n = 469) and Validation (n = 474) framework. RESULTS: Tumors containing a von-Hippel Lindau (VHL) mutation alone were associated with significantly improved outcomes in comparison with tumors containing a VHL plus additional mutations. Within the Discovery cohort, those with VHL+0, VHL+1, VHL+2, and VHL+≥3 tumors had disease-free survival (DFS) rates of 90.8%, 80.1%, 68.2%, and 50.7% respectively, at 5 years. This trend was replicated in the Validation cohort. Notably, these genomically defined groups were independent of tumor mutational burden. Amongst patients eligible for adjuvant therapy, those with a VHL+0 tumor (29%) had a 5-year DFS rate of 79.3% and could, therefore, potentially be spared further treatment. Conversely, patients with VHL+2 and VHL+≥3 tumors (32%) had equivalent DFS rates of 45.6% and 35.3%, respectively, and should be prioritized for adjuvant therapy. CONCLUSIONS: Genomic characterization of ccRCC identified biologically distinct groups of patients with divergent relapse rates. These groups account for the ∼80% of cases with VHL mutations and could be used to personalize adjuvant treatment discussions with patients as well as inform future adjuvant trial design.

The Effects of Meldonium on the Renal Acute Ischemia/Reperfusion Injury in Rats.

Acute renal ischemia/reperfusion (I/R) injury is a clinical condition that is challenging to treat. Meldonium is an anti-ischemic agent that shifts energy production from fatty acid oxidation to less oxygen-consuming glycolysis. Thus, in this study we investigated the effects of a four-week meldonium pre-treatment (300 mg/kg b.m./day) on acute renal I/R in male rats (Wistar strain). Our results showed that meldonium decreased animal body mass gain, food and water intake, and carnitine, glucose, and lactic acid kidney content. In kidneys of animals subjected to I/R, meldonium increased phosphorylation of mitogen-activated protein kinase p38 and protein kinase B, and increased the expression of nuclear factor erythroid 2-related factor 2 and haeme oxygenase 1, causing manganese superoxide dismutase expression and activity to increase, as well as lipid peroxidation, cooper-zinc superoxide dismutase, glutathione peroxidase, and glutathione reductase activities to decrease. By decreasing the kidney Bax/Bcl2 expression ratio and kidney and serum high mobility group box 1 protein content, meldonium reduced apoptotic and necrotic events in I/R, as confirmed by kidney histology. Meldonium increased adrenal noradrenaline content and serum, adrenal, hepatic, and renal ascorbic/dehydroascorbic acid ratio, which caused complex changes in renal lipidomics. Taken together, our results have confirmed that meldonium pre-treatment protects against I/R-induced oxidative stress and apoptosis/necrosis.

Impact of acute hyperglycemia on layer-specific left ventricular strain in asymptomatic diabetic patients: an analysis based on two-dimensional speckle tracking echocardiography.

BACKGROUND: Hyperglycemia has detrimental effect on ischemic myocardium, but the impact of acute hyperglycemia on the myocardium in asymptomatic diabetic patients has not been fully elucidated. Thus, this follow-up study was aimed to investigate the effects and reversibility of acute hyperglycemia on regional contractile function of left ventricle (LV) in diabetic patients without cardiovascular disease. METHODS: The two-dimensional speckle tracking echocardiography (2D-STE), including multilayer strain analysis, was used for evaluation of global and regional LV function in asymptomatic, normotensive patients with uncomplicated diabetes, with acute hyperglycemia ( ≥ 11.1 mmol/l) (Group A, n = 67), or with optimal metabolic control (fasting plasma glucose < 7 mmol/l and HbA1c < 7%) (Group B, n = 20), while 20 healthy individuals served as controls (Group C). In group A, after 72 h of i.v. continuous insulin treatment (at the time euglycemia was achieved) (second examination) and after 3 months following acute hyperglycemia (third examination) 2D-STE was repeated. RESULTS: Global longitudinal strain (GLS) (- 19.6 ± 0.4%) in Group A was significantly lower in comparison to both groups B (- 21.3 ± 0.4%; p < 0.05) and C (- 21.9 ± 0.4%; p < 0.01) at baseline, while we could not detect the differences between groups B and C. Peak systolic longitudinal endocardial (Endo), mid-myocardial (Mid) and epicardial (Epi) layer strain were significantly lower in group A at baseline compared to both groups B and C. Deterioration in peak systolic circumferential strain was observed at basal LV level, in all three layers (Endo, Mid and Epi) and in mid-cavity LV level in Epi layer in group A in comparison to group C. Moreover, in group A, after euglycemia was achieved (at second and third examination) GLS, as well as peak longitudinal and circumferential strain remain the same. CONCLUSION: Acute hyperglycemia in asymptomatic diabetic patients has significant negative effects on systolic LV myocardial mechanics primarily by reducing GLS and multilayer peak systolic longitudinal and circumferential strain which was not reversible after three months of good glycemic control.

Concomitance of Polymorphisms in Glutathione Transferase Omega Genes Is Associated with Risk of Clear Cell Renal Cell Carcinoma.

Glutathione S-transferases (GSTs), a superfamily of multifunctional enzymes, play an important role in the onset and progression of renal cell carcinoma (RCC). However, novel GST omega class (GSTO), consisting of GSTO1-1 and GSTO2-2 isoenzymes, has not been studied in RCC yet. Two coding single nucleotide polymorphisms (SNPs) supposedly affect their functions: GSTO1*C419A (rs4925) causing alanine to aspartate substitution (*A140D) and GSTO2*A424G (rs156697) causing asparagine to aspartate substitution (*N142D), and have been associated with several neurodegenerative diseases and cancers. Functional relevance of yet another GSTO2 polymorphism, identified at the 5' untranslated (5'UTR) gene region (GSTO2*A183G, rs2297235), has not been clearly discerned so far. Therefore, we aimed to assess the effect of specific GSTO1 and GSTO2 gene variants, independently and in interaction with established risk factors (smoking, obesity and hypertension) on the risk for the most aggressive RCC subtype, the clear cell RCC (ccRCC). Genotyping was performed in 239 ccRCC patients and 350 matched controls, while plasma levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG), a biomarker of oxidative DNA damage, were determined by ELISA. As a result, combined effect of all three variant genotypes exhibited almost 3-fold risk of RCC development. Additionally, this association was confirmed at the haplotype level [variant GSTO1*A/GSTO2*G (rs156697)/GSTO2*G (rs2297235) haplotype], suggesting a potential role of those variants in propensity to RCC. Regarding the gene-environment interactions, variant GSTO2*G (rs156697) homozygous smokers are at higher ccRCC risk. Association in terms of oxidative DNA damage was found for GSTO2 polymorphism in 5'UTR and 8-OHdG. In conclusion, the concomitance of GSTO polymorphisms may influence ccRCC risk.

Immunohistochemical study of cyclin A and p16 expression in patients with renal cell carcinoma.

PURPOSE: Renal cell carcinoma (RCC) is the most common malignant kidney tumor in adults. Dysregulation of the cell cycle can lead to cancer development. In this study, the mitosis-associated cyclin A and p16, a negative controller, were investigated as potential key points in the RCC development. METHODS: This retrospective study included 74 patients with RCC. The expression of cyclin A and p16 and their correlation to histopathological parameters (TNM stage, histological subtype, nuclear grade, tumor size), gender, age, and clinical outcome were studied and analyzed. RESULTS: The highest median value for cyclin A (40%; range 0-70)) and for p16 (57.5%); range 35-80) were found in the papillary histological subtype. Survival analysis showed that in the group of patients that had died before September 2015, the median value for cyclin A was 20% (range 0-60), which was significantly higher than 5% (range 0-70), found in the group of patients that survived (p=0.019). CONCLUSIONS: In relation to the histological subtype, the papillary type of RCC was associated with a significantly higher expression of cyclin A and p16 compared to other subtypes of RCC. High expression of cyclin A indicated worse prognosis, therefore cyclin A could be considered to be a significant prognostic marker.

Polymorphisms of survivin -31 G/C gene are associated with risk of urothelial carcinoma in Serbian population.

PURPOSE: Survivin is thought to play an important role in carcinogenesis and is found to be associated with poor clinical outcome in various malignancies. Gene -31 G/C polymorphism has been identified as a risk factor for the development of several types of tumors. The purpose of this study was to investigate the association between survivin gene promoter -31C/G polymorphism and urothelial carcinoma (UC) risk in Serbian population and to compare the different expressions of survivin in UC of different disease stages, histological grades and tumor location in the upper or lower urinary tract. METHODS: DNA from 94 patients with primary UC and from 82 healthy subjects was subjected to PCR restriction fragment length polymorphism analysis (PCR-RFLP) to identify individual genotypes. UC samples were subjected to immunohistochemical analysis to assess survivin expression in these lesions. RESULTS: It was observed that the frequency of G/G genotype was greater in patients with UC (58.7%) than in controls (32%). Compared with study subjects carrying the C/G or C/C genotypes, significantly increased UC risk was found for individuals carrying the G/G genotype. Those carrying the G/G genotype had a significantly increased UC risk compared with those with C/G or C/C genotypes. Patients with UC carrying the G/G genotype had a greater prevalence of muscle-invading (stage T2-T4), high-grade (G2) tumor and immunohistochemicaly overexpressed survivin compared with those carrying the C/G or C/C genotypes. CONCLUSIONS: G/G genotype of the -31C/G polymorphism might be a risk factor for UC development.

Combined GSTM1-Null, GSTT1-Active, GSTA1 Low-Activity and GSTP1-Variant Genotype Is Associated with Increased Risk of Clear Cell Renal Cell Carcinoma.

The aim of this study was to evaluate specific glutathione S-transferase (GST) gene variants as determinants of risk in patients with clear cell renal cell carcinoma (cRCC), independently or simultaneously with established RCC risk factors, as well as to discern whether phenotype changes reflect genotype-associated risk. GSTA1, GSTM1, GSTP1 and GSTT1 genotypes were determined in 199 cRCC patients and 274 matched controls. Benzo(a)pyrene diolepoxide (BPDE)-DNA adducts were determined in DNA samples obtained from cRCC patients by ELISA method. Significant association between GST genotype and risk of cRCC development was found for the GSTM1-null and GSTP1-variant genotype (p = 0.02 and p<0.001, respectively). Furthermore, 22% of all recruited cRCC patients were carriers of combined GSTM1-null, GSTT1-active, GSTA1-low activity and GSTP1-variant genotype, exhibiting 9.32-fold elevated cRCC risk compared to the reference genotype combination (p = 0.04). Significant association between GST genotype and cRCC risk in smokers was found only for the GSTP1 genotype, while GSTM1-null/GSTP1-variant/GSTA1 low-activity genotype combination was present in 94% of smokers with cRCC, increasing the risk of cRCC up to 7.57 (p = 0.02). Furthermore, cRCC smokers with GSTM1-null genotype had significantly higher concentration of BPDE-DNA adducts in comparison with GSTM1-active cRCC smokers (p = 0.05). GSTM1, GSTT1, GSTA1 and GSTP1 polymorphisms might be associated with the risk of cRCC, with special emphasis on GSTM1-null and GSTP1-variant genotypes. Combined GSTM1-null, GSTT1-active, GSTA1 low activity and GSTP1-variant genotypes might be considered as "risk-carrying genotype combination" in cRCC.

Acute Renal Failure in Different Malignant Tumors.

Acute renal failure (ARF) represents a severe complication of malignancies, that causes significant morbidity and mortality. ARF is a common part of multiple organ dysfunction in critically ill patients with cancer with reported mortality rates from 72% to 85% in patients who need renal replacement therapy. The pathways leading to ARF in cancer patients are common to the development of ARF in other conditions. However, certain factors leading to the development of ARF may be associated to the tumor or to the tumor therapy. The purpose of this review is to give specific aspects of renal disease in critically ill cancer patients (CICPs), to overview the causes of ARF in CICPs and to describe recent progress in the management of these complications, including treatment toxicity and bone marrow transplantation (BMT). The prevention of ARF is obligatory and therefore the possible treatments of ARF in CICPs are also discussed.

Cytomegalovirus induces Interleukin-6 mediated inflammatory response in salivary gland cancer.

PURPOSE: The purpose of this study was to examine whether cytomegalovirus (CMV) is present in different histological types of salivary gland cancer (SGC) by detecting CMV immediate-early (IE) and early gene products, and to determine the presence of its association with the overexpression of interleukin (IL)-6. METHODS: Immunohistochemical analysis of 92 cases of different histological types of SGC was performed to determine the presence of IL-6 and CMV antigen and its intensity in tumor tissue. Twenty samples of normal salivary gland tissue obtained during autopsy served as healthy controls. RESULTS: CMV antigens were not found in healthy acinar tissue of salivary glands, but were expressed in epithelium of salivary gland ducts. Negative expression of CMV antigens was also found in salivary gland tissue surrounding tumors. On the other hand, CMV was detected in 65/92 SGC cases (70.6%). Higher expression of IL-6 was found in SGC (70.7%) than in normal tissue (20%). There was a high association of CMV antigen presence with the presence of IL-6, and with the IL-6 expression intensity. CONCLUSIONS: Positive expression of CMV antigens in a high percentage of SGC cells suggests that it might play an important role in carcinogenesis by increasing IL-6 production and leading to inhibition of apoptosis and tumor development.

Survivin gene promoter -31 G/C polymorphism is associated with Wilms tumor susceptibility in Serbian children.

Survivin, an apoptotic inhibitor, is overexpressed in various types of cancer. Mechanisms of survivin upregulation are still poorly understood, but single nucleotide polymorphisms in the survivin gene promoter have been shown to modulate survivin expression and consequently the risk for some types of cancer. The aim of the present study was to investigate whether survivin promoter -31 G/C and -241 C/T polymorphisms could represent susceptibility factors for Wilms tumor (WT) development in Serbian population. Genotype and allele frequencies for the 2 polymorphisms in survivin promoter have been analyzed by polymerase chain reaction/restriction fragment length polymorphism in 59 WT patients and 82 controls. The frequencies of alleles and genotypes were significantly different between patients and controls for the -31 G/C polymorphism. Individuals with CC and CG genotypes had significantly decreased risk of WT compared with GG individuals (odds ratio 0.26, 95% confidence interval, 0.07-0.96; odds ratio 0.30, 95% confidence interval, 0.15-0.60). There was also a statistically significant difference in genotype frequencies between intermediate and high-risk prognostic groups (P=0.015). The -241 C/T polymorphism did not show association with WT susceptibility. Our findings suggest that the G allele at -31 survivin gene promoter position is associated with a significantly higher cancer risk in Serbian children, with a gene dosage effect.

[The significance of Goodpasture antigen in hereditary nephritis].

INTRODUCTION: Two types of hereditary nephritis, nonprogressive and progressive, clinically present as asymptomatic haematuria, sometimes combined with proteinuria. At the onset, in both types, light microscopic changes are minimal, immunofluorescence findings are negative, and diagnosis can be made only upon electron microscopic findings that are considered to be specific. OBJECTIVE: The aim of this study was to determine the significance of Goodpasture antigen detection in diagnosis of progressive and nonprogressive hereditary nephritis in its early phase. METHOD: Analysis of renal biopsy specimens was done in patients with hereditary nephritis that were followed from 1990 to 2005. Progression of renal disease was examined in 14 patients with Alport's syndrome, 10 patients with thin basement membrane disease, and 6 patients with unclassified hereditary nephritis diagnosed. For all these cases, indirect immunofluorescence study with serum from a patient with high titer of Goodpasture autoantibodies that recognize the antigenic determinants in human glomerular and tubular basement membrane was performed. RESULTS: In 11 out of 14 cases diagnosed as Alport's syndrome, there was negative staining with Goodpasture serum, and in 3 additional cases with Alport's syndrome, expression of Goodpasture antigen in glomerular basement membrane and thin basement membrane was highly reduced. In all 10 patients with thin basement membrane disease, immunofluorescence showed intensive, bright linear staining with Goodpasture serum along glomerular and tubular basement membrane. In 2 out of 6 patients with unclassified hereditary nephritis, Goodpasture antigen expression was very strong, in one patient it was very reduced, and in 3 patients it was negative. CONCLUSION: The results of our study show that Goodpasture antigen detection plays a very important role in differential diagnosis of progressive and nonpregressive hereditary nephritis, particularly in early phases of the disease.

[Polycystic kidney disease--autopsy review from the period 1987-2007].

INTRODUCTION: Polycystic kidney disease is an inherited kidney disease that affects both kidneys and it is characterized by diffuse replacement of renal parenchyma by thousands of microcysts. In time, renal insufficiency develops. There are two forms of PKD: ADPKD, which is detected in adults (children are rarely affected), and ARPK, which is detected in neonates (later presentations do occur, but rarely). OBJECTIVE: The aim of this study was to analyse frequency of polycystic kidney disease, clinical data and morphological characteristics. METHOD: At the Institute of Pathology, School of Medicine, Belgrade, there were detected 33 cases of ADPKD and 20 cases of ARPKD between 1987 and 2007. RESULTS: There were no differences between incidence of ADPKD in males and females. Average age of patients with ADPKD was 52 years. In 20 (66.7%) cases of ADPKD there were neither extrarenal cysts nor extrarenal manifestations detected. In other 13 cases, we detected extrarenal cysts: hepatic cysts in 8 cases, pancreatic cysts in 5 cases. In two cases, hepatic cysts were associated with intracranial (arachnoid cysts) and extracranial aneurysms. The most frequent cause of death in patients with ADPKD was end-stage disease. ARPKD affects more often male children compared to female. 70% of children with ARPKD were male. The mean age of patients with ARPKD was 1 month. 5 patients (40%) had hepatic fibrosis. The most frequent cause of death was respiratory insufficiency (75%). In 25% of patients, the cause of death was sepsis and renal insufficiency. CONCLUSION: Morphological and clinical manifestations of the analysed cases of both types of PKD are fairly consistent with literature data. Better knowing of aethiopathogenesis of PKD will facilitate early diagnosis, based on clinical and morphological characteristics and better management of the disease.

[Malignant diseases in medicolegal practice].

INTRODUCTION: In many cases of natural death (so-called obscure natural death), medicolegal assessment and elucidation are required. Although malignant tumors may be the cause of obscure natural death, their forensic significance has not been particularly studied in our population. OBJECTIVE: The objective of the study was to provide a general report on medicolegal importance of malignant diseases. METHOD: Autopsy material of the Institute of Forensic Medicine, Belgrade, from 1990 to 2000, was analyzed. The data were obtained from autopsy records, investigation reports, family members of the deceased and available medical documentation. RESULTS: The malignant tumor was the cause of death in 81 cases (0.69% out of a total of 11771 autopsies), in 52 males and 29 females, respectively. The incidence of malignant diseases increased with age. The most frequent cause of death was the lung cancer--diagnosed in 33 cases (40.7% out of 81), with somewhat higher percentage in females (41.4%) than in males (40.4%), but this difference was not statistically significant (chi2 = 0.13; p > 0.05). Other locations of malignancies were less frequently found: brain (7 cases), colon (6), stomach (5) and pancreas (4). In 67 cases, widespread tumor with metastases was identified as an immediate cause of death, in 8 cases there was a hemorrhage from eroded blood vessels, and in 6 cases peritonitis due to gastric or intestinal leakage into the abdominal cavity at the site of wall perforation. In 61.7% cases, fatal tumor was not diagnosed during the life, and was recorded more frequently in female group (75.9%) than in males (53.8%), but this difference was not statistically significant (chi2 = 2.71; p > 0.05). In most cases of lung cancer (22 or 66.7% out of 33), which was predominant type in the analyzed sample, malignancy remained unrecognized during the life. CONCLUSION: The most important medicolegal problems regarding fatal malignant diseases are associated with exclusion of violent death in cases that are characterized as obscure (suspicious) natural death, as well as assessment of possible legal responsibility of medical staff in cases in which malignant tumor has not been diagnosed during the life.

[Malignant diseases as suicidal motives].

INTRODUCTION: Suicide is a conscious and intentional destruction of one's own life, which occurs as a result of mutual influence of a person's disposition and motives (facts inspiring the commitment of suicide). It is well known that various diseases, including malignancies, could be important and in some cases the only motive for committing suicide. OBJECTIVE: The purpose of the study was to analyze in detail suicides of persons whose only motive was an established malignant disease. METHOD: The analysis was performed using the autopsy material of the Institute of Forensic Medicine, School of Medicine, University of Belgrade, during the period from 1990 to 2004. The reports on performed medico-legal autopsies were used, as well as history data obtained from the family members of suicidal persons, investigation reports and the available medical documents. RESULTS: In 1931 cases there was established suicidal nature of a violent death. Neoplasms were the suicidal motive in 37 persons (1.9%). The basic characteristics of the analyzed sample were predominance of males (26:11, ratio 2.4:1), the age of over 70 years and the highest incidence of malignant lung and breast tumours. Almost all cases were the persons who underwent treatment for malignant neoplasms over a longer period of time. During 19 autopsies (51.3% out of 37), a progressive phase of malignancy was established, i.e. metastases. The data on prior oral announcement of suicide intention were obtained for 70.3% (26 cases), and on previous suicidal attempts only for 13.5% (5) cases. In the majority of cases (78.4%) the place of committed suicide was the person's home. In 16 cases (43.2%) the suicide was committed with a firearm. Hanging as a manner of destroying one's own life was chosen by 12 persons (32.4%), while other ways were less frequently used. CONCLUSION: Although malignancies were not present with high incidence as a suicidal motive in our analyzed sample, such cases require particular care of health workers in order to enable the application of adequate measures in the prevention of suicides in persons with malignant diseases. The suicide analyses performed so far indicated that a malignant disease was the motive. Doctors have to show maximal interest for the patient. Prevention is crucial, so the patient should be provided with such surroundings in which the person will not feel alone and abandoned.