Identification of Driving ALK Fusion Genes and Genomic Landscape of Medullary Thyroid Cancer.

The genetic landscape of medullary thyroid cancer (MTC) is not yet fully understood, although some oncogenic mutations have been identified. To explore genetic profiles of MTCs, formalin-fixed, paraffin-embedded tumor tissues from MTC patients were assayed on the Ion AmpliSeq Cancer Panel v2. Eighty-four sporadic MTC samples and 36 paired normal thyroid tissues were successfully sequenced. We discovered 101 hotspot mutations in 18 genes in the 84 MTC tissue samples. The most common mutation was in the ret proto-oncogene, which occurred in 47 cases followed by mutations in genes encoding Harvey rat sarcoma viral oncogene homolog (N = 14), serine/threonine kinase 11 (N = 11), v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (N = 6), mutL homolog 1 (N = 4), Kiesten rat sarcoma viral oncogene homolog (N = 3) and MET proto-oncogene (N = 3). We also evaluated anaplastic lymphoma kinase (ALK) rearrangement by immunohistochemistry and break-apart fluorescence in situ hybridization (FISH). Two of 98 screened cases were positive for ALK FISH. To identify the genomic breakpoint and 5' fusion partner of ALK, customized targeted cancer panel sequencing was performed using DNA from tumor samples of the two patients. Glutamine:fructose-6-phosphate transaminase 1 (GFPT1)-ALK and echinoderm microtubule-associated protein-like 4 (EML4)-ALK fusions were identified. Additional PCR analysis, followed by Sanger sequencing, confirmed the GFPT1-ALK fusion, indicating that the fusion is a result of intra-chromosomal translocation or deletion. Notably, a metastatic MTC case harboring the EML4-ALK fusion showed a dramatic response to an ALK inhibitor, crizotinib. In conclusion, we found several genetic mutations in MTC and are the first to identify ALK fusions in MTC. Our results suggest that the EML4-ALK fusion in MTC may be a potential driver mutation and a valid target of ALK inhibitors. Furthermore, the GFPT1-ALK fusion may be a potential candidate for molecular target therapy.

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.

Estimation of carrier frequencies of six autosomal-recessive Mendelian disorders in the Korean population.

Although many studies have been performed to identify mutations in Korean patients with various autosomal-recessive Mendelian disorders (AR-MDs), little is known about the carrier frequencies of AR-MDs in the Korean population. Twenty common mutations from six AR-MDs, including Wilson disease (WD), non-syndromic hearing loss (NSHL), glycogen storage disease type Ia (GSD Ia), phenylketonuria (PKU), congenital hypothyroidism (CH), and congenital lipoid adrenal hyperplasia (CLAH) were selected to screen for based on previous studies. A total of 3057 Koreans were genotyped by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry followed by confirmation using the Sanger sequencing. We found 201 and 8 carriers with either one or two mutations in different genes, respectively, yielding a total carrier frequency of 1 in 15 (6.7%). Of the six AR-MDs, NSHL has the highest carrier frequency followed by WD, CH, CLAH, GSD Ia, and PKU. As carrier screening tests are becoming prevalent and the number of mutations known and tested is rising, a priori data on the carrier frequencies in different ethnic groups is mandatory to plan a population screening program and to estimate its efficiency. In light of this, the present results can be used as a basis to establish a screening policy for common AR-MRs in the Korean population.

Polymorphisms of interferon-gamma and interferon-gamma receptor 1 genes and non-tuberculous mycobacterial lung diseases.

Interferon-gamma (IFN-gamma) is crucial for host defense against mycobacterial infections. Recent studies have indicated that IFN-gamma and IFN-gamma receptor 1 (IFN-gammaR1) gene polymorphisms are associated with susceptibility to pulmonary tuberculosis. The aim of this study was to test the hypothesis that IFN-gamma and IFN-gammaR1 gene polymorphisms influence susceptibility to lung disease caused by non-tuberculous mycobacteria (NTM). Seventy-six patients with the nodular bronchiectatic form of NTM lung disease (37 patients with Mycobacterium avium complex infection and 39 patients with Mycobacterium abscessus infection) and 80 controls were included. Polymorphisms of the IFN-gamma gene at position +874 were determined by the amplification refractory mutation system (ARMS) polymerase chain reaction assay and IFN-gammaR1 gene at positions -611, -270, -56 and +95 was genotyped by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry using genomic DNA. IFN-gammaR1 -270 and +95 polymorphisms were not present in any of the patients or controls. The patients with NTM lung disease showed no significant difference from controls in genotype and allele frequencies of the IFN-gamma +874 and IFN-gammaR1 -611 and -56 polymorphisms. The IFN-gamma +874 and IFN-gammaR1 -611 and -56 polymorphisms do not appear to be responsible for host susceptibility to NTM lung disease, at least in the Korean population.

Deamination Effects in Formalin-Fixed, Paraffin-Embedded Tissue Samples in the Era of Precision Medicine.

Deamination of nucleotides causes C:G>T:A changes in formalin-fixed, paraffin-embedded (FFPE) tissue samples and produces false positives during next-generation sequencing (NGS). Uracil DNA glycosylase (UDG) helps eliminate this issue, but the effect of UDG in different tissue preparation conditions has not been rigorously studied. To investigate whether UDG can reduce false-positive single-nucleotide variant (SNV) calls, we used tumor and normal tissues from gastric adenocarcinoma patients prepared using different fixation times and pH conditions. FFPE tumor blocks >10 years were also evaluated for the comparison. We performed semiconductor-based NGS to evaluate nucleotide changes and used UDG to test deamination-related effects. Sequencing quality parameters mildly worsened with prolonged fixation time, acidic pH, and delayed fixation. SNV calls and C:G>T:A changes increased after >48 hours of fixation. In both recently prepared and old FFPE tissue blocks, UDG treatment reduced deamination-induced nucleotide changes. In the recently prepared samples, both high-quality SNVs and mean target coverage were remarkably increased on treatment with UDG. However, the quality of NGS results from old-age samples varied irrespective of UDG treatment. In conclusion, based on our findings, we believe that when performing NGS on recently embedded blocks, it is important to consider that certain poorly fixed samples may be at the risk of being deaminated, which can be corrected with UDG treatment.

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.

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.

Sequential array comparative genomic hybridization analysis identifies copy number changes during blastic transformation of chronic myeloid leukemia.

The present study was performed to provide direct evidence on copy number changes during progression from chronic phase (CP) to blastic phase (BP) in chronic myeloid leukemia (CML) through a longitudinal follow-up study. Matched CP and BP samples in three patients were analyzed using high-resolution array comparative genomic hybridization (aCGH) chips. During blastic transformation, loss of large genomic segments including 6q14.1-q22.31, chromosome 7 and 9p13.2-p21.3 were noted. Furthermore, small-sized copy number changes involving cancer-associated genes were observed. In addition, we identified a novel fusion gene consisted of PAX5 and MLLT3 (AF9). It is likely that blastic transformation of CML is a multi-step process associated accumulation of several genomic events which may largely overlap with those found in acute leukemias.

Polymorphisms of interferon-gamma and interferon-gamma receptor 1 genes and pulmonary tuberculosis in Koreans.

BACKGROUND AND OBJECTIVE: Many genetic variations are thought to be risk factors for the development of pulmonary tuberculosis (TB). The association of interferon-gamma (IFN-gamma) and IFN-gamma receptor 1 (IFN-gammaR1) gene polymorphisms with pulmonary TB is controversial. This study examined the association between IFN-gamma and IFN-gammaR1 gene polymorphisms and pulmonary TB among Koreans. METHODS: Eighty patients with culture-confirmed pulmonary TB and 80 controls were studied. Polymorphisms of the IFN-gamma gene at position +874 were determined using the amplification refractory mutation system PCR assay, and the IFN-gammaR1 gene was genotyped at positions -611, -270, -56 and +95 employing matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using genomic DNA. RESULTS: The genotype and allele frequencies of the IFN-gamma and IFN-gammaR1 gene polymorphisms did not differ significantly between the patients with pulmonary TB and controls. CONCLUSIONS: The IFN-gamma and IFN-gammaR1 gene polymorphisms do not appear to be responsible for host susceptibility to pulmonary TB in the Korean population.