Patient-derived cell models as preclinical tools for genome-directed targeted therapy.

BACKGROUND: In this study, we established patient-derived tumor cell (PDC) models using tissues collected from patients with metastatic cancer and assessed whether these models could be used as a tool for genome-based cancer treatment. METHODS: PDCs were isolated and cultured from malignant effusions including ascites and pleural fluid. Pathological examination, immunohistochemical analysis, and genomic profiling were performed to compare the histological and genomic features of primary tumors, PDCs. An exploratory gene expression profiling assay was performed to further characterize PDCs. RESULTS: From January 2012 to May 2013, 176 samples from patients with metastatic cancer were collected. PDC models were successfully established in 130 (73.6%) samples. The median time from specimen collection to passage 1 (P1) was 3 weeks (range, 0.5-4 weeks), while that from P1 to P2 was 2.5 weeks (range, 0.5-5 weeks). Sixteen paired samples of genomic alterations were highly concordant between each primary tumor and progeny PDCs, with an average variant allele frequency (VAF) correlation of 0.878. We compared genomic profiles of the primary tumor (P0), P1 cells, P2 cells, and patient-derived xenografts (PDXs) derived from P2 cells and found that three samples (P0, P1, and P2 cells) were highly correlated (0.99-1.00). Moreover, PDXs showed more than 100 variants, with correlations of only 0.6-0.8 for the other samples. Drug responses of PDCs were reflective of the clinical response to targeted agents in selected patient PDC lines. CONCLUSION(S): Our results provided evidence that our PDC model was a promising model for preclinical experiments and closely resembled the patient tumor genome and clinical response.

Recapitulation of previous genome-wide association studies with two distinct pathophysiological entities of gastric cancer in the Korean population.

Gastric cancer (GC) is the most common malignancy. The incidence rates remain remarkably high in East Asians. Although genome-wide association studies in the Han Chinese and Japanese populations have so far yielded susceptibility loci for GC, these findings need to be validated in an independent ethnic group. To identify the potential heterogeneity by histological classified subtypes (intestinal and diffuse), we examined the previously reported associations in the Korean population. PRKAA1 at 5p13.1 was found to be more strongly associated with intestinal type (odds ratio, OR=1.39, 95% CI (confidence interval) =1.22-1.58, P=3.77 × 10(-7)) than diffuse type. In addition, PSCA at 8q23.3 was significantly replicated in diffuse type (OR=1.49, 95% CI=1.32-1.67, P=2.43 × 10(-11)) but far less significant in intestinal type. In conclusion, these findings could bring additional insights into the etiologic heterogeneity in gastric carcinogenesis mechanisms.

A functional single nucleotide polymorphism at the promoter region of cyclin A2 is associated with increased risk of colon, liver, and lung cancers.

BACKGROUND: The objective of this was to identify functional single nucleotide polymorphisms (SNPs) in cyclin-dependent kinases (CDKs) and cyclins that are associated with risk of human cancer. METHODS: First, 45 SNPs in CDKs and cyclins were analyzed in 106 lung cancers and 108 controls for a pilot study. One SNP (reference SNP [rs] 769236, +1 guanine to adenine [G→A]) at the promoter region of cyclin A2 (CCNA2) also was analyzed in 1989 cancers (300 breast cancers, 450 colorectal cancers, 450 gastric cancers, 367 hepatocellular carcinomas, and 422 lung cancers) and in 1096 controls. Genotyping was performed using matrix-assisted laser desorption-ionization/time-of-flight mass spectrometry. Transcriptional activity of the SNP according to the cell cycle was analyzed by using a luciferase reporter assay and fluorescence-activated cell sorting analysis in NIH3T3 cells. RESULTS: In the pilot study, the SNP (rs769236) was associated significantly with the risk of lung cancer. In the expanded study, multivariate logistic regression indicated that the AA homozygous variant of the SNP was associated significantly with the development of lung cancer (P < .0001; codominant model), colorectal cancer (P < .0001), and hepatocellular carcinoma (P = .02) but not with breast cancer or gastric cancer. The luciferase activity of a 300-base pair construct that contained the A allele was 1.5-fold greater than the activity of a construct with the G allele in NIH3T3 cells. The high luciferase activity of constructs that contained the A allele did not change with cell cycle progression. CONCLUSIONS: The current results suggested that an SNP (rs769236) at the promoter of CCNA2 may be associated significantly with increased risk of colon, liver, and lung cancers.

Associations between DRDs and schizophrenia in a Korean population: multi-stage association analyses.

The dysregulation of the dopaminergic system has been implicated in the pathophysiology of major psychosis, including schizophrenia, with dopamine receptor genes (DRDs) presently targeted as the most promising candidate genes. We investigated DRD1-5 for association with schizophrenia using a multi-stage approach in a Korean sample. One hundred forty-two SNPs in DRD1-5 were selected from the dbSNP, and the associations of each SNP were then screened and typed by MALDI-TOF mass spectrometry using pooled DNA samples from 150 patients with major psychosis and 150 controls. Each of the suggested SNPs was then genotyped and tested for an association within the individual samples comprising each pool. Finally, the positively associated SNPs were genotyped in an extended sample of 270 patients with schizophrenia and 350 controls. Among the 142 SNPs, 88 (62%) SNPs in our Korean population were polymorphic. At the pooling stage, 10 SNPs (DRD1: 2, DRD2: 3, and DRD4: 5) were identified (P<0.05). SNPs rs1799914 of DRD1 (P=0.046) and rs752306 of DRD4 (P=0.017) had significantly different allele frequencies in the individually genotyped samples comprising the pool. In the final stage, with the extended sample, the suggestive association of DRD4 with rs752306 was lost, but the association of DRD1 with rs1799914 gained greater significance (P=0.017). In these large-scale multi-stage analyses, we were able to find a possible association between DRD1 and schizophrenia. These findings suggested the potential contribution of a multi-step strategy for finding genes related to schizophrenia.

Candidate gene polymorphisms for diabetes mellitus, cardiovascular disease and cancer are associated with longevity in Koreans.

Long-lived people may have a unique genetic makeup that makes them more resistant than the general population to prevalent age-related diseases; however, not much is known about genes involved in the longevity. To identify susceptibility variants controlling longevity, we performed a high-throughput candidate gene study using 137 Koreans over 90 yr old and 213 young healthy Koreans. We evaluated 463 informative markers located in 176 candidate genes mostly for diabetes mellitus, cardiovascular disease and cancer under five genetic models. We estimated the odds ratios for each allele, genotype, haplotype, and gene-gene interaction using logistic regression analysis. Associations between 13 genes and longevity were detected at a P-value less than 0.01. Particularly, the rs671 (A) allele of the aldehyde dehydrogenase 2 family (mitochondrial) (ALDH2) gene was associated with longevity only in men (OR 2.11, P =0.008). Four genes, proprotein convertase subtilisin/kexin type 1 (PCSK1, P=0.008), epidermal growth factor receptor (EGFR, P=0.003), paired box 4 (PAX4, P=0.008), and V-yes-1 Yamaguchi sarcoma viral related oncogene homolog (LYN, P=0.002) consistently yielded statistical evidence for association with longevity. The findings of the current study may provide a starting point for future studies to unravel genetic factors controlling longevity in Koreans.

Association of interindividual differences in p14ARF promoter methylation with single nucleotide polymorphism in primary colorectal cancer.

BACKGROUND: CpG island hypermethylation has been reported at the promoter region of many tumor suppressor genes in colorectal cancers. However, there are significant interindividual differences in the degree of DNA methylation in colorectal cancers. The objective of the current study was to understand whether single nucleotide polymorphisms (SNPs) around the promoter of a gene are implicated in the interindividual differences of CpG island hypermethylation. METHODS: Promoter methylation of the p14(ARF) gene and messenger RNA (mRNA) expression levels of p14(ARF), DNA methyltransferase 1 (DNMT1), and DNMT3b were investigated by using methylation-specific polymerase chain reaction (PCR) analysis (MSP) and quantitative real-time PCR analysis in fresh tissues from 188 patients with colorectal cancer. SNPs around the p14(ARF) promoter were genotyped in DNA from peripheral blood lymphocytes in 300 healthy individuals and in 188 patients with colorectal cancer by using matrix-assisted laser desorption/ionization mass spectrometry. RESULTS: p14(ARF) methylation was present in 61 of 188 colorectal cancers (32%). Fourteen SNPs among the 20 candidate SNPs were identified as monomorphic in the Korean population studied. Two individual SNPs (-4256 thymine to cytosine [T-->C] and -1477 guanine to adenine [G-->A]), which were in strong linkage disequilibrium (|D'|=0.99; correlation coefficient [r(2)]=0.95), were associated significantly with p14(ARF) methylation. Patients who had the CC variant at the-4256 locus or the AA variant at the -1477 locus had 2.42 times (95% confidence interval [95% CI], 1.07-5.46; P = .03) and 2.47 times (95% CI, 1.09-5.56; P= .03) greater risk of p14(ARF) methylation than patients who had the TT or GG homozygote, respectively, after adjusting for mRNA levels of DNMTs. Four major haplotypes were identified within a block (-4256 T-->C, -3631 T-->C, -1477 G-->A, and +20,188 T-->C). p14(ARF) promoter methylation also was associated significantly with the CCAT haplotype (odds ratio [OR], 8.31; 95% CI, 2.43-28.41; P= .0007) and the CTAC haplotype (OR, 9.71; 95% CI, 1.09-86.24; P= .04). CONCLUSIONS: The current results suggested that SNPs around the p14(ARF) promoter region may be responsible for the interindividual susceptibility to p14(ARF) promoter methylation among individuals with colorectal cancer.

A case-control association study of the polymorphism at the promoter region of the DRD4 gene in Korean boys with attention deficit-hyperactivity disorder: evidence of association with the -521 C/T SNP.

Recent genetic studies at the 5' end of the dopamine D4 receptor (DRD4) gene have identified several polymorphisms having a possible relationship with attention deficit-hyperactivity disorder (ADHD). This study examined the association between the -521 and -376 promoter single nucleotide polymorphisms (SNPs) of the DRD4 gene and ADHD through a case-control association study in Korean boys, who constitute a single ethnic population. Ninety-four ADHD and ninety-five control boys were enrolled in this study. All of the ADHD subjects completed a comprehensive and standardized diagnostic and psychological evaluation battery including the ADHD Rating Scale-IV (ARS). Genotyping for the 2 promoter SNPs was performed. There were significant differences in the genotype and allele frequencies of the -521 C/T SNP between the ADHD and control groups (chi2=6.28, p=0.043 and chi2=6.22, p=0.013, respectively). However, the distribution of the -376 C/T genotypes and alleles were similar in the ADHD and control groups. The subtypes of ADHD were not related to either of these two SNPs. In the ADHD subjects, the -521 TT genotype group had a higher score in the inattentive subscale and a lower score in the hyperactive subscale of the parents version of ARS, although these differences did not attain statistical significance (p=0.146, p=0.082). In conclusion, there was a significant association between the -521 C/T SNP and ADHD in Korean boys. These results suggest a role of the -521 C/T SNP in the susceptibility for ADHD.

Relationship of Ras association domain family 1 methylation and K-ras mutation in primary non-small cell lung cancer.

Recently, several groups have reported that Ras association domain family 1 (RASSF1A) interacts with Ras and mediates Ras-dependent apoptosis. However, the mechanism by which RASSF1A plays a role as a tumor suppressor in human cancer is unclear. In this study, we investigated the relationship between the RASSF1A methylation and K-ras mutation and their effects on patient's survival in 242 primary non-small cell lung cancers (NSCLCs) to understand the role of RASSF1A in Ras-mediated oncogenic transformation. RASSF1A methylation was not found to be associated with the K-ras mutation in NSCLCs (P = 0.37). For patients with stage I adenocarcinoma, those with RASSF1A methylation and K-ras mutation had a poorer prognosis than those with either RASSF1A methylation or K-ras mutation (P = 0.001). In stage II-III adenocarcinoma patients, the median survival of those with RASSF1A methylation and K-ras mutation was 9 months, and this was poorer than that of those with either RASSF1A methylation or K-ras mutation (P = 0.001). The hazard of failure for those with RASSF1A methylation and K-ras mutation was approximately 2.94 times higher compared with that of those with neither K-ras mutation nor RASSF1A methylation (95% confidence interval = 1.67-9.42; P = 0.01). Our results suggest that RASSF1A methylation and K-ras mutation are not mutually exclusive in NSCLC. In addition, RASSF1A methylation, in combination with K-ras mutation, may have an adverse synergistic effect on patient's survival in NSCLCs.

Hypermethylation of RASSF1A promoter is associated with the age at starting smoking and a poor prognosis in primary non-small cell lung cancer.

Cigarette smoking is the most common cause of lung cancer. The greatest risk of lung cancer is among those who started smoking early in life and continued throughout their lives. However, the molecular mechanisms responsible for the susceptibility to lung cancer in the young smoker are not clear. Recently, several groups have reported that the hypermethylation of CpG islands is associated with exposure to tobacco smoke. We studied the association between the age at starting smoking and hypermethylation of p14, p16, and RASSF1A promoters in 204 primary non-small cell lung cancer patients. We also examined whether hypermethylation of the RASSF1A promoter is an independent prognostic factor. Methylation rates in the 204 samples were detected in 9% for p14, 27% for p16, and 32% for RASSF1A. There was no relationship between the hypermethylation of p14 and p16 and the age at starting smoking. However, hypermethylation of the RASSF1A promoter was found to be significantly associated with the age at starting smoking (P = 0.001). No relationship was found between the methylation status of the RASSF1A promoter and other smoking variables, such as pack-years, smoking status, and the duration of smoking. The age at starting smoking in patients with hypermethylation of RASSF1A was earlier than that of patients without hypermethylation of the RASSF1A promoter (19 +/- 8 versus 25 +/- 7; P = 0.001). Young smokers who started smoking before age 19 were 4.23 times [95% confidence interval (CI) = 1.03-9.67; P = 0.001] more likely to have hypermethylation of the RASSF1A promoter than smokers who started smoking after the age of 19. Furthermore, hypermethylation of the RASSF1A promoter was found to be associated with a poor prognosis in non-small cell lung cancer patients at stages 1 and 2 (P = 0.02 and 0.01, respectively; Log-rank test). The hazard of failure was approximately 3.27 times higher for patients with hypermethylation of the RASSF1A promoter than for those without hypermethylation of the RASSF1A promoter (95% CI = 1.42-8.71; P = 0.01). Young smokers who started the habit before the age of 19 also had a poorer prognosis than those who started after the age of 19 (hazard ratio = 2.14, 95% CI = 1.22-9.11; P = 0.02). Our results suggest that starting cigarette smoking at an early age is associated with hypermethylation of the RASSF1A promoter and that hypermethylation of the RASSF1A promoter may be an independent prognostic factor in primary non-small cell lung cancer.