DNA damage response as a candidate anti-cancer barrier in early human tumorigenesis.

During the evolution of cancer, the incipient tumour experiences 'oncogenic stress', which evokes a counter-response to eliminate such hazardous cells. However, the nature of this stress remains elusive, as does the inducible anti-cancer barrier that elicits growth arrest or cell death. Here we show that in clinical specimens from different stages of human tumours of the urinary bladder, breast, lung and colon, the early precursor lesions (but not normal tissues) commonly express markers of an activated DNA damage response. These include phosphorylated kinases ATM and Chk2, and phosphorylated histone H2AX and p53. Similar checkpoint responses were induced in cultured cells upon expression of different oncogenes that deregulate DNA replication. Together with genetic analyses, including a genome-wide assessment of allelic imbalances, our data indicate that early in tumorigenesis (before genomic instability and malignant conversion), human cells activate an ATR/ATM-regulated DNA damage response network that delays or prevents cancer. Mutations compromising this checkpoint, including defects in the ATM-Chk2-p53 pathway, might allow cell proliferation, survival, increased genomic instability and tumour progression.

The association between genetic variants in hMLH1 and hMSH2 and the development of sporadic colorectal cancer in the Danish population.

BACKGROUND: Mutations in the mismatch repair genes hMLH1 and hMSH2 predispose to hereditary non-polyposis colorectal cancer (HNPCC). Genetic screening of more than 350 Danish patients with colorectal cancer (CRC) has led to the identification of several new genetic variants (e.g. missense, silent and non-coding) in hMLH1 and hMSH2. The aim of the present study was to investigate the frequency of these variants in hMLH1 and hMSH2 in Danish patients with sporadic colorectal cancer and in the healthy background population. The purpose was to reveal if any of the common variants lead to increased susceptibility to colorectal cancer. METHODS: Associations between genetic variants in hMLH1 and hMSH2 and sporadic colorectal cancer were evaluated using a case-cohort design. The genotyping was performed on DNA isolated from blood from the 380 cases with sporadic colorectal cancer and a sub-cohort of 770 individuals. The DNA samples were analyzed using Single Base Extension (SBE) Tag-arrays. A Bonferroni corrected Fisher exact test was used to test for association between the genotypes of each variant and colorectal cancer. Linkage disequilibrium (LD) was investigated using HaploView (v3.31). RESULTS: Heterozygous and homozygous changes were detected in 13 of 35 analyzed variants. Two variants showed a borderline association with colorectal cancer, whereas the remaining variants demonstrated no association. Furthermore, the genomic regions covering hMLH1 and hMSH2 displayed high linkage disequilibrium in the Danish population. Twenty-two variants were neither detected in the cases with sporadic colorectal cancer nor in the sub-cohort. Some of these rare variants have been classified either as pathogenic mutations or as neutral variants in other populations and some are unclassified Danish variants. CONCLUSION: None of the variants in hMLH1 and hMSH2 analyzed in the present study were highly associated with colorectal cancer in the Danish population. High linkage disequilibrium in the genomic regions covering hMLH1 and hMSH2, indicate that common genetic variants in the two genes in general are not involved in the development of sporadic colorectal cancer. Nevertheless, some of the rare unclassified variants in hMLH1 and hMSH2 might be involved in the development of colorectal cancer in the families where they were originally identified.

Emmprin and survivin predict response and survival following cisplatin-containing chemotherapy in patients with advanced bladder cancer.

PURPOSE: Cisplatin-containing chemotherapy is the standard of care for patients with locally advanced and metastatic transitional cell carcinoma of the urothelium. The response rate is approximately 50% and tumor-derived molecular prognostic markers are desirable for improved estimation of response and survival. EXPERIMENTAL DESIGN: Affymetrix GeneChip expression profiling was carried out using tumor material from 30 patients. A set of genes with an expression highly correlated to survival time after chemotherapy was identified. Two genes were selected for validation by immunohistochemistry in an independent material of 124 patients receiving cisplatin-containing therapy. RESULTS: Fifty-five differentially expressed genes correlated significantly to survival time. Two of the protein products (emmprin and survivin) were validated using immunohistochemistry. Multivariate analysis identified emmprin expression (hazard ratio, 2.23; P < 0.0001) and survivin expression (hazard ratio, 2.46; P < 0.0001) as independent prognostic markers for poor outcome, together with the presence of visceral metastases (hazard ratio, 2.62; P < 0.0001). In the clinical good prognostic group of patients without visceral metastases, both markers showed significant discriminating power as supplemental risk factors (P < 0.0001). Within this group of patients, the subgroups of patients with no positive, one positive, or two positive immunohistochemistry scores (emmprin and survivin) had estimated 5-year survival rates of 44.0%, 21.1%, and 0%, respectively. Response to chemotherapy could also be predicted with an odds ratio of 4.41 (95% confidence interval, 1.91-10.1) and 2.48 (95% confidence interval, 1.1-5.5) for emmprin and survivin, respectively. CONCLUSIONS: Emmprin and survivin proteins were identified as strong independent prognostic factors for response and survival after cisplatin-containing chemotherapy in patients with advanced bladder cancer.

High-density single nucleotide polymorphism array defines novel stage and location-dependent allelic imbalances in human bladder tumors.

Bladder cancer is a common disease characterized by multiple recurrences and an invasive disease course in more than 10% of patients. It is of monoclonal or oligoclonal origin and genomic instability has been shown at certain loci. We used a 10,000 single nucleotide polymorphism (SNP) array with an average of 2,700 heterozygous SNPs to detect allelic imbalances (AI) in 37 microdissected bladder tumors from 17 patients. Eight tumors represented upstaging from Ta to T1, eight from T1 to T2+, and one from Ta to T2+. The AI was strongly stage-dependent as four chromosomal arms showed AI in > 50% of Ta samples, eight in T1, and twenty-two in T2+ samples. The tumors showed stage-dependent clonality as 61.3% of AIs were reconfirmed in later T1 tumors and 84.4% in muscle-invasive tumors. Novel unstable chromosomal areas were identified at chromosomes 6q, 10p, 16q, 20p, 20q, and 22q. The tumors separated into two distinct groups, highly stable tumors (all Ta tumors) and unstable tumors (2/3 muscle-invasive). All 11 unstable tumors had lost chromosome 17p areas and 90% chromosome 8 areas affecting Netrin-1/UNC5D/MAP2K4 genes as well as others. AI was present at the TP53 locus in 10 out of 11 unstable tumors, whereas 6 had homozygous TP53 mutations. Tumor distribution pattern reflected AI as seven out of eight patients with additional upper urinary tract tumors had genomic stable bladder tumors (P < 0.05). These data show the power of high-resolution SNP arrays for defining clinically relevant AIs.

Aberrations of the Chk2 tumour suppressor in advanced urinary bladder cancer.

Checkpoint kinase 2 (Chk2) is a tumour suppressor and signal transducer in genome integrity checkpoints that coordinate cell-cycle progression with DNA repair or cell death in response to DNA damage. Defects of Chk2 occur in subsets of diverse sporadic malignancies and predispose to several types of hereditary carcinomas. However, the status of Chk2 in tumours of the urinary bladder remains unknown. Here, we report that among 58 advanced (grade T2-T4) human bladder carcinomas, immunohistochemical analysis revealed tumour-specific reduction or lack of Chk2 protein in 6 (10.3%) cases. Genetic analysis of the latter subset showed that a Chk2-negative carcinoma #668 harboured a truncating mutation 1100delC, in one Chk2 allele and loss of the corresponding second allele. The 1100delC mutation was also found in the germ line of this patient. Sequencing of TP53 in tumour #668 identified two missense mutations. Furthermore, the vast majority of the tumours showed 'unscheduled' activatory phosphorylation on Thr68 of Chk2 in the absence of any DNA-damaging treatment. Our results indicate that the otherwise dormant DNA damage signal transducer Chk2 is aberrantly and constitutively activated in invasive urinary bladder carcinomas, and that such likely proapoptotic checkpoint signalling can be disabled by inactivation of Chk2 and/or p53 tumour suppressors in subsets of these tumours.