Cheol Keun Park

Phase III Trial Comparing Capecitabine Plus Cisplatin Versus Capecitabine Plus Cisplatin With Concurrent Capecitabine Radiotherapy in Completely Resected Gastric Cancer With D2 Lymph Node Dissection: The ARTIST Trial

PURPOSE The ARTIST (Adjuvant Chemoradiation Therapy in Stomach Cancer) trial was the first study to our knowledge to investigate the role of postoperative chemoradiotherapy therapy in patients with curatively resected gastric cancer with D2 lymph node dissection. This trial was designed to compare postoperative treatment with capecitabine plus cisplatin (XP) versus XP plus radiotherapy with capecitabine (XP/XRT/XP). PATIENTS AND METHODS The XP arm received six cycles of XP (capecitabine 2,000 mg/m2 per day on days 1 to 14 and cisplatin 60 mg/m2 on day 1, repeated every 3 weeks) chemotherapy. The XP/XRT/XP arm received two cycles of XP followed by 45-Gy XRT (capecitabine 1,650 mg/m2 per day for 5 weeks) and two cycles of XP. RESULTS Of 458 patients, 228 were randomly assigned to the XP arm and 230 to the XP/XRT/XP arm. Treatment was completed as planned by 75.4% of patients (172 of 228) in the XP arm and 81.7% (188 of 230) in the XP/XRT/XP arm. Overall, the addition of XRT to XP chemotherapy did not significantly prolong disease-free survival (DFS; P = .0862). However, in the subgroup of patients with pathologic lymph node metastasis at the time of surgery (n = 396), patients randomly assigned to the XP/XRT/XP arm experienced superior DFS when compared with those who received XP alone (P = .0365), and the statistical significance was retained at multivariate analysis (estimated hazard ratio, 0.6865; 95% CI, 0.4735 to 0.9952; P = .0471). CONCLUSION The addition of XRT to XP chemotherapy did not significantly reduce recurrence after curative resection and D2 lymph node dissection in gastric cancer. A subsequent trial (ARTIST-II) in patients with lymph node-positive gastric cancer is planned.

Epigenetic Changes Induced by Reactive Oxygen Species in Hepatocellular Carcinoma: Methylation of the E-cadherin Promoter

BACKGROUND & AIMS In addition to genetic alterations, epigenetic changes underlie tumor progression and metastasis. Promoter methylation can silence tumor suppressor genes, and reactive oxygen species (ROS) promote DNA damage, although the relationship between ROS and epigenetic changes in cancer cells is not clear. We sought to determine whether ROS promote hypermethylation of the promoter region of E-cadherin, a regulator of the epithelial-to-mesenchymal transition, in hepatocellular carcinoma (HCC) cells. METHODS HCC cells were exposed to H(2)O(2) or stably transfected to express Snail, a transcription factor that down-regulates E-cadherin expression. E-cadherin and Snail expression levels were examined by real-time reverse-transcriptase polymerase chain reaction and immunoblot analyses. The methylation status of E-cadherin was examined by methyl-specific polymerase chain reaction, bisulfite sequencing, and chromatin immunoprecipitation. The interactions between Snail, histone deacetylase 1, and DNA methyltransferase 1 were assessed by immunoprecipitation/immunoblot and immunofluorescence analyses. ROS-induced stress, E-cadherin expression, Snail expression, and E-cadherin promoter methylation were confirmed in HCC tissues by immunoblot, immunohistochemistry, and methyl-specific polymerase chain reaction analyses. RESULTS We demonstrated that ROS induce hypermethylation of the E-cadherin promoter by increasing Snail expression. Snail induced DNA methylation of the E-cadherin promoter by recruiting histone deacetylase 1 and DNA methyltransferase 1. In human HCC tissues, we observed a correlation among ROS induction, E-cadherin down-regulation, Snail up-regulation, and E-cadherin promoter methylation. CONCLUSIONS These findings provide novel mechanistic insights into epigenetic modulations induced by ROS in the process of carcinogenesis. They are potentially relevant to understanding the activity of ROS in silencing various tumor suppressor genes and in subsequent tumor progression and metastasis.

Molecular changes from dysplastic nodule to hepatocellular carcinoma through gene expression profiling

Progression of hepatocellular carcinoma (HCC) is a stepwise process that proceeds from pre‐neoplastic lesions—including low‐grade dysplastic nodules (LGDNs) and high‐grade dysplastic nodules (HGDNs)—to advanced HCC. The molecular changes associated with this progression are unclear, however, and the morphological cues thought to distinguish pre‐neoplastic lesions from well‐differentiated HCC are not universally accepted. To understand the multistep process of hepato‐carcinogenesis at the molecular level, we used oligo‐nucleotide microarrays to investigate the transcription profiles of 50 hepatocellular nodular lesions ranging from LGDNs to primary HCC (Edmondson grades 1‐3). We demonstrated that gene expression profiles can discriminate not only between dysplastic nodules and overt carcinoma but also between different histological grades of HCC via unsupervised hierarchical clustering with 10,376 genes. We identified 3,084 grade‐associated genes, correlated with tumor progression, using one‐way ANOVA and a one‐versus‐all unpooled t test. Functional assignment of these genes revealed discrete expression clusters representing grade‐dependent biological properties of HCC. Using both diagonal linear discriminant analysis and support vector machines, we identified 240 genes that could accurately classify tumors according to histological grade, especially when attempting to discriminate LGDNs, HGDNs, and grade 1 HCC. In conclusion, a clear molecular demarcation between dysplastic nodules and overt HCC exists. The progression from grade 1 through grade 3 HCC is associated with changes in gene expression consistent with plausible functional consequences. Supplementary material for this article can be found on the HEPATOLOGY website (http://www.interscience.wiley.com/jpages/0270‐9139/suppmat/index.html). (HEPATOLOGY 2005;42:809–818.)

Lysophosphatidylcholine as a death effector in the lipoapoptosis of hepatocytes

The pathogenesis of nonalcoholic steatohepatitis (NASH) is unclear, despite epidemiological data implicating FFAs. We studied the pathogenesis of NASH using lipoapoptosis models. Palmitic acid (PA) induced classical apoptosis of hepatocytes. PA-induced lipoapoptosis was inhibited by acyl-CoA synthetase inhibitor but not by ceramide synthesis inhibitors, suggesting that conversion products other than ceramide are involved. Phospholipase A2 (PLA2) inhibitors blocked PA-induced hepatocyte death, suggesting an important role for PLA2 and its product lysophosphatidylcholine (LPC). Small interfering RNA for Ca2+-independent phospholipase A2 (iPLA2) inhibited the lipoapoptosis of hepatocytes. PA increased LPC content, which was reversed by iPLA2 inhibitors. Pertussis toxin or dominant-negative Gαi mutant inhibited hepatocyte death by PA or LPC acting through G-protein-coupled receptor (GPCR)/Gαi. PA decreased cardiolipin content and induced mitochondrial potential loss and cytochrome c translocation. Oleic acid inhibited PA-induced hepatocyte death by diverting PA to triglyceride and decreasing LPC content, suggesting that FFAs lead to steatosis or lipoapoptosis according to the abundance of saturated/unsaturated FFAs. LPC administration induced hepatitis in vivo. LPC content was increased in the liver specimens from NASH patients. These results demonstrate that LPC is a death effector in the lipoapoptosis of hepatocytes and suggest potential therapeutic values of PLA2 inhibitors or GPCR/Gαi inhibitors in NASH.

Progressive methylation during the serrated neoplasia pathway of the colorectum

Serrated adenoma is a recently described entity characterized by having combined architectural features of hyperplastic polyps and classical adenoma. To understand the role of gene regulation in the progression of the serrated neoplasia pathway, we examined the methylation profiles of the promoter regions of 19 genes, DNA ploidy, and mutator phenotype status. In all, 40 sporadic, classical serrated adenomas were pathologically reviewed and divided into four pathologic groups according to their histologic grades. Methylation-specific PCR was performed using primers for p16, hMLH1, RASSF1A, APC, HIC-1, DAPK, MGMT, SLC5A8, RB1, H-Cadherin, E-Cadherin, TIMP3, PTEN, THBS1, LKB1, p14, p15, FHIT, and VHL. Dual flow-cytometric analyses using cytokeratin and DAPI and MSI studies using BAT26 were also performed. Methylation was observed in 2.5–82.5% (mean 33.9%) of the CpG islands in the promoter regions of 16 genes. The tumors with higher histologic grades, including carcinomas, showed more extensive methylation compared to those with lower grades, and serrated adenomas in the right colon showed more frequent methylation than those in the left (P<0.05). Tumor-specific promoter methylation of SLC5A8 was observed in 33 (82.5%) of the serrated adenomas. Aneuploidization with near-diploid DNA indices was detected in four out of 28 cases examined (14.3%); two were low-grade serrated adenomas and two were carcinomas in the left colon. The high mutator phenotype was not observed in any of the cases examined. Our results indicate that: (1) aberrant, widespread methylation of CpG islands increases with the histological progression of serrated adenomas; (2) methylation of SLC5A8 is an early event; and (3) additional methylation of the p16, p14, MGMT, TIMP3, and FHIT genes are important tumorigenic steps in the serrated neoplasia pathway.

Liver X receptor mediates hepatitis B virus X protein–induced lipogenesis in hepatitis B virus–associated hepatocellular carcinoma

Although hepatitis B virus X protein (HBx) has been implicated in abnormal lipid metabolism in hepatitis B virus (HBV)–associated hepatic steatosis, its underlying molecular mechanism remains unclear. Liver X receptor (LXR) plays an important role in regulating the expression of genes involved in hepatic lipogenesis. Here we demonstrate that LXRα and LXRβ mediate HBV‐associated hepatic steatosis. We have found that HBx induces the expression of LXR and its lipogenic target genes, such as sterol regulatory element binding protein‐1c (SREBP‐1c), fatty acid synthase (FAS), and peroxisome proliferator‐activated receptor, and this is accompanied by the accumulation of lipid droplets. RNA interference with LXR expression decreases the amount of lipid droplets as well as the expression of the lipogenic genes, and this indicates that HBx‐induced lipogenesis is LXR‐dependent. LXRα and HBx colocalize in the nucleus and are physically associated. HBx induces the transactivation function of LXRα by recruiting CREB binding protein to the promoter of the target gene. Furthermore, we have observed that expression of LXR is increased in the livers of HBx‐transgenic mice. Finally, there is a significant increase in the expression of LXRβ (P = 0.036), SREBP‐1c (P = 0.008), FAS, and stearoyl–coenyzme A desaturase‐1 (P = 0.001) in hepatocellular carcinoma (HCC) in comparison with adjacent nontumorous nodules in human HBV‐associated HCC specimens. Conclusion: Our results suggest a novel association between HBx and LXR that may represent an important mechanism explaining HBx‐induced hepatic lipogenesis during HBV‐associated hepatic carcinogenesis. (HEPATOLOGY 2009.)

Gadoxetic Acid-Enhanced Hepatobiliary Phase MRI and High- b-Value Diffusion-Weighted Imaging to Distinguish Well-Differentiated Hepatocellular Carcinomas From Benign Nodules in Patients With Chronic Liver Disease

OBJECTIVE The purpose of this article is to evaluate the value of gadoxetic acid-enhanced hepatobiliary phase imaging and high-b-value diffusion-weighted imaging (DWI) for distinguishing well-differentiated hepatocellular carcinomas (HCCs) from benign hepatocellular nodules in patients with chronic liver disease using 3-T MRI. MATERIALS AND METHODS Forty-five patients with 46 well-differentiated HCCs (mean, 2.3 cm) and 21 patients with 24 benign hepatocellular nodules (five large regenerative nodules and 19 dysplastic nodules; mean, 1.8 cm) were included in this study. Diagnosis of well-differentiated HCCs and benign hepatocellular nodules was made histopathologically by percutaneous biopsy (n = 12 and n = 11, respectively) or surgical resection (n = 34 and n = 13, respectively). Gadoxetic acid-enhanced MRI was performed for all patients, and DWI (b values of 0 and 800 s/mm(2)) was performed for 31 well-differentiated HCCs and 11 benign hepatocellular nodules. Two radiologists performed a consensus review of the MRI scans for signal intensity compared with that of the surrounding liver parenchyma on hepatobiliary phase images and DWI (b value, 800 s/mm(2)) for qualitative analysis. The contrast-to-noise ratio (CNR) and relative contrast enhancement of lesions on hepatobiliary phase images and the apparent diffusion coefficient (ADC) values were assessed for quantitative analysis. RESULTS In the qualitative analysis, 39 well-differentiated HCCs (85%) and 14 benign hepatocellular nodules (58%) were hypointense on hepatobiliary phase images, and seven well-differentiated HCCs (15%) and 10 benign hepatocellular nodules (42%) were iso- or hyperintense (p = 0.04). Twenty-five well-differentiated HCCs (81%) and three benign hepatocellular nodules (27%) were hyperintense on DWI, with b value of 800 s/mm(2), and six well-differentiated HCCs (19%) and eight benign hepatocellular nodules (73%) were iso- or hypointense (p = 0.01). When lesion hypointensity on hepatobiliary phase images or hyperintensity on DWI were considered signs of HCC in cirrhotic liver, our results yielded sensitivities of 85% and 81% and specificities of 42% and 73%, respectively. In the quantitative analysis, the mean (± SD) relative contrast enhancement ratio of the well-differentiated HCCs (0.76 ± 2.30) was significantly higher than that of benign hepatocellular nodules (0.25 ± 0.97) (p = 0.02). The lesion-to-liver CNRs and the mean ADC values were not significantly different between the two groups (p > 0.05). CONCLUSION Hypointensity on gadoxetic acid-enhanced hepatobiliary phase images and hyperintensity on high-b-value DWI to surrounding liver parenchyma suggest well-differentiated HCCs rather than benign hepatocellular nodules in chronic liver disease.

Mutational analysis of EGFR and K-RAS genes in lung adenocarcinomas

Both epidermal growth factor receptor (EGFR) and RAS gene mutations contribute to the development of non-small cell lung cancer (NSCLC). Because RAS is one of the downstream molecules in the EGFR signal transduction, the association between the somatic mutations of EGFR and RAS may be important in the pathogenesis of NSCLC . However, to date, such data are lacking. In this study, we analyzed the hotspot regions of K-RAS gene (codons 12, 13, 59 and 61) and EGFR gene (exons 18, 19 and 21) in 153 NSCLC tissue samples including 69 adenocarcinomas. Overall, we detected 30 EGFR mutations (19.6%) and 6 K-RAS mutations (3.9%) in the 153 NSCLCs. In the 69 adenocarcinomas, 26 EGFR mutations (37.7%) and six K-RAS mutations (8.7%) were detected. Of note, the 26 tumors with EGFR mutations did not harbor any K-RAS mutations, and the six tumors with K-RAS mutations did not harbor any EGFR mutations. Inverse relationship between K-RAS and EGFR mutations in the lung adenocarcinoma was statistically significant (P=0.046, χ2 test). As regards smoking history, EGFR mutation was significantly associated with never-smoking history, whereas K-RAS mutation was significantly associated with smoking history. Our data suggest that mutations of EGFR and K-RAS genes might separately, but not cooperatively, contribute to lung adenocarcinoma pathogenesis, and that EGFR and K-RAS mutants could separately be anti-neoplastic targets in lung adenocarcinomas.

Molecular features of colorectal hyperplastic polyps and sessile serrated adenoma/polyps from Korea.

Abundant recent data suggest that sessile serrated adenoma/polyp (SSA/P) is an early precursor lesion in the serrated pathway of carcinogenesis. It is believed that SSA/Ps develop cancer by an SSA/P-dysplasia-carcinoma sequence. Hyperplastic polyps (HPs) share some histologic and molecular characteristics with SSA/P, but it is unclear whether SSA/Ps are derived from HPs or whether they develop by a different pathogenetic pathway. Previous studies have shown that serrated polyps from Korean patients show different prevalence rates of certain molecular abnormalities compared with similar lesions from American patients, and this suggests that lifestyle and dietary factors may influence the serrated neoplasia pathway. The purpose of this study was to evaluate the molecular features of HPs and SSA/Ps, the latter both with and without dysplasia, from Korean patients and to compare the findings with similar lesions from American patients. One hundred and eleven serrated polyps, consisting of 45 HPs (30 microvesicular, 11 goblet cell, 4 mucin depleted) and 56 SSA/Ps (36 with dysplasia, 20 without dysplasia), were retrieved from the pathology files of a large medical center in Korea and 38 SSA/P from American patients were evaluated for BRAF and KRAS mutations, microsatellite instability, and hypermethylation of O6-methylguanine-DNA methyltransferase (MGMT), hMLH1, adenomatous polyposis coli (APC), p16, methylated in tumor-1 (MINT-1), MINT2, and MINT31. Methylation of hMLH1 was performed using 2 different sets of primers. Twenty-three conventional adenomas from Korean patients were included as controls. The data were compared between polyp subtypes and between polyps in the right versus the left colon. With regard to HP, KRAS mutations were present in 31.1% of polyps and BRAF mutations in 46.7% of polyps. KRAS mutations were significantly more common in goblet cell HP and BRAF in microvesicular HP (MVHP). Methylation of MGMT, hMLH1, APC, p16, MINT1, MINT2, and MINT31 were present in 42.2%, 64.4% (and 24.4%), 37.8%, 60%, 68.9%, 51.1%, and 60% of HPs. CpG island methylator phenotype high was noted in 60% of HPs. Methylation of hMLH1, p16, MINT2, and MINT31 were more frequent in MVHPs compared with other types of HPs. In contrast, SSA/Ps showed KRAS and BRAF mutations in 12.5% and 60.7% of cases, respectively. Methylation of all tumor-related genes, except hMLH1 (23.2% using 1 type of primers) and APC (37.5%), occurred in >50% of lesions, and CpG island methylator phenotype (CIMP) high was noted in 76.8% of cases. None of the molecular findings were significantly more common in SSA/P with, versus those without, dysplasia, but only 2 of the 36 polyps with dysplasia were of the conventional adenomatous type; the remainder (34 of 36) was of the serrated type. Nevertheless, both SSA/P with conventional adenomatous dysplasia showed methylation of MGMT, APC, MINT1, and MINT31 and were CIMP high. BRAF mutations, methylation of most tumor related genes, and CIMP high occurred more frequently in HPs and SSA/Ps in the right colon, compared with the left colon. In fact, no significant differences were observed between HPs and SSPs of the right colon and HPs and SSA/Ps from the left colon. Furthermore, compared with American patients, Korean male individuals were affected more frequently than female individuals, and both BRAF mutations and hMLH1 methylation were less frequent in the latter compared with the former. We conclude that HPs and SSA/Ps in Korean patients share some, but not all, clinical and molecular characteristics to those that occur in Americans. The data support the theory that the right and left colon are biologically different with regard to susceptibility to serrated cancer, and that anatomic location (right vs. left) may be a more significant risk factor of progression than the histologic type of polyp. Our data also support the theory that right-sided MVHPs may be a precursor to SSA/P.

Multiple gastrointestinal stromal tumors: Clinicopathologic and genetic analysis of 12 patients.

Multiple gastrointestinal stromal tumors (GISTs) are extremely rare and usually associated with type 1 neurofibromatosis and familial GIST. The aim of this study was to investigate the clinical, phenotypic, and genetic characteristics of multiple GISTs to gain insights into their underlying pathogenesis and clinical behavior. Forty-seven paraffin blocks of multiple GISTs from 12 patients were analyzed. Genomic DNA was extracted from the tumor and normal mucosa and mutations for 4 exons of KIT gene and 3 exons of PDGFRA gene were determined. Among 12 patients with multiple GISTs, 5 were sporadic, 2 were familial with germline mutations of KIT gene, and 5 were associated with type 1 neurofibromatosis. All but 1 sporadic and familial multiple GISTs showed mutations of KIT gene shared by the same mutation on each GIST mass within a patient. But in 1 sporadic case, different types of KIT mutations were observed. Two familial multiple GIST cases showed diffuse involvement of the gastrointestinal tract with diffuse hyperplasia of interstitial cell of Cajal. Multiple GISTs associated with type 1 neurofibromatosis were located in the jejunum and harbored no mutations of KIT or PDGFRA. Different types of KIT gene mutation found in our case raise a possibility that recurrence of GISTs within a gastrointestinal tract may have a chance to be a rare occurrence of multiple primary GISTs instead of true recurrence. Multiple GISTs show unique clinical, phenotypic, and genotypic characteristics that are dependent on the particular underlying mechanisms, but the overall prognosis is favorable regardless of the numbers or phenotype of GISTs.