Autophagy-Related Gene ATG7 Polymorphism Could Potentially Serve as a Biomarker of the Progression of Atrophic Gastritis.

Cytotoxin-associated gene A (CagA) is an oncoprotein that H. pylori injects into the host's gastric epithelial cells and that induces proinflammatory cytokines, such as interleukin (IL)-18 and IL-1ß. As a result, it leads to atrophic gastritis (AG), a precancerous lesion of gastric cancer. On the other hand, host cells degrade CagA using autophagy systems. However, few studies exist about the single nucleotide polymorphisms (SNPs) in MAP1LC3A, MAP1LC3B, ATG4A, ATG4B, ATG4C, ATG7, and ATG13, which belong to the autophagy-related genes concerning AG. This study aimed to detect biomarkers associated with AG. Herein, H. pylori-positive subjects (n = 200) were divided into the AG (n = 94) and non-AG (n = 106) groups. Thirty tag SNPs were selected from the above seven candidate genes. The SNP frequency between the two groups was analyzed. The frequency of the C/T or T/T genotype at rs4683787 of ATG7 was significantly lower in the AG group than in the non-AG group (p = 0.034, odds ratio = 0.535). Based on multivariate analysis, the C/C genotype of rs4684787 and age were independently associated with gastric mucosal atrophy. This finding helps stratify the patients needing timely endoscopic screening or early eradication of H. pylori.

ATG16L1 and ATG12 Gene Polymorphisms Are Involved in the Progression of Atrophic Gastritis.

Helicobacter pylori (H. pylori) infection causes a progression to atrophic gastritis and results in gastric cancer. Cytotoxin-associated gene A (CagA), a major virulence factor of H. pylori, is injected into gastric epithelial cells using the type IV secretion system. On the other hand, gastric epithelial cells degrade CagA using an autophagy system, which is strictly regulated by the autophagy-related (ATG) genes. This study aimed to identify SNPs in ATG5, ATG10, ATG12, and ATG16L1 associated with gastric mucosal atrophy (GMA). Here, two-hundred H. pylori-positive participants without gastric cancer were included. The degree of GMA was evaluated via the pepsinogen method. Twenty-five SNPs located in the four candidate genes were selected as tag SNPs. The frequency of each SNP between the GMA and the non-GMA group was evaluated. The rs6431655, rs6431659, and rs4663136 in ATG16L1 and rs26537 in ATG12 were independently associated with GMA. Of these four SNPs, the G/G genotype of rs6431659 in ATG16L1 has the highest odd ratio (Odds ratio = 3.835, 95% confidence intervals = 1.337-1.005, p = 0.008). Further functional analyses and prospective analyses with a larger sample size are required.

Polymorphism in autophagy-related genes LRP1 and CAPZA1 may promote gastric mucosal atrophy.

BACKGROUND: Helicobacter pylori secretes cytotoxin-associated gene A (CagA) into the gastric epithelium, causing gastric mucosal atrophy (GMA) and gastric cancer. In contrast, host cells degrade CagA via autophagy. However, the association between polymorphisms in autophagy-related genes and GMA must be fully elucidated. RESULTS: We evaluated the association between single nucleotide polymorphisms (SNPs) in autophagy-related genes (low-density lipoprotein receptor-related protein 1, LRP1; capping actin protein of muscle Z-line alpha subunit 1, CAPAZ1; and lysosomal-associated membrane protein 1, LAMP1) and GMA in 200 H. pylori-positive individuals. The frequency of the T/T genotype at rs1800137 in LRP1 was significantly lower in the GMA group than in the non-GMA group (p = 0.018, odds ratio [OR] = 0.188). The frequencies of the G/A or A/A genotype at rs4423118 and T/A or A/A genotype at rs58618380 of CAPAZ1 in the GMA group were significantly higher than those in the non-GMA group (p = 0.029 and p = 0.027, respectively). Multivariate analysis revealed that C/C or C/T genotype at rs1800137, T/A or A/A genotype at rs58618380, and age were independent risk factors for GMA (p = 0.038, p = 0.023, and p = 0.006, respectively). Furthermore, individuals with the rs1800137 C/C or C/T genotype of LRP1 had a 5.3-fold higher susceptibility to GMA. These genetic tests may provide future directions for precision medicine for individuals more likely to develop GMA. CONCLUSION: LRP1 and CAPZA1 polymorphisms may be associated with the development of GMA.

Echinocandin Resistance in Candida auris Occurs in the Murine Gastrointestinal Tract Due to FKS1 Mutations.

Candida auris is resistant to multiple antifungal agents. This study investigated its antifungal susceptibility and explored FKS1 mutations across the isolates from mice enterically colonized with wild-type C. auris and treated with echinocandin. Resistant C. auris with FKS1 mutations, including S639F, S639Y, D642Y, R1354H, or R1354Y, were isolated and found to be micafungin- and caspofungin-resistant in vivo; however, the MICs of isolates with mutation in R1354 remained below the micafungin breakpoint in vitro.

SNP rs2920280 in PSCA Is Associated with Susceptibility to Gastric Mucosal Atrophy and Is a Promising Biomarker in Japanese Individuals with Helicobacter pylori Infection.

Helicobacter pylori infection results in gastric cancer (GC) with gastric mucosal atrophy (GMA). Some single-nucleotide polymorphisms (SNPs) in the prostate stem cell antigen gene (PSCA) are associated with GC and duodenal ulcers. However, the relationship of other identified SNPs in PSCA with these diseases remains unclear. Herein, the association between PSCA SNPs and GMA among 195 Japanese individuals with H. pylori infection was evaluated. The definition of GMA or non-GMA was based on serum pepsinogen levels or endoscopic findings. Five tag PSCA SNPs were analyzed using PCR high-resolution melting curve analysis with nonlabelled probes. The frequencies of alleles and the genotypes of each tag SNP were compared between the GMA and non-GMA groups. Subsequently, a genetic test was performed using associated SNPs as biomarkers to detect patients developing GMA. Two tag PSCA SNPs (rs2920280 and rs2294008) were related to GMA susceptibility. Individuals with the rs2920280 G/G genotype or the rs2294008 T/T genotype in PSCA had 3.5- and 2.1-fold susceptibility to GMA, respectively. In conclusion, SNP rs2920280 is a possible biomarker for detecting individuals developing GMA. PSCA polymorphisms may be useful biomarkers for predicting GMA linked to GC risk and a screening endoscopy strategy to detect GC related to early stage H. pylori associated GMA.

Isolation and characterization of fetal nucleated red blood cells from maternal blood as a target for single cell sequencing-based non-invasive genetic testing.

PURPOSE: Although non-invasive prenatal testing (NIPT) based on cell-free DNA (cfDNA) in maternal plasma has been prevailing worldwide, low levels of fetal DNA fraction may lead to false-negative results. Since fetal cells in maternal blood provide a pure source of fetal genomic DNA, we aimed to establish a workflow to isolate and sequence fetal nucleated red blood cells (fNRBCs) individually as a target for NIPT. METHODS: Using male-bearing pregnancy cases, we isolated fNRBCs individually from maternal blood by FACS, and obtained their genomic sequence data through PCR screening with a Y-chromosome marker and whole-genome amplification (WGA)-based whole-genome sequencing. RESULTS: The PCR and WGA efficiencies of fNRBC candidates were consistently lower than those of control cells. Sequencing data analyses revealed that although the majority of the fNRBC candidates were confirmed to be of fetal origin, many of the WGA-based genomic libraries from fNRBCs were considered to have been amplified from a portion of genomic DNA. CONCLUSIONS: We established a workflow to isolate and sequence fNRBCs individually. However, our results demonstrated that, to make cell-based NIPT targeting fNRBCs feasible, cell isolation procedures need to be further refined such that the nuclei of fNRBCs are kept intact.

JAK/STAT3 and NF-κB Signaling Pathways Regulate Cancer Stem-Cell Properties in Anaplastic Thyroid Cancer Cells.

Background: Anaplastic thyroid carcinoma (ATC) is one of the most aggressive and refractory cancers, and a therapy with a new concept needs to be developed. Recently, research on cancer stem cells (CSCs) has progressed, and CSCs have been suggested to be responsible for metastasis, recurrence, and therapy resistance. In ATC-CSCs, aldehyde dehydrogenase (ALDH) activity is the most reliable marker to enrich CSCs. However, it is just a marker and is not involved in CSC properties. The present study therefore aimed to identify key signaling pathways specific for ATC-CSCs. Methods: A small interfering RNA library targeting 719 kinases was used in a sphere formation assay and cell survival assay using ATC cell lines to select target molecules specific for CSC properties. The functions of the selected candidates were confirmed by sphere formation, cell survival, soft agar, and nude mice xenograft assays using small compound inhibitors. Results: The study focused on PDGFR, JAK, and PIM, whose small interfering RNAs had a higher inhibitory effect on sphere formation, as well as a lower or no effect on regular cell growth in both FRO and KTC3 cells. Next, inhibitors of PDGFR, JAK, STAT3, PIM and NF-κB were used, and all of them successfully suppressed sphere formation in a dose-dependent manner but not regular cell growth, confirming the screening results. Inhibition of the JAK/STAT3 and NF-κB pathways also reduced anchorage-independent growth in soft agar and tumor growth in nude mice. Conclusions: These results suggest that JAK/STAT3 and NF-κB signals play important roles in ATC-CSCs. Targeting these signaling pathways may be a promising approach to treat ATC.

Genetic variants of SMAD2/3/4/7 are associated with susceptibility to ulcerative colitis in a Japanese genetic background.

PURPOSE: Inflammatory bowel disease (IBD), which includes Crohn's disease (CD) and ulcerative colitis (UC), is attributed to inappropriate inflammatory response in intestinal mucosa. Transforming growth factor ß (TGF-ß)/SMAD signaling plays key role in differentiation of naïve CD4+ T cells to T helper 17 (Th17) cells or regulatory T (Treg) cells. This study aimed to investigate associations between single nucleotide polymorphisms (SNPs) of SMAD family genes and susceptibility to IBD in a Japanese cohort to elucidate genetic determinants of IBD. METHODS: This study included 81 patients with CD, 108 patients with UC, and 199 healthy subjects as controls. A total of 21 SNPs in four genes (SMAD2, SMAD3, SMAD4, and SMAD7) involved in the TGF-ß/SMAD signaling pathway were genotyped by polymerase chain reaction (PCR)-restriction fragment length polymorphism, PCR-direct DNA sequencing, or PCR-high resolution melting curve analysis. RESULTS: Four SNPs (rs13381619, rs9955626, rs1792658, and rs1792671) within SMAD2, one SNP within SMAD3 (rs41473580), two SNPs within SMAD4 (rs7229678 and rs9304407), and one SNP within SMAD7 (rs12956924) were significantly associated with susceptibility only to UC. rs13381619 within SMAD2, rs4147358 within SMAD3, rs9304407 within SMAD4, and rs12956924 within SMAD7 exhibited the strongest association (p < 0.001, p = 0.021, p = 0.005, and p = 0.001, respectively). Furthermore, rs4147358 of SMAD3 altered the expression of a luciferase reporter gene in Jurkat T cell line in vitro. CONCLUSIONS: Genetic variants of several SMAD family of genes might alter the balance of differentiation between Th17 and Treg, resulting in the development of IBD, especially UC.

Blockade of analgesic effects following systemic administration of N-methyl-kyotorphin, NMYR and arginine in mice deficient of preproenkephalin or proopiomelanocortin gene.

Kyotorphin is a unique biologically active neuropeptide (l-tyrosine-l-arginine), which is reported to have opioid-like analgesic actions through a release of Met-enkephalin from the brain slices. N-methyl-l-tyrosine-l-arginine (NMYR), an enzymatically stable mimetic of kyotorphin, successfully caused potent analgesic effects in thermal and mechanical nociception tests in mice when it was given through systemic routes. NMYR analgesia was abolished in µ-opioid receptor-deficient (MOP-KO) mice, and by intracerebroventricular (i.c.v.) injection of naloxone and of N-methyl l-leucine-l-arginine (NMLR), a kyotorphin receptor antagonist. In the Ca2+-mobilization assay using CHO cells expressing Gαqi5 and hMOPr or hDOPr, however, the addition of kyotorphin neither activated MOPr-mechanisms, nor affected the concentration-dependent activation of DAMGO- or Met-Enkephalin-induced MOPr activation, and Met-enkephalin-induced DOPr activation. NMYR-analgesia was significantly attenuated in preproenkephalin (PENK)- or proopioimelanocortin (POMC)-KO mice. The systemic administration of arginine, which is reported to elevate the level of endogenous kyotorphin selectively in midbrain and medulla oblongata, pain-related brain regions, caused significant analgesia, and the analgesia was reversed by i.c.v. injection of NMLR or naloxone. In addition, PENK- and POMC-KO mice also attenuated the arginine-induced analgesia. All these findings suggest that NMYR and arginine activate brain kyotorphin receptor in direct and indirect manner, respectively and both compounds indirectly cause the opioid-like analgesia through the action of endogenous opioid peptides.

Epidermal growth factor signals regulate dihydropyrimidine dehydrogenase expression in EGFR-mutated non-small-cell lung cancer.

BACKGROUND: It has been shown that epidermal growth factor receptor (EGFR) mutation status is associated with 5-fluorouracil (5-FU) sensitivity in non-small-cell lung cancer (NSCLC). However, the relationship between EGFR mutation status and dihydropyrimidine dehydrogenase (DPD), a 5-FU degrading enzyme, is unknown. METHODS: We elucidated the crosstalk among the EGFR signal cascade, the DPD gene (DPYD), and DPD protein expression via the transcription factor Sp1 and the effect of EGFR mutation status on the crosstalk. RESULTS: In the PC9 (exon19 E746-A750) study, EGF treatment induced up-regulation of both Sp1 and DPD; gefitinib, an EGFR-tyrosine kinase inhibitor (EGFR-TKI), and mithramycin A, a specific Sp-1 inhibitor, suppressed them. Among EGFR-mutated (PC9, HCC827; exon19 E746-A750 and H1975; exon21 L858R, T790M, gefitinib resistant) and -non-mutated (H1437, H1299) cell lines, EGF administration increased DPYD mRNA expression only in mutated cells (p < 0.05). Accordingly, gefitinib inhibited DPD protein expression only in PC9 and HCC827 cells, and mithramycin A inhibited it in EGFR-mutated cell lines, but not in wild-type. FU treatment decreased the level of cell viability more in gefitinib-treated EGFR-TKI sensitive cell lines. Further, combination treatment of FU and mithramycin A suppressed cell viability even in a gefitinib resistant cell line. CONCLUSIONS: The EGFR signal cascade regulates DPD expression via Sp1 in EGFR mutant cells. These results might be a step towards new therapies targeting Sp1 and DPD in NSCLC with different EGFR mutant status.

A Phase II Study of S-1 for Previously Untreated Elderly Patients with Advanced Non-Small Cell Lung Cancer.

BACKGROUND: S-1, a novel oral fluoropyrimidine, is active in the treatment of non-small cell lung cancer (NSCLC). However, data on S-1 for elderly patients with NSCLC are insufficient. METHODS: Eligibility criteria were no prior chemotherapy, stage IIIB or IV NSCLC, performance status 0-1, age >70 years, and adequate hematological, hepatic, and renal functions. Patients received S-1 (40 mg/m(2) twice a day) for 28 consecutive days. This schedule was repeated every 6 weeks. The primary end point was the tumor response rate. RESULTS: Thirty-two patients were enrolled and 31 patients were evaluable for response. The patients' median age was 80 years (range: 71-88). The response rate was 22.6% (95% CI: 11-38). Neutropenia, anemia, thrombocytopenia, febrile neutropenia, and diarrhea of grade ≥ 3 occurred in 6, 6, 10, 3, and 3%, respectively. CONCLUSIONS: In elderly patients with previously untreated advanced NSCLC, S-1 appears to be well tolerated and demonstrates encouraging activity.

Drug fever after cancer chemotherapy is most commonly observed on posttreatment days 3 and 4.

BACKGROUND: This study was undertaken to analyze the characteristics of fever after cancer chemotherapy in order to reduce unnecessary medical care. METHODS: Retrospectively, 1016 consecutive cycles of cancer chemotherapy were analyzed. Fever was defined as a temperature of ≥ 37.5 °C lasting for 1 h. Age, sex, tumor histology, the treatment regimen, the timing of fever onset, the number of days for which the fever persisted, the cause of the fever, the presence or absence of radiotherapy, and the use of granulocyte colony-stimulating factor (G-CSF) were examined. RESULTS: The patients included 748 males and 268 females (median age = 68, range = 29-88), of whom 949, 52, and 15 were suffering from lung cancer, malignant pleural mesothelioma, and other diseases, respectively. Fever was observed in 367 cycles (36 %), including 280 cycles (37 %) involving males and 87 cycles (32 %) involving females. Fever occurred most commonly in the first cycles and was higher than later cycles (41 vs. 30 %, p < 0.001). Fever occurred most frequently on posttreatment days 4 (8 %), 3 (7 %), and 12 (7 %), and the distribution of fever episodes exhibited two peaks on posttreatment days 3 and 4 and 10-14. Fever on posttreatment days 3 and 4 was most commonly observed in patients treated with gemcitabine (20 %) or docetaxel (18 %). The causes of fever included infection (47 %; including febrile neutropenia [24 %]), adverse drug effects (24 %), unknown causes (19 %), and tumors (7 %). Radiotherapy led to a significant increase in the frequency of fever (46 vs. 34 %, p < 0.001). Thirty-three percent of patients received G-CSF, and the incidence ratios of fever in patients who received G-CSF were higher than those who did not receive G-CSF (44 vs. 31 %, p < 0.001). CONCLUSION: The febrile episodes that occurred on posttreatment days 3 and 4 were considered to represent adverse drug reactions after cancer chemotherapy. Physicians should be aware of this feature of chemotherapy-associated fever and avoid unnecessary examination and treatments including prescribing antibiotics.

Genetic Polymorphisms of IL-17F and TRAF3IP2 Could Be Predictive Factors of the Long-Term Effect of Infliximab against Crohn's Disease.

BACKGROUND: We aimed to identify certain genes related to response to infliximab (IFX) and biomarkers to predict the IFX effect for Japanese Crohn's disease (CD) patients by performing an association study of single nucleotide polymorphisms (SNPs) in candidate genes in the interleukin- (IL-) 17 signaling pathway with response to IFX after 1 year of treatment. METHODS: A total of 103 patients were divided into two groups, responders and nonresponders. Twenty-eight tag SNPs in 5 genes were genotyped. The frequencies of alleles and genotypes of each SNP were compared between responders and nonresponders in three different inheritance models. A genetic test was performed using a combination of the associated SNPs as biomarkers. RESULTS: Multivariate logistic regression analysis indicated that the four variable factors, concomitant use of immunomodulators, penetrating disease, a G/G genotype of rs766748 in IL-17F, and a C/C or C/A genotype of rs1883136 in TRAF3IP2, independently contributed to response to IFX after 1 year of treatment. Genetic test using the polymorphisms of these genes perfectly predicted the responder and nonresponder CD patients with both concomitant use of immunomodulators and penetrating disease. CONCLUSION: IL17F and TRAF3IP2 are one of IFX-related genes, useful as biomarkers of IFX response, and may be target molecules for new therapeutic drugs.

A phase II study of amrubicin and carboplatin for previously untreated patients with extensive-disease small cell lung cancer.

BACKGROUND: Amrubicin is active in the treatment of extensive-disease small cell lung cancer (ED-SCLC), and carboplatin is an analogue of cisplatin with less non-hematological toxicity. PURPOSE: The purpose of this study was to determine the efficacy and toxicity of amrubicin and carboplatin combination chemotherapy for previously untreated patients with ED-SCLC. PATIENTS AND METHODS: Eligibility criteria were chemotherapy-naïve ED-SCLC patients, performance status 0-1, age ≤75, and adequate hematological, hepatic and renal function. Based on the phase I study, the patients received amrubicin 35 mg/m(2) i.v. infusion on days 1, 2, and 3, and carboplatin AUC 5 i.v. infusion on day 1. Four cycles of chemotherapy were repeated every 3 weeks. RESULTS: Thirty-five patients were enrolled, and 34 patients were eligible and assessable for response, toxicity, and survival. Patients' characteristics were as follows: male/female = 26/8; performance status 0/1 = 4/30; median age (range) = 64 (41-75); stage IV = 34. Evaluation of responses was 6 complete response, 21 partial response, and 7 stable disease (response rate 79.4 %, 95 % CI 63.6-88.5 %). Grade 3 and 4 leukopenia, neutropenia, and thrombocytopenia occurred in 59, 82, and 26 %, respectively. There were no treatment-related deaths or pneumonitis. Three patients experienced hypotension as an amrubicin infusion reaction. The median progression-free survival time was 6.5 months. The median overall survival time and 1-, 2-, and 3-year survival rates were 15.6 months, and 63, 28, and 7 %, respectively. CONCLUSIONS: Amrubicin and carboplatin were effective and tolerable as chemotherapy for previously untreated patients with ED-SCLC. Further investigation of amrubicin and carboplatin is warranted.

High expression of dihydropyrimidine dehydrogenase in lung adenocarcinoma is associated with mutations in epidermal growth factor receptor: implications for the treatment of non--small-cell lung cancer using 5-fluorouracil.

BACKGROUND: It has been shown that 5-fluorouracil (5-FU) sensitivity in patients with non-small-cell lung cancer (NSCLC) is associated with epidermal growth factor receptor (EGFR) mutation status. However, the relationship between dihydropyrimidine dehydrogenase (DPD), a 5-FU degrading enzyme, and EGFR mutation status is unknown. Here, we focus on clinicopathologic factors and in vitro correlations between DPD expression and EGFR mutation status. PATIENTS AND METHODS: EGFR mutations and messenger RNA (mRNA) levels of DPD and thymidylate synthase (TS) were analyzed in 47 resected NSCLC tumors by laser-capture microdissection. In addition, relationships between EGFR mutation status and the immunohistochemical expression of DPD and TS in 49 patients with primary NSCLC who were treated with a 5-FU derivative of S-1 postoperatively were examined. Correlations among clinicopathologic factors were evaluated. The effect of epidermal growth factor on DPD expression was also investigated in vitro in various cell lines. RESULTS: Adenocarcinoma in situ showed significantly higher DPD mRNA levels and more EGFR mutation frequency than other histological types (P < .05). DPD immunopositive cases were more frequently observed in adenocarcinoma, in females, and in nonsmokers. DPD immunopositive cases were correlated with EGFR mutation status (P < .003). The prognoses of wild-type EGFR and mutated EGFR populations were similarly favorable with postoperative S-1 treatment, which overcomes the problem of 5-FU degradation in mutated EGFR. In vitro, EGFR-mutated cell lines showed high DPD mRNA and protein expression. CONCLUSION: High DPD expression was shown to be correlated with EGFR mutation in adenocarcinoma cells and tissues. Clinicians should take this finding into consideration when using 5-FU to treat patients with NSCLC.

Phase II trial of erlotinib in patients with advanced non­small­cell lung cancer harboring epidermal growth factor receptor mutations: additive analysis of pharmacokinetics.

BACKGROUND: We conducted a phase II trial of erlotinib in patients with advanced non-small-cell lung cancer (NSCLC) harboring epidermal growth factor receptor (EGFR) mutations and evaluated the relationship between plasma concentration and efficacy of erlotinib. METHODS: Patients who were previously treated but naive to epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs), with advanced NSCLC harboring EGFR mutations, were enrolled. Erlotinib was given at 150 mg once daily until disease progression. The primary end point was objective response rate (ORR). Plasma trough levels of erlotinib were measured on Days 2 (D2) and 8 (D8) by high-performance liquid chromatography. RESULTS: In total, 29 patients were enrolled from September 2008 to January 2011. ORR was 61.5 % (95 % confidence interval [CI] 40.57­79.8) of 26 assessable patients. The median progression-free survival (PFS) and overall survival (OS) were 6.3 months and 16.9 months, respectively. Skin rash was observed in 24 patients, mostly at grade 1 or 2. Grade 2 pneumonitis was observed in one patient. We collected blood samples from 16 patients. The median PFS of the high and low D8/D2 ratio group was 11.2 months and 5.7 months, respectively (p = 0.044, hazard ratio = 0.301, 95 % CI 0.094­0.968). CONCLUSION: Erlotinib showed an ORR comparable to that seen in previous studies for patients with NSCLC harboring EGFR mutations, although response, the primary end point, did not reach the predetermined threshold level. The D8/D2 ratio of erlotinib plasma trough levels might be a predictive factor for PFS.

Analysis of intratumor heterogeneity of EGFR mutations in mixed type lung adenocarcinoma.

BACKGROUND: Epidermal growth factor receptor mutations are predictive of the success of EGFR tyrosine kinase inhibitor treatment in patients with advanced non--small-cell lung cancer. As with other solid tumors, lung cancer is thought to be the result of an accumulation of genetic alterations after exposure to carcinogens. The aim of the present study was to clarify the relationship between multistep carcinogenesis and the accumulation of EGFR mutations. PATIENTS AND METHODS: The intratumor heterogeneity of EGFR mutations was analyzed in 38 patients with resected mixed-type lung adenocarcinoma according to histological patterns, and the clinical features of the patients harboring intratumor heterogeneity of EGFR mutations were evaluated. RESULTS: Intratumor heterogeneity of EGFR mutations was detected in 9 of 38 tumors. EGFR mutations were more common in the bronchioloalveolar (lepidic) carcinoma pattern than in the papillary and acinar patterns, although this difference was not significant. However, there was a significant correlation between intratumor heterogeneity of EGFR mutations and smoking history (P < .043). CONCLUSION: Intratumor heterogeneity of EGFR mutations correlates with the distribution of histological subtype in mixed type adenocarcinoma and is associated with smoking history.

Novel serine/threonine kinase 11 gene mutations in Peutz-Jeghers syndrome patients and endoscopic management.

AIM: To explore mutations in serine/threonine kinase 11 (STK11) gene in Peutz-Jeghers syndrome (PJS) with gastrointestinal (GI) hamartomatous polyps. METHODS: Six Japanese PJS patients in 3 families were enrolled in this study. Each of the cases had hamartomatous polyposis in the gastrointestinal tract, including the small intestine, along with mucocutaneous hyperpigmentation. Narrow-band imaging (NBI)-magnification endoscopy was employed to detect microvascular and microsurface irregularities in the GI lesions. NBI magnification findings could be classified into three groups (type A, type B, or type C). Endoscopic polypectomy was performed using double-balloon enteroscopy or colonoscopy. Genomic DNA was extracted from a whole blood sample from each subject. All of the coding exons of STK11 gene, its boundary regions, and the promoter region containing the polymorphic regions were amplified by polymerase chain reaction, and direct sequencing was performed to assess the germline mutations. RESULTS: NBI-magnification endoscopic observation could detect the abnormalities in microvessels and microsurface structures of GI polyps. Overall, we found 5 cases of type A and one case without the examination for the gastric polyps, while there were 4 cases of type B and 2 case of type A for the colorectal polyps. Seventy-nine small-bowel and 115 colorectal polyps over 27 sessions for each were resected endoscopically without significant complications. The only delayed complication included the occurrence of bleeding in a case, and this was successfully managed with hemoclips. Resected polyps contained no malignant components. Based on mutation analysis, all 3 cases in Family I exhibited the +658C>T nonsense mutation in exon 5, which resulted in the production of a truncated protein (Q220X). In Family II, a case had -252C>A and -193C>A in the promoter region. In Family III, a case was found to have the +1062C>G (F342L) mutation in exon 8. CONCLUSION: We found two novel mutations of STK11 in association with PJS. Endoscopic polypectomy of GI polyps in PJS patients appears to be useful to prevent emergency laparotomies and reduce the cancer risk.

Association of genes involved in bile acid synthesis with the progression of primary biliary cirrhosis in Japanese patients.

BACKGROUND: Patients with primary biliary cirrhosis (PBC) exhibit a variety of clinical manifestations and patterns of disease progression. The aim of this study was to identify genetic determinants of PBC progression. METHODS: A total of 52 tag single nucleotide polymorphisms (SNPs) of 11 candidate genes involved in regulating bile acid synthesis were analyzed by polymerase chain reaction (PCR)-restriction fragment length polymorphism, -high resolution melting curve analysis, or -direct DNA sequencing in 315 Japanese patients with PBC. RESULTS: In this study, four tag SNPs of CYP7A1 (rs1457043, rs8192870, rs3808607, and rs3824260), two tag SNPs of HNF4A (rs6017340 and 6031587), and one SNP of PPARGC1A (rs8192678) showed a significant association with PBC progression. In addition, a dual luciferase assay revealed that the polymorphism of rs3808607 in CYP7A1 altered the expression of CYP7A1 in HepG2. Specifically, the CYP7A1 promoter carrying the risk G allele for PBC progression induced higher expression of CYP7A1 under both the normal and cholestatic conditions in vitro as compared to another promoter carrying the non-risk T allele. CONCLUSION: These results suggested that the genetic variants of CYP7A1 and its transcriptional activators (HNF4A and PPARGC1A) may activate bile acid synthesis, resulting in the accumulation of bile acids in hepatocytes and eventually leading to the predisposition to PBC progression. Thus, the regulation of CYP7A1 expression may represent an attractive therapeutic target for cholestatic liver diseases including PBC.

Phase II study of irinotecan and cisplatin with concurrent split-course radiotherapy in limited-disease small cell lung cancer.

BACKGROUND: Irinotecan and cisplatin are one of active regimens for patients with extensive-stage small cell lung cancer (SCLC). To determine the efficacy and toxicity of irinotecan and cisplatin with concurrent split-course thoracic radiotherapy in limited-disease (LD) SCLC, we conducted a phase II study. PATIENTS AND METHODS: Thirty-four patients fulfilling the following eligibility criteria were enrolled: chemotherapy-naïve, good performance status (PS 0-1), age ≤75, LD-SCLC, and adequate organ function. The patients received irinotecan 40 mg/m(2) i.v. on days 1, 8, and 15, and cisplatin 60 mg/m(2) i.v. on day 1. Four cycles of chemotherapy were repeated every 4 weeks. Split-course thoracic radiotherapy of once-daily 2 Gy/day commenced on day 2 of each chemotherapy cycle, with 26 and 24 Gy administered in the first and second cycles, respectively. RESULTS: Thirty-four patients were eligible and assessable for response, toxicity, and survival. Patients' characteristics were as follows: male/female = 29/5; PS 0/1 = 18/16; median age (range) = 67 (50-73); and stage IB/IIA/IIB/IIIA/IIIB = 2/2/3/16/11. The overall response was 100 % (CR 8, PR 26). Grade 4 leukopenia, neutropenia, grade 3-5 pneumonitis, diarrhea, and esophagitis occurred in 24, 38, 6, 3, and 0 %, respectively. There were 2 treatment-related deaths from pneumonitis. The median time to tumor progression was 14.3 months. The median overall survival time and the 2- and 5-year survival rates were 44.5 months, 66.7 and 46.1 %, respectively. No tumor progression was observed in patients with CR. CONCLUSION: Irinotecan plus cisplatin with concurrent split-course thoracic radiotherapy was effective and tolerable in untreated LD-SCLC.