稲村 健太郎

Marine ω-3 polyunsaturated fatty acids (PUFAs), including eicosapentaenoic acid, docosahexaenoic acid, and docosapentaenoic acid, possess potent immunomodulatory activity and may protect against cancer development. However, evidence relating marine ω-3 PUFAs to colorectal cancer (CRC) risk remains inconclusive.|To test the hypothesis that marine ω-3 PUFA intake may be associated with lower risk of CRC subsets characterized by immune infiltrate.|This prospective cohort study was conducted among participants in the Nurses' Health Study (1984-2010) and Health Professionals Follow-up Study (1986-2010).|Intake of marine ω-3 PUFAs.|Incidence of CRC characterized by CD3+, CD8+, CD45RO (PTPRC)+, or FOXP3+ T-cell densities in tumor tissue, measured by immunohistochemical and computer-assisted image analysis.|Among 173 229 predominantly white participants, 125 172 with 2 895 704 person-years of follow-up provided data about marine ω-3 PUFA intake every 4 years through a validated food frequency questionnaire and followed up for incident CRC evaluation. Of 2504 CRC cases, we documented 614 (252 men, 362 women) from which we could assess T-cell infiltration in the tumor microenvir

Higher body mass index (BMI), higher body adiposity and obesity have been associated with increased risk of colorectal cancer. Evidence suggests that excess energy balance may influence systemic immune and inflammatory status. Thus, we hypothesized that the positive association between BMI and colorectal cancer risk might differ according to colorectal carcinoma subtypes according to levels of histopathological lymphocytic reaction to tumor. We collected biennial questionnaire data on weight and baseline height information in two prospective cohort studies, the Nurses' Health Study (1980-2010) and the Health Professionals Follow-up Study (1986-2010). Utilizing duplication-method Cox proportional hazards regression models, we prospectively assessed the association between BMI and risk of colorectal cancer subtypes according to the degree of Crohn's-like lymphoid reaction, peritumoral lymphocytic reaction, intratumoral periglandular reaction, tumor-infiltrating lymphocytes, the overall lymphocytic reaction score, or T-cell [CD3(+) , CD8(+) , CD45RO (PTPRC)(+) or FOXP3(+) ] density in tumor tissue. Statistical significance level was adjusted for multiple hypotheses testing by Bonferroni correction. During follow up of 1,708,029 men and women (over 3,346,752 person-years), we documented 1,436 incident rectal and colon cancer cases with available formalin-fixed paraffin-embedded tumor tissue materials and pathological immunity data. BMI was significantly associated with higher risk of overall colorectal cancer (Ptrend  < 0.001); however, the association of BMI with colorectal carcinoma risk did not significantly differ by the level of lymphocytic reaction or T-cell infiltration in tumor tissue status (Pheterogeneity  > 0.10). BMI may be associated with risk of colorectal cancer regardless of levels of lymphocytic response to tumor.

Large-scale genomic characterization of tumors from prospective cohort studies may yield new insights into cancer pathogenesis. We performed whole-exome sequencing of 619 incident colorectal cancers (CRCs) and integrated the results with tumor immunity, pathology, and survival data. We identified recurrently mutated genes in CRC, such as BCL9L, RBM10, CTCF, and KLF5, that were not previously appreciated in this disease. Furthermore, we investigated the genomic correlates of immune-cell infiltration and found that higher neoantigen load was positively associated with overall lymphocytic infiltration, tumor-infiltrating lymphocytes (TILs), memory T cells, and CRC-specific survival. The association with TILs was evident even within microsatellite-stable tumors. We also found positive selection of mutations in HLA genes and other components of the antigen-processing machinery in TIL-rich tumors. These results may inform immunotherapeutic approaches in CRC. More generally, this study demonstrates a framework for future integrative molecular epidemiology research in colorectal and other malignancies.

Background: Low levels of adiponectin (ADIPOQ; HGNC ID; HGNC: 13633), an adipokine, are associated with obesity, adiposity, excess energy balance, and increased risk of colorectal neoplasia. Given the reported association of increased body mass index (BMI) and low-level physical activity with KRAS-mutated colorectal tumor, we hypothesized that low-level plasma adiponectin might be associated with increased risk of KRAS-mutant colorectal carcinoma but not with risk of KRAS wild-type carcinoma. Methods: We conducted molecular pathological epidemiology research using a nested case-control study design (307 incident rectal and colon cancer case patients and 593 matched control individuals) within prospective cohort studies, the Nurses' Health Study (152 case patients and 297 control individuals, with blood collection in 1989-1990) and the Health Professionals Follow-up Study (155 case patients and 296 control individuals, with blood collection in 1993-1995). Multivariable conditional logistic regression models and two-sided likelihood ratio tests were used to assess etiologic heterogeneity of the associations. Results: The association of low-level plasma adiponectin with colorectal cancer risk statistically significantly differed by KRAS mutation status (P-heterogeneity = .004). Low levels of plasma adiponectin were associated with KRAS-mutant colorectal cancer (for the lowest vs highest tertile: multivariable odds ratio [OR] = 2.83, 95% confidence interval [CI] = 1.50 to 5.34, P-trend = .002) but not with KRAS wild-type cancer (for the lowest vs highest tertile: multivariable OR = 0.83, 95% CI = 0.49 to 1.43, P-trend = .48). In secondary analyses, the association between plasma adiponectin and colorectal cancer did not appreciably differ by BRAF or PIK3CA oncogene mutation status. Conclusions: Low-level plasma adiponectin is associated with KRAS-mutant colorectal cancer risk but not with KRAS wildtype cancer risk.

Lung cancer is the leading cause of cancer death in men and women worldwide. The lack of specific and sensitive tools for early diagnosis as well as still-inadequate targeted therapies contribute to poor outcomes. MicroRNAs are small non-coding RNAs, which regulate gene expression post-transcriptionally by translational repression or degradation of target mRNAs. A growing body of evidence suggests various roles of microRNAs including development and progression of lung cancer. In lung cancer, several studies have showed that certain microRNA profiles classified lung cancer subtypes, and that specific microRNA expression signatures distinguished between better-prognosis and worse-prognosis lung cancers. Furthermore, microRNAs circulate in body fluids, and therefore may serve as promising biomarkers for early diagnosis of lung cancer as well as for predicting prognosis of patients. In the present review, we briefly summarize microRNAs in the development and progression of lung cancer, focusing on possible applications of microRNAs as novel biomarkers and tools for treatment.

Purpose: We hypothesized that adverse prognostic associations of specific tumor molecular factors vary by patient age at colorectal cancer diagnosis. Experimental Design: We examined the prognostic associations and interactions by age at colorectal cancer diagnosis (&lt;60 vs. 60-74 vs. &gt;= 75 years old) of key molecular factors-CpG island methylator phenotype (CIMP), microsatellite instability (MSI), KRAS, BRAF, and PIK3CA mutations, and nuclear CTNNB1 expression status-on colorectal cancer-specific survival (CSS) and overall survival (OS), using 1,280 incident colorectal cancer cases (median age, 69 years; range, 38-91 years) within the Nurses' Health Study and Health Professionals Follow-up Study cohorts. Results: MSI-high was associated with better survival, whereas BRAF mutation was associated with worse survival, but these associations did not appreciably differ by age group. Status of CIMP, KRAS mutation, or PIK3CA mutation was not associated with prognosis regardless of age. Nuclear CTNNB1 expression was associated with a trend toward worse prognosis among older adults [age &gt;= 75 years; multivariate HR, 1.67; 95% confidence interval (CI), 0.89-3.13 (for CSS); multivariate HR, 1.44; 95% CI, 0.93-2.24 (for OS)] but not among younger patients, and there was a statistically significant interaction by age (P-interaction = 0.03 for CSS; P-interaction = 0.007 for OS). Conclusions: Tumor nuclear CTNNB1 expression may be associated with higher mortality among older patients with colorectal cancer but not among younger patients. Our findings need to be confirmed in independent datasets. Detailed exploration of tumor molecular signatures in older patients with colorectal cancer in large populations is warranted. (C) 2015 AACR.

Background. High-level physical activity is associated with lower colorectal cancer (CRC) mortality, likely through insulin sensitization. Insulin receptor substrate 1 (IRS1) is a mediator of insulin and insulin-like growth factor (IGF) signaling pathways, and its down-regulation is associated with insulin resistance. Therefore, we hypothesized that tumor IRS1 expression status might modify cellular sensitivity to insulin and IGF, and the prognostic association of physical activity. Methods. We assessed IRS1 expression level in 371 stage I-III rectal and colon cancers in the Nurses' Health Study and the Health Professionals Follow-up Study by immunohistochemistry. In survival analysis, Cox proportional hazards model was used to assess an interaction between post-diagnosis physical activity (ordinal scale of sex-specific quartiles Q1 to Q4) and IRS1 expression (ordinal scale of negative, low, and high), controlling for potential confounders, including microsatellite instability, CpG island methylator phenotype, long interspersed nucleotide element-1 (LINE-1) methylation level, and KRAS, BRAF, and PIK3CA mutation status. Results. There was a statistically significant interaction between post-diagnosis physical activity and tumor IRS1 expression in CRC-specific mortality analysis (P-interaction = 0.005). Multivariable hazard ratio (95 % confidence interval) for higher post-diagnosis physical activity (Q3-Q4 vs. Q1-Q2) was 0.15 (0.02-1.38) in the IRS1-negative group, 0.45 (0.19-1.03) in the IRS1-low group, and 1.32 (0.50-3.53) in the IRS1-high group. Conclusions. The association of post-diagnosis physical activity with colorectal carcinoma patient survival may differ by tumor IRS1 expression level. If validated, tumor IRS1 expression status may serve as a predictive marker to identify subgroups of patients who might gain greater survival benefit from an increased level of exercise.

Objective Evidence suggests protective effects of vitamin D and antitumour immunity on colorectal cancer risk. Immune cells in tumour microenvironment can convert 25-hydroxyvitamin D [25(OH)D] to bioactive 1 alpha, 25-dihydroxyvitamin D-3, which influences neoplastic and immune cells as an autocrine and paracrine factor. Thus, we hypothesised that the inverse association between vitamin D and colorectal cancer risk might be stronger for cancers with high-level immune response than those with low-level immune response. Design We designed a nested case-control study (318 rectal and colon carcinoma cases and 624 matched controls) within the Nurses' Health Study and Health Professionals Follow-up Study using molecular pathological epidemiology database. Multivariable conditional logistic regression was used to assess the association of plasma 25 (OH) D with tumour subtypes according to the degree of lymphocytic reaction, tumour-infiltrating T cells (CD3+, CD8+, CD45RO+ (PTPRC) and FOXP3+ cells), microsatellite instability or CpG island methylator phenotype. Results The association of plasma 25(OH)D with colorectal carcinoma differed by the degree of intratumoural periglandular reaction (p for heterogeneity=0.001); high 25(OH)D was associated with lower risk of tumour with high-level reaction (comparing the highest versus lowest tertile: OR 0.10; 95% CI 0.03 to 0.35; p for trend&lt;0.001), but not risk of tumour with lower-level reaction (p for trend&gt;0.50). A statistically non-significant difference was observed for the associations of 25(OH)D with tumour subtypes according to CD3+ T cell density (p for heterogeneity= 0.03; adjusted statistical significance level of alpha=0.006). Conclusions High plasma 25(OH)D level is associated with lower risk of colorectal cancer with intense immune reaction, supporting a role of vitamin D in cancer immunoprevention through tumour-host interaction.

The complex interactions between colorectal neoplasia and immune cells in the tumor microenvironment remain to be elucidated. Experimental evidence suggests that microRNA MIR21 (miR-21) suppresses antitumor T-cell-mediated immunity. Thus, we hypothesized that tumor MIR21 expression might be inversely associated with T-cell density in colorectal carcinoma tissue. Using 538 rectal and colon cancer cases from the Nurses' Health Study and the Health Professionals Follow-up Study, we measured tumor MIR21 expression by a quantitative reverse-transcription PCR assay. Densities of CD3(+), CD8(+), CD45RO (PTPRC)(+), and FOXP3(+) cells in tumor tissue were determined by tissue microarray immunohistochemistry and computer-assisted image analysis. Ordinal logistic regression analysis was conducted to assess the association of MIR21 expression (ordinal quartiles as a predictor variable) with T-cell density (ordinal quartiles as an outcome variable), adjusting for tumor molecular features, including microsatellite instability; CpG island methylator phenotype; KRAS, BRAF, and PIK3CA mutations; and LINE-1 methylation. We adjusted the two-sided a level to 0.012 for multiple hypothesis testing. Tumor MIR21 expression was inversely associated with densities of CD3(+) and CD45RO(+) cells (P-trend &lt; 0.0005). The multivariate odds ratio of the highest versus lowest quartile of MIR21 for a unit increase in quartile categories of CD3(+) or CD45RO(+) cells was 0.44 [95% confidence interval (CI), 0.28 to 0.68] or 0.41 (95% CI, 0.26-0.64), respectively. Our data support a possible role of tumor epigenetic deregulation by noncoding RNA in suppressing the antitumor T-cell-mediated adaptive immune response and suggest MIR21 as a potential target for immunotherapy and prevention in colorectal cancer. (C) 2015 AACR.

Background: Loss of CDH1 (E-cadherin) expression in cancer cells may promote cell migration and invasion. Therefore, we hypothesised that loss of CDH1 expression in colorectal carcinoma might be associated with aggressive features and clinical outcome. Methods: Utilising molecular pathological epidemiology database of 689 rectal and colon cancer cases in the Nurses' Health Study and the Health Professionals Follow-up Study, we assessed tumour CDH1 expression by immunohistochemistry. Multivariate logistic regression analysis was conducted to assess association of CDH1 loss with tumour growth pattern (expansile-intermediate vs infiltrative) and lymph node metastasis and distant metastasis, controlling for potential confounders including microsatellite instability, CpG island methylator phenotype, LINE-1 methylation, and PIK3CA, BRAF and KRAS mutations. Mortality according to CDH1 status was assessed using Cox proportional hazards model. Results: Loss of tumour CDH1 expression was observed in 356 cases (52%), and associated with infiltrative tumour growth pattern (odds ratio (OR), 2.02; 95% confidence interval (CI), 1.23-3.34; P = 0.006) and higher pN stage (OR, 1.73; 95% CI, 1.23-2.43; P = 0.001). Tumour CDH1 expression was not significantly associated with distant metastasis or prognosis. Conclusions: Loss of CDH1 expression in colorectal cancer is associated with infiltrative tumour growth pattern and lymph node metastasis.

Background: Observational data have suggested that intakes of nutrients involved in one-carbon metabolism are inversely associated with risk of colorectal carcinoma and adenomas. In contrast, results from some preclinical studies and cardiovascular and chemoprevention trials have raised concerns that high folate intake may promote carcinogenesis by facilitating the progression of established neoplasia. Objective: We tested the hypothesis that higher total folate intake (including food folate and folic acid from fortified foods and supplements) or other one-carbon nutrient intakes might be associated with poorer survival after a diagnosis of colorectal cancer. Design: We used rectal and colon cancer cases within the following 2 US prospective cohort studies: the Nurses' Health Study and the Health Professionals Follow-Up Study. Biennial questionnaires were used to gather information on medical history and lifestyle factors, including smoking and alcohol consumption. B-vitamin and methionine intakes were derived from food-frequency questionnaires. Data on tumor molecular characteristics (including microsatellite instability, CpG island methylator phenotype, KRAS, BRAF, and PIK3CA mutations, and long interspersed nucleotide element 1 methylation level) were available for a subset of cases. We assessed colorectal cancer-specific mortality according to postdiagnostic intakes of one-carbon nutrients with the use of multivariable Cox proportional hazards regression models. Results: In 1550 stage I-III colorectal cancer cases with a median follow-up of 14.9 y, we documented 641 deaths including 176 colorectal cancer-specific deaths. No statistically significant associations were observed between postdiagnostic intakes of folate or other one-carbon nutrients and colorectal cancer-specific mortality (multivariate P-trend &gt;= 0.21). In an exploratory molecular pathologic epidemiology survival analysis, there was no significant interaction between one-carbon nutrients or alcohol and any of the tumor molecular biomarkers examined. Conclusions: Higher postdiagnostic intakes of one-carbon nutrients are not associated with the prognosis in stage colorectal cancer. Our findings do not support the hypothesis that higher folate intake after colorectal cancer diagnosis might increase risk of cancer-related death.

IMPORTANCE Evidence indicates a complex link between gut microbiome, immunity, and intestinal tumorigenesis. To target the microbiota and immunity for colorectal cancer prevention and therapy, a better understanding of the relationship between microorganisms and immune cells in the tumor microenvironment is needed. Experimental evidence suggests that Fusobacterium nucleatum may promote colonic neoplasia development by downregulating antitumor T cell-mediated adaptive immunity. OBJECTIVE To test the hypothesis that a greater amount of F nucleatum in colorectal carcinoma tissue is associated with a lower density of T cells in tumor tissue. DESIGN, SETTING, AND PARTICIPANTS A cross-sectional analysis was conducted on 598 rectal and colon carcinoma cases in 2 US nationwide prospective cohort studies with follow-up through 2006, the Nurses' Health Study (participants enrolled in 1976) and the Health Professionals Follow-up Study (participants enrolled in 1986). Tissue collection and processing were performed from 2002 through 2008, and immunity assessment, 2008 through 2009. From 2013 through 2014, the amount of F nucleatum in colorectal carcinoma tissue was measured by quantitative polymerase chain reaction assay; we equally dichotomized positive cases (high vs low). Multivariable ordinal logistic regression analysis was conducted in 2014 to assess associations of the amount of F nucleatum with densities (quartiles) of T cells in tumor tissue, controlling for clinical and tumor molecular features, including microsatellite instability, CpG island methylator phenotype, long interspersed nucleotide element-1 (LINE-1) methylation, and KRAS, BRAF, and PIK3CA mutation status. We adjusted the 2-sided a level to.013 for multiple hypothesis testing. MAIN OUTCOMES AND MEASURES Densities of CD3(+), CD8(+), CD45RO (protein tyrosine phosphatase receptor type C [PTPRC])(+), and FOXP3(+) T cells in tumor tissue, determined by means of tissue microarray immunohistochemical analysis and computer-assisted image analysis. RESULTS F nucleatum was detected in colorectal carcinoma tissue in 76 (13%) of 598 cases. Compared with F nucleatum-negative cases, F nucleatum-high cases were inversely associated with the density of CD3(+) T cells (for a unit increase in quartile categories of CD3(+) T cells as an outcome: multivariable odds ratio, 0.47 [95% CI, 0.26-0.87]; P for trend =.006). The amount of F nucleatum was not significantly associated with the density of CD8(+), CD45RO(+), or FOXP3(+) T cells (P for trend =.24,.88, and.014, respectively). CONCLUSIONS AND RELEVANCE The amount of tissue F nucleatum is inversely associated with CD3(+) T-cell density in colorectal carcinoma tissue. On validation, our human population data may provide an impetus for further investigations on potential interactive roles of Fusobacterium and host immunity in colon carcinogenesis.

Lung adenocarcinomas in situ (AISs) often occur in individuals who have never smoked, although smoking is one of the main causes of lung cancer. To characterize AIS and, in particular, determine how AIS might be related to smoking, we collected a large number of AIS cases and examined clinicopathologic features, EGFR and KRAS mutation status, and activation status of receptor tyrosine kinase downstream signal pathways, including pAkt, pERK, and pStat3, using immunohistochemistry. We identified 110 AISs (36 smokers and 74 nonsmokers) among 1549 adenocarcinomas resected surgically during 1995 to 2010. Between the AIS of smokers and nonsmokers, only the sex ratio was significantly different; all the other clinicopathologic factors including TTF-1 and driver mutations were not significantly different: EGFR and KRAS mutation rates (smokers:nonsmokers) were 61:58 (%) (P=0.7) and 6.1:1.4 (%) (P=0.2), respectively, whereas, in invasive adenocarcinomas, the rates were 41:69 (%) (P<0.001) and 9.4:2.3 (%) (P<0.04), respectively. For pAkt and pERK, around 40% to 50% of AISs were positive, and for pStat3, >80% were positive, with no significant differences between smokers and nonsmokers with AIS. Mucinous AIS (n=8) rarely harbored KRAS mutations and expressed significantly less pStat3 (P<0.001) than nonmucinous AIS. Taken together, AIS occurs predominantly in female individuals and nonsmokers. However, characteristics of AIS arising in smokers and nonsmokers were similar in terms of cell lineage, driver mutations, and receptor tyrosine kinase pathway activation. Our results suggest that smoking is not a major cause of AIS. Rather, smoking may play a role in progression of AIS to invasive adenocarcinoma with AIS features.

Colorectal cancer (CRC) remains a major contributor to cancer-related mortality. LIN28A and LIN28B are highly related RNA-binding protein paralogs that regulate biogenesis of let-7 microRNAs and influence development, metabolism, tissue regeneration, and oncogenesis. Here we demonstrate that overexpression of either LIN28 paralog cooperates with the Wnt pathway to promote invasive intestinal adenocarcinoma in murine models. When LIN28 alone is induced genetically, half of the resulting tumors harbor Ctnnb1 (beta-catenin) mutation. When overexpressed in Apc(Min/+) mice, LIN28 accelerates tumor formation and enhances proliferation and invasiveness. In conditional genetic models, enforced expression of a LIN28-resistant form of the let-7 microRNA reduces LIN28-induced tumor burden, while silencing of LIN28 expression reduces tumor volume and increases tumor differentiation, indicating that LIN28 contributes to tumor maintenance. We detected aberrant expression of LIN28A and/or LIN28B in 38% of a large series of human CRC samples (n = 595), where LIN28 expression levels were associated with invasive tumor growth. Our late-stage CRC murine models and analysis of primary human tumors demonstrate prominent roles for both LIN28 paralogs in promoting CRC growth and progression and implicate the LIN28/let-7 pathway as a therapeutic target.

Colorectal carcinoma (CRC) represents a group of histopathologically and molecularly heterogeneous diseases, which may contain signet-ring cell component and/or mucinous component to a varying extent under pathology assessment. However, little is known about the prognostic significance of those components, independent of various tumor molecular features. Utilizing a molecular pathological epidemiology database of 1,336 rectal and colon cancers in the Nurses' Health Study and the Health Professionals Follow-up Study, we examined patient survival according to the proportion of signet-ring cell and mucinous components in CRCs. Cox proportional hazards models were used to compute hazard ratio (HR) for mortality, adjusting for potential confounders including stage, microsatellite instability, CpG island methylator phenotype, LINE-1 methylation, and KRAS, BRAF, and PIK3CA mutations. Compared to CRC without signet-ring cell component, 1-50 % signet-ring cell component was associated with multivariate CRC-specific mortality HR of 1.40 [95 % confidence interval (CI) 1.02-1.93], and &gt; 50 % signet-ring cell component was associated with multivariate CRC-specific mortality HR of 4.53 (95 % CI 2.53-8.12) (P (trend) &lt; 0.0001). Compared to CRC without mucinous component, neither 1-50 % mucinous component (multivariate HR 1.04; 95 % CI 0.81-1.33) nor &gt; 50 % mucinous component (multivariate HR 0.82; 95 % CI 0.54-1.23) was significantly associated with CRC-specific mortality (P (trend) &lt; 0.57). Even a minor (50 % or less) signet-ring cell component in CRC was associated with higher patient mortality, independent of various tumor molecular and other clinicopathological features. In contrast, mucinous component was not associated with mortality in CRC patients.

The microRNA-34 family (miR-34a, -34b and -34c) have been reported to be tumor suppressor microRNAs (miRNAs) that are regulated by the TP53 and DNA hypermethylation. However, the expression, regulation, and prognostic value of the miR-34 family have not been systematically studied in colon cancer. To elucidate the roles of miR-34 family in colon carcinogenesis, miR-34a/b/c were measured in tumors and adjacent noncancerous tissues from 159 American and 113 Chinese colon cancer patients using quantitative RT-PCR, and we examined associations between miR-34a/b/c expression with TNM staging, cancer-specific mortality, TP53 mutation status and Affymetrix microarray data. All miR-34 family members were significantly increased in colon tumors, counter to the proposed tumor suppressor role for these miRNAs. Increased miR-34b/c were observed in more advanced tumors in two independent cohorts and increased expression of miR-34b/c was associated with poor cancer-specific mortality. While the expression of miR-34 family was not associated with TP53 mutation status, TP53 transcriptional activity was associated with miR-34a/b/c expression that is consistent with the proposed regulation of miR-34a/b/c by TP53. To examine where the miR-34 family is expressed, the expression of miR-34 family was compared between epitheliums and stromal tissues using laser microdissection technique. The expression of miR-34b/c was increased significantly in stromal tissues, especially in cancer stroma, compared with epithelial tissue. In conclusion, increased miR-34b/c predominantly expressed in stromal tissues is associated with poor prognosis in colon cancer. MiR-34 may contribute to cancer-stromal interaction associated with colon cancer progression.

Smoothened, frizzled family receptor (SMO) is an important component of the hedgehog signaling pathway, which has been implicated in various human carcinomas. However, clinical, molecular, and prognostic associations of SMO expression in colorectal cancer remain unclear. Using a database of 735 colon and rectal cancers in the Nurse's Health Study and the Health Professionals Follow-up Study, we examined the relationship of tumor SMO expression (assessed by immunohistochemistry) to prognosis, and to clinical, pathological, and tumor molecular features, including mutations of KRAS, BRAF, and PIK3CA, microsatellite instability, CpG island methylator phenotype (CIMP), LINE-1 methylation, and expression of phosphorylated AKT and CTNNB1. SMO expression was detected in 370 tumors (50 %). In multivariate logistic regression analysis, SMO expression was independently inversely associated with phosphorylated AKT expression [odds ratio (OR) 0.48; 95 % confidence interval (CI) 0.34-0.67] and CTNNB1 nuclear localization (OR 0.48; 95 % CI 0.35-0.67). SMO expression was not significantly associated with colorectal cancer-specific or overall survival. However, in CIMP-high tumors, but not CIMP-low/0 tumors, SMO expression was significantly associated with better colorectal cancer-specific survival (log-rank P = 0.012; multivariate hazard ratio, 0.36; 95 % CI 0.13-0.95; P (interaction) = 0.035, for SMO and CIMP status). Our data reveal novel potential associations between the hedgehog, the WNT/CTNNB1, and the PI3K (phosphatidylinositol-4,5-bisphosphonate 3-kinase)/AKT pathways, supporting pivotal roles of SMO and hedgehog signaling in pathway networking. SMO expression in colorectal cancer may interact with tumor CIMP status to affect patient prognosis, although confirmation by future studies is needed.

Background: Although a higher consumption of alcohol, which is a methyl-group antagonist, was previously associated with colorectal cancer risk, mechanisms remain poorly understood. Objective: We hypothesized that excess alcohol consumption might increase risk of colorectal carcinoma with hypomethylation of insulin-like growth factor 2 (IGF2) differentially methylated region-0 (DMR0), which was previously associated with a worse prognosis. Design: With the use of a molecular pathologic epidemiology database in 2 prospective cohort studies, the Nurses' Health Study and Health Professionals Follow-up Study, we examined the association between alcohol intake and incident colorectal cancer according to the tumor methylation level of IGF2 DMR0. Duplication-method Cox proportional cause-specific hazards regression for competing risk data were used to compute HRs and 95% CIs. In addition, we investigated intakes of vitamin B-6, vitamin B-12, methionine, and folate as exposures. Results: During 3,206,985 person-years of follow-up, we identified 993 rectal and colon cancer cases with an available tumor DNA methylation status. Compared with no alcohol consumption, the consumption of &gt;= 15 g alcohol/d was associated with elevated risk of colorectal cancer with lower levels of IGF2 DMR0 methylation [within the first and second quartiles: HRs of 1.55 (95% CI: 1.08, 2.24) and 2.11(95% CI: 1.44, 3.07), respectively]. By contrast, alcohol consumption was not associated with cancer with higher levels of IGF2 DMR0 methylation. The association between alcohol and cancer risk differed significantly by IGF2 DMR0 methylation level (P-heterogeneity = 0.006). The association of vitamin B-6, vitamin B-12, and folate intakes with cancer risk did not significantly differ according to IGF2 DMR0 methylation level (P-heterogeneity &gt; 0.2). Conclusions: Higher alcohol consumption was associated with risk of colorectal cancer with IGF2 DMR0 hypomethylation but not risk of cancer with high-level IGF2 DMR0 methylation. The association between alcohol intake and colorectal cancer risk may differ by tumor epigenetic features.

Background Hypomethylation in long interspersed nucleotide element-1 (LINE-1) and high-degree microsatellite instability (MSI-high) in colorectal cancer (CRC) have been associated with inferior and superior survival, respectively; however, it remains uncertain whether the prognostic association of LINE-1 hypomethylation differs by MSI status. We hypothesized that the adverse prognostic association of LINE-1 hypomethylation might be stronger in MSI-high CRCs than in microsatellite stable (MSS) CRCs. Methods Utilizing 1211 CRCs in the Nurses' Health Study and the Health Professionals Follow-up Study, we examined patient survival according to LINE-1 hypomethylation status in strata of MSI status. A Cox proportional hazards model was used to compute multivariable CRC-specific mortality hazard ratios (HRs) for a 10% decrease in LINE-1 methylation level (range = 23.1-93.1%), adjusting for potential confounders, including CpG island methylator phenotype, and KRAS, BRAF, and PIK3CA mutations. Statistical tests (log-rank test, chi-square test, and likelihood ratio test) were two-sided. Results In MSI-high cancers, the association of LINE-1 hypomethylation with higher mortality (HR = 2.45, 95% confidence interval [CI] = 1.64 to 3.66, P &lt; .001) was stronger than that in MSS cancers (HR = 1.10, 95% CI = 0.98 to 1.24, P = .11) (Pinteraction &lt; .001, between LINE-1 and MSI statuses). In MSI-high cases with CRC family history, the association of LINE-1 hypomethylation with higher mortality (HR = 5.13, 95% CI = 1.99 to 13.2; P &lt; .001) was stronger than that in MSI-high cases without CRC family history (HR = 1.62, 95% CI = 0.89 to 2.94, P = .11) (Pinteraction = .02, between LINE-1 and CRC family history statuses). Conclusions The association of LINE-1 hypomethylation with inferior survival is stronger in MSI-high CRCs than in MSS CRCs. Tumor LINE-1 methylation level may be a useful prognostic biomarker to identify aggressive carcinomas among MSI-high CRCs.

Background: Despite accumulating evidence for the preventive effect of vitamin D on colorectal carcinogenesis, its precise mechanisms remain unclear. We hypothesized that vitamin D was associated with a lower risk of colorectal cancer with high-level vitamin D receptor (VDR) expression, but not with risk of tumor with low-level VDR expression. Methods: Among 140,418 participants followed from 1986 through 2008 in the Nurses' Health Study and the Health Professionals' Follow-up Study, we identified 1,059 incident colorectal cancer cases with tumor molecular data. The predicted 25-hydroxyvitamin D [25(OH)D] score was developed using the known determinants of plasma 25(OH)D. We estimated the HR for cancer subtypes using the duplication method Cox proportional hazards model. Results: Ahigher predicted 25(OH)D score was associated with a lower risk of colorectal cancer irrespective of VDR expression level (P-heterogeneity for subtypes = 0.75). Multivariate HRs (95% confidence intervals) comparing the highest with the lowest quintile of predicted 25(OH)D scores were 0.48 (0.30-0.78) for VDR-negative tumor and 0.56 (0.42-0.75) for VDR-positive tumor. Similarly, the significant inverse associations of the predicted 25(OH)D score with colorectal cancer risk did not significantly differ by KRAS, BRAF, or PIK3CA status (P-heterogeneity for subtypes &gt;= 0.22). Conclusions: Ahigher predicted vitamin Dscore was significantly associated with a lower colorectal cancer risk, regardless of VDR status and other molecular features examined. Impact: The preventive effect of vitamin D on colorectal carcinogenesis may not totally depend on tumor factors. Host factors (such as local and systemic immunity) may need to be considered. (C) 2014 AACR.

The carcinogens in cigarette smoke are distinct from asbestos. However, an understanding of their differential effects on lung adenocarcinoma development remains elusive. We investigated loss of heterozygosity (LOH) and the p53 mutation in 132 lung adenocarcinomas, for which asbestos body burden (AB; in numbers per gram of dry lung) was measured using adjacent normal lung. All cases were classified into 9 groups based on a matrix of cumulative smoking (CS in pack-years; CS=0, 0&lt;CS&lt;25, &gt;= 25 CS) and AB (AB=0, 0&lt;AB&lt;1,000 &gt;= 1,000 AB). AB=0 indicates a lower level than the detection limit of similar to 100. LOH frequency increased only slightly with the elevation of CS in the AB=0 groups. In the AB&gt;0 groups, LOH frequency increased as AB and/or CS was elevated and was significantly higher in the &gt;= 1,000 AB, &gt;= 25 CS group (p=0.032). p53 mutation frequency was the lowest in the AB=0, CS=0 group, increased as AB and/or CS rose, and was significantly higher in the &gt;= 1,000 AB, &gt;= 25 CS group (p=0.039). p53 mutations characteristic of smoking were frequently observed in the CS&gt;0 groups contrary to non-specific mutations in the CS=0, AB&gt;0 groups. Combined effects of asbestos and smoking were suggested by LOH and p53 analyses. Sole exposure to asbestos did not increase LOH frequency but increased non-specific p53 mutations. These findings indicate that the major carcinogenic mechanism of asbestos may be tumor promotion, acting in an additive or synergistic manner, contributing to the genotoxic effect of smoking. Since this study was based on a general cancer center's experience, the limited sample size did not permit the consideration that the result was conclusive. Further investigation with a large sample size is needed to establish the mechanism of asbestos-induced lung carcinogenesis.

Patients with small cell lung carcinoma (SCLC) rarely demonstrate long-term survival. We previously reported that gene expression profiling identified a subset of SCLC with good prognosis in surgical cases. To find an easier way to routinely identify SCLC belonging to this subset, we conducted the present study with a hypothesis that neuroendocrine (NE) or basaloid (BA) phenotypes may influence prognosis. To confirm the subset, we used an array platform to analyze fresh samples. Because inoperable cases may differ from surgical cases, we enrolled 51 biopsy cases and 43 resected samples. To evaluate NE and BA phenotypes, we used NE (synaptophysin, chromogranin A, and CD56) and BA (p63 and CK34 beta E12) markers. To varying extents, expression profiling based on the "array platform duplicated the subsets. For NE phenotypes, 77% of surgical cases and 100% of biopsy cases were positive for at least 1 marker. For BA phenotypes, only 19% of surgical cases were positive for at least 1 marker, whereas there were no positive biopsy cases. Cases undergoing surgery were categorized based on NE and BA immunoreactivity; 58% into NE+BA, 19% into NE+BA+, 23% into NE-BA-, and 0 into NE-BA+ groups. NE patients (n = 10) demonstrated a significantly better prognosis (P =.0306) than their NE+ counterparts (n = 33), whereas no survival difference was evident between the BA+ and BA groups. Multivariate analyses showed that NE positivity was an independent prognostic factor. In conclusion, the SCLC subset with good prognosis is identified by low NE marker expression, which was found only in surgical cases. (C) 2014 The Authors. Published by Elsevier Inc. All rights reserved.

BackgroundColonoscopy and sigmoidoscopy provide protection against colorectal cancer, but the magnitude and duration of protection, particularly against cancer of the proximal colon, remain uncertain. MethodsWe examined the association of the use of lower endoscopy (updated biennially from 1988 through 2008) with colorectal-cancer incidence (through June 2010) and colorectal-cancer mortality (through June 2012) among participants in the Nurses' Health Study and the Health Professionals Follow-up Study. ResultsAmong 88,902 participants followed over a period of 22 years, we documented 1815 incident colorectal cancers and 474 deaths from colorectal cancer. With endoscopy as compared with no endoscopy, multivariate hazard ratios for colorectal cancer were 0.57 (95% confidence interval [CI], 0.45 to 0.72) after polypectomy, 0.60 (95% CI, 0.53 to 0.68) after negative sigmoidoscopy, and 0.44 (95% CI, 0.38 to 0.52) after negative colonoscopy. Negative colonoscopy was associated with a reduced incidence of proximal colon cancer (multivariate hazard ratio, 0.73; 95% CI, 0.57 to 0.92). Multivariate hazard ratios for death from colorectal cancer were 0.59 (95% CI, 0.45 to 0.76) after screening sigmoidoscopy and 0.32 (95% CI, 0.24 to 0.45) after screening colonoscopy. Reduced mortality from proximal colon cancer was observed after screening colonoscopy (multivariate hazard ratio, 0.47; 95% CI, 0.29 to 0.76) but not after sigmoidoscopy. As compared with colorectal cancers diagnosed in patients more than 5 years after colonoscopy or without any prior endoscopy, those diagnosed in patients within 5 years after colonoscopy were more likely to be characterized by the CpG island methylator phenotype (CIMP) (multivariate odds ratio, 2.19; 95% CI, 1.14 to 4.21) and microsatellite instability (multivariate odds ratio, 2.10; 95% CI, 1.10 to 4.02). ConclusionsColonoscopy and sigmoidoscopy were associated with a reduced incidence of cancer of the distal colorectum; colonoscopy was also associated with a modest reduction in the incidence of proximal colon cancer. Screening colonoscopy and sigmoidoscopy were associated with reduced colorectal-cancer mortality; only colonoscopy was associated with reduced mortality from proximal colon cancer. Colorectal cancer diagnosed within 5 years after colonoscopy was more likely than cancer diagnosed after that period or without prior endoscopy to have CIMP and microsatellite instability.

Background: A subset of lung adenocarcinomas harboring an EML4-ALK fusion gene resulting in dominant oncogenic activity has emerged as a target for specific therapy. EML4-ALK fusion confers a characteristic histology and is detected more frequently in never or light smokers and younger patients. Methods: To gain insights into etiology and carcinogenic mechanisms we conducted analyses to compare allelotypes of 35 ALK fusion-positive and 95 -negative tumours using single nucleotide polymorphism (SNP) arrays and especially designed software which enabled precise global genomic profiling. Results: Overall aberration numbers (gains + losses) of chromosomal alterations were 8.42 and 9.56 in tumours with and without ALK fusion, respectively, the difference not being statistically significant, although patterns of gain and loss were distinct. Interestingly, among selected genomic regions, oncogene-related examples such as 1p34.3 (MYCL1), 7q11.2(EGFR), 7p21.1, 8q24.21(MYC), 16p13.3, 17q12(ERBB2) and 17q25.1 showed significantly less gain. Also, changes in tumour suppressor gene-related regions, such as 9p21.3 (CDKN2A) 9p23-24.1 (PTPRD), 13q14.2 (RB1), were significantly fewer in tumours with ALK fusion. Conclusion: Global genomic comparison with SNP arrays showed tumours with ALK fusion to have fewer alterations in oncogenes and suppressor genes despite a similar overall aberration frequency, suggesting very strong oncogenic potency of ALK activation by gene fusion.

Background: Lung adenocarcinoma is heterogeneous regarding histology, etiology and prognosis. Although there have been several attempts to find a subgroup with poor prognosis, it is unclear whether or not adenocarcinoma with neuroendocrine (NE) nature has unfavorable prognosis. Materials and methods: To elucidate whether a subtype of adenocarcinoma with NE nature has poor prognosis, we performed gene expression profiling by cDNA microarray for 262 Japanese lung cancer and 30 normal lung samples, including 171 adenocarcinomas, 56 squamous cell carcinomas and 35 NE tumors. A co-expression gene set with ASCL1, an NE master gene, was utilized to classify tumors by non-negative matrix factorization, followed by validation using an ASCL1 knock-down gene set in DMS79 cells as well as an independent cohort (n = 139) derived from public microarray databases as a test set. Results: The co-expression gene set classified the adenocarcinomas into alveolar cell (AL), squamoid, and NE subtypes. The NE subtype, which clustered together almost all the NE tumors, had significantly poorer prognosis than the AL subtype that clustered with normal lung samples (p = 0.0075). The knock-down gene set also classified the 171 adenocarcinomas into three subtypes and this NE subtype also had the poorest prognosis. The co-expression gene set classified the independent database-derived American cohort into two subtypes, with the NE subtype having poorer prognosis. None of the single NE gene expression was found to be linked to survival difference. Conclusion: Co-expression gene set with ASCH, rather than single NE gene expression, successfully identifies an NE subtype of lung adenocarcinoma with poor prognosis. (C) 2011 Elsevier Ireland Ltd. All rights reserved.

Renal cell carcinoma (RCC) with t(6; I I) translocation, involving the transcription factor EB (TFEB) and Alpha, also known as MALATI, (TFEB RCC), is extremely rare, with only 20 cases reported to date. It may be frequently misdiagnosed because of a lack of established characteristics. TFEB RCCs are predominantly seen in younger patients and are generally indolent, with only 2 cases of metastasis. Genetic analysis has been limited, showing break points upstream of TFEB exon 3, yielding only a single transcript. We examined 3 new adult Japanese TFEB RCC cases by means of precise clinicopathologic, immunohistochemical, cytogenctic, and molecular analyses and compared them with 200 ordinary RCCs. A 57-year-old man was the oldest patient with TFEB RCC at the time of this study. Although the tumor had histology typical of translocation RCC, its fusion points were different between the genomic and transcript coordinates. A 37-year-old man had an aggressive course resulting in death. The tumor had 2 variants of messenger ribonucleic acid. A 47-year-old man showed borderline histologic and immunohistochemical features between TFEB RCC and chromophobe-type RCC. The tumor had a fusion point in TFEB exon 4, downstream of the wild-type ATG in exon 3. Nuclear expression of the TFEB protein was detected, and a Western blotting analysis identified a protein similar in size to the wild-type TFEB protein. Immunohistochemistry is useful for the diagnosis of these tumors, and TFEB RCCs have heterogeneous clinicopathologic features and more diverse fusion patterns than previously thought, requiring attention to polymerase chain reaction experiments for diagnosis. Our study will contribute to the correct diagnosis of TFEB RCC.

OBJECTIVES To investigate the correlations between the initial tumor size and size reduction rate in patients treated with targeted agents. To select the patients who can benefit the most from treatment with targeted agents, it will be necessary to find a tumor characteristic that predicts their effectiveness. METHODS The data from 139 metastatic and 16 primary lesions treated with the targeted agents were retrospectively analyzed. They consisted of 86 sunitinib-treated and 69 sorafenib-treated lesions in 54 patients with metastatic renal cell carcinoma who had undergone treatment from April 2008 to July 2010. The relationship between the longest tumor diameter at baseline and the rate of reduction in tumor size was assessed using the Spearmancorrelation test. RESULTS A linear, moderate to strong association between the initial tumor size and tumor size reduction rate was shown (correlation coefficient -0.441, P &lt; .001). When these tumors were divided into 2 groups at the threshold value (23.95 mm), which was decided by the receiver operating characteristic curve analysis, the smaller tumors demonstrated a significantly greater size reduction than the larger tumors according to the Mann-Whitney U test (P &lt; .001). Both univariate and multivariate linear regression analyses revealed that only the initial tumor size was associated with the rate of reduction in individual tumors (P &lt; .001). CONCLUSIONS The initial tumor size was a good predictor of the tumor size reduction. This simple observation could be useful for physicians who treat patients with metastatic renal cell carcinoma. In addition, in assessing clinical trials of targeted agents for metastatic renal cell carcinoma using the ResponseEvaluation Criteria in Solid Tumors, perhaps this association should be considered. UROLOGY 77: 831-835, 2011. (c) 2011 Elsevier Inc.

Mixed squamous cell and glandular papilloma (mixed papilloma) of the lung is an extremely rare neoplasm, with only 10 cases reported so far in the English literature. We present a case study of endobronchial mixed papilloma with immunohistochemical and etiological investigations. A 49-year-old male with a smoking history complained of hemoptysis, presented with a lung mass closely adjacent to large vessels in the computed tomography findings, and underwent lobectomy. The 3.0-cm sized polypoid tumor was histologically diagnosed as endobronchial mixed papilloma. Immunohistochemically, intracellular mucin was positive for MUC5AC, which is expressed in tracheobronchial goblet cells. CAM5.2 and CK19 were diffusely positive, indicating that the tumor originated from the columnar epithelium by squamous metaplasia. CEA and CA19-9 were focally positive. A human papillomavirus (HPV) investigation with in situ hybridization using a wide spectrum probe and a newly-developed PCR system did not detect any HPV infection. Including this case with a detailed HPV investigation, all of the reported cases of mixed papilloma were HPV-negative, and a literature review including newly-reported cases indicated a high frequency of smoking in such cases. Endobronchial mixed papillomas might have a smoking-related etiology.

The activation status of signal transduction pathways involving receptor tyrosine kinases and its association with EGFR or KRAS mutations have been widely studied using cancer cell lines, although it is still uncertain in primary tumors. To study the activation status of main components of growth factor-induced pathways, phosphorylated Akt (pAkt), extracellular signal-regulated kinases 1 and 2 (pERK) and other downstream proteins were immunohistochemically examined using surgical samples of 193 primary lung adenocarcinomas. Also, thyroid transcription factor-1 (TTF-1) expression and mutation status of EGFR and KRAS were examined. Advanced tumor stages (p &lt; 0.001), negative TTF-1 expression (p &lt; 0.001) and Akt activation (p = 0.015) were independent and significant poor prognostic markers. Akt activation related to advanced stage (p = 0.021), invasiveness (p = 0.004), and not to mutations. TTF-1 expression associated with never-smoker (p = 0.013), pre- or minimally invasiveness (p &lt; 0.001) and EGFR mutations (p = 0.017) as well as with pERK (p = 0.039) expression. EGFR mutations did not correlated with pAkt and pERK expression, which was different from the results based on cultured cells, while KRAS mutations were solely and significantly linked to ERK activation (p = 0.009). In lung adenocarcinoma, tumors with TTF-1 expression have distinct characteristics regarding mutations, signal protein activation and clinical issues. Moreover, this property was revealed to be important in outcome estimation at any tumor stage, whereas Akt activation is abnormally affected according to the tumor stage regardless of their cell origin. The signal proteins were differently related to mutation status from cultured cells. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

Lung cancer is the leading cause of cancer death in men and women worldwide. Since the occurrence of metastases in distant organs is the major reason for mortality of cancer patients, we need to elucidate the underlying mechanisms. Many studies featuring analysis of gene expression, comparative genomic hybridization and loss of heterozygosity analysis have been performed and generated support for the hypothesis that metastatic potential is acquired early in tumorigenesis. Furthermore, it is now clear that the majority of tumor cells have the potential to metastasize. Although many changes in gene expression profiles have been established retrospectively, translational research is now a high priority to enable clinical application and treatment based on laboratory findings.

Context.-Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors are molecular-targeted drugs that are innovatively effective for non-small cell lung carcinomas with EGFR mutations. Epidermal growth factor receptor is a transmembrane receptor forming dimers on ligand binding. These then stimulate signals by activating receptor autophosphorylation through tyrosine kinase activity. Autophosphorylation triggers intracellular pathways facilitating malignant conversion. The most clinically advanced EGFR inhibition strategies include small-molecule inhibition of the intracellular tyrosine kinase domain (gefitinib and erlotinib) and monoclonal antibody-mediated blockade of the extracellular ligand-binding domain (cetuximab). Lung cancers with EGFR mutations are prevalent among patients who are female, of Asian ethnicity, and nonsmokers; thus, they can obtain benefit from EGFR tyrosine kinase inhibitors. Objective.-To survey histopathologic findings and examine correlations with EGFR mutations. We mainly focused on component cell types (hobnail, columnar, and polygonal) and presence or absence of bronchioloalveolar carcinoma elements and a micropapillary pattern. Although EGFR mutations can be detected by various methods, including polymerase chain reaction-Invader assay or direct sequencing, these are inconvenient. Data Sources.-Review of the published literature. Conclusion.-Detailed pathologic examination showed significant genotype-phenotype correlations between EGFR mutations and presence of a bronchioloalveolar carcinoma component, a micropapillary pattern, and the hobnail cell type. We conclude that these characteristic histologic features are good predictors of EGFR mutations, and patients with these features might be good candidates for and could benefit from therapy with EGFR tyrosine kinase inhibitors. (Arch Pathol Lab Med. 2010; 134: 66-72)

Purpose: EML4-ALK is a transforming fusion tyrosine kinase, several isoforms of which have been identified in lung cancer. Immunohistochemical detection of EML4-ALK has proved difficult, however, likely as a result of low transcriptional activity conferred by the promoter-enhancer region of EML4. The sensitivity of EML4-ALK detection by immunohistochemistry should be increased adequately. Experimental Design: We developed an intercalated antibody-enhanced polymer (iAEP) method that incorporates an intercalating antibody between the primary antibody to ALK and the dextran polymer-based detection reagents. Results: Our iAEP method discriminated between tumors positive or negative for EML4-ALK in a test set of specimens. Four tumors were also found to be positive for ALK in an archive of lung adenocarcinoma (n = 130) and another 4 among fresh cases analyzed in a diagnostic laboratory. These 8 tumors were found to include 1 with EML4-ALK variant 1, 1 with variant 2, 3 with variant 3, and 2 with previously unidentified variants (designated variants 6 and 7). Inverse reverse transcription-PCR analysis revealed that the remaining tumor harbored a novel fusion in which intron 24 of KIF5B was ligated to intron 19 of ALK. Multiplex reverse transcription-PCR analysis of additional archival tumor specimens identified another case of lung adenocarcinoma positive for KIF5B-ALK. Conclusions: The iAEP method should prove suitable for immunohistochemical screening of tumors positive for ALK or ALK fusion proteins among pathologic archives. Coupling of PCR-based detection to the iAEP method should further facilitate the rapid identification of novel ALK fusion genes such as KIF5B-ALK.

A subset of lung cancers harbors a small inversion within chromosome 2p, giving rise to a transforming fusion gene, EML4-ALK (echinoderm microtubule-associated protein-like 4 gene and the anaplastic lymphoma kinase gene), which encodes an activated tyrosine kinase. We have earlier examined the presence of EML4-ALK by multiplex reverse transcription-polymerase chain reaction in 363 specimens of lung cancer, identifying 11 adenocarcinoma cases featuring the fusion gene. In this study, we clinicopathologically examined the characteristics of the EML4-ALK-positive cases, including the mutation status of EGFR, KRAS, and TP53, and whether they were of thyroid transcription factor-1 (TTF-1) cell lineage or not. Of 11 patients, 4 (36%) with EML4-ALK-positive lung adenocarcinomas who were below 50 years of age were affected by these diseases, as compared with 12 of 242 patients (5.0%) with EML4-ALK-negative lung adenocarcinomas (P=0.00038). EML4-ALK-positive lung adenocarcinomas were characterized by less-differentiated grade (P=0.0082) and acinar-predominant structure (P &lt; 0.0001) in histology. Furthermore, the presence of EML4-ALK appears to be mutually exclusive for EGFR and KRAS mutations (P=0.00018), whereas coexisting with TP53 mutations at a low frequency (1/11=9.1%), and correlating with non- or light smoking (P=0.040), in line with the TTF-1 immunoreactivity. Thus, EML4-ALK-positive tumors may form a distinct entity among lung adenocarcinomas, characterized by young onset, acinar histology, no or rare mutations in EGFR, KRAS, and TP53, and a TTF-1 cell lineage, all in agreement with the prevalence in non- or light smokers.

The presence of epidermal growth factor receptor (EGFR) tyrosine kinase (TK) mutations significantly correlates with tumor sensitivity to TK inhibitors, particularly in lung adenocarcinomas, the predominant histological subtype in Japan and the United States. To clarify links between EGFR mutations and pathological findings in Japanese lung cancer, detailed pathological features of adenocarcinomas were examined using the WHO criteria as well as our cell type classification (hobnail, columnar and polygonal). Medical records were reviewed for a total of 107 surgically resected tumors. Clinicopathological factors were examined and correlations with EGFR status were evaluated. EGFR mutations were found in 63 patients (59%) distributed through all four exons examined (through exons 18-21). EGFR mutations were significantly associated with female gender (P=0.003), non-smoker status (P=0.008) and hobnail cell morphology (P<0.00001). In addition, detailed pathological examination showed significant associations with bronchioloalveolar carcinoma (BAC) component and a micropapillary pattern (MPP) (P=0.012 and 0.043, respectively). We conclude that characteristic histological features, i.e. the hobnail cell morphology and the presence of BAC component and MPP are good predictors of EGFR mutations in lung adenocarcinoma.

Purpose: EML4-ALK is a fusion-type protein tyrosine kinase that is generated by inv(2) (p21p23) in the genome of non - small cell lung cancer (NSCLC). To allow sensitive detection of EML4-ALK fusion transcripts, we have now developed a multiplex reverse transcription-PCR (RT-PCR) system that captures all in-frame fusions between the two genes. Experimental Design: Primers were designed to detect all possible in-frame fusions of EML4 to exon 20 of ALK, and a single-tube multiplex RT-PCR assay was done with total RNA from 656 solid tumors of the lung (n = 364) and 10 other organs. Results: From consecutive lung adenocarcinoma cases (n = 253), we identified 11 specimens (4.35%) positive for fusion transcripts, 9 of which were positive for the previously identified variants 1, 2, and 3. The remaining two specimens harbored novel transcript isoforms in which exon 14 (variant 4) or exon 2 (variant 5) of EML4 was connected to exon 20 of ALK. No fusion transcripts were detected for other types of lung cancer (n = 111) or for tumors from 10 other organs (n = 292). Genomic rearrangements responsible for the fusion events in NSCLC cells were confirmed by genomic PCR analysis and fluorescence in situ hybridization. The novel isoforms of EML4-ALK manifested marked oncogenic activity, and they yielded a pattern of cytoplasmic staining with fine granular foci in immunohistochemical analysis of NSCLC specimens. Conclusions: These data reinforce the importance of accurate diagnosis of EML4-ALK - positive tumors for the optimization of treatment strategies.

The genome of a subset of non-small-cell lung cancers (NSCLC) harbors a small inversion within chromosome 2 that gives rise to a transforming fusion gene, EML4-ALK, which encodes an activated protein tyrosine kinase. Although breakpoints within EML4 have been identified in introns 13 and 20, giving rise to variants 1 and 2, respectively, of EML4-ALK, it has remained unclear whether other isoforms of the fusion gene are present in NSCLC cells. We have now screened NSCLC specimens for other in-frame fusion cDNAs that contain both EML4 and ALK sequences. Two slightly different fusion cDNAs in which exon 6 of EML4 was joined to exon 20 of ALK were each identified in two individuals of the cohort. Whereas one cDNA contained only exons 1 to 6 of EML4 (variant 3a), the other also contained an additional 33-bp sequence derived from intron 6 of EML4 (variant 3b). The protein encoded by the latter cDNA thus contained an insertion of 11 amino acids between the EML4 and ALK sequences of that encoded by the former. Both variants 3a and 3b of EML4-ALK exhibited marked transforming activity in vitro as well as oncogenic activity in vivo. A lung cancer cell line expressing endogenous variant 3 of EML4-ALK underwent cell death on exposure to a specific inhibitor of ALK catalytic activity. These data increase the frequency of EML4-ALK-positive NSCLC tumors and bolster the clinical relevance of this oncogenic kinase.

Background: Previously, using microarray and real-time RT-PCR analysis, we established that HOXB2 is an adverse prognostic indicator for Stage I lung adenocarcinomas. HOXB2 is one of the homeobox master development-controlling genes regulating morphogenesis and cell differentiation. Materials and Methods: The molecular functions of HOXB2 were analyzed with a small interfering RNA (siRNA) approach in HOP-62 human non-small cell lung cancer (NSCLC) cells featuring high HOXB2 expression. Matrigel invasion assays and microarray gene expression analysis were compared between the HOXB2-siRNA cells and the control cells. Results: The Matrigel invasion assays showed attenuation of HOXB2 expression by siRNA to result in a significant decrease of invasiveness compared to the control cells (p=0.0013, paired t-test). On microarray gene expression analysis, up-regulation of many metastasis-related genes and others correlating with HOXB2 expression was observed in the control case. With attenuation of HOXB2 expression, down-regulation was noted for laminins alpha 4 and 5, involved in enriched signaling, and for Mac-2BP (Mac-2 binding protein) and integrin beta 4 amongst the genes having an enriched glycoprotein ontology. Conclusion: HOXB2 promotes invasion of lung cancer cells through the regulation of metastasis-related genes.

Introduction: Very recently, we have found a novel fusion product between the echinoderm microtubule-associated protein-like4 (EML4) and the anaplastic lymphoma kinase (ALK) in non-small cell lung cancers (NSCLCs). Tumors featuring EML4-ALK fusion constitute one subtype of NSCLC that might be highly sensitive to ALK inhibitors. Herein, we present results of a first large scale study of EML4-ALK fusion in lung cancers. Methods: Using reverse transcription-polymerase chain reaction for EML4-ALK fusion mRNA, we investigated 149 lung adenocarcinomas, 48 squamous cell carcinomas, 3 large-cell neuroendocrine carcinomas, and 21 small-cell carcinomas. For EML4-ALK-positive cancers, we further investigated the presence of ALK fusion proteins by immunohistochemistry. Results: Five of 149 adenocarcinomas (3.4%) showed EML4-ALK fusion mRNA, this being totally lacking in carcinomas of other types (0/72). In all the fusion-positive cases, ALK fusion protein could be detected in the cytoplasm immunohistochemically. The five fusion cases featured two EML4-ALK variant 1 fusions and three variant 2 fusions. Histologically, both variant 1 cases were mixed type adenocarcinomas, showing papillary with bronchioloalveolar components. Interestingly, all three variant 2 cases were acinar adenocarcinomas, the link being statistically significant (p = 0.00018). None of the five fusion-positive cases demonstrated any mutations of EGFR or KRAS, pointing to a mutually exclusive relationship (p = 0.014). There was no association with smoking habits. Conclusions: In the present first investigation of EML4-ALK fusion in a large study of lung cancers (5/221), we found an interesting histotype-genotype relationship. Furthermore, we could detect the fusion protein by immunohistochemistry, pointing to possible clinical applications.