Prevalence of rare F5 variants in general population from Bosnia and Herzegovina.

Human gene F5, encoding coagulation factor V, was previously reported to be highly polymorphic. Apart from FV Leiden, several other rare variants have been detected in clinical practice and associated with thrombotic events, especially in cases when patient's phenotype and FV Leiden genotype were not in agreement. In this study, the prevalence of 17 rare F5 variants has been studied on a sample of 130 healthy adult individuals from the general Bosnian-Herzegovinian population. DNA was isolated from buccal swab samples, while genotyping was performed using MALDI-TOF MS method. The results have shown that Asp2194Gly and Met2120Thr are polymorphic in the study population with minor allele frequencies of 0.077 and 0.073, respectively. Additionally, these two variants were mutually exclusive with FV Leiden and none of them was positively associated with participants' family history of cardiovascular or cerebrovascular diseases. While the obtained results are in agreement with previously reported data for the general Caucasian populations, it is worth noting that only two rare F5 variants were detected in the study population, albeit at considerable frequencies. Still, scientific information on rare F5 variants is rather scarce and further studies aiming to assess functional importance of these variants, as well as their role as prothrombotic factors are necessary.

Population study of thrombophilic markers and pharmacogenetic markers of warfarin prevalence in Bosnia and Herzegovina.

AIM: To investigate the prevalence of common genetic variants that can serve as markers of thrombophilia and warfarin pharmacogenetics in Bosnia and Herzegovina. METHODS: The study was performed between August and October 2017 on 130 healthy unrelated adult volunteers from Bosnian-Herzegovinian population sample. The prevalence of the following genetic variants was determined: F5 c.1601G>A (factor V Leiden), F2 c.*97G>A (factor II or prothrombin mutation), F13A1 (factor XIII) c.103G>T, MTHFR (methylenetetrahydrofolate reductase) c.665C>T and c.1286A>C, as well as PAI-1 (plasminogen activator inhibitor 1) c.-816A>G and c.-844G>A as markers of thrombophilia risk, and *2 and *3 alleles of CYP2C9 (cytochrome P450 2C9) and five variants of VKORC1 (vitamin K epoxide reductase complex subunit 1) as markers of warfarin pharmacogenetics. DNA was isolated from buccal swabs using salting out method, while genotyping was performed using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. RESULTS: Minor allele frequencies for two main thrombophilia risk factors, F5 c.1601G>A and F2 c.*97G>A were 0.023 and 0.008, respectively. Combined data for the markers of warfarin pharmacogenetics imply that 57.4% study participants can be expected to metabolize warfarin at an extensive, 40.3% at intermediate, and 2.3% at a poor rate. CONCLUSION: This study reports the first extensive population genetic data for thrombophilia and warfarin pharmacogenetic markers in Bosnia and Herzegovina. Allele frequencies of genetic variants are within the general average for European populations, and their presence implies the necessity of introduction of personalized medicine in warfarin-mediated antithrombotic therapy.

Cystic fibrosis presentation in del. F508 and p. Tyr109Glyfs compound heterozygote CFTR state: a case report.

The diagnosis of cystic fibrosis (CF) is commonly confirmed by molecular genetics with the presence of specific mutations of cystic fibrosis transmembrane conductance regulator (CFTR) gene. We report a case of cystic fibrosis (CF) in a 15-year-old female patient who is a compound heterozygote for CFTR gene, with delta F508 and Tyr109Glyfs mutations detected. This is the first detailed description of such a case in the medical literature. The primary CF presentation occurred at the age of 9 in the form of gastrointestinal symptoms including greasy, bulky, and foul-smelling stool. The patient exhibited delayed growth, with her height and weight being below the 5th centile for age according to the World Health Organization growth curves. Pancreatic enzyme supplement treatment was started immediately, alongside high-fat and high-calorie diet, resulting in patient's recovery and development. DNA analysis of CFTR gene demonstrated the presence of del. F508 mutation and a rare combining deletion and insertion mutation p. Tyr109Glyfs. The combination of the two mutations is very rare in CF patients and is therefore valuable to document this case in order to provide information on disease progression, therapy options, and outcomes. With standard treatment and early diagnosis, the patient is currently doing well and is not restricted by the disease in her daily and sports activities.

Evaluating Guideline Adherence for T1 Bladder Cancer Treatment and Surveillance: A Retrospective German Multicenter Observation.

INTRODUCTION: To mirror guideline-adherence for pT1 bladder cancer treatment in Northern Germany. MATERIALS AND METHODS: Overall, 111 patients with pT1 diagnosis were treated at 4 institutions. Guideline-adherence was defined as repeat resection, instillation, and quarterly cystoscopy. Patient characteristics and pathological parameters were assessed. We summarized patients using descriptive analyses and evaluated guideline-adherence within selected subgroups. We created a multivariable model to identify predictors of guideline-adherence. RESULTS: Median age was 75 years (range 39-94 years), multifocal tumors were found in 44.1%, early instillation was performed in 33.3%, and repeat resection was performed in 77.5%. Of 62.2% who underwent instillation, 59.4% received BCG, while 40.6% received Mitomycin C or other agents. Cystoscopic follow-up was performed in 81.8%. Guideline-adherence was met in 56.8%. Patients aged below the median met adherence metrics more often compared to those above the median (66.7 vs. 46.3%; p = 0.030). Men more frequently met adherence metrics compared to women (62.1 vs. 37.5%; p = 0.038). More patients with multifocal tumors met all 3 adherence metrics (69.4 vs. 48.0%; p = 0.050), as compared to those with unifocal lesions. In multivariable analyses, age-adjusted comorbidity (OR 0.75; 95% CI 0.59-0.94; p = 0.011) and multifocality (OR 2.62; 95% CI 1.09-6.27; p = 0.031) were predictors of guideline-adherence. CONCLUSIONS: We found non-adherence in more than one-third of patients and disparities among patients of different age and according to tumor focality. Larger samples and prospective studies are needed to delineate and eradicate treatment disadvantages in these high-risk patients.

PTEN deletions are related to disease progression and unfavourable prognosis in early bladder cancer.

AIMS: This study aimed to determine the prevalence and clinical significance of deletions of the tumour suppressor gene PTEN in bladder cancer. METHODS AND RESULTS: A tissue microarray with 686 bladder cancers was analysed for PTEN deletions by fluorescence in-situ hybridization. PTEN mutations were analysed in nine tumours with heterozygous PTEN deletion. Heterozygous PTEN deletions were present in 16.5% of tumours and were associated with grade (P = 0.0024) and p53 status (P = 0.0141), but not linked to stage (P = 0.0965). PTEN deletions were seen in 5.8% of pTaG1, 10.9% of pTaG2, 29.0% of pTaG3, 16.7% of pT1G2, 22.2% of pT1G3, 17.7% of pT2-4G2 and 20.9% of pT2-4G3 tumours (P = 0.0235). PTEN deletions were associated significantly with recurrences in pTa tumours (P = 0.0173), progression in pT1 tumours (P = 0.0016), but not with overall or cancer-specific survival in pT2 tumours. Multivariate analyses including grade and PTEN deletions revealed that PTEN deletions but not grade were associated independently with recurrence in pTa tumours (P = 0.0377) and progression in pT1 tumours (P = 0.0030). No inactivating PTEN mutations were found. CONCLUSIONS: PTEN is linked to aggressive tumour phenotype and to unfavourable outcome in early bladder cancer. Heterozygous PTEN loss, i.e. reduced PTEN gene dosage, might be sufficient to cause aggressive tumour behaviour in bladder cancer cells.

Sequence analysis of CYP21A1P in a German population to aid in the molecular biological diagnosis of congenital adrenal hyperplasia.

BACKGROUND: The high homology between the CYP21A2 (cytochrome P450, family 21, subfamily A, polypeptide 2) and CYP21A1P (cytochrome P450, family 21, subfamily A, polypeptide 1 pseudogene) genes is the major obstacle to risk-free genetic diagnosis of congenital adrenal hyperplasia, especially regarding the quantification of gene dosage. Because of the lack of a comprehensive study providing useful information about the detailed genetic structure of CYP21A1P, we used a large data set to analyze and characterize this pseudogene. METHODS: We amplified and directly sequenced the CYP21A1P and CYP21A2 genes of 200 unrelated individuals. The resulting sequence data were aligned against the manually curated transcript ENST0000448314 from Havana/Vega matching to the genebuild ENSG00000198457; all differences were documented. Copy number was measured by multiplex ligation-dependent probe amplification when necessary. RESULTS: We found that 40 potentially variable positions in CYP21A2 were conserved in CYP21A1P in all study participants. In addition, we detected 14 CYP21A1P variants that were not previously reported in either CYP21A2 or CYP21A1P. Unlike CYP21A2, CYP21A1P possessed certain mutation haplotypes. CONCLUSIONS: The genetic structure of CYP21A1P and the potential risks of false conclusions it may introduce are essential considerations in designing a PCR-based diagnosis procedure for congenital adrenal hyperplasia.

Allyl-, butyl- and phenylethyl-isothiocyanate activate Nrf2 in cultured fibroblasts.

The isothiocyanate sulforaphane (SFN) has been shown to induce phase 2 and antioxidant enzymes in cultured cells and in vivo via a Nrf2 dependent signal transduction pathway. However, little is known regarding the effect of structurally related compounds such as allyl isothiocyanate (AITC), butyl isothiocyanate (BITC) and phenylethyl isothiocyanate (PEITC) on Nrf2 target gene expression. In this study AITC, BITC and PEITC significantly increased phosphorylation of ERK1/2, an upstream target of Nrf2 in NIH3T3 fibroblasts. EKR1/2 phosphorylation was accompanied by an increased nuclear translocation and transactivation of Nrf2. AITC, BITC and PEITC significantly enhanced mRNA and protein levels of the Nrf2 targets γ-glutamyl cysteine synthetase (γGCS), heme oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase (NQO1). HO-1 and γGCS both contain CpG islands within their promoter region. However, analysis of DNA methylation status in NIH3T3 cells indicated that expression of these genes may not be dependant on promoter methylation. Current data indicate that not only SFN but also other aliphatic and aromatic isothiocyanates such as AITC, BITC and PEITC induce phase 2 and antioxidant enzymes in cultured fibroblasts.

Mismatch repair deficiency occurs very rarely in seminomas.

BACKGROUND: Dense tumor-associated lymphocyte infiltration is linked to mismatch repair (MMR) deficiency in colorectal and endometrial cancer. MMR deficiency is of high clinical importance as MMR deficient cancers tend to react favorably to treatment with immune checkpoint inhibitors. Strong lymphocytic infiltration is a morphological hallmark of seminomas. We thus asked whether seminomas may exhibit MMR deficiency at relevant frequency. METHODS: To screen for tumors with MMR deficiency, protein expression of MLH1, PMS2, MSH2, and MSH6 was analyzed by immunohistochemistry (IHC) on a tissue microarray (TMA) containing 574 seminomas. RESULTS: In total, 536 cases were evaluable resulting in 481 seminomas with unequivocally intact MMR protein expression. In 55 cancers, one or several IHC stains were equivocal and lacked detectable MMR protein in both tumor and stromal cells. Large section IHC analysis of all 55 equivocal cases demonstrated substantial staining issues due to improper fixation in 54 cases and identified one tumor with clear-cut MLH1 and PMS2 protein loss. This seminoma showed homogeneous loss of MLH1 and PMS2 in the entire tumor mass whereas minor adjacent foci of associated germ cell neoplasia in situ (GCNIS) were MMR intact. Polymerase chain reaction (PCR) analysis using the 5 microsatellite loci of the "Bethesda Panel" revealed instability in 1 of 4 interpretable loci ("MSI-low") and additional instability of the complex tetra-penta repeat locus MYCL1 in this tumor. CONCLUSIONS: In summary, one single seminoma with MMR deficiency, characterized by protein loss of MLH1 and PMS2, was identified among 536 interpretable seminomas (0.19%). MMR deficiency is not a relevant determinant of lymphocyte influx in seminoma.

Tumor cell PD-L1 expression is a strong predictor of unfavorable prognosis in immune checkpoint therapy-naive clear cell renal cell cancer.

BACKGROUND: PD-L1 expression predicts response to immune checkpoint inhibitors in renal cell carcinomas (RCC), but has also been suggested to be linked to poor patient outcome. METHODS: We analyzed PD-L1 in > 1400 RCC in a tissue microarray format by immunohistochemistry. Results were compared with histological tumor type, parameters of cancer aggressiveness, and intratumoral CD8+ cytotoxic cells. RESULT: At a cut-off level of 5% PD-L1 positive tumor cells, PD-L1 positivity was seen in 6.3% of 633 clear cell RCC (ccRCC), 18.2% of 165 papillary RCC, 18.8% of 64 chromophobe RCC, and 41.7% of 103 oncocytomas. In ccRCC, PD-L1 positivity was significantly linked to high ISUP (p < 0.0001), Fuhrman (p < 0.0001), Thoenes grade (p < 0.0001), distant metastasis (p = 0.0042), short recurrence-free (p < 0.0001), and overall survival (p = 0.0002). Intratumoral CD8+ lymphocytes were more frequent in PD-L1 positive (1055 ± 109) than in PD-L1 negative ccRCC (407 ± 28; p < 0.0001). PD-L positive immune cells were seen in 8.2% of all RCC and 13.9% of papillary RCC. In ccRCC, PD-L1 positive immune cells were linked to high numbers of tumor-infiltrating CD8+ cells (p < 0.0001), high ISUP (p < 0.0001), Fuhrman (p = 0.0027), and Thoenes grade (p < 0.0001), and poor tumor-specific survival (p = 0.0280). CONCLUSIONS: These data suggest that PD-L1 expression in highly immunogenic RCCs facilitates immune evasion and contributes to cancer aggressiveness.

Association analysis of serotonin receptor 1B (HTR1B) and brain-derived neurotrophic factor gene polymorphisms in Borderline personality disorder.

The purpose of this study was to test for association between Borderline personality disorder (BPD) and variants of the HTR1B and the brain-derived neurotrophic factor (BDNF) gene. We genotyped four HTR1B and the functional BDNF G196A marker in 161 Caucasian BPD patients and 156 healthy controls. There were no significant differences between groups in genotype or haplotype distribution of HTR1B markers or in genotype distribution of the BDNF marker. Logistic regression analyses revealed an over-representation of the BDNF 196A allele in HTR1B A-161 allele carrying BPD patients.

Interaction between gene variants of the serotonin transporter promoter region (5-HTTLPR) and catechol O-methyltransferase (COMT) in borderline personality disorder.

Borderline personality disorder (BPD) is characterized by a heterogeneous symptomatology with instability in impulse control, interpersonal relationships and self-image. BPD patients display repeated self-injury, chronic suicidal tendencies and emotional dysregulation, mainly dysregulation of negative affect. In its etiology, genetic and environmental factors have been suggested. Recently, an investigation in male healthy volunteers found gene-gene effects of the catechol-O-methyl-transferase (COMT) low-activity (Met(158)) and the low-expression allele of the deletion/insertion (short/long or S/L, respectively) polymorphism in the serotonin transporter-linked promoter region (5-HTTLPR) on the central processing of aversive stimuli. The purpose of the present study was to test for association between BPD and the COMT Val(158)Met single nucleotide polymorphism (SNP), the 5-HTTLPR S/L variant and the interaction of these two gene variants. One hundred sixty one well-defined Caucasian BPD patients and 156 healthy controls were recruited from central Germany. In BPD patients, the genotype COMT Met(158)Met was over-represented compared to healthy controls (P = 0.0085; adjusted P = 0.034). We observed no differences in 5-HTTLPR genotypes between BPD and controls (P = 0.286). Additionally, the COMT Met(158)Met genotype was significantly over-represented in BPD patients carrying at least one 5-HTTLPR S allele (P = 0.0007; adjusted P = 0.028). Logistic regression analysis confirmed an interaction of the COMT Met(158) and the 5-HTTLPR S allele (P = 0.001). These data suggest an involvement of altered dopaminergic and/or noradrenergic neurotransmission as well as an interactive effect of COMT and 5-HTTLPR gene variants in the etiology of BPD, and underline the usefulness of analyses of gene-gene effects in diseases of complex inheritance with multiple genes involved.

Association analysis of SCN9A gene variants with borderline personality disorder.

Borderline personality disorder (BPD) is a serious psychiatric disorder affecting about 1-2% of the general population. Key features of BPD are emotional instability, strong impulsivity, repeated self-injurious behavior (SIB) and dissociation. In the etiology of BPD and its predominant symptoms, genetic factors have been suggested. The voltage-gated sodium channel Nav1.7 is expressed in sensory neurons and in the hippocampus, a key region of the limbic system probably dysfunctional in BPD and dissociative disorders. The alpha-subunit of Nav1.7 is encoded by the SCN9A gene on chromosome 2 and variations of SCN9A can lead to complete inability to sense pain. The aim of the present study was to test for associations between SCN9A gene variants and BPD as well as BPD-related phenotypes. We genotyped ten tagging single nucleotide polymorphisms (SNPs) within the SCN9A gene in 161 well-defined Caucasian BPD patients and 156 healthy controls. We found no globally significant association of SCN9A markers with BPD at level 5%. However, in the female and in the male subsample, different SCN9A markers and individual haplotypes showed uncorrected p-values<0.05. In addition, p-values<0.05 were observed in the analysis of associations between SCN9A markers and dissociative symptoms. Although our results were largely negative, replication studies in an independent sample are warranted to follow up on the potential role of SCN9A gene variants in BPD and dissociative symptoms, paying special attention to a possible gender different etiology.

Impact of the Ki-67 labeling index and p53 expression status on disease-free survival in pT1 urothelial carcinoma of the bladder.

BACKGROUND: The identification of protein biomarkers to guide treatment decisions regarding adjuvant therapies for high-risk non-muscle-invasive bladder cancer (NMIBC) has been of increasing interest. Evidence of the impact of tumor suppressor gene product p53 and cell proliferation marker Ki-67 on oncologic outcomes in bladder cancer patients at highest risk of recurrence and progression is partially contradictory. We sought to mirror contemporary expression patterns of p53 and Ki-67 in a select cohort of patients with pT1 bladder cancer. METHODS: Patients from four Northern German institutions with a primary diagnosis of pT1 bladder cancer between 2009 and 2016 and complete data regarding p53 or Ki-67 expression status were included for final analyses. Baseline patient characteristics (age, gender, age-adjusted Charlson comorbidity index) and tumor characteristics [diagnostic sequence, tumor focality, concomitant carcinoma in situ, 1973 World Health Organization (WHO) grading, lymphovascular invasion, adjuvant instillation therapy] were abstracted by retrospective chart review. Immunohistochemistry for detection of p53 and Ki-67 expression was performed according to standardized protocols. Microscopic analyses were performed by central pathologic review. First, we compared patients with positive vs. negative p53 expression and Ki-67 labeling index [>40% vs. ≤40%; cutoffs based on best discriminative ability in univariable Cox regression analysis with disease-free survival (DFS) as endpoint] with regard to baseline and tumor characteristics. Second, we evaluated the effect of biomarker positivity on DFS by plotting univariable Kaplan-Meier curves and performing uni- and multivariable Cox regression analyses. RESULTS: Of 102 patients with complete information on p53 status, 44 (43.1%) were p53 positive, and they more often harbored concomitant carcinoma in situ (50.0% vs. 27.6%; P=0.032) and 1973 WHO grade 3 (97.7% vs. 69.0%; P=0.001) compared to their p53 negative counterparts. Of 79 patients with complete information on Ki-67 expression status, 30 (38.0%) had a labeling index >40%. Mean Ki-67 labeling index was higher in WHO grade 3 vs. grade 2 tumors (45.8 vs. 29.7; P=0.004). At a median follow-up of 51.0 months, 31/91 patients with complete follow-up information (34.1%) suffered from disease recurrence or progression. In univariable Kaplan-Meier analyses, no difference regarding DFS was found in p53 positive vs. negative (P=0.8) or Ki-67 labeling index >40% vs. ≤40% (P=0.078) patients. In multivariable analyses, Ki-67 labeling index >40% remained an independent predictor of DFS [hazard ratio (HR), 2.66; 95% confidence interval (CI), 1.02-6.95; P=0.046], after adjusting for p53 expression and lymphovascular invasion. However, p53 status was not associated with our endpoint (P=0.8). CONCLUSIONS: While we found an association of a Ki-67 labeling index >40% and shorter DFS in pT1 bladder cancer patients, this did not hold true for p53 positivity. Future research is needed to identify additional microscopic and molecular risk factors and biomarker panels to improve risk stratification and guide adjuvant therapies in those patients.

Severe form of thyroid hormone resistance in a patient with homozygous/hemizygous mutation of T3 receptor gene.

Resistance to thyroid hormone syndrome (RTH) is a rare disorder, usually inherited as an autosomal dominant trait. Patients with RTH are usually euthyroid but can occasionally present with signs and symptoms of thyrotoxicosis or rarely with hypothyroidism. Affected individuals are usually heterozygous for mutations in the thyroid hormone receptor beta gene (TR-beta). We present a patient with RTH found to be homo-/hemizygous for a mutation in the TR-beta gene. The single nucleotide substitution I280S (1123T-->G) was present either on both alleles or in a hemizygous form with complete deletion of the second allele. The I280S mutation was recently reported in a heterozygous patient. The severe phenotype with seriously impaired intellectual development, hyperkinetic behaviour, tachycardia, hearing and visual impairment is probably due to the dominant negative effect of the I280S mutant protein and the absence of any functional TR-beta.

Expression of the cholecystokinin 2-receptor in normal human thyroid gland and medullary thyroid carcinoma.

OBJECTIVE: The cholecystokinin(2)-receptor (CCK(2)R) promotes secretion and cell growth induced by its ligands cholecystokinin (CCK) and gastrin. The receptor has recently been shown to be expressed in human medullary thyroid carcinomas (MTCs). The objective of this study was to analyze CCK(2)R expression in MTC samples of different tumor stages as well as in non-malignant thyroid tissues. DESIGN AND METHODS: Using RT-PCR we investigated 19 MTC samples and TT-cells (a human MTC cell line), as well as samples of normal thyroid. In addition, we performed immunohistochemistry using calcitonin- and CCK(2)R-specific antibodies on MTCs and samples of C-cell hyperplasia. RESULTS: We demonstrate for the first time that CCK(2)R is expressed not only in MTCs but in all samples of normal thyroid tissue. Using immunohistochemistry the receptor could be localized on calcitonin-secreting C-cells. The highest incidence of CCK(2)R expression in MTCs was observed in early-tumor stages, whereas CCK(2)R could not be detected in advanced or metastasized tumors. CONCLUSIONS: The expression of CCK(2)R in C-cells suggests a physiological function for gastrin and/or CCK in the regulation of calcitonin release, presumably related to bone and calcium metabolism. Moreover, these ligands might act as growth factors in MTCs. Efforts in the development of CCK(2)R scintigraphy for the detection of MTC lesions might have to consider a lower incidence of the receptor in advanced tumor stages.

Reduced membranous MET expression is linked to bladder cancer progression.

The MET protein is involved in the malignant progression of different tumors. This study aimed to analyze the relationship of MET expression with tumor phenotype and clinical outcome in bladder cancer and the role of gene amplification for MET overexpression. A bladder cancer tissue microarray containing 686 bladder cancers was analyzed by immunohistochemistry and by fluorescence in situ hybridization. MET immunostaining was seen in normal urothelium and was recorded in 459 of 560 analyzable urothelial carcinomas (82.0%). Low MET staining was associated with a more unfavorable tumor phenotype. MET staining was seen in 89.8% of 266 pTa, 81.1% of 132 pT1, and 69.4% of 160 pT2-4 cancers (P < 0.0001). MET staining was detectable in 92.4% of 66 grade 1, 85.6% of 257 grade 2, and 75.1% of 237 grade 3 cancers (P = 0.001). MET expression status was not associated with overall or tumor-specific survival in muscle-invasive cancers (pT2-4), tumor progression in pT1 cancers, or recurrences in pTa tumors. Only four of the analyzed tumors (0.8%) showed amplification of the MET gene. We conclude that MET is not overexpressed in urothelial cancer but rather downregulated in a fraction of cancers. Accordingly, rare amplification of the genomic area including the MET gene was not associated with MET protein overexpression.

MALDI imaging-based identification of prognostically relevant signals in bladder cancer using large-scale tissue microarrays.

OBJECTIVE: Although most patients with urinary bladder cancer present with noninvasive and low-malignant stages of the disease, about 20% eventually develop life-threatening metastatic tumors. This study was designed to evaluate the potential of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) to identify molecular markers predicting the clinical course of bladder cancer. MATERIALS AND METHODS: We employed MALDI-MSI to a bladder cancer tissue microarray including paraffin-embedded tissue samples from 697 patients with clinical follow-up data to search for prognostically relevant associations. RESULTS: Analysis of our MALDI imaging data revealed 40 signals in the mass spectra (m/z signals) associated with epithelial structures. The presence of numerous m/z signals was statistically related to one or several phenotypical findings including tumor aggressiveness (stage, grade, or nodal status; 30 signals), solid (5 signals) or papillary (3 signals) growth patterns, and increased (6 signals) or decreased (12 signals) cell proliferation, as determined by Ki-67 immunohistochemistry. Two signals were linked with tumor recurrence in noninvasive (pTa category) tumors, of which one was also related to progression from pTa-category to pT1-category disease. The absence of one m/z signal was linked with decreased survival in the subset of 102 muscle-invasive cancers. CONCLUSION: Our data demonstrate the suitability of combining MSI and large-scale tissue microarrays to simultaneously identify and validate clinically useful molecular markers in urinary bladder cancer.

Molecular epidemiology of multiple endocrine neoplasia 2: implications for RET screening in the new millenium.

OBJECTIVE: Twenty years ago, the groundbreaking discovery that rearranged during transfection (RET) mutations underlie multiple endocrine neoplasia 2 (MEN2) and familial medullary thyroid cancer (FMTC) ushered in the era of personalized medicine. MEN2-associated signs, taking time to manifest, can be subtle. This study sought to clarify to what extent conventional estimates of 1:200 000-500 000 underestimate the incidence of RET mutations in the population. DESIGN: Included in this retrospective investigation were 333 RET carriers born between 1951 and 2000 and operated on at the largest German surgical referral center (286 carriers) or elsewhere (47 carriers). METHODS: To estimate the incidence of RET mutations, the number of RET carriers born in Germany in five decades (1951-1960, 1961-1970, 1971-1980, 1981-1990, and 1991-2000) was divided by the corresponding number of German live births. RESULTS: Owing to improved diagnosis and capture of FMTC and MEN2 patients, minimum incidence estimates increased over time: overall from 5.0 (1951-1960) to 9.9 (1991-2000) per million live births and year (P=0.008), and by American Thyroid Association/ATA class from 1.7 to 3.7 for ATA class C (P=0.008); from 1.8 to 2.7 for ATA class A (P=0.017); from 1.5 to 2.2 for ATA class B (P=0.20); and from 0 to 1.4 for ATA class D mutations per million live births and year (P=0.008). Based on 1991-2000 incidence estimates the prevalence in Germany is ∼1:80 000 inhabitants. CONCLUSIONS: The molecular minimum incidence estimate of ≈1:100 000 was two- to fivefold greater than conventional estimates of 1:200 000-500 000.

Effects on transcriptional regulation and lipid droplet characteristics in the liver of female juvenile pigs after early postnatal feed restriction and refeeding are dependent on birth weight.

Epidemiological and experimental data indicate that caloric restriction in early postnatal life may improve liver lipid metabolism in low birth weight individuals. The present study investigated transcriptional and metabolic responses to low (U) and normal (N) birth weight (d 75, T1) and postnatal feed restriction (R, 60% of controls, d 98, T2) followed by subsequent refeeding until d 131 of age (T3). Liver tissue studies were performed with a total of 42 female pigs which were born by multiparous German landrace sows. Overall, 194 genes were differentially expressed in the liver of U vs. N (T1) animals with roles in lipid metabolism. The total mean area and number of lipid droplets (LD) was about 4.6- and 3.7 times higher in U compared to N. In U, the mean LD size (µm(2)) was 24.9% higher. 3-week feed restriction reduced total mean area of LDs by 58.3 and 72.7% in U and N, respectively. A functional role of the affected genes in amino acid metabolism was additionally indicated. This was reflected by a 17.0% higher arginine concentration in the liver of UR animals (vs. NR). To evaluate persistency of effects, analyses were also done after refeeding period at T3. Overall, 4 and 22 genes show persistent regulation in U and N animals after 5 weeks of refeeding, respectively. These genes are involved in e.g. processes of lipid and protein metabolism and glucose homeostasis. Moreover, the recovery of total mean LD area in U and N animals back to the previous T1 level was observed. However, when compared to controls, the mean LD size was still reduced by 23.3% in UR, whereas it was increased in NR (+24.7%). The present results suggest that short-term postnatal feed restriction period programmed juvenile U animals for an increased rate of hepatic lipolysis in later life.