HopAZ1, a type III effector of Pseudomonas amygdali pv. tabaci, induces a hypersensitive response in tobacco wildfire-resistant Nicotiana tabacum 'N509'.

Pseudomonas amygdali pv. tabaci (formerly Pseudomonas syringae pv. tabaci; Pta) is a gram-negative bacterium that causes bacterial wildfire disease in Nicotiana tabacum. The pathogen establishes infections by using a type III secretion system to inject type III effector proteins (T3Es) into cells, thereby interfering with the host__s immune system. To counteract the effectors, plants have evolved disease-resistance genes and mechanisms to induce strong resistance on effector recognition. By screening a series of Pta T3E-deficient mutants, we have identified HopAZ1 as the T3E that induces disease resistance in N. tabacum 'N509'. Inoculation with the Pta ∆hopAZ1 mutant did not induce resistance to Pta in N509. We also found that the Pta ∆hopAZ1 mutant did not induce a hypersensitive response and promoted severe disease symptoms in N509. Furthermore, a C-terminal truncated HopAZ1 abolished HopAZ1-dependent cell death in N509. These results indicate that HopAZ1 is the avirulence factor that induces resistance to Pta by N509.

Durvalumab-associated Late-onset Myocarditis Successfully Treated with Corticosteroid Therapy.

We herein report a 66-year-old man with locally advanced non-small-cell lung cancer (NSCLC) who developed durvalumab-associated myocarditis. The patient underwent durvalumab administration every two weeks following concurrent chemoradiotherapy, without any adverse events or apparent disease progression. He presented with fatigue and dyspnea on exertion seven months after the first administration. Myocarditis was suspected based on laboratory data, an electrocardiogram, echocardiography, and magnetic resonance imaging findings. The definitive diagnosis was confirmed by a myocardial biopsy. Myocarditis was alleviated by cessation of durvalumab and corticosteroid therapy. This is a noteworthy case to describe late-onset myocarditis following the administration of durvalumab for NSCLC.

Complete Genome Sequence of Pseudomonas amygdali pv. tabaci Strain 6605, a Causal Agent of Tobacco Wildfire Disease.

Pseudomonas amygdali pv. tabaci strain 6605 is the bacterial pathogen causing tobacco wildfire disease that has been used as a model for elucidating virulence mechanisms. Here, we present the complete genome sequence of P. amygdali pv. tabaci 6605 as a circular chromosome from reads using a PacBio sequencer.

Role of Two Sets of RND-Type Multidrug Efflux Pump Transporter Genes, mexAB-oprM and mexEF-oprN, in Virulence of Pseudomonas syringae pv. tabaci 6605.

Pseudomonas syringae pv. tabaci 6605 has two multidrug resistance (MDR) efflux pump transporters, MexAB-OprM and MexEF-OprN. To understand the role of these MDR efflux pumps in virulence, we generated deletion mutants, ∆mexB, ∆mexF, and ∆mexB∆mexF, and investigated their sensitivity to plant-derived antimicrobial compounds, antibiotics, and virulence. Growth inhibition assays with KB soft agar plate showed that growth of the wild-type (WT) was inhibited by 5 µl of 1 M catechol and 1 M coumarin but not by other plant-derived potential antimicrobial compounds tested including phytoalexins. The sensitivity to these compounds tended to increase in ∆mexB and ∆mexB∆mexF mutants. The ∆mexB∆mexF mutant was also sensitive to 2 M acetovanillone. The mexAB-oprM was constitutively expressed, and activated in the ∆mexF and ∆mexB∆mexF mutant strains. The swarming and swimming motilities were impaired in ∆mexF and ∆mexB∆mexF mutants. The flood inoculation test indicated that bacterial populations in all mutant strains were significantly lower than that of WT, although all mutants and WT caused similar disease symptoms. These results indicate that MexAB-OprM extrudes plant-derived catechol, acetovanillone, or coumarin, and contributes to bacterial virulence. Furthermore, MexAB-OprM and MexEF-OprN complemented each other's functions to some extent.

Molecular Scoring of Hepatocellular Carcinoma for Predicting Metastatic Recurrence and Requirements of Systemic Chemotherapy.

Hepatocellular carcinoma (HCC) causes one of the most frequent cancer-related deaths; an HCC subset shows rapid progression that affects survival. We clarify molecular features of aggressive HCC, and establish a molecular scoring system that predicts metastasis after curative treatment. In total, 125 HCCs were examined for TP53, CTNNB1, and TERT promoter mutation, methylation of 8 tumor suppressor genes, and 3 repetitive DNA sequences to estimate promoter hypermethylation and global hypomethylation. A fractional allelic loss (FAL) was calculated to represent chromosomal instability through microsatellite analysis. Molecular subclasses were determined using corresponding and hierarchical clustering analyses. Next, twenty-five HCC patients who underwent liver transplantation were analyzed for associations between molecular characteristics and metastatic recurrence; survival analyses were validated using a publicly available dataset of 376 HCC cases from the Cancer Genome Atlas (TCGA). An HCC subtype characterized by TP53 mutation, high FAL, and global hypomethylation was associated with aggressive tumor characteristics, like vascular invasion; CTNNB1 mutation was a feature of the less-progressive phenotype. A number of molecular risk factors, including TP53 mutation, high FAL, significant global hypomethylation, and absence of CTNNB1 mutation, were noted to predict shorter recurrence-free survival in patients who underwent liver transplantation (p = 0.0090 by log-rank test). These findings were validated in a cohort of resected HCC cases from TCGA (p = 0.0076). We concluded that molecular risks determined by common genetic and epigenetic alterations could predict metastatic recurrence after curative treatments, and could be a marker for considering systemic therapy for HCC patients.

A case of neuroendocrine carcinoma in the hepatic hilar lymph nodes concomitant with an adenocarcinoma of the gallbladder.

BACKGROUND: Neuroendocrine tumors (NETs) are rare especially in the gallbladder. They have not been elucidated in the pathogenesis, clinicopathological characteristics, and treatment options. CASE PRESENTATION: We present a 76-year-old woman with a gallbladder tumor and hepatic hilar lymph node swelling. The lymph node biopsy demonstrated neuroendocrine carcinoma (NEC). We performed cholecystectomy, hepatic hilar lymphadenectomy, extrahepatic biliary duct resection, and hepaticojejunostomy prior to chemotherapy. Pathological examination revealed the gallbladder mass was an adenocarcinoma invading to the muscular layer without any NEC components, whereas the hepatic hilar lymph nodes were filled with high-grade NEC cells with negligible area of adenocarcinoma. The patient received general chemotherapy consisting of carboplatin and etoposide, but a recurrence in the para-aortic lymph nodes occurred 4 months after surgery. CONCLUSIONS: We report a rare case of NEC of the hepatic hilar lymph nodes that were concomitant with an adenocarcinoma of the gallbladder. High-grade NEC generally has an aggressive behavior and an optimal treatment strategy should be chosen for each patient.

Successful treatment of arrhythmia-induced cardiomyopathy in an infant with tuberous sclerosis complex.

BACKGROUND: Tuberous sclerosis complex (TSC) is an autosomal-dominant tumor suppressor gene syndrome that is characterized by the development of distinctive benign tumors and malformations in multiple organ systems (N Eng J Med 355:1345-1356, 2006). Cardiac rhabdomyomas are intracavitary or intramural tumors observed in 50-70 % of infants with TSC but only cause serious clinical problems in a very small fraction of these patients (N Eng J Med 355:1345-1356, 2006; Pediatrics 118:1146-1151, 2006; Eur J Pediatr 153:155-7, 1994); most individuals have no clinical symptoms and their tumors spontaneously regress. However, despite being clinically silent, these lesions can provoke arrhythmias and heart failure (Pediatrics 118:1146-1151, 2006; Eur J Pediatr 153:155-7, 1994). CASE PRESENTATION: We here report the clinical findings of an infant suffering from TSC complicated with dilated cardiomyopathy (DCM) after the regression of cardiac rhabdomyomas. Although his tumors improved spontaneously, tachycardia and irregular heart rate due to frequent premature ventricular and supraventricular contractions persisted from the newborn period and were refractory to several medications. His cardiomyopathy was suspected to have been induced by the tachycardia or arrhythmia. We found carvedilol therapy to be safe and highly effective in treating the cardiomyopathy. To our knowledge, this is the first case report of TSC with DCM after regression of cardiac tumors and its successful treatment. CONCLUSION: The patient's clinical course suggests that careful life-long disease management is important, even in TSC patients without apparent symptoms.

An increase in circulating B cell-activating factor in childhood-onset ocular myasthenia gravis.

BACKGROUND: Myasthenia gravis is a B cell-mediated autoimmune disorder. The pathophysiology of childhood-onset ocular myasthenia gravis remains unclear. We investigated serum B cell-activating factor levels and other immunological parameters in child patients with ocular myasthenia gravis. METHODS: Blood samples were obtained from 9 children with ocular myasthenia gravis and 20 age-matched controls. We assayed serum concentrations of B cell-activating factor, anti-acetylcholine receptor antibody titers, 7 types of cytokines (interleukins-2, -4, -6, -10, and -17A; interferon-γ; tumor necrosis factor-α) as well as the percentages of peripheral blood CD4+, CD8+, and CD19+ cells. RESULTS: Serum B cell-activating factor levels were significantly higher before immunosuppressive therapy in patients with childhood-onset ocular myasthenia gravis than in controls and decreased after immunosuppressive therapy. A significant positive correlation was observed between serum B cell-activating factor levels and anti-acetylcholine receptor antibody titers in patients with myasthenia gravis. Serum B cell-activating factor concentrations did not correlate with the percentages of CD4+, CD8+, and CD19+ cells or the CD4+/CD8+ ratio. No significant differences were observed in the levels of the 7 different types of cytokines examined, including interleukin-17A, between preimmunosuppressive therapy myasthenia gravis patients and controls. CONCLUSIONS: Circulating B cell-activating factor may play a key role in the pathophysiology of childhood-onset ocular myasthenia gravis.

Reduction in peripheral regulatory T cell population in childhood ocular type myasthenia gravis.

OBJECTIVE: Myasthenia gravis (MG) is a T-cell dependent and antibody mediated autoimmune disease. Recent studies of adult patients and animal models have shown that regulatory T cells (Tregs) play an important role in the pathogenesis of MG, but little is known about MG in children. This study evaluated the role of peripheral blood Tregs in childhood ocular MG and assessed if Tregs could be an index for estimating immunological status. PATIENTS AND METHODS: Clinical data and peripheral lymphocytes were obtained from 13 children with serum AChR antibody-positive ocular type MG and 18 age-matched controls. Committed cells from MG patients were divided into two clinical stages: active (n=12) and remission (n=11). Tregs and Th17 cells were analyzed by flow cytometric analysis based on CD4(+)CD25(+) intracellular Foxp3(+) and CD4(+) intracellular IL-17A(+) fractions, respectively. RESULTS: The percentage of Tregs among peripheral blood CD4(+) T cells in active stage, remission stage, and control groups was 3.3±1.3%, 4.8±1.7%, and 5.0±0.6%, respectively. The Treg population was significantly lower in the active stage than in the remission stage and controls. Furthermore, Treg percentage was significantly lower during relapse of myasthenia symptoms. We witnessed no remarkable associations between the percentage of Tregs and immune suppressant dosages. CONCLUSIONS: A significant reduction in the peripheral Treg population is considered to contribute to the pathophysiology of ocular type childhood MG and may be a marker of immunological state in these patients.

Successful treatment for West syndrome with severe combined immunodeficiency.

Several immune mechanisms are suspected in the unknown etiology of West syndrome (WS). We report a male infant who suffered from WS and X-linked T-B+NK- severe combined immunodeficiency (X-SCID) with a missense mutation of the IL2RG gene (c.202G>A, p.Glu68Lys). He promptly began vitamin B6 and valproic acid treatment, but infantile spasms (IS) and hypsarrhythmia persisted. Administration of intravenous immunoglobulin and the change to topiramate (TPM) at 7 months of age resulted in the rapid resolution of IS. The CD4/8 ratio in his peripheral blood increased from 0.04-0.09 to 0.20-1.95 following unrelated cord blood transplantation (UCBT). In vitro lymphocyte proliferation in response to phytohemagglutinin or concanavalin A and the ability of B lymphocytes to produce antibodies improved as well. Electroencephalogram findings became normal 1 month after UCBT. Thus, we consider that T-cell dysfunction and/or impairments in T-B cell interactions due to X-SCID may have played important roles in the onset of WS. Immune-modulating therapies along with the administration of TPM effectively treated this severe epileptic syndrome in our patient.

Prognostic significance of preoperative mean platelet volume in resected non-small-cell lung cancer.

An increased mean platelet volume (MPV) is an early marker of platelet activation. MPV was also shown to be associated with the pathophysiological characteristics of various types of cancer. A previous study demonstrated that MPV was significantly associated with the overall survival (OS) of patients with advanced non-small-cell lung cancer (NSCLC). However, there has been no analysis of the prognostic effect of MPV on patients with resected NSCLC. The aim of this study was to evaluate the contribution of MPV to the survival of patients with completely resected NSCLC. We retrospectively analyzed 308 consecutive patients with NSCLC who underwent curative resection at Kitano Hospital. The associations between MPV and clinicopathological factors were assessed. We also evaluated the effect of MPV on survival, using the two-tailed log-rank test and the Cox proportional hazards model. A MPV value of 8.50 fl was considered to be the optimal cut-off value for prognosis. A low MPV was not associated with any other clinicopathological factors. The two-tailed log-rank test demonstrated that patients with a low MPV experienced a shorter disease-free survival (DFS) and overall survival (OS) (P=0.011 and 0.001, respectively), compared to those with a high MPV. The multivariate analysis demonstrated that a low MPV was an independent unfavorable prognostic factor for DFS and OS [hazard ratio (HR)=1.713; 95% confidence interval (CI): 1.070-2.742, P=0.025; and HR=2.835; 95% CI: 1.304-6.163, P=0.009, respectively)]. Therefore, we demonstrated that a low MPV predicted an unfavorable prognosis in patients with NSCLC following curative resection.

Genome-wide profiling of DNA methylation and tumor progression in human hepatocellular carcinoma.

OBJECTIVE: To clarify the progression pattern of abnormal DNA methylation during the development of hepatocellular carcinoma (HCC) using a comprehensive methylation assay. METHODS: We used an Infinium HumanMethylation450 BeadChip array that can analyze >485,000 CpG sites distributed throughout the genome for a comprehensive methylation study of 117 liver tissues consisting of 59 HCC and 58 noncancerous livers. Altered DNA methylation patterns during tumor progression were also analyzed. RESULTS: We identified 38,330 CpG sites with significant differences in methylation levels between HCCs and noncancerous livers (DM-CpGs) using strict criteria. Of the DM-CpGs, 92% were hypomethylated and only 3,051 CpGs (8%) were hypermethylated in HCC. The DM-CpGs were more prevalent within intergenic regions with isolated CpGs. In contrast, DM-CpGs that were hypermethylated in HCC were predominantly located within promoter regions and CpG islands (p < 0.0001). The association between methylation profiles of DM-CpGs and tumor size was statistically significant, especially in hepatitis C virus (HCV)-positive cases (p = 0.0001). CONCLUSIONS: We clarified the unique characteristics of DM-CpGs in human HCCs. The stepwise progression of alterations in DNA methylation was a common feature of HCV-related hepatocarcinogenesis.

Prognostic significance of combined pulmonary fibrosis and emphysema in patients with resected non-small-cell lung cancer: a retrospective cohort study.

OBJECTIVES: Combined pulmonary fibrosis and emphysema (CPFE) is a unique disorder that is usually diagnosed on the basis of high resolution computed tomography (HRCT) findings. It is unclear whether CPFE is an independent prognostic factor in patients with non-small-cell lung cancer (NSCLC). Therefore, we conducted a retrospective analysis to assess the impact of CPFE on the prognosis of patients with completely resected NSCLC. METHODS: We retrospectively reviewed 365 patients diagnosed with NSCLC who underwent complete resection at the Tazuke Kofukai Medical Research Institute, Kitano Hospital between January 2007 and December 2012. Patients were classified into four groups according to chest HRCT findings: those with CPFE, those with fibrosis, those with emphysema or those with a normal lung except for the presence of a tumour. We evaluated disease-free survival (DFS) and overall survival (OS) using the two-tailed log-rank test and the Cox proportional hazards model. RESULTS: The two-tailed log-rank test demonstrated that the four groups had significantly different DFS and OS (P < 0.01). In the multivariate analysis, CPFE was found to be an independent prognostic factor for DFS and OS compared with a normal lung [hazard ratio (HR): 2.52; 95% confidence interval (CI): 1.24-5.13; P = 0.01 and HR: 4.53; 95% CI: 1.91-10.7; P < 0.01, respectively]. CONCLUSIONS: CPFE is a significant, unfavourable prognostic factor for NSCLC patients after curative resection.

Prognostic impact of preoperative monocyte counts in patients with resected lung adenocarcinoma.

OBJECTIVES: Increasing evidence suggests that an elevated peripheral monocyte count at presentation predicts a poor prognosis in various types of malignancy, including malignant lymphoma. In lung adenocarcinoma, tumor-associated macrophages (TAMs) were reported to be associated with a poor prognosis. However, it is unknown if an elevated peripheral monocyte count is associated with a poor prognosis in lung adenocarcinoma. This study assessed the prognostic impact of the preoperative peripheral monocyte count in lung adenocarcinoma. MATERIALS AND METHODS: We retrospectively analyzed 302 consecutive patients with lung adenocarcinoma who received curative resection at Kitano Hospital. The receiver operating characteristic (ROC) curve for the peripheral monocyte count was used to determine the cut-off value. The relations between peripheral monocyte counts and clinicopathological factors were assessed. We also evaluated the impacts of possible prognostic factors including the preoperative peripheral monocyte count on survival, using the two-tailed log-rank test and Cox proportional hazards model. In addition, immunohistochemical staining for CD68 was performed to evaluate the monocytes in primary tumors. RESULTS: A peripheral monocyte count of 430mm(-3) was the optimal cut-off value for prognosis. An elevated peripheral monocyte count was significantly associated with sex, performance status, smoking history, chronic obstructive pulmonary disease and interstitial lung disease. The two-tailed log-rank test demonstrated that patients with an elevated peripheral monocyte count experienced a poorer recurrence-free survival (RFS) and overall survival (OS) (P=0.0063, P<0.0001, respectively). In the multivariate analysis an elevated peripheral monocyte count was shown to be an independent prognostic factor for the RFS and OS (HR: 1.765; 95% CI: 1.071-2.910; P=0.0258, HR: 4.339; 95% CI: 2.032-9.263; P=0.0001, respectively). Furthermore, numbers of the monocytes in primary tumors significantly correlated with peripheral monocyte counts (r=0.627, P<0.0001). CONCLUSION: The preoperative peripheral monocyte count is an important prognostic factor for patients with lung adenocarcinoma after curative resection.

Comparative outcomes between initially unresectable and recurrent cases of advanced pancreatic cancer following palliative chemotherapy.

OBJECTIVES: The objective of this study was to compare the clinical outcomes between initially unresectable and recurrent advanced pancreatic cancer (APC) patients after palliative chemotherapy. METHODS: Data of a total of consecutive 269 patients with pathologically confirmed APC patients who received palliative chemotherapy between January 2006 and April 2012 were reviewed. Patients were classified into initially unresectable and recurrent group, and overall survival (OS) was compared between the 2 groups. RESULTS: The median OS was significantly longer in the recurrent group compared with the initially unresectable group (383 vs 308 days; hazard ratio [HR], 0.59; 95% confidence interval, 0.44-0.80; P < 0.01). After adjustment for distant metastasis, performance status, and levels of carbohydrate antigen 19-9, carcinoembryonic antigen, C-reactive protein, and lactate dehydrogenase, the status of recurrent or unresectable disease remained as an independent prognostic factor with a clinically relevant HR value (HR, 0.66; 95% confidence interval, 0.48-0.90; P = 0.01). In addition, the 2-year OS rate of the recurrent group was significantly higher than that of the unresectable group (24.2% vs 9.6%, P = 0.01). CONCLUSIONS: Our results suggested that the status of recurrent or initially unresectable disease was an independent prognostic factor in APC patients receiving palliative chemotherapy.

Neutrophil-to-lymphocyte ratio for predicting palliative chemotherapy outcomes in advanced pancreatic cancer patients.

Several previous studies reported that the neutrophil-to-lymphocyte ratio (NLR) could be a promising prognostic factor for patients with cancer. We aimed to determine the prognostic value of NLR in patients with advanced pancreatic cancer (APC) following palliative chemotherapy. We retrospectively reviewed 252 consecutive APC patients receiving palliative chemotherapy between January 2006 and December 2012. We classified the patients according to the pretreatment NLR values (≤ 5 or >5) into two groups and investigated the difference in treatment outcomes, including time to treatment failure (TTF) and overall survival (OS). A total of 212 patients had pretreatment NLR values of ≤ 5 (group A), while 40 patients had an NLR of >5 (group B). TTF and OS were significantly shorter in group B than in group A (3.1 vs. 8.7 months and 6.0 vs. 12.8 months, respectively; both P < 0.01). After adjustment for putative prognostic factors, including distant metastasis, status of recurrent/unresectable disease, pretreatment carbohydrate antigen 19-9 levels, and carcinoembryonic antigen levels using the Cox regression model, elevated pretreatment NLR remained an independent poor prognostic factor for OS (hazard ratio, 1.92; 95% confidence interval, 1.27-2.90; P < 0.01). In addition, patients in group B whose NLR dropped to ≤ 5 before the second cycle of chemotherapy showed longer TTF and OS compared with those whose NLR remained at >5. Our results support the idea that NLR can be a promising prognostic and predictive marker for APC patients receiving palliative chemotherapy.

[Efficacy of visual cognitive function tests in health examinations of five-year-old children].

OBJECTIVE: Health examination programs for five-year-old children are aimed at effectively detecting developmental disorders, such as attention deficit/hyperactivity disorders (AD/HD), learning disorders (LD), higher functioning autistic spectrum disorders (HFASD), and other abnormalities. Tests usually include a questionnaire and observation of group playing, verbal communication, and soft neurological signs; however, it is often difficult to detect children who have LD with visual cognitive dysfunctions through such conventional examination techniques. Here, we analyzed the efficacy of using a battery of visual cognitive function tests to identify such cases. METHODS: We employed four simple tests to evaluate visual cognitive function in addition to a standard health examination for five-year-old between April 2008 and March 2010. To analyze visual cognitive function tests, the results were scored and the applicability of these tests was verified by comparisons with established tests. RESULTS: A total of 653 five-year-old children underwent health examinations, and 48 children were referred to the hospital for further examinations. As a result, 34 children were newly diagnosed with developmental disorders, including HFASD, AD/HD, LD, and mild intellectual disturbances. Strong correlations were seen between the scores of these four examinations and those of other established tests, such as the performance intelligence score, the perceptual organization index of WISC-III, and the Frostig visual development test score. An additional benefit of our method was that parents could easily recognize developmental disorders in their children through direct observation of these examinations. CONCLUSIONS: We concluded that the battery of visual cognitive function tests was simple and useful for detecting developmental disorders in the health examinations of five-year-old children.

Unique association between global DNA hypomethylation and chromosomal alterations in human hepatocellular carcinoma.

Global DNA hypomethylation is a characteristic feature of cancer cells that closely associates with chromosomal instability (CIN). However, the association between these characteristics during hepatocarcinogenesis remains unclear. Herein, we determined the relationship between hypomethylation and CIN in human hepatocellular carcinoma (HCC) by analyzing 179 HCCs, 178 matched non-tumor livers and 23 normal liver tissues. Hypomethylation at three different repetitive DNA (rDNA) sequences and hypermethylation of 12 CpG loci, including 11 tumor suppressor gene (TSG) promoters, were quantified using MethyLight or combined bisulfite restriction analysis. Fractional allelic loss (FAL) was used as a marker for CIN, calculated by analyzing 400 microsatellite markers. Gains and losses at each chromosome were also determined using semi-quantitative microsatellite analysis. The associations between rDNA hypomethylation and FAL, as well as between TSG hypermethylation and FAL were investigated. Significantly more hypomethylation was observed in HCC tissues than in normal liver samples. Progression of hypomethylation during carcinogenesis was more prominent in hepatitis C virus (HCV)-negative cases, which was in contrast to our previous reports of significantly increased TSG methylation levels in HCV-positive tumors. Absence of liver cirrhosis and higher FAL scores were identified as independent contributors to significant hypomethylation of rDNA in HCC. Among the chromosomal alterations frequently observed in HCC, loss of 8p, which was unique in the earliest stages of hepatocarcinogenesis, was significantly associated with hypomethylation of rDNA by multivariable analysis (p=0.0153). rDNA hypomethylation was also associated with a high FAL score regardless of tumor differentiation (p=0.0011, well-differentiated; p=0.0089, moderately/poorly-differentiated HCCs). We conclude that DNA hypomethylation is an important cause of CIN in the earliest step of HCC, especially in a background of non-cirrhotic liver.