Radiolabelled somatostatin analogue scintigraphy in oncology

Somatostatin analogue scintigraphy represents a new technique employing radiolabelled peptides to detect specific receptor-bearing lesions. 111Indium diethylenetriaminopentaacetic acid-linked octreotide (111In-DTPA-D-Phe1 octreotide), also known as [111In]pentetreotide or OctreoScan, is now established in the management of patients with neuroendocrine gastrointestinal tract and pancreatic tumours, and has proved effective in localizing disease sites in lung, breast and medullary thyroid carcinomas, lymphomas, meningiomas and others. In these conditions (a) the imaging of all disease sites at a single sitting (in a proportion of patients) thereby making further investigations unnecessary, (b) the localization of otherwise unexpected metastatic deposits and (c) the detection of residual disease not found by other means suggest that [111In]pentetreotide may be a useful adjunct in the diagnostic evaluation of patients with somatostatin receptor-bearing tumours.

Somatostatin receptor expression in lung cancer

Experimental evidence suggests that somatostatin analogues may have a role to play in the management of lung tumours. We evaluated membrane preparations of nine small cell lung cancer (SCLC) cell lines and of tumour samples from 3 patients with non-small cell lung cancer (NSCLC), 1 patient with an atypical carcinoid and another with a bronchial carcinoid for the presence of specific binding sites for RC-160, a potent growth inhibitory octapeptide analogue of somatostatin. Specific binding was noted on six of nine SCLC lines. Radio-receptor assay on the cell line NCI H 69 showed evidence of two specific binding sites for RC-160, one with high affinity and the other with low affinity. Binding sites were also found on all five tumour samples. Scatchard analysis indicated the presence of a single class of receptors with high affinity in each case. Histological assessment of the resected specimens before binding assay showed them to be comprised of tumour cells and necrotic tissue, stroma and/or inflammatory cells. Therefore, the specific binding of RC-160 may be to tissues other than the tumour cells. In 3 patients, from whom the tumour samples were obtained, radiolabelled somatostatin analogue scintigraphy using [111In] pentetreotide was performed prior to surgery. In all cases, the radiolabel localised the disease. This study demonstrates the presence of specific binding sites for RC-160 in SCLC. Furthermore, the detection of specific binding in vitro and in vivo in NSCLC and intrapulmonary carcinoids demonstrates that these tumours contain cells which express specific binding sites for somatostatin. These results suggest that RC-160 may have a role toplay as a therapeutic agent in lung cancer.

Clinical skin examination outcomes after a video-based behavioral intervention analysis from a randomized clinical trial

Importance Older men are at risk of dying of melanoma. Objective To assess attendance at and clinical outcomes of clinical skin examinations (CSEs) in older men exposed to a video-based behavioral intervention. Design, Setting, and Participants This was a behavioral randomized clinical trial of a video-based intervention in men aged at least 50 years. Between June 1 and August 31, 2008, men were recruited, completed baseline telephone interviews, and were than randomized to receive either a video-based intervention (n = 469) or brochures only (n = 461; overall response rate, 37.1%) and were again interviewed 7 months later (n = 870; 93.5% retention). Interventions Video on skin self-examination and skin awareness and written informational materials. The control group received written materials only. Main Outcomes and Measures Participants who reported a CSE were asked for the type of CSE (skin spot, partial body, or whole body), who initiated it, whether the physician noted any suspicious lesions, and, if so, how lesions were managed. Physicians completed a case report form that included the type of CSE, who initiated it, the number of suspicious lesions detected, how lesions were managed (excision, nonsurgical treatment, monitoring, or referral), and pathology reports after lesion excision or biopsy. Results Overall, 540 of 870 men (62.1%) self-reported a CSE since receiving intervention materials, and 321 of 540 (59.4%) consented for their physician to provide medical information (received for 266 of 321 [82.9%]). Attendance of any CSE was similar between groups (intervention group, 246 of 436 [56.4%]; control group, 229 of 434 [52.8%]), but men in the intervention group were more likely to self-report a whole-body CSE (154 of 436 [35.3%] vs 118 of 434 [27.2%] for control group; P = .01). Two melanomas, 29 squamous cell carcinomas, and 38 basal cell carcinomas were diagnosed, with a higher proportion of malignant lesions in the intervention group (60.0% vs 40.0% for controls; P = .03). Baseline attitudes, behaviors, and skin cancer history were associated with higher odds of CSE and skin cancer diagnosis. Conclusions and Relevance A video-based intervention may increase whole-body CSE and skin cancer diagnosis in older men. Trial Registration: anzctr.org.au Identifier: ACTRN12608000384358

Occurrence of urinary tract tumors in miners highly exposed to dinitrotoluene

Between 1984 and 1997, six cases of urothelial cancer and 14 cases of renal cell cancer occurred in a group of 500 underground mining workers in the copper-mining industry of the former German Democratic Republic, with high exposures to explosives containing technical dinitrotoluene. Exposure durations ranged from 7 to 37 years, and latency periods ranged from 21 to 46 years. The incidences of both urothelial and renal cell tumors in this group were much higher than anticipated on the basis of the cancer registers of the German Democratic Republic by factors of 4.5 and 14.3, respectively. The cancer cases and a representative group of 183 formerly dinitrotoluene- exposed miners of this local industry were interviewed for their working history and grouped into four exposure categories. This categorization of the 14 renal cell tumor cases revealed no dose-dependency concerning explosives in any of the four exposure categories and was similar to that of the representative group of employees, whereas the urothelial tumor cases were predominantly confined to the high-exposure categories. Furthermore, all identified tumor patients were genotyped by polymerase chain reaction, using lymphocyte DNA, regarding their genetic status of the polymorphic xenobiotic metabolizing enzymes, including the N-acetyltransferase 2 and the glutathione-S-transferases M1 and T1. This genotyping revealed remarkable distributions only for the urothelial tumor cases, who were exclusively identified as 'slow acetylators.' This points to the possibility of human carcinogenicity of dinitrotoluene, with regard to the urothelium as the target tissue.

Glutathione transferase activities in renal carcinomas and adjacent normal renal tissues : factors influencing renal carcinogenesis induced by xenobiotics

In general, the biological activation of nephrocarcinogenic chlorinated hydrocarbons proceeds via conjugatiton with glutathione. It has mostly been assamed that the main site of initial conjugation is the liver, followed by a mandatory transfer of intermediates to the kidney. It was therefore of interest to study the enzyme activities of subgroups of glutathione transferases (GSTs) in renal cancers and the surrounding normal renal tissues of the same individuals (n = 21). For genotyping the individuals with respect to known polymorphic GST isozymes the following substrates with differential specificity were used: 1-chloro-2,4-dinitrobenzene for overall GST activity (except GST θ); 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole for GST α; 1,2-dichloro-4-nitro-benzene for GST μ; ethacrynic acid and 4-vinylpyridine for GST π; and methyl chloride for GST θ. In general, the normal tissues were able to metabolize the test substrates. A general decrease in individual GST enzyme activities was apparent in the course of cancerization, and in some (exceptional) cases individual activities, expressed in the normal renal tissue, were lost in the tumour tissue. The GST enzyme activities in tumours were independent of tumour stage, or the age and gender of the patients. There was little influence of known polymorphisms of GSTM1, GSTM3 and GSTP1 upon the activities towards the test substrates, whereas the influence of GSTT1 polymorphism on the activity towads methyl chloride was straightforward. In general, the present findings support the concept that the initial GST-dependent bioactivation step of nephrocarcinogenic chlorinated hydrocarbons may take place in the kidney itself. This should be a consideration in toxicokinetic modelling.

Occupational and non-occupational risk factors in bladder cancer patients in an industrialized area located in former East-Germany [Berufliche und außerberufliche Risikofaktoren für das Harnblasenkarzinom in einem ehemaligen Industriegebiet der neuen Bundesländer]

Purpose: Several occupational carcinogens are metabolized by polymorphic enzymes. The distribution of the polymorphic enzymes N-acetyltransferase 2 (NAT2; substrates: aromatic amines), glutathione S-transferase M1 (GSTM1; substrates: e.g., reactive metabolites of polycyclic aromatic hydrocarbons), and glutathione S-transferase T1 (GSTT1; substrates: small molecules with 1-2 carbon atoms) were investigated. Material and Methods: At the urological department in Lutherstadt Wittenberg, 136 patients with a histologically proven transitional cell cancer of the urinary bladder were investigated for all occupations performed for more than 6 months. Several occupational and non-occupational risk factors were asked. The genotypes of NAT2, GSTM1, and GSTT1 were determined from leucocyte DNA by PCR. Results: Compared to the general population in Middle Europe, the percentage of GSTT1 negative persons (22.1 %) was ordinary; the percentage of slow acetylators (59.6%) was in the upper normal range, while the percentage of GSTM1 negative persons (58.8%) was elevated in the entire group. Shifts in the distribution of the genotypes were observed in subgroups who had been exposed to asbestos (6/6 GSTM1 negative, 5/6 slow acetylators), rubber manufacturing (8/10 GSTM1 negative), and chlorinated solvents (9/15 GSTM1 negative). Conclusions: The overrepresentation of GSTM1 negative bladder cancer patients also in this industrialized area and more pronounced in several occupationally exposed subgroups points to an impact of the GSTM1 negative genotype in bladder carcinogenesis. [Article in German]

Common variants near ATM are associated with glycemic response to metformin in type 2 diabetes

Metformin is the most commonly used pharmacological therapy for type 2 diabetes. We report a genome-wide association study for glycemic response to metformin in 1,024 Scottish individuals with type 2 diabetes with replication in two cohorts including 1,783 Scottish individuals and 1,113 individuals from the UK Prospective Diabetes Study. In a combined meta-analysis, we identified a SNP, rs11212617, associated with treatment success (n = 3,920, P = 2.9 P×-9, odds ratio = 1.35, 95% CI 1.22-1.49) at a locus containing ATM, the ataxia telangiectasia mutated gene. In a rat hepatoma cell line, inhibition of ATM with KU-55933 attenuated the phosphorylation and activation of AMP-activated protein kinase in response to metformin. We conclude that ATM, a gene known to be involved in DNA repair and cell cycle control, plays a role in the effect of metformin upstream of AMP-activated protein kinase, and variation in this gene alters glycemic response to metformin. © 2011 Nature America, Inc. All rights reserved.

Targeted delivery of hepatitis C virus-specific short hairpin RNA in mouse liver using Sendai virosomes

Internal ribosome entry site (IRES)-mediated translation of input viral RNA is the initial required step for the replication of the positive-stranded genome of hepatitis C virus (HCV). We have shown previously the importance of the GCAC sequence near the initiator AUG within the stem and loop IV (SLIV) region in mediating ribosome assembly on HCV RNA. Here, we demonstrate selective inhibition of HCV-IRES-mediated translation using short hairpin (sh)RNA targeting the same site within the HCV IRES. sh-SLIV showed significant inhibition of viral RNA replication in a human hepatocellular carcinoma (Huh7) cell line harbouring a HCV monocistronic replicon. More importantly, co-transfection of infectious HCV-H77s RNA and sh-SLIV in Huh7.5 cells successfully demonstrated a significant decrease in viral RNA in HCV cell culture. Additionally, we report, for the first time, the targeted delivery of sh-SLIV RNA into mice liver using Sendai virosomes and demonstrate selective inhibition of HCV-IRES-mediated translation. Results provide the proof of concept that Sendai virosomes could be used for the efficient delivery of shRNAs into liver tissue to block HCV replication.

Roles for Transcription Factors Sp1, NF-kappa B, IRF3, and IRF7 in Expression of the Human IFNL4 Gene

The expression of a biologically active human IFN4 depends on the presence of a frameshift deletion polymorphism within the first exon of the interferon lambda 4 (IFNL4) gene. In this report, we use the lung carcinoma-derived cell line, A549, which is genetically viable to express a functional IFN4, to address transcriptional requirements of the IFNL4 gene. We show that the GC-rich DNA-binding transcription factor (TF) specificity protein 1 (Sp1) is recruited to the IFNL4 promoter and has a role in induction of gene expression upon stimulation with viral RNA mimic poly(I:C). By using RNAi and overexpression strategies, we also show key roles in IFNL4 gene expression for the virus-inducible TFs, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-B), IFN regulatory factor 3 (IRF3), and IRF7. Interestingly, we also observe that overexpression of IFN4 influences IFNL4 promoter activity, which may further be dependent on the retinoic acid-inducible gene-I (RIG-I)-like receptor pathway. Together, our work for the first time reports on the functional characterization of the human IFNL4 promoter.

MicroRNAs in breast cancer progression and DNA damage response

Os tumores de mama são caracterizados pela sua alta heterogeneidade. O câncer de mama é uma doença complexa, que possui o seu desenvolvimento fortemente influenciado por fatores ambientais, combinada a uma progressiva acumulação de mutações genéticas e desregulação epigenética de vias críticas. Alterações nos padrões de expressão gênica podem ser resultado de uma desregulação no controle de eventos epigenéticos, assim como, na regulação pós-transcricional pelo mecanismo de RNA de interferência endógeno via microRNA (miRNA). Estes eventos são capazes de levar à iniciação, à promoção e à manutenção da carcinogênese, como também ter implicações no desenvolvimento da resistência à terapia Os miRNAs formam uma classe de RNAs não codificantes, que durante os últimos anos surgiram como um dos principais reguladores da expressão gênica, através da sua capacidade de regular negativamente a atividade de RNAs mensageiros (RNAms) portadores de uma seqüencia parcialmente complementar. A importância da regulação mediada por miRNAs foi observada pela capacidade destas moléculas em regular uma vasta gama de processos biológicos incluindo a proliferação celular, diferenciação e a apoptose. Para avaliar a expressão de miRNAs durante a progressão tumoral, utilizamos como modelo experimental a série 21T que compreende 5 linhagens celulares originárias da mesma paciente diagnosticada com um tumor primário de mama do tipo ErbB2 e uma posterior metástase pulmonar. Essa série é composta pela linhagem obtida a partir do tecido normal 16N, pelas linhagens correspondentes ao carcinoma primário 21PT e 21NT e pelas linhagens obtidas um ano após o diagnóstico inicial, a partir da efusão pleural no sítio metastatico 21MT1 e 21MT2. O miRNAoma da série 21T revelou uma redução significativa nos níveis de miR-205 e nos níveis da proteina e-caderina e um enriquecimento do fator pró-metastático ZEB-1 nas células 21MT. Considerando a importância dos miRNAs na regulação da apoptose, e que a irradiação em diferentes espectros é comumente usada em procedimentos de diagnóstico como mamografia e na radioterapia, avaliamos a expressão de miRNAs após irradiação de alta e baixa energia e do tratamento doxorrubicina. Para os ensaios foram utilizados as linhagens não tumorais MCF-10A e HB-2 e as linhagens de carcinoma da mama MCF-7 e T-47D. Observou-se que raios-X de baixa energia são capazes de promover quebras na molécula do DNA e apoptose assim como, alterar sensivelmente miRNAs envolvidos nessas vias como o let-7a, miR-34a e miR-29b. No que diz respeito à resposta a danos genotóxicos, uma regulação positiva sobre a expressão de miR-29b, o qual em condições normais é regulado negativamente foi observada uma regulação positiva sobre miR-29b expressão após todos os tratamentos em células tumorais. Nossos resultados indicam que miR-29b é um possível biomarcador de estresse genotóxico e que miR-205 pode participar no potencial metastático das células 21T.

Epithelial-mesenchymal transitions and hepatocarcinogenesis.

Epithelial-mesenchymal transitions (EMTs) are believed to play a role in invasion and metastasis of many types of tumors. In this issue of the JCI, Chen et al. show that a gene that has been associated with aggressive biology in hepatocellular carcinomas initiates a molecular cascade that results in EMT.

An active learning approach for rapid characterization of endothelial cells in human tumors.

Currently, no available pathological or molecular measures of tumor angiogenesis predict response to antiangiogenic therapies used in clinical practice. Recognizing that tumor endothelial cells (EC) and EC activation and survival signaling are the direct targets of these therapies, we sought to develop an automated platform for quantifying activity of critical signaling pathways and other biological events in EC of patient tumors by histopathology. Computer image analysis of EC in highly heterogeneous human tumors by a statistical classifier trained using examples selected by human experts performed poorly due to subjectivity and selection bias. We hypothesized that the analysis can be optimized by a more active process to aid experts in identifying informative training examples. To test this hypothesis, we incorporated a novel active learning (AL) algorithm into FARSIGHT image analysis software that aids the expert by seeking out informative examples for the operator to label. The resulting FARSIGHT-AL system identified EC with specificity and sensitivity consistently greater than 0.9 and outperformed traditional supervised classification algorithms. The system modeled individual operator preferences and generated reproducible results. Using the results of EC classification, we also quantified proliferation (Ki67) and activity in important signal transduction pathways (MAP kinase, STAT3) in immunostained human clear cell renal cell carcinoma and other tumors. FARSIGHT-AL enables characterization of EC in conventionally preserved human tumors in a more automated process suitable for testing and validating in clinical trials. The results of our study support a unique opportunity for quantifying angiogenesis in a manner that can now be tested for its ability to identify novel predictive and response biomarkers.

Inactivation of the von Hippel-Lindau tumor suppressor leads to selective expression of a human endogenous retrovirus in kidney cancer.

A human endogenous retrovirus type E (HERV-E) was recently found to be selectively expressed in most renal cell carcinomas (RCCs). Importantly, antigens derived from this provirus are immunogenic, stimulating cytotoxic T cells that kill RCC cells in vitro and in vivo. Here, we show HERV-E expression is restricted to the clear cell subtype of RCC (ccRCC) characterized by an inactivation of the von Hippel-Lindau (VHL) tumor-suppressor gene with subsequent stabilization of hypoxia-inducible transcription factors (HIFs)-1α and -2α. HERV-E expression in ccRCC linearly correlated with HIF-2α levels and could be silenced in tumor cells by either transfection of normal VHL or small interfering RNA inhibition of HIF-2α. Using chromatin immunoprecipitation, we demonstrated that HIF-2α can serve as transcriptional factor for HERV-E by binding with HIF response element (HRE) localized in the proviral 5' long terminal repeat (LTR). Remarkably, the LTR was found to be hypomethylated only in HERV-E-expressing ccRCC while other tumors and normal tissues possessed a hypermethylated LTR preventing proviral expression. Taken altogether, these findings provide the first evidence that inactivation of a tumor suppressor gene can result in aberrant proviral expression in a human tumor and give insights needed for translational research aimed at boosting human immunity against antigenic components of this HERV-E.

Cytochrome P450 1B1 expression in rat esophageal tumorigenesis promoted by gastric and duodenal reflux.

Cytochrome P450 1B1 (CYP1B1) mRNA is constitutively expressed in most normal extra-hepatic tissues; however the protein is not detectable in these tissues but is expressed in a wide variety of tumors. CYP1B1 is responsible for the activation of a number of carcinogens present in tobacco smoke and food. A surgical model of rat esophageal tumorigenesis, promoted by gastric or duodenal reflux was used to determine CYP1B1 expression in premalignant esophageal tissue. Immunohistochemistry was performed using a modified amplified fluorescein tyramide protocol. CYP1B1 was not observed in normal esophageal mucosa, submucosa, or muscularis mucosa. Animals exposed to gastric reflux developed mild hyperplasia. Varying degrees of hyperplasia were observed in the duodenal reflux group. All regions of hyperplasia showed moderate or strong CYP1B1 immunoreactivity. Duodenal reflux induced a small number of premalignant changes: immunoreactivity was absent from the epithelium of squamous dysplasia (0/10), Barrett's esophagus (0/7), and majority of dysplastic Barrett's esophagus (1/4). Moderate or strong immunoreactivity was observed in the majority (7/8) of squamous cell carcinomas (SCCs) in situ. Immunoreactivity was also observed in the lamina propria and submucosa in association with inflammation, regardless of the severity of inflammation. The expression of CYP1B1 in hyperplasia, SCCs in situ, or in association with inflammation may increase the production of carcinogenic metabolites, which may promote esophageal tumorigenesis.

Identification of a methylation hotspot in the death receptor Fas/CD95 in bladder cancer

We characterized Fas immunoreactivity, functionality and its role in the response to mitomycin-C (MMC) chemotherapy in vitro in cell lines and in vivo in bladder washings from 23 transitional cell carcinoma of the bladder (TCCB) patients, harvested prior to and during MMC intravesical treatment. Having established the importance of functional Fas, we investigated the methylation and exon 9 mutation as mechanisms of Fas silencing in TCCB. For the first time, we report p53 up-regulation in 9/14 and Fas up-regulation in 7/9 TCCB patients during intravesical MMC treatment. Fas immunoreactivity was strong in the TCCB cell line T24 and in 17/20 (85%) tumor samples from patients with advanced TCCB. T24 and HT1376 cells were resistant to MMC and recombinant Fas ligand, whilst RT4 cells were responsive to Fas ligand and MMC. Using RT4 cells as a model, siRNA targeting p53 significantly reduced MMC-induced p53 and Fas up-regulation and stable DN-FADD transfection decreased MMC-induced apoptosis, suggesting that functional Fas enhances chemotherapy responses in a p53-dependent manner. In HT1376 cells, 5-aza-2-deoxycytidine (12 µM) induced Fas immunoreactivity and reversed methylation at CpG site -548 within the Fas promoter. This site was methylated in 13/24 (54%) TCCB patient samples assessed using Methylation-Specific Polymerase Chain Reaction. There was no methylation at either the p53 enhancer region within the first intron or at the SP-1 binding region in the promoter and no mutation within exon 9 in tumor DNA extracted from 38 patients. Methylation at CpG site -548 is a potential target for demethylating drugs.