Marcus Bettstetter

Smoking and cancer-related gene expression in bronchial epithelium and non-small-cell lung cancers.

Tobacco smoking is the leading cause of lung cancer worldwide. Gene expression in surgically resected and microdissected samples of non‐small‐cell lung cancers (18 squamous cell carcinomas and nine adenocarcinomas), matched normal bronchial epithelium, and peripheral lung tissue from both smokers (n = 22) and non‐smokers (n = 5) was studied using the Affymetrix U133A array. A subset of 15 differentially regulated genes was validated by real‐time PCR or immunohistochemistry. Hierarchical cluster analysis clearly distinguished between benign and malignant tissue and between squamous cell carcinomas and adenocarcinomas. The bronchial epithelium and adenocarcinomas could be divided into the two subgroups of smokers and non‐smokers. By comparison of the gene expression profiles in the bronchial epithelium of non‐smokers, smokers, and matched cancer tissues, it was possible to identify a signature of 23 differentially expressed genes, which might reflect early cigarette smoke‐induced and cancer‐relevant molecular lesions in the central bronchial epithelium of smokers. Ten of these genes are involved in xenobiotic metabolism and redox stress (eg AKR1B10, AKR1C1, and MT1K). One gene is a tumour suppressor gene (HLF); two genes act as oncogenes (FGFR3 and LMO3); two genes are involved in matrix degradation (MMP12 and PTHLH); three genes are related to cell differentiation (SPRR1B, RTN1, and MUC7); and five genes have not been well characterized to date. By comparison of the tobacco‐exposed peripheral alveolar lung tissue of smokers with non‐smokers and with adenocarcinomas from smokers, it was possible to identify a signature of 27 other differentially expressed genes. These genes are involved in the metabolism of xenobiotics (eg GPX2 and FMO3) and may represent cigarette smoke‐induced, cancer‐related molecular targets that may be utilized to identify smokers with increased risk for lung cancer. Copyright

AFV1, a novel virus infecting hyperthermophilic archaea of the genus acidianus.

We describe a novel virus, AFV1, of the hyperthermophilic archaeal genus Acidianus. Filamentous virions are covered with a lipid envelope and contain at least five different proteins with molecular masses in the range of 23-130 kDa and a 20.8-kb-long linear double-stranded DNA. The virus has been assigned to the family Lipothrixviridae on the basis of morphotypic characteristics. Host range is confined to several strains of Acidianus and the virus persists in its hosts in a stable carrier state. The latent period of virus infection is about 4 h. Viral DNA was sequenced and sequence similarities were found to the lipothrixvirus SIFV, the rudiviruses SIRV1 and SIRV2, as well as to conjugative plasmids and chromosomes of the genus Sulfolobus. Exceptionally for the linear genomes of archaeal viruses, many short direct repeats, with the sequence TTGTT or close variants thereof, are closely clustered over 300 bp at each end of the genome. They are reminiscent of the telomeric ends of linear eukaryal chromosomes.

Remarkable morphological diversity of viruses and virus-like particles in hot terrestrial environments.

Summary. Electron microscopic studies of the viruses in two hot springs (85 °C, pH 1.5–2.0, and 75–93 °C, pH 6.5) in Yellowstone National Park revealed particles with twelve different morphotypes. This diversity encompassed known viruses of hyperthermophilic archaea, filamentous Lipothrixviridae, rod-shaped Rudiviridae, and spindle-shaped Fuselloviridae, and novel morphotypes previously not observed in nature. Two virus types resembled head-and-tail bacteriophages from the families Siphoviridae and Podoviridae, and constituted the first observation of these viruses in a hydrothermal environment. Viral hosts in the acidic spring were members of the hyperthermophilic archaeal genus Acidianus.

Distinction of hereditary nonpolyposis colorectal cancer and sporadic microsatellite-unstable colorectal cancer through quantification of MLH1 methylation by real-time PCR.

Purpose: Promoter hypermethylation occurs frequently in tumors and leads to silencing of tumor-relevant genes like tumor suppressor genes. In a subset of sporadic colorectal cancers (CRC), inactivation of the mismatch repair gene MLH1 due to promoter methylation causes high level of microsatellite instability (MSI-H). MSI-H is also a hallmark of hereditary nonpolyposis colorectal cancer (HNPCC) in which mismatch repair inactivation results from germ-line mutations. For differentiation of sporadic and hereditary MSI-H tumor patients, MLH1 promoter methylation analysis is a promising tool but is not yet used in daily diagnostics because only qualitative techniques without standardization are available. The aim of this study is to establish a reliable and quantitative MLH1 methylation analysis technique and to define valid MLH1 methylation cutoff values for HNPCC diagnostics. Experimental Design: We developed a new real-time PCR–based technique to detect and quantify methylation of both proximal and distal hMLH1 promoter regions. We established and validated this technique in a cohort of 108 CRCs [94 MSI-H and 16 microsatellite stable (MSS) cases] comprising a reference (n = 58) and a tester tumor group (n = 50). Results: The reference tumor group contained 28 HNPCC with proven germ-line mutations or positive Amsterdam I criteria (median age, 37 years) and loss of MLH1 expression, 14 sporadic MSI-H CRC tumors with loss of MLH1 expression and BRAF V600E mutation (median age, 80.5 years), and 16 sporadic MSS CRC (median age, 76.5 years). No MLH1 promoter methylation could be found in any MSS tumors. HNPCC patients showed no or low level of MLH1 promoter methylation. A cutoff value of 18% methylation extent could be determined in this study to define MLH1 hypermethylation specific for sporadic MSI-H cases. Methylation could also be verified qualitatively by melting point analysis. BRAF V600E mutations were not detected in any HNPCC patients (n = 22 informative cases). Conclusion: According to the present data, quantitative MLH1 methylation analysis in MSI-H CRC is a valuable molecular tool to distinguish between HNPCC and sporadic MSI-H CRC. The detection of a BRAF V600E mutation further supports the exclusion of HNPCC.

Elevated nuclear maspin expression is associated with microsatellite instability and high tumour grade in colorectal cancer.

Maspin, a member of the serpin family, has been reported to suppress metastasis and angiogenesis in breast and prostate cancers. Overexpression of maspin was associated with adverse prognostic features in several other tumours. In this study, expression of maspin was analysed in 41 colorectal carcinomas with high‐frequency microsatellite instability (MSI‐H) and 159 microsatellite stable colorectal cancers (MSS/MSI‐L) by immunohistochemistry (IHC) and partly by relative quantitative real‐time RT‐PCR and western blot analyses. Significant upregulation of maspin expression was found in MSI‐H tumours compared to both MSS/MSI‐L tumours and matched benign colonic mucosa. Increased maspin expression was also found in three MSI‐H colon cancer cell lines, but not in three MSS colon cancer cell lines by RT‐PCR and western blot analyses. Regulation of maspin expression depended on promoter methylation as tissue specimens and cell lines expressing maspin showed unmethylated maspin promoters, whereas promoter hypermethylation was found in specimens with loss of maspin expression. Intense nuclear maspin immunostaining was seen specifically in MSI‐H tumours (p = 0.013), de‐differentiated tumours (p = 0.006), and at the invasion front. These findings provide new insights into the role of maspin in colorectal cancer progression and may be useful for diagnosis and treatment strategies. Copyright

Intraductal papillary neoplasms of the bile duct: stepwise progression to carcinoma involves common molecular pathways

Intraductal papillary neoplasms of the bile duct are still poorly characterized regarding (1) their molecular alterations during the development to invasive carcinomas, (2) their subtype stratification and (3) their biological behavior. We performed a multicenter study that analyzed these issues in a large European cohort. Intraductal papillary neoplasms of the bile duct from 45 patients were graded and subtyped using mucin markers and CDX2. In addition, tumors were analyzed for common oncogenic pathways, and the findings were correlated with subtype and grade. Data were compared with those from 22 extra- and intrahepatic cholangiocarcinomas. Intraductal papillary neoplasms showed a development from preinvasive low- to high-grade intraepithelial neoplasia to invasive carcinoma. Molecular and immunohistochemical analysis revealed mutated KRAS, overexpression of TP53 and loss of p16 in low-grade intraepithelial neoplasia, whereas loss of SMAD4 was found in late phases of tumor development. Alterations of HER2, EGFR, β-catenin and GNAS were rare events. Among the subtypes, pancreato-biliary (36%) and intestinal (29%) were the most common, followed by gastric (18%) and oncocytic (13%) subtypes. Patients with intraductal papillary neoplasm of the bile duct showed a slightly better overall survival than patients with cholangiocarcinoma (hazard ratio (cholangiocarcinoma versus intraductal papillary neoplasm of the bile duct): 1.40; 95% confidence interval: 0.46–4.30; P=0.552). The development of biliary intraductal papillary neoplasms of the bile duct follows an adenoma-carcinoma sequence that correlates with the stepwise activation of common oncogenic pathways. Further large trials are needed to investigate and verify the finding of a better prognosis of intraductal papillary neoplasms compared with conventional cholangiocarcinoma.

Nuclear Maspin expression is associated with response to adjuvant 5-fluorouracil based chemotherapy in patients with stage III colon cancer.

Maspin, a member of the Serpin protease inhibitor family, is overexpressed in poorly differentiated colorectal tumors and more frequently found in tumors with microsatellite instability. Immunohistochemical nuclear Maspin staining is predominantly seen in tumor cells at the invasion front of such cancers, suggesting that this molecule is associated with local tumor cell infiltration and aggressiveness. In a retrospective study, we studied nuclear Maspin expression as a potential prognostic tool in a total of 172 primary stage III colon cancers by immunohistochemistry. Of those 172 patients, 76 were treated by surgery only, and 96 patients received additional adjuvant 5‐fluorouracil (5‐FU) based chemotherapy. Nuclear Maspin expression was an independent adverse prognostic factor for overall survival in our patient cohort (hazard ratio 2.08; 95% CI, 1.13–3.81; p = 0.018). However, patients with primary tumors expressing Maspin in the nucleus showed a significant treatment benefit from 5‐FU chemotherapy (hazard ratio 0.384; 95% CI, 0.188–0.784; p = 0.009) compared to adjuvantly treated patients whose tumors did not express this molecule. Nuclear Maspin expression is highly predictive of 5‐FU chemotherapy response in patients with advanced stage colon cancer. Patients with negative immunhistochemical Maspin expression do not benefit from 5‐FU treatment and may be candidates for an alternative (non‐5‐FU based) adjuvant therapy regime.

Expression of the MAP kinase phosphatase DUSP4 is associated with microsatellite instability in colorectal cancer (CRC) and causes increased cell proliferation

DUSP4 (MKP‐2), a member of the mitogen‐activated protein kinase phosphatase (MKP) family and potential tumor suppressor, negatively regulates the MAPKs (mitogen‐activated protein kinases) ERK, p38 and JNK. MAPKs play a crucial role in cancer development and progression. Previously, using microarray analyses we found a conspicuously frequent overexpression of DUSP4 in colorectal cancer (CRC) with high frequent microsatellite instability (MSI‐H) compared to microsatellite stable (MSS) CRC. Here we studied DUSP4 expression on mRNA level in 38 CRC (19 MSI‐H and 19 MSS) compared to matched normal tissue as well as in CRC cell lines by RT‐qPCR. DUSP4 was overexpressed in all 19 MSI‐H tumors and in 14 MSS tumors. Median expression levels in MSI‐H tumors were significantly higher than in MSS‐tumors (p < 0.001). Consistently, MSI‐H CRC cell lines showed 6.8‐fold higher DUSP4 mRNA levels than MSS cell lines. DUSP4 expression was not regulated by promoter methylation since no methylation was found by quantitative methylation analysis of DUSP4 promoter in CRC cell lines neither in tumor samples. Furthermore, no DUSP4 mutation was found on genomic DNA level in four CRC cell lines. DUSP4 overexpression in CRC cell lines through DUSP4 transfection caused upregulated expression of MAPK targets CDC25A, CCND1, EGR1, FOS, MYC and CDKN1A in HCT116 as well as downregulation of mismatch repair gene MSH2 in SW480. Furthermore, DUSP4 overexpression led to increased proliferation in CRC cell lines. Our findings suggest that DUSP4 acts as an important regulator of cell growth within the MAPK pathway and causes enhanced cell growth in MSI‐H CRC.

Epidermal growth factor receptor, phosphatidylinositol-3-kinase catalytic subunit/PTEN, and KRAS/NRAS/BRAF in primary resected esophageal adenocarcinomas: loss of PTEN is associated with worse clinical outcome.

Alterations of the epidermal growth factor receptor (EGFR) can be observed in a significant subset of esophageal adenocarcinomas (EACs), and targeted therapy against EGFR may become an interesting approach for the treatment of these tumors. Mutations of KRAS, NRAS, BRAF, and phosphatidylinositol-3-kinase catalytic subunit (PIK3CA) and deregulation of PTEN expression influence the responsiveness against anti-EGFR therapy in colorectal carcinomas. We investigated the prevalence of these events in a collection of 117 primary resected EACs, correlated the findings with EGFR expression and amplification, and determined their clinicopathologic impact. KRAS mutations were detected in 4 (3%) of 117 tumors (3× G12D and 1 G12V mutation). One tumor had a PIK3CA E545K mutation. Neither NRAS nor BRAF mutations were detected. Sixteen (14%) of 117 cases were negative for PTEN expression, determined by immunohistochemistry. Loss of PTEN was observed predominantly in advanced tumor stages (P = .004). There was no association between PTEN and EGFR status. Loss of PTEN was associated with shorter overall and disease-free survival (P < .001 each) and also an independent prognostic factor in multivariate analysis (P = .015). EGFR status had no prognostic impact in this case collection. In summary, loss of PTEN can be detected in a significant subset of EAC and is associated with an aggressive phenotype. Therefore, PTEN may be useful as a prognostic biomarker. In contrast, mutations of RAS/RAF/PIK3CA appear only very rarely, if at all, in EAC. A possible predictive role of PTEN in anti-EGFR treatment warrants further investigations, whereas determination of RAS/RAF/PIK3CA mutations may only have a minor impact in this context.

MethyQESD, a robust and fast method for quantitative methylation analyses in HNPCC diagnostics using formalin-fixed and paraffin-embedded tissue samples.

Promoter hypermethylation occurs in various tumors and leads to silencing of tumor-relevant genes. Thus, promoter methylation analysis (MA) has been established as an important tool in cancer research and diagnostics. Here we present MethyQESD (methylation-quantification of endonuclease-resistant DNA) as a fast, easy, precise and reliable method for quantitative MA without the need of bisulfite-treatment or fluorescent probes. Though MethyQESD principally works with any gene promoter we established MethyQESD for the mismatch repair gene MLH1 and tested its utility to differentiate between sporadic microsatellite unstable (MSI-H) colorectal cancer and hereditary nonpolyposis colorectal cancer (HNPCC) by quantitative MLH1 MA. We investigated formalin-fixed and paraffin-embedded tissue samples from a previously published, well-characterized tumor collective comprising 25 HNPCC, 14 sporadic MSI-H CRC and 16 sporadic microsatellite stable (MSS) CRC. We found a high accuracy of MethyQESD by spiking experiments with dilution series of methylated (SW48 cancer cell line) and unmethylated (blood) DNA (Pearsons r=0.9997 (proximal MLH1 promoter region), r=0.9976 (distal MLH1 promoter region)). MethyQESD and conventional quantitative MA using of 96 formalin-fixed and paraffin-embedded CRC showed a high degree of concordance of both methods (Pearsons r=0.885). HNPCC tumors showed either null MLH1 methylation or a significantly lower degree of MLH1 methylation than sporadic MSI-H CRC (P<0.001). MLH1 methylation was negative in all MSS tumors. Receiver operating characteristic (ROC) curve analyses defined a cutoff value of 16.5% MLH1 methylation for specific and sensitive identification of sporadic MSI-H CRC (area under ROC curve: 1.000; asymptotic significance: P<0.001). Thus, quantitative MLH1 MA by MethyQESD provides a simple, fast and valuable tool to identify HNPCC candidates. Furthermore, MethyQESD works reliably with formalin-fixed paraffin-embedded tissue and simplifies DNA MA both for research and diagnostic purposes.