Teppei Morikawa

Drug resistance presents a challenge to the treatment of cancer patients. Many studies have focused on cell-autonomous mechanisms of drug resistance. By contrast, we proposed that the tumour micro-environment confers innate resistance to therapy. Here we developed a co-culture system to systematically assay the ability of 23 stromal cell types to influence the innate resistance of 45 cancer cell lines to 35 anticancer drugs. We found that stroma-mediated resistance is common, particularly to targeted agents. We characterized further the stroma-mediated resistance of BRAF-mutant melanoma to RAF inhibitors because most patients with this type of cancer show some degree of innate resistance. Proteomic analysis showed that stromal cell secretion of hepatocyte growth factor (HGF) resulted in activation of the HGF receptor MET, reactivation of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-OH kinase (PI(3)K)-AKT signalling pathways, and immediate resistance to RAF inhibition. Immunohistochemistry experiments confirmed stromal cell expression of HGF in patients with BRAF-mutant melanoma and showed a significant correlation between HGF expression by stromal cells and innate resistance to RAF inhibitor treatment. Dual inhibition of RAF and either HGF or MET resulted in reversal of drug resistance, suggesting RAF plus HGF or MET inhibitory combination therapy as a potential therapeutic strategy for BRAF-mutant melanoma. A similar resistance mechanism was uncovered in a subset of BRAF-mutant colorectal and glioblastoma cell lines. More generally, this study indicates that the systematic dissection of interactions between tumours and their micro-environment can uncover important mechanisms underlying drug resistance.

Colonoscopy and sigmoidoscopy provide protection against colorectal cancer, but the magnitude and duration of protection, particularly against cancer of the proximal colon, remain uncertain.We 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.Among 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).Colonoscopy 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. (Funded by the National Institutes of Health and others.).

Regular use of aspirin after a diagnosis of colon cancer has been associated with a superior clinical outcome. Experimental evidence suggests that inhibition of prostaglandin-endoperoxide synthase 2 (PTGS2) (also known as cyclooxygenase-2) by aspirin down-regulates phosphatidylinositol 3-kinase (PI3K) signaling activity. We hypothesized that the effect of aspirin on survival and prognosis in patients with cancers characterized by mutated PIK3CA (the phosphatidylinositol-4,5-bisphosphonate 3-kinase, catalytic subunit alpha polypeptide gene) might differ from the effect among those with wild-type PIK3CA cancers.We obtained data on 964 patients with rectal or colon cancer from the Nurses' Health Study and the Health Professionals Follow-up Study, including data on aspirin use after diagnosis and the presence or absence of PIK3CA mutation. We used a Cox proportional-hazards model to compute the multivariate hazard ratio for death. We examined tumor markers, including PTGS2, phosphorylated AKT, KRAS, BRAF, microsatellite instability, CpG island methylator phenotype, and methylation of long interspersed nucleotide element 1.Among patients with mutated-PIK3CA colorectal cancers, regular use of aspirin after diagnosis was associated with superior colorectal cancer-specific survival (multivariate hazard ratio for cancer-related death, 0.18; 95% confidence interval [CI], 0.06 to 0.61; P<0.001 by the log-rank test) and overall survival (multivariate hazard ratio for death from any cause, 0.54; 95% CI, 0.31 to 0.94; P=0.01 by the log-rank test). In contrast, among patients with wild-type PIK3CA, regular use of aspirin after diagnosis was not associated with colorectal cancer-specific survival (multivariate hazard ratio, 0.96; 95% CI, 0.69 to 1.32; P=0.76 by the log-rank test; P=0.009 for interaction between aspirin and PIK3CA variables) or overall survival (multivariate hazard ratio, 0.94; 95% CI, 0.75 to 1.17; P=0.96 by the log-rank test; P=0.07 for interaction).Regular use of aspirin after diagnosis was associated with longer survival among patients with mutated-PIK3CA colorectal cancer, but not among patients with wild-type PIK3CA cancer. The findings from this molecular pathological epidemiology study suggest that the PIK3CA mutation in colorectal cancer may serve as a predictive molecular biomarker for adjuvant aspirin therapy. (Funded by The National Institutes of Health and others.).

Colorectal cancer is typically classified into proximal colon, distal colon and rectal cancer. Tumour genetic and epigenetic features differ by tumour location. Considering a possible role of bowel contents (including microbiome) in carcinogenesis, this study hypothesised that tumour molecular features might gradually change along bowel subsites, rather than change abruptly at splenic flexure.Utilising 1443 colorectal cancers in two US nationwide prospective cohort studies, the frequencies of molecular features (CpG island methylator phenotype (CIMP), microsatellite instability (MSI), LINE-1 methylation and BRAF, KRAS and PIK3CA mutations) were examined along bowel subsites (rectum, rectosigmoid junction, sigmoid, descending colon, splenic flexure, transverse colon, hepatic flexure, ascending colon and caecum). The linearity and non-linearity of molecular relations along subsites were statistically tested by multivariate logistic or linear regression analysis.The frequencies of CIMP-high, MSI-high and BRAF mutations gradually increased from the rectum (<2.3%) to ascending colon (36-40%), followed by falls in the caecum (12-22%). By linearity tests, these molecular relations were significantly linear from rectum to ascending colon (p<0.0001), and there was little evidence of non-linearity (p>0.09). Caecal cancers exhibited the highest frequency of KRAS mutations (52% vs 27-35% in other sites; p<0.0001).The frequencies of CIMP-high, MSI-high and BRAF mutations in cancer increased gradually along colorectum subsites from the rectum to ascending colon. These novel data challenge the common conception of discrete molecular features of proximal versus distal colorectal cancers, and have a substantial impact on clinical, translational and epidemiology research, which has typically been performed with the dichotomous classification of proximal versus distal tumours.

A remarkable feature of regenerative processes is their ability to halt proliferation once an organ's structure has been restored. The Wnt signalling pathway is the major driving force for homeostatic self-renewal and regeneration in the mammalian intestine. However, the mechanisms that counterbalance Wnt-driven proliferation are poorly understood. Here we demonstrate in mice and humans that yes-associated protein 1 (YAP; also known as YAP1)--a protein known for its powerful growth-inducing and oncogenic properties--has an unexpected growth-suppressive function, restricting Wnt signals during intestinal regeneration. Transgenic expression of YAP reduces Wnt target gene expression and results in the rapid loss of intestinal crypts. In addition, loss of YAP results in Wnt hypersensitivity during regeneration, leading to hyperplasia, expansion of intestinal stem cells and niche cells, and formation of ectopic crypts and microadenomas. We find that cytoplasmic YAP restricts elevated Wnt signalling independently of the AXIN-APC-GSK-3β complex partly by limiting the activity of dishevelled (DVL). DVL signals in the nucleus of intestinal stem cells, and its forced expression leads to enhanced Wnt signalling in crypts. YAP dampens Wnt signals by restricting DVL nuclear translocation during regenerative growth. Finally, we provide evidence that YAP is silenced in a subset of highly aggressive and undifferentiated human colorectal carcinomas, and that its expression can restrict the growth of colorectal carcinoma xenografts. Collectively, our work describes a novel mechanistic paradigm for how proliferative signals are counterbalanced in regenerating tissues. Additionally, our findings have important implications for the targeting of YAP in human malignancies.

BRAF mutation in colorectal cancer is associated with microsatellite instability (MSI) through its relationship with high-level CpG island methylator phenotype (CIMP) and MLH1 promoter methylation. MSI and BRAF mutation analyses are routinely used for familial cancer risk assessment. To clarify clinical outcome associations of combined MSI/BRAF subgroups, we investigated survival in 1253 rectal and colon cancer patients within the Nurses' Health Study and Health Professionals Follow-up Study with available data on clinical and other molecular features, including CIMP, LINE-1 hypomethylation, and KRAS and PIK3CA mutations. Compared with the majority subtype of microsatellite stable (MSS)/BRAF-wild-type, MSS/BRAF-mutant, MSI-high/BRAF-mutant, and MSI-high/BRAF-wild-type subtypes showed multivariable colorectal cancer-specific mortality hazard ratios of 1.60 (95% confidence interval [CI] =1.12 to 2.28; P = .009), 0.48 (95% CI = 0.27 to 0.87; P = .02), and 0.25 (95% CI = 0.12 to 0.52; P < .001), respectively. No evidence existed for a differential prognostic role of BRAF mutation by MSI status (P(interaction) > .50). Combined BRAF/MSI status in colorectal cancer is a tumor molecular biomarker for prognosic risk stratification.

Abstract To understand the genetic drivers of immune recognition and evasion in colorectal cancer, we analyzed 1,211 colorectal cancer primary tumor samples, including 179 classified as microsatellite instability–high (MSI-high). This set includes The Cancer Genome Atlas colorectal cancer cohort of 592 samples, completed and analyzed here. MSI-high, a hypermutated, immunogenic subtype of colorectal cancer, had a high rate of significantly mutated genes in important immune-modulating pathways and in the antigen presentation machinery, including biallelic losses of B2M and HLA genes due to copy-number alterations and copy-neutral loss of heterozygosity. WNT/β-catenin signaling genes were significantly mutated in all colorectal cancer subtypes, and activated WNT/β-catenin signaling was correlated with the absence of T-cell infiltration. This large-scale genomic analysis of colorectal cancer demonstrates that MSI-high cases frequently undergo an immunoediting process that provides them with genetic events allowing immune escape despite high mutational load and frequent lymphocytic infiltration and, furthermore, that colorectal cancer tumors have genetic and methylation events associated with activated WNT signaling and T-cell exclusion. Significance: This multi-omic analysis of 1,211 colorectal cancer primary tumors reveals that it should be possible to better monitor resistance in the 15% of cases that respond to immune blockade therapy and also to use WNT signaling inhibitors to reverse immune exclusion in the 85% of cases that currently do not. Cancer Discov; 8(6); 730–49. ©2018 AACR. This article is highlighted in the In This Issue feature, p. 663

Abstract CpG island promoter methylation of tumor suppressor genes is one of the most characteristic abnormalities in EBV-associated gastric carcinoma (GC). Aberrant promoter methylation and expression loss of PTEN were evaluated in cancer tissues of GC by methylation-specific PCR and immunohistochemistry, respectively, showing that both abnormalities occurred concurrently in EBV-associated GC. PTEN abnormalities were reiterated in GC cell lines MKN-1 and MKN-7 infected with recombinant EBV, and DNA methyltransferase 1 (DNMT1) was commonly overexpressed in both cell lines. Stable and transient transfection systems in MKN-1 similarly showed that viral latent membrane protein 2A (LMP2A) up-regulated DNMT1, leading to an increase in methylation of the PTEN promoter. Importantly, the level of phosphorylated signal transducer and activator of transcription 3 (pSTAT3) increased in the nuclei of LMP2A-expressing GC cells, and knockdown of STAT3 counteracted LMP2A-mediated DNMT1 overexpression. Immunohistochemistry for both pSTAT3 and DNMT1 showed diffuse labeling in the nuclei of the cancer cells in GC tissues, especially in EBV-associated GC. Taken together, LMP2A induces the phosphorylation of STAT3, which activates DNMT1 transcription and causes PTEN expression loss through CpG island methylation of the PTEN promoter in EBV-associated GC. LMP2A plays an essential role in the epigenetic abnormalities in host stomach cells and in the development and maintenance of EBV-associated cancer. [Cancer Res 2009;69(7):2766–74]

Mutations in PIK3CA [the gene encoding the p110α catalytic subunit of phosphatidylinositide-3-kinase (PI3K)] play an important role in colorectal carcinogenesis. Experimental evidence suggests that PIK3CA exon 9 and exon 20 mutations trigger different biologic effects, and that concomitant mutations in both exons 9 and 20 synergistically enhance tumorigenic effects. Thus, we hypothesized that PIK3CA exon 9 and exon 20 mutations might have differential effects on clinical outcome in colorectal cancer, and that concomitant PIK3CA exon 9 and 20 mutations might confer aggressive tumor behavior.We sequenced PIK3CA by pyrosequencing in 1,170 rectal and colon cancers in two prospective cohort studies, and found 189 (16%) PIK3CA mutated tumors. Mortality HR according to PIK3CA status was computed using Cox proportional hazards model, adjusting for clinical and molecular features, including microsatellite instability, CpG island methylator phenotype, LINE-1 methylation, and BRAF and KRAS mutations.Compared with PIK3CA wild-type cases, patients with concomitant PIK3CA mutations in exons 9 and 20 experienced significantly worse cancer-specific survival [log-rank P = 0.031; multivariate HR = 3.51; 95% confidence interval (CI): 1.28-9.62] and overall survival (log-rank P = 0.0008; multivariate HR = 2.68; 95% CI: 1.24-5.77). PIK3CA mutation in either exon 9 or 20 alone was not significantly associated with patient survival. No significant interaction of PIK3CA mutation with BRAF or KRAS mutation was observed in survival analysis.Coexistence of PIK3CA (the PI3K p110α subunit) exon 9 and 20 mutations, but not PIK3CA mutation in either exon 9 or 20 alone, is associated with poor prognosis of colorectal cancer patients.

To assess prognostic roles of various KRAS oncogene mutations in colorectal cancer, BRAF mutation status must be controlled for because BRAF mutation is associated with poor prognosis, and almost all BRAF mutants are present among KRAS wild-type tumors. Taking into account experimental data supporting a greater oncogenic effect of codon 12 mutations compared with codon 13 mutations, we hypothesized that KRAS codon 12-mutated colorectal cancers might behave more aggressively than KRAS wild-type tumors and codon 13 mutants.Using molecular pathological epidemiology database of 1,261 rectal and colon cancers, we examined clinical outcome and tumor biomarkers of KRAS codon 12 and 13 mutations in 1,075 BRAF wild-type cancers (i.e., controlling for BRAF status). Cox proportional hazards model was used to compute mortality HR, adjusting for potential confounders, including stage, PIK3CA mutations, microsatellite instability, CpG island methylator phenotype, and LINE-1 methylation.Compared with patients with KRAS wild-type/BRAF wild-type cancers (N = 635), those with KRAS codon 12 mutations (N = 332) experienced significantly higher colorectal cancer-specific mortality [log-rank P = 0.0001; multivariate HR, 1.30; 95% confidence interval (CI), 1.02-1.67; P = 0.037], whereas KRAS codon 13-mutated cases (N = 108) were not significantly associated with prognosis. Among the seven most common KRAS mutations, c.35G>T (p.G12V; N = 93) was associated with significantly higher colorectal cancer-specific mortality (log-rank P = 0.0007; multivariate HR, 2.00; 95% CI, 1.38-2.90, P = 0.0003) compared with KRAS wild-type/BRAF wild-type cases.KRAS codon 12 mutations (in particular, c.35G>T), but not codon 13 mutations, are associated with inferior survival in BRAF wild-type colorectal cancer. Our data highlight the importance of accurate molecular characterization in colorectal cancer.

Colorectal cancer comprises a heterogeneous group of diseases that arise through varying molecular carcinogenic pathways, involving complex interactions between tumour cells and the host microenvironment.1 2 It has long been appreciated that developmental and physiological differences exist between anatomic segments of the colorectum, and that colorectal cancers occur with distinctly different frequencies at different subsites.3 While approaches to surgical and adjuvant therapy have set rectal cancer as a separate entity, colon cancers still tend to get grouped together. In the 1980s, epidemiological and molecular data suggested that cancers arising at different subsites may be biologically disparate, implying different cancer aetiologies or evolutions. These observations prompted the suggestion that the proximal and distal colon should be considered separately in aetiological studies, with the splenic flexure as a demarcation point.3 This dichotomisation has been propagated by subsequent clinical, translational and epidemiological studies,4–10 while evolving molecular data have galvanised and lent further support to the two-colon concept.11–14 It remains uncertain, however, whether the splenic flexure represents a genuine or arbitrary divide in the aetiological spectrum of colon cancers. Notably, our recent study,15 demonstrating that the frequencies of key tumour molecular features change gradually along the length of the colon (rather than abruptly at the splenic flexure), challenges the current two-colon paradigm. The aim of this article is to review data relevant to the current concept of distinct molecular pathogeneses for proximal versus distal colon cancers, to describe the impact of our recent findings and to discuss the implications for future research. ### Historical overture There are incontrovertible differences between the ontogeny, morphology, biochemistry and physiology of the proximal and distal colon.11–14 Furthermore, gross macroscopic differences are well described between proximal and distal colorectal tumours.9 11–14 In …

Cushing's syndrome is caused by excess cortisol production from the adrenocortical gland. In corticotropin-independent Cushing's syndrome, the excess cortisol production is primarily attributed to an adrenocortical adenoma, in which the underlying molecular pathogenesis has been poorly understood. We report a hotspot mutation (L206R) in PRKACA, which encodes the catalytic subunit of cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA), in more than 50% of cases with adrenocortical adenomas associated with corticotropin-independent Cushing's syndrome. The L206R PRKACA mutant abolished its binding to the regulatory subunit of PKA (PRKAR1A) that inhibits catalytic activity of PRKACA, leading to constitutive, cAMP-independent PKA activation. These results highlight the major role of cAMP-independent activation of cAMP/PKA signaling by somatic mutations in corticotropin-independent Cushing's syndrome, providing insights into the diagnosis and therapeutics of this syndrome.

Abstract Purpose: STAT3 is a transcription factor that is constitutively activated in some cancers. It seems to play crucial roles in cell proliferation and survival, angiogenesis, tumor-promoting inflammation, and suppression of antitumor host immune response in the tumor microenvironment. Although the STAT3 signaling pathway is a potential drug target, clinical, pathologic, molecular, or prognostic features of STAT3-activated colorectal cancer remain uncertain. Experimental Design: Utilizing a database of 724 colon and rectal cancer cases, we evaluated phosphorylated STAT3 (p-STAT3) expression by immunohistochemistry. The Cox proportional hazards model was used to compute mortality HR, adjusting for clinical, pathologic, and molecular features, including microsatellite instability (MSI), the CpG island methylator phenotype (CIMP), LINE-1 methylation, 18q LOH, TP53 (p53), CTNNB1 (β-catenin), JC virus T-antigen, and KRAS, BRAF, and PIK3CA mutations. Results: Among the 724 tumors, 131 (18%) showed high-level p-STAT3 expression (p-STAT3-high), 244 (34%) showed low-level expression (p-STAT3-low), and the remaining 349 (48%) were negative for p-STAT3. p-STAT3 overexpression was associated with significantly higher colorectal cancer–specific mortality [log-rank P = 0.0020; univariate HR (p-STAT3-high vs. p-STAT3-negative): 1.85, 95% CI: 1.30–2.63, Ptrend = 0.0005; multivariate HR: 1.61, 95% CI: 1.11–2.34, Ptrend = 0.015]. p-STAT3 expression was positively associated with peritumoral lymphocytic reaction (multivariate OR: 3.23; 95% CI: 1.89–5.53, P &amp;lt; 0.0001). p-STAT3 expression was not associated with MSI, CIMP, or LINE-1 hypomethylation. Conclusions: STAT3 activation in colorectal cancer is associated with adverse clinical outcome, supporting its potential roles as a prognostic biomarker and a chemoprevention and/or therapeutic target. Clin Cancer Res; 17(6); 1452–62. ©2011 AACR.

Alterations of the WNT signaling pathway and cadherin-associated protein β 1 (CTNNB1 or β-catenin) have been implicated in colorectal carcinogenesis and metabolic diseases.To test the hypothesis that CTNNB1 activation in colorectal cancer modifies prognostic associations of body mass index (BMI) and level of postdiagnosis physical activity.Two US prospective cohort studies (Nurses' Health Study and the Health Professionals Follow-up Study) were used to evaluate CTNNB1 localization by immunohistochemistry in 955 patients with stage I, II, III, or IV colon and rectal cancer from 1980 through 2004. A Cox proportional hazards model was used to compute the hazard ratio (HR) for mortality, adjusting for clinical and tumor features, including microsatellite instability, CpG island methylator phenotype, level of long interspersed nucleotide element 1 methylation, mutations in KRAS, BRAF, or PIK3CA, and tumor protein p53.Colorectal cancer-specific mortality and overall mortality through June 30, 2009.In obese patients (BMI ≥30), positive status for nuclear CTNNB1 was associated with significantly better colorectal cancer-specific survival (adjusted HR, 0.24 [95% confidence interval {CI}, 0.12-0.49], P <.001 for interaction; 5-year survival: 0.85 for patients with positive nuclear CTNNB1 status vs 0.78 for those with negative status) and overall survival (adjusted HR, 0.56 [95% CI, 0.35-0.90], P = .03 for interaction; 5-year survival: 0.77 for patients with positive nuclear CTNNB1 status vs 0.74 for those with negative status), while CTNNB1 status was not associated with prognosis among nonobese patients (BMI <30). Among patients with negative status for nuclear CTNNB1 and cancer in stages I, II, or III, postdiagnosis physical activity was associated with better colorectal cancer-specific survival (adjusted HR, 0.33 [95% CI, 0.13-0.81], P = .05 for interaction; 5-year survival: 0.97 for ≥18 vs 0.89 for <18 metabolic equivalent task hours/week), while postdiagnosis physical activity was not associated with colorectal cancer-specific survival among patients with positive status for nuclear CTNNB1 (adjusted HR, 1.07 [95% CI, 0.50-2.30]).Among obese patients only, activation of CTNNB1 was associated with better colorectal cancer-specific survival and overall survival. Postdiagnosis physical activity was associated with better colorectal cancer-specific survival only among patients with negative status for nuclear CTNNB1. These molecular pathological epidemiology findings suggest that the effects of alterations in the WNT-CTNNB1 pathway on outcome are modified by BMI and physical activity.

Aspirin use reduces the risk of colorectal carcinoma. Experimental evidence implicates a role of RAF kinases in up-regulation of prostaglandin-endoperoxide synthase 2 (PTGS2, cyclooxygenase 2), suggesting that BRAF-mutant colonic cells might be less sensitive to the antitumor effects of aspirin than BRAF-wild-type neoplastic cells.To examine whether the association of aspirin intake with colorectal cancer risk differs according to status of tumor BRAF oncogene mutation.We collected biennial questionnaire data on aspirin use and followed up participants in the Nurses' Health Study (from 1980) and the Health Professionals Follow-up Study (from 1986) until July 1, 2006, for cancer incidence and until January 1, 2012, for cancer mortality. Duplication-method Cox proportional cause-specific hazards regression for competing risks data was used to compute hazard ratios (HRs) for colorectal carcinoma incidence according to BRAF mutation status.Incidence of colorectal cancer cases according to tumor BRAF mutation status.Among 127,865 individuals, with 3,165,985 person-years of follow-up, we identified 1226 incident rectal and colon cancers with available molecular data. Compared with nonuse, regular aspirin use was associated with lower BRAF-wild-type cancer risk (multivariable HR, 0.73; 95% CI, 0.64 to 0.83; age-adjusted incidence rate difference [RD], -9.7; 95% CI, -12.6 to -6.7 per 100,000 person-years). This association was observed irrespective of status of tumor PTGS2 expression or PIK3CA or KRAS mutation. In contrast, regular aspirin use was not associated with a lower risk of BRAF-mutated cancer (multivariable HR, 1.03; 95% CI, 0.76 to 1.38; age-adjusted, incidence RD, 0.7; 95% CI, -0.3 to 1.7 per 100,000 person-years: P for heterogeneity = .037, between BRAF-wild-type vs BRAF-mutated cancer risks). Compared with no aspirin use, aspirin use of more than 14 tablets per week was associated with a lower risk of BRAF-wild-type cancer (multivariable HR, 0.43; 95% CI, 0.25 to 0.75; age-adjusted incidence RD, -19.8; 95% CI, -26.3 to -13.3 per 100,000 person-years). The relationship between the number of aspirin tablets per week and colorectal cancer risk differed significantly by BRAF mutation status (P for heterogeneity = .005).Regular aspirin use was associated with lower risk of BRAF-wild-type colorectal cancer but not with BRAF-mutated cancer risk. These findings suggest that BRAF-mutant colon tumor cells may be less sensitive to the effect of aspirin. Given the modest absolute risk difference, further investigations are necessary to determine clinical implications of our findings.

Integrated sequencing analysis identified a group of tumors among clear cell renal cell carcinomas characterized by hotspot mutations in TCEB1 (a gene that contributes to the VHL complex to ubiquitinate hypoxia-inducible factor). We analyzed 11 tumors from two distinct cohorts with TCEB1 mutations along with an expanded cohort to assess whether these should be considered an entity distinct from clear cell renal cell carcinoma and clear cell papillary renal cell carcinoma. All tumors were characterized by hotspot mutations in TCEB1 Y79C/S/F/N or A100P. Morphological and immunohistochemical characteristics of the tumors were assessed by two experienced genitourinary pathologists. Clinical and pathological variables, copy number alterations, mutations, and expression signatures were compared with a cohort of TCEB1 wild-type tumors. All TCEB1-mutated tumors were VHL and PBRM1 wild type and contained distinct copy number profiles including loss of heterozygosity of chromosome 8, the location of TCEB1 (8q21.11). All tumors lacked the clear cell renal cell carcinoma signature 3p loss and contained distinct gene expression signatures. None of the clear cell papillary tumors harbored TCEB1 mutations. Pathologically, all TCEB1-mutated tumors shared characteristic features including thick fibromuscular bands transecting the tumor, pure clear cell cytology frequently with cells showing voluminous cytoplasm, and clear cell renal cell carcinoma-like acinar areas associated with infolding tubular and focally papillary architecture. The presence of voluminous cytoplasm, absence of luminal polarization of tumor nuclei, and lack of extensive cup-like distribution of carbonic anhydrase-IX expression distinguish it from clear cell papillary carcinoma. None of the patients developed metastases at last follow-up (median 48 months). In sum, TCEB1-mutated renal cell carcinoma is a distinct entity with recurrent hotspot mutations, specific copy number alterations, pathway activation, and characteristic morphological features. Further clinical follow-up is needed to determine whether these tumors are more indolent compared with the conventional clear cell renal cell carcinoma.

Interstitial cystitis (IC) is a chronic bladder disease with urinary frequency, bladder discomfort or bladder pain of unknown etiology. Based on cystoscopic findings, patients with IC are classified as either Hunner-type/classic IC (HIC), presenting with a specific Hunner lesion, or non-Hunner-type IC (NHIC), presenting with no Hunner lesion, but post-hydrodistension mucosal bleeding. Inflammatory cell infiltration, composed predominantly of lymphocytes, plasma cells and epithelial denudation, has in the past been documented as a major pathological IC finding. However, the significance of the pathological evaluation of IC, especially with regard to the difference between HIC and NHIC, has been downplayed in recent years. In this study, we performed immunohistochemical quantification of infiltrating T-lymphocytes, B-lymphocytes and plasma cells, and measured the amount of residual epithelium in urinary bladder biopsy specimens taken from patients with HIC and NHIC, and those with no IC, using image analysis software. In addition, in situ hybridization of the light chains was performed to examine clonal B-cell expansion. Lymphoplasmacytic infiltration was significantly more severe in HIC specimens than in NHIC specimens (P <0.0001). Substantial lymphoplasmacytic inflammation (≥200 cells/mm2) was observed in 93% of HIC specimens, whereas only 8% of NHIC specimens were inflamed. Plasmacytic infiltration was more prominent in HIC specimens compared with NHIC and non-IC cystitis specimens (P <0.005). Furthermore, expansion of light-chain-restricted B-cells was observed in 31% of cases of HIC. The amount of residual epithelium was decreased in HIC specimens compared with NHIC specimens and non-IC cystitis specimens (P <0.0001). These results suggest that NHIC and HIC are distinct pathological entities, with the latter characterized by pancystitis, frequent clonal B-cell expansion and epithelial denudation. An abnormality in the B-cell population may be involved in the pathogenesis of HIC.

Colorectal cancers (CRC) express the WNT effector protein β-catenin in a heterogeneous subcellular pattern rather than uniformly in the nucleus. In this study, we investigated this important aspect of molecular heterogeneity in CRCs by analyzing its basis and relationship with tumor-initiating capability. CRC cells with the highest WNT levels showed only a marginal increase in tumor initiation capacity. Notably, high WNT activity correlated with a coincident activation of robust mitogen-activated protein kinase (MAPK) signaling, which when upregulated by KRAS expression or downregulated by epidermal growth factor receptor inhibition elicited parallel effects on WNT activity. These findings suggested that on its own high WNT activity may not be a reliable signifier of tumor-initiating potential or stem-like potential. Furthermore, they suggest that MAPK signaling is a critical modifier of intratumoral heterogeneity that contributes significantly to determining the impact of WNT activity on stemness phenotypes in colon cancer cells.

Oncological outcomes of lateral pelvic lymph node metastasis in rectal cancer treated with preoperative chemoradiotherapy remain to be elucidated.The purpose of this study was to clarify the therapeutic effect of chemoradiotherapy on lateral pelvic lymph node metastasis, the risk factors of lateral pelvic lymph node metastasis, and oncological outcomes of lateral pelvic lymph node dissection after chemoradiotherapy.This was a nonrandomized, retrospective study.The study was conducted at a tertiary referral university hospital.Patients with rectal cancer treated with chemoradiotherapy and radical surgery from 2003 to 2015 (N = 222) were included.Radiation (total, 50.4 Gy in 28 fractions) with concomitant fluorouracil-based chemotherapy was administered. Lateral pelvic lymph nodes with a diameter of ≥8 mm before chemoradiotherapy were selectively dissected.Frequency and risk factors of lateral pelvic lymph node metastasis were examined.Lateral pelvic lymph node dissection was performed in 31 patients (14.0%), and 16 (51.6%) of these patients were pathologically diagnosed as positive for metastasis. Among the patients treated with total mesorectal excision alone (n = 191), 2 (0.9%) had recurrence in the lateral pelvic lymph node area, which was pathologically confirmed after salvage R0 resection. T category downstaging (73.3% vs 12.5%; p < 0.01) and high histological regression of the primary lesion (73.3% vs 18.8%; p < 0.01) were more frequent in patients with pathologically negative lateral pelvic lymph nodes than in those with positive lateral pelvic lymph nodes. Young age, short distance from the anal verge, and enlarged lateral pelvic lymph node before chemoradiotherapy were associated with lateral pelvic lymph node metastasis.The study was limited by its retrospective nature and small study population.The incidence of lateral pelvic lymph node metastasis after chemoradiotherapy was estimated to be 8.1% (18/222). Young age, short distance from the anal verge, and enlarged lateral pelvic lymph node before chemoradiotherapy were risk factors of lateral pelvic lymph node metastasis after chemoradiotherapy.

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 (β-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.

YAP 1 , the main Hippo pathway effector, is a potent oncogene and is overexpressed in non‐small‐cell lung cancer ( NSCLC ); however, the YAP 1 expression pattern in small‐cell lung cancer ( SCLC ) has not yet been elucidated in detail. We report that the loss of YAP 1 is a special feature of high‐grade neuroendocrine lung tumors. A hierarchical cluster analysis of 15 high‐grade neuroendocrine tumor cell lines containing 14 SCLC cell lines that depended on the genes of Hippo pathway molecules and neuroendocrine markers clearly classified these lines into two groups: the YAP 1 ‐negative and neuroendocrine marker‐positive group ( n = 11), and the YAP 1 ‐positive and neuroendocrine marker‐negative group ( n = 4). Among the 41 NSCLC cell lines examined, the loss of YAP 1 was only observed in one cell line showing the strong expression of neuroendocrine markers. Immunostaining for YAP 1, using the sections of 189 NSCLC , 41 SCLC , and 30 large cell neuroendocrine carcinoma ( LCNEC ) cases, revealed that the loss of YAP 1 was common in SCLC (40/41, 98%) and LCNEC (18/30, 60%), but was rare in NSCLC (6/189, 3%). Among the SCLC and LCNEC cases tested, the loss of YAP 1 correlated with the expression of neuroendocrine markers, and a survival analysis revealed that YAP 1‐negative cases were more chemosensitive than YAP 1‐positive cases. Chemosensitivity test for cisplatin using YAP 1‐positive/ YAP 1‐negative SCLC cell lines also showed compatible results. YAP 1 ‐sh‐mediated knockdown induced the neuroendocrine marker RAB 3a , which suggested the possible involvement of YAP 1 in the regulation of neuroendocrine differentiation. Thus, we showed that the loss of YAP 1 has potential as a clinical marker for predicting neuroendocrine features and chemosensitivity.

Chronic inflammation plays a role in the development of colorectal cancer (CRC). The novel plasma inflammatory biomarker macrophage inhibitory cytokine-1 (MIC-1, GDF15) may have a direct mechanistic role in colorectal carcinogenesis. We conducted a prospective, nested, case–control study of incident CRC among men and women who provided a prediagnostic blood specimen. We used an enzyme-linked immunosorbent assay to measure MIC-1 and examined associations between quintiles of MIC-1 and CRC using logistic regression adjusted for matching factors (age and date of blood draw), risk factors, and other plasma inflammatory markers. We also assessed the relationship between MIC-1 levels and prostaglandin-endoperoxide synthase 2 (PTGS2)/cyclooxygenase-2 (COX-2) enzyme status in tumors with available tissue for analysis. All statistical tests were two-sided. Compared with men and women within the lowest quintile of plasma MIC-1, the multivariable relative risk (RR) for CRC was 1.93 (95% confidence interval [CI] = 1.27 to 2.94) for the highest quintile (Plinear trend = .004). In an exploratory analysis, we found that among individuals with high plasma MIC-1 levels (quintiles 2–5), compared with nonuse, regular use of aspirin and nonsteroidal anti-inflammatory drugs (NSAIDs) was associated with a lower risk of PTGS2-positive CRC (multivariable RR = 0.60; 95% confidence interval = 0.41 to 0.88) but not PTGS2-negative CRC (multivariable RR = 1.21; 95% CI = 0.71 to 2.07). In contrast, among individuals with low MIC-1 levels (quintile 1), aspirin and NSAID use was not associated with a lower risk of PTGS2-positive CRC (multivariable RR = 0.57; 95% CI = 0.21 to 1.54) or PTGS2-negative CRC (multivariable RR = 1.41; 95% CI = 0.47 to 4.23). Our results support an association between higher levels of circulating MIC-1 (GDF15) and CRC. Aspirin/NSAID use appeared to lower risk of PTGS2-positive cancers, particularly among individuals with high levels of circulating MIC-1.

Upper urinary tract urothelial carcinoma (UTUC) is one of the common urothelial cancers. Its molecular pathogenesis, however, is poorly understood, with no useful biomarkers available for accurate diagnosis and molecular classification. Through an integrated genetic study involving 199 UTUC samples, we delineate the landscape of genetic alterations in UTUC enabling genetic/molecular classification. According to the mutational status of TP53, MDM2, RAS, and FGFR3, UTUC is classified into five subtypes having discrete profiles of gene expression, tumor location/histology, and clinical outcome, which is largely recapitulated in an independent UTUC cohort. Sequencing of urine sediment-derived DNA has a high diagnostic value for UTUC with 82.2% sensitivity and 100% specificity. These results provide a solid basis for better diagnosis and management of UTUC.

Prostate cancer (PCa) is a common malignant tumor among adult males, and convenient intraoperative detection of PCa would reduce the risk of leaving positive surgical margins, especially during nerve-sparing procedures. To achieve rapid, fluorescence-based visualization of PCa, we focused on the glutamate carboxypeptidase (CP) activity of prostate-specific membrane antigen (PSMA), a type II transmembrane glycoprotein that is attracting attention as a PCa biomarker. Based on our finding that aryl glutamate conjugates with an azoformyl linker are recognized by PSMA and have a sufficiently low LUMO (lowest unoccupied molecular orbital) energy level to quench the fluorophore through photoinduced electron transfer, we designed and synthesized a first-in-class activatable fluorescence probe for CP activity of PSMA. The developed probe allowed us to visualize the CP activity of PSMA in living cells and in clinical specimens from PCa patients and is expected to be useful for rapid intraoperative detection and diagnosis of PCa.

c-Met is often overexpressed in non-small cell lung cancer, but it remains unsolved whether its overexpression leads to its activation. We used an antibody specific to phospho-c-Met (Tyr1235) to investigate c-Met activation immunohistochemically in 130 surgically resected lung adenocarcinomas. The expression of c-Met and hepatocyte growth factor (HGF) was also investigated. Phospho-c-Met was positive in 21.5% (28/130) of cases. c-Met was positive in 74.6% of cases (97/130) and was expressed at high levels in 36.1% of cases (47/130). HGF was expressed at high levels in 31.5% of cases (41/130). Phospho-c-Met was correlated with high levels of HGF (P =0.0010) and high levels c-Met expression (P = 0.0303), but it was also found to be positive in 12 cases with little to no HGF expression. Phospho-c-Met expression was significantly associated with tumor differentiation (P = 0.0023) and papillary histology (P = 0.0011), but not with pathological stage, lymph node metastasis or survival. High levels of c-Met and HGF were also associated with papillary histology (P = 0.0056 and P = 0.0396, respectively), but not with tumor differentiation. Phospho-c-Met was correlated with phospho-Akt (P = 0.0381), but not with phospho-Erk or phospho-Stat3. Phospho-Akt expression was marginally correlated with the expression of phospho-epidermal growth factor receptor (EGFR) (P = 0.0533) and, importantly, it was strongly correlated with the expression of either phospho-c-Met or phospho-EGFR (P = 0.0013). The data suggest that in lung adenocarcinoma tissue, c-Met activation may take place either ligand-dependently or ligand-independently via c-Met overexpression. c-Met activation may play special roles in the papillary subtype and in well differentiated lung adenocarcinomas.

Clinical studies have implicated the mechanistic target of rapamycin (serine/threonine kinase; MTOR) pathway in the regulation of neuroendocrine tumor (NET) growth. We explored whether expression of MTOR pathway components has prognostic significance in NET patients.We evaluated immunohistochemical expression of MTOR and phospho (p) -MTOR; its downstream targets RPS6KB1, RPS6, and EIF4EBP1; and its upstream regulators, in a cohort of 195 archival neuroendocrine tumors. We correlated expression levels with clinical outcomes, after adjusting for other prognostic variables.We observed anticipated correlations between expression of upstream components of the MTOR pathway and their downstream targets. Expression of PIK3CA, MTOR, or p-EIF4EBP1 was associated with high MKI67 (Ki-67) labeling index. We failed to identify clinical correlations associated with expression of the upstream regulators TSC1, TSC2, AKT, p-AKT, PDPK1, PTEN, PIK3R1, or PIK3CA. In contrast, high expression of MTOR or its activated downstream targets p-RPS6KB1, p-RPS6, or p-EIF4EBP1 was associated with adverse clinical outcomes.Our observations suggest that expression of MTOR or its downstream targets may be adverse prognostic factors in neuroendocrine tumors.

The effect of duration of cigarette smoking cessation on colorectal cancer risk by molecular subtypes remains unclear.Using duplication-method Cox proportional-hazards regression analyses, we examined associations between duration of smoking cessation and colorectal cancer risk according to status of CpG island methylator phenotype (CIMP), microsatellite instability, v-raf murine sarcoma viral oncogene homolog B1 (BRAF) mutation, or DNA methyltransferase-3B (DNMT3B) expression.Follow-up of 134,204 individuals in 2 US nationwide prospective cohorts (Nurses' Health Study (1980-2008) and Health Professionals Follow-up Study (1986Study ( -2008))) resulted in 1,260 incident rectal and colon cancers with available molecular data.Compared with current smoking, 10-19, 20-39, and ≥40 years of smoking cessation were associated with a lower risk of CIMP-high colorectal cancer, with multivariate hazard ratios (95% confidence intervals) of 0.53 (0.29, 0.95), 0.52 (0.32, 0.85), and 0.50 (0.27, 0.94), respectively (P trend = 0.001), but not with the risk of CIMP-low/CIMP-negative cancer (P trend = 0.25) (P heterogeneity = 0.02, between CIMP-high and CIMP-low/CIMP-negative cancer risks).Differential associations between smoking cessation and cancer risks by microsatellite instability (P heterogeneity = 0.02), DNMT3B expression (P heterogeneity = 0.03), and BRAF (P heterogeneity = 0.10) status appeared to be driven by the associations of CIMP-high cancer with microsatellite instability-high, DNMT3B-positive, and BRAF-mutated cancers.These molecular pathological epidemiology data suggest a protective effect of smoking cessation on a DNA methylation-related carcinogenesis pathway leading to CIMP-high colorectal cancer.

Morikawa T, Maeda D, Kume H, Homma Y &amp; Fukayama M (2010) Histopathology 57, 885–892 Ribonucleotide reductase M2 subunit is a novel diagnostic marker and a potential therapeutic target in bladder cancer Aims: To examine the immunohistochemical expression and function of ribonucleotide reductase M2 subunit (RRM2), a gemcitabine‐related molecule, in bladder cancer. Methods and results: One hundred and seventeen bladder specimens on a tissue microarray were immunostained for RRM2. Positive RRM2 staining was observed in none of 14 examples of non‐neoplastic urothelium, none of four low‐grade urothelial carcinoma (UC), 69 of 83 (83%) high‐grade UC, three of three (100%) squamous cell carcinoma and 12 of 13 (92%) lymph node metastasis of UC. RRM2 overexpression was associated significantly with muscularis propria invasion in UC patients who had undergone radical cystectomy ( P = 0.005). Immunohistochemistry for RRM2 was then applied to small biopsy specimens of 15 cystitis with reactive atypia cases and 25 urothelial carcinoma in‐situ (CIS) cases. Positive RRM2 staining was found in one of 15 (6.7%) cystitis with reactive atypia cases and in 24 of 25 (96%) CIS cases. Finally, UM‐UC‐3 bladder cancer cells were transfected with RRM2 siRNAs and cell growth was evaluated. Knockdown of RRM2 protein markedly inhibited cell growth. Conclusions: We have shown frequent overexpression of RRM2 protein and its possible role in bladder cancer. Our results suggest that RRM2 is a novel diagnostic marker and a potential therapeutic target in bladder cancer.

Infiltrative growth pattern at the tumor margin has been associated with shorter patient survival. However, little is known about the prognostic significance of tumor growth pattern, independent of tumoral molecular alterations and other histologic features. Utilizing a database of 1139 colon and rectal cancer patients in two prospective cohort studies, histologic features including tumor growth pattern, tumor differentiation, lymphocytic reaction, mucinous component, and signet ring cell component were recorded by a single pathologist. Cox proportional hazard model was used to compute mortality hazard ratio, adjusting for clinical, pathologic, and tumor molecular features, including microsatellite instability, the CpG island methylator phenotype, long interspersed nucleotide element 1 (LINE-1) methylation, and KRAS, BRAF, and PIK3CA mutations. Among 1139 colorectal cancers, we observed expansile growth pattern in 372 tumors (33%), intermediate growth pattern in 610 tumors (54%), and infiltrative growth pattern in 157 tumors (14%). Compared to patients with expansile growth pattern, those with infiltrative growth pattern experienced shorter cancer-specific survival (log rank P < 0.0001; multivariate hazard ratio 1.74; 95% confidence interval 1.22–2.47) and overall survival (log rank P < 0.0001; multivariate hazard ratio 1.78; 95% confidence interval 1.33–2.39). The prognostic association of infiltrative growth pattern was confined to patients with stage I–III disease (P interaction with stage = 0.0001). Infiltrative growth pattern was associated with worse prognosis among stage I–III colorectal cancer patients, independent of other clinical, pathologic, and molecular characteristics.

Evidence suggests that insulin-like growth factor 2 messenger RNA binding protein 3 (IGF2BP3, also known as IMP3) represents a promising cancer biomarker. However, the clinical, pathological, molecular and prognostic features of IGF2BP3-positive colorectal cancers remain uncertain.We evaluated IGF2BP3 expression by immunohistochemistry in 671 rectal and colon cancer cases that form part of a molecular pathological epidemiology database. Cox proportional hazards regression models were used to compute mortality hazard ratio (HR), adjusting for clinical, pathological and molecular features, including microsatellite instability, the CpG island methylator phenotype, LINE-1 methylation and KRAS, BRAF and PIK3CA mutations.Among 671 colorectal cancers, 234 (35%) tumours were positive for IGF2BP3. In contrast, normal colorectal epithelium was negative for IGF2BP3 in all 403 specimens of normal mucosa adjacent to carcinoma. IGF2BP3 positivity was associated with poor differentiation (p=0.0003), stage III-IV disease (p=0.0081), BRAF mutation (p=0.031), and LINE-1 hypomethylation (p=0.020). IGF2BP3 positivity was significantly associated with shorter colorectal cancer-specific [log-rank p<0.0001; multivariate HR, 1.37; 95% confidence interval (CI), 1.02-1.84] and overall survival (log-rank p=0.0004; multivariate HR, 1.32; 95% CI, 1.05-1.66).IGF2BP3 expression in colorectal cancer is associated with adverse clinical outcome. Our findings support a role for IGF2BP3 as a diagnostic and/or prognostic biomarker in colorectal cancer.

Significance Increasing evidence supports the notion that an aberrant microenvironment facilitates cancer development. The mechanisms underlying the phenotypic changes and the tumor-promoting function of cancer-associated stroma remain largely unknown. Overexpression of the chromatin remodeling protein Hmga2 in the stroma leads to the formation of multifocal prostatic precancerous lesions in the neighboring naïve epithelium in a paracrine Wnt-dependent manner. The potent growth-promoting effects of Hmga2-modified stroma collaborate significantly with enhanced stromal AR signaling to drive the development of prostate adenocarcinoma. Our findings provide compelling evidence for the critical contribution of epigenetic changes in stromal cells to cancer initiation and progression.

Although protein arginine methyltransferase 5 (PRMT5) has been implicated in various cancers, its expression pattern in lung adenocarcinoma cell lines and tissues has not been elucidated enough. In this study, microarray analysis of 40 non–small-cell lung carcinoma cell lines showed that PRMT5 was a candidate histone methyltransferase gene that correlated with epithelial-mesenchymal transition. Immunocytochemical analysis of these cell lines indicated that the expression of PRMT5 was localized to the cytoplasm of E-cadherin–low and vimentin-high cell lines, whereas it was predominant in the nucleus and faint in the cytoplasm of E-cadherin–high and vimentin-low cell lines. Immunohistochemical analysis of lung adenocarcinoma cases (n = 130) revealed that the expression of PRMT5 was high in the cytoplasm of 47 cases (36%) and the nuclei of 34 cases (26%). The marked cytoplasmic expression of PRMT5 was frequently observed in high-grade subtypes (1 of 17 low grade, 21 of 81 intermediate grade, and 25 of 32 high grade; P < .0001) such as solid adenocarcinoma with the low expression of thyroid transcription factor 1 (the master regulator of lung) and low expression of cytokeratin 7 and E-cadherin (2 markers for bronchial epithelial differentiation), whereas the high nuclear expression of PRMT5 was frequently noted in adenocarcinoma in situ, a low-grade subtype (6 of 17 low grade, 25 of 81 intermediate grade, and 3 of 32 high grade; P = .0444). The cytoplasmic expression of PRMT5 correlated with a poor prognosis (P = .0089). We herein highlighted the importance of PRMT5 expression, especially its cytoplasmic expression, in the process of epithelial-mesenchymal transition and loss of the bronchial epithelial phenotype of lung adenocarcinoma.

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.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.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.Loss of CDH1 expression in colorectal cancer is associated with infiltrative tumour growth pattern and lymph node metastasis.

Dysregulation of the WNT/β-catenin (CTNNB1) signaling pathway is implicated in colorectal carcinoma and metabolic diseases. Considering these roles and cancer prevention, we hypothesized that tumor CTNNB1 status might influence cellular sensitivity to obesity and physical activity. In clinical follow-up of 109,046 women in the Nurses' Health Study and 47,684 men in the Health Professionals Follow-up Study, there were 861 incident rectal and colon cancers with tissue immunohistochemistry data on nuclear CTNNB1 expression. Using this molecular pathological epidemiology database, we conducted Cox proportional hazards regression analysis using data duplication method to assess differential associations of body mass index (BMI) or exercise activity with colorectal cancer risk according to tumor CTNNB1 status. Greater BMI was associated with a significantly higher risk of CTNNB1-negative cancer [multivariate HR = 1.34; 95% confidence interval (CI), 1.18-1.53 for 5.0 kg/m(2) increment; Ptrend = 0.0001] but not with CTNNB1-positive cancer risk (multivariate HR = 1.07; 95% CI, 0.92-1.25 for 5.0 kg/m(2) increment; Ptrend = 0.36; Pheterogeneity = 0.027, between CTNNB1-negative and CTNNB1-positive cancer risks). Physical activity level was associated with a lower risk of CTNNB1-negative cancer (multivariate HR = 0.93; 95% CI, 0.87-1.00 for 10 MET-h/wk increment; Ptrend = 0.044) but not with CTNNB1-positive cancer risk (multivariate HR = 0.98; 95% CI, 0.91-1.05 for 10 MET-h/wk increment; Ptrend = 0.60). Our findings argue that obesity and physical inactivity are associated with a higher risk of CTNNB1-negative colorectal cancer but not with CTNNB1-positive cancer risk. Furthermore, they suggest that energy balance and metabolism status exerts its effect in a specific carcinogenesis pathway that is less likely dependent on WNT/CTNNB1 activation. Cancer Res; 73(5); 1600-10. ©2012 AACR.

Regular aspirin use reduces the risk for colorectal cancer (CRC), possibly through inhibition of WNT/cadherin-associated protein β1 (CTNNB1 or β-catenin) signaling. The single nucleotide polymorphism (SNP) rs6983267 on chromosome 8q24 is a CRC susceptibility locus that affects binding activity of transcription factor 7 like-2 (TCF7L2) to CTNNB1, thereby altering expression of target oncogenes, including MYC.We evaluated regular aspirin use and CRC risk according to genotypes of SNP rs6983267 and CTNNB1 expression status in two prospective case-control studies (840 CRC case patients and 1686 age- and race-matched control subjects) nested within the Nurses' Health Study and the Health Professionals Follow-up Study. We estimated odds ratios (ORs) and 95% confidence intervals (CIs) using unconditional logistic regression models. All statistical tests were two-sided.A lower risk of CRC was associated with regular aspirin use and the T allele of rs6983267. The effect of aspirin was confined to individuals with protective T allele of rs6983267 (additive matching factors-adjusted OR for T allele = 0.83; 95% CI = 0.74 to 0.94; P trend = .002; P interaction = .01). Additionally, the T allele of rs6983267 was associated with a reduced expression of MYC oncogene (P trend = .03). Moreover, among individuals with protective T allele, the effect of regular aspirin use was limited to those with positive nuclear CTNNB1 expression. In a functional analysis, in vitro treatment of LS174T cells (a cell line heterozygous for rs6983267) with aspirin was statistically significantly associated with higher G/T allelic ratio of TCF7L2 immunoprecipitated DNA (P = .03).Our results support an influence of aspirin on WNT/CTNNB1 signaling and suggest that aspirin chemoprevention may be tailored according to rs6983267 genotype.

This study aimed to characterize the FDG uptake of renal cell carcinoma (RCC) by the pathological subtype and nuclear grade.We retrospectively identified patients who underwent F-FDG PET and subsequent partial or radical nephrectomy for renal tumors. The relationships of the SUV of renal tumor with subtypes, nuclear grade, and clinicopathological variables were investigated.Ninety-two tumors were analyzed, including 52 low-grade (G1 and G2) and 18 high-grade (G3 and G4) clear cell RCC; 7 chromophobe, 5 papillary, and 1 unclassified RCC; and 9 benign tumors (7 angiomyolipoma and 2 oncocytoma). The SUVs of high-grade clear cell RCC (mean ± SD, 6.8 ± 5.1) and papillary RCC (6.6 ± 3.7) were significantly higher than that of the controls (2.2 ± 0.3). The SUV of high-grade clear cell RCC was higher than that of low-grade tumors (median, 4.0 vs. 2.2; P < 0.001). The optimal SUV cutoff value of 3.0 helped to differentiate high-grade from low-grade clear cell RCC, with 89% sensitivity and 87% specificity. On multiple regression analysis, a high grade was the most significant predictor of SUV for clear cell RCC.FDG uptake higher than that observed in normal kidney tissues suggests a high-grade clear cell RCC or papillary RCC subtype. FDG-PET using SUV may have a role in prediction of pathological grade of renal tumor.

Aspirin use reduces colorectal cancer risk. Aspirin, a nonsteroidal anti-inflammatory drug, inhibits prostaglandin-endoperoxide synthase 2 (PTGS2 or cyclooxygenase-2); PTGS2 promotes inflammation and suppresses T-cell-mediated adaptive immunity. We investigated whether the inverse association of aspirin use with colorectal carcinoma risk was stronger for tumors with lower degrees of lymphocytic infiltrates than for tumors with higher degrees of lymphocytic infiltrates.We collected aspirin use data biennially from participants in the Nurses' Health Study and Health Professionals Follow-up Study. Participants were asked whether they took aspirin in most weeks, the number of tablets taken per week, and years of aspirin use. We collected available tumor specimens (n = 1458) from pathology laboratories in the United States. A pathologist confirmed the diagnosis of colorectal adenocarcinoma (excluding anal squamous cell carcinoma), and evaluated histopathology features, including patterns and degrees of lymphocytic infiltrates within and around tumor areas. Person-years of follow-up evaluation were accrued from the date of return of questionnaires until dates of colorectal cancer diagnosis, death, or the end of follow-up evaluation (June 2010). Duplication-method Cox proportional hazards regression was used to assess the association of aspirin with the incidence of colorectal carcinoma subgroups according to the degree of tumor-infiltrating lymphocytes (TILs), intratumoral periglandular reaction, peritumoral reaction, or Crohn's-like reaction.We documented 1458 rectal and colon cancers. The inverse association between regular aspirin use and colorectal cancer risk significantly differed by concentrations of TILs (Pheterogeneity = .007). Compared with nonregular use, regular aspirin use was associated with a lower risk of tumors that had low levels of TILs (relative risk, 0.72; 95% confidence interval, 0.63-0.81), and strength of the association depended on aspirin dose and duration (both Ptrend < .001). In contrast, aspirin use was not associated with a risk of tumors having intermediate or high levels of TILs. This differential association was consistent regardless of the status of tumor microsatellite instability, mutations in BRAF, or expression of PTGS2. Regular aspirin use was associated with a lower risk of tumors that contained low levels of CD3+ T cells, CD8+ T cells, or CD45RO (PTPRC)+ T cells (measured by immunohistochemistry and computer-assisted image analysis).Based on data from the prospective cohort studies, regular use of aspirin is associated with a lower risk of colorectal carcinomas with low concentrations of TILs. These findings indicate that the immune response in the tumor microenvironment could be involved in the chemopreventive effects of aspirin.

Prostaglandin-endoperoxide synthase 2 (PTGS2, cyclooxygenase-2; a target of aspirin) produces inflammatory mediator prostaglandin E2 (PGE2), and contributes to colorectal neoplasia development. PTGS2-driven inflammatory responses can induce tumor expression of microRNA MIR21 (miR-21) that can increase local PGE2 level by downregulating PGE2-metabolizing enzymes. We hypothesized that the prognostic association of tumor MIR21 expression level in colorectal carcinoma might depend on inflammatory tumor microenvironment and be stronger in tumors expressing high-level PTGS2.Utilizing 765 rectal and colon cancer specimens in the Nurses' Health Study and the Health Professionals Follow-up Study, we measured MIR21 expression by quantitative reverse transcription PCR, and PTGS2 expression by immunohistochemistry. Cox proportional hazards regression model was used to assess statistical interaction between MIR21 and PTGS2 in colorectal cancer-specific survival analysis, controlling for potential confounders including microsatellite instability, CpG island methylator phenotype, LINE-1 methylation level, and KRAS, BRAF, and PIK3CA mutations.Tumor MIR21 expression level was associated with higher colorectal cancer-specific mortality (Ptrend = 0.029), and there was a statistically significant interaction between MIR21 and PTGS2 (Pinteraction = 0.0004). The association between MIR21 expression and colorectal cancer-specific mortality was statistically significant in PTGS2-high cancers (multivariable hazard ratio of the highest vs. lowest quartile of MIR21, 2.28; 95% confidence interval, 1.42-3.67; Ptrend = 0.0004) but not in PTGS2-absent/low cancers (Ptrend = 0.22).MIR21 expression level in colorectal carcinoma is associated with worse clinical outcome, and this association is stronger in carcinomas expressing high-level PTGS2, suggesting complex roles of immunity and inflammation in tumor progression. Clin Cancer Res; 22(15); 3841-8. ©2016 AACR.

Inhibitors targeting the PDCD1 (programmed cell death 1, PD-1) immune checkpoint pathway have revolutionized cancer treatment strategies. The TIME (Tumor Immunity in the MicroEnvironment) classification based on tumor CD274 (PDCD1 ligand 1, PD-L1) expression and tumor-infiltrating lymphocytes (TIL) has been proposed to predict response to immunotherapy. It remains to be determined clinical, pathological, and molecular features of TIME subtypes of colorectal cancer. Using 812 colon and rectal carcinoma cases from the Nurses' Health Study and Health Professionals Follow-up Study, we examined the association of tumor characteristics and survival outcomes with four TIME subtypes (TIME 1, CD274low/TILabsent; TIME 2, CD274high/TILpresent; TIME 3, CD274low/TILpresent; and TIME 4, CD274high/TILabsent). In survival analyses, Cox proportional hazards models were adjusted for potential confounders, including microsatellite instability (MSI) status, CpG island methylator phenotype (CIMP) status, LINE-1 methylation level, and KRAS, BRAF, and PIK3CA mutation status. TIME subtypes 1, 2, 3 and 4 had 218 (27%), 117 (14%), 103 (13%), and 374 (46%) colorectal cancer cases, respectively. Compared with TIL-absent subtypes (TIME 1 and 4), TIL-present subtypes (TIME 2 and 3) were associated with high-level MSI, high-degree CIMP, BRAF mutation, and higher amounts of neoantigens (p < 0.001). TIME subtypes were not significantly associated with colorectal cancer-specific or overall survival. In conclusion, TIL-present TIME subtypes of colorectal cancer are associated with high levels of MSI and neoantigen load, supporting better responsiveness to cancer immunotherapy. Further studies examining tumor molecular alterations and additional factors in the tumor microenvironment may inform development of immunoprevention and immunotherapy strategies.

To facilitate the optimization of cancer immunotherapy lacking immune-related adverse events, we performed TCR repertoire analysis of tumor-infiltrating CD8+ T-cells in B16 melanoma-bearing mice receiving anti-PD-1, anti-CTLA-4, anti-4-1BB, anti-CD4 or a combination of anti-PD-1 and 4-1BB antibodies. Although CD8+ T-cells in the tumor were activated and expanded to a greater or lesser extent by these therapies, tumor growth suppression was achieved only by anti-PD-1, anti-PD-1/4-1BB combined, or by anti-CD4 treatment, but not by anti-CTLA-4 or anti-4-1BB monotherapy. Increased CD8+ T cell effector function and TCR diversity with enrichment of certain TCR clonotypes in the tumor was associated with anti-tumor effects. In contrast, polyclonal activation of T-cells in the periphery was associated with tissue damage. Thus, optimal combination therapy increases TCR diversity with extended activation of selective CD8+ T-cells specifically in the tumor but not in the periphery. Incorporation of the concept of evenness for the TCR diversity is proposed.

No AccessJournal of UrologyAdult Urology1 Aug 2019Molecular Taxonomy of Interstitial Cystitis/Bladder Pain Syndrome Based on Whole Transcriptome Profiling by Next-Generation RNA Sequencing of Bladder Mucosal Biopsies Yoshiyuki Akiyama, Daichi Maeda, Hiroto Katoh, Teppei Morikawa, Aya Niimi, Akira Nomiya, Yusuke Sato, Taketo Kawai, Akiteru Goto, Tetsuya Fujimura, Hiroshi Fukuhara, Tohru Nakagawa, Yasuhiko Igawa, Shumpei Ishikawa, Masashi Fukayama, Haruki Kume, and Yukio Homma Yoshiyuki AkiyamaYoshiyuki Akiyama *Correspondence: Department of Urology, Graduate School of Medicine, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan telephone: +81-35-800-8753 [03-5800-8753]; FAX: +81-35-800-6435 [03-5800-6435]; E-mail Address: [email protected] Department of Urology, Graduate School of Medicine, University of Tokyo, Tokyo More articles by this author , Daichi MaedaDaichi Maeda Department of Clinical Genomics, Graduate School of Medicine, Osaka University, Osaka More articles by this author , Hiroto KatohHiroto Katoh Department of Genomic Pathology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo More articles by this author , Teppei MorikawaTeppei Morikawa Department of Diagnostic Pathology, NTT Medical Center Tokyo, Tokyo More articles by this author , Aya NiimiAya Niimi Department of Urology, National Center for Global Health and Medicine, Tokyo More articles by this author , Akira NomiyaAkira Nomiya Department of Urology, Graduate School of Medicine, University of Tokyo, Tokyo More articles by this author , Yusuke SatoYusuke Sato Department of Urology, Graduate School of Medicine, University of Tokyo, Tokyo More articles by this author , Taketo KawaiTaketo Kawai Department of Urology, Graduate School of Medicine, University of Tokyo, Tokyo More articles by this author , Akiteru GotoAkiteru Goto Department of Cellular and Organ Pathology, Graduate School of Medicine, Akita University, Akita More articles by this author , Tetsuya FujimuraTetsuya Fujimura Department of Urology, Jichi Medical University, Tochigi, Japan More articles by this author , Hiroshi FukuharaHiroshi Fukuhara Kyorin University School of Medicine, Tokyo More articles by this author , Tohru NakagawaTohru Nakagawa Teikyo University School of Medicine, Tokyo More articles by this author , Yasuhiko IgawaYasuhiko Igawa Department of Continence Medicine, Graduate School of Medicine, University of Tokyo, Tokyo More articles by this author , Shumpei IshikawaShumpei Ishikawa Department of Genomic Pathology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo More articles by this author , Masashi FukayamaMasashi Fukayama Department of Pathology, Graduate School of Medicine, University of Tokyo, Tokyo More articles by this author , Haruki KumeHaruki Kume Department of Urology, Graduate School of Medicine, University of Tokyo, Tokyo More articles by this author , and Yukio HommaYukio Homma Japanese Red Cross Medical Center, Tokyo More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000234AboutFull TextPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract Purpose: We systematically characterized gene expression, inflammation and neovascularization in patients with interstitial cystitis/bladder pain syndrome to obtain biological evidence supporting diagnosis and classification. Materials and Methods: We sequenced RNA obtained from bladder mucosal biopsies of 33 patients with 3 subtypes of interstitial cystitis/bladder pain syndrome, including Hunner lesions in 12, no Hunner lesions in 11 but with glomerulations and neither Hunner lesions nor glomerulations in 10, and 9 controls. Differentially expressed genes of each subtype were searched to identify subtype specific biological pathways and candidate genes important for pathogenesis. Candidate genes were validated by quantitative polymerase chain reaction and immunohistochemistry. Digital immunohistochemical quantification was performed to assess subepithelial lymphoplasmacytic cell and microvessel density. Relationships between candidate gene over expression and symptom severity were explored. Results: Patients with Hunner lesions showed a distinct gene expression profile associated with significant up-regulation of biological processes involving immune responses and infection, and an increase in subepithelial lymphoplasmacytic cell and microvessel density. Over expression of 2 candidate genes, VEGF and BAFF, correlated with symptom severity. Meanwhile, the gene expression profiles of patients with the 2 subtypes without Hunner lesions were similar to those of controls. No difference in biological pathways or subepithelial lymphoplasmacytic cell and microvessel density were detected between these 2 subtypes and controls. Conclusions: Interstitial cystitis/bladder pain syndrome with Hunner lesions shows distinct genomic and histological features associated with immune responses and infection. In addition, VEGF and BAFF are potential disease biomarkers and therapeutic targets. This subtype should be considered separate from the syndrome. 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Supported by the Practical Research Project for Rare/Intractable Diseases of the Japan AMED (Agency for Medical Research and Development), the Japan Intractable Diseases Research Foundation, the Asahi Kasei Pharma Urological Academy and KAKENHI Grants-in-Aid from the PSPS (Japanese Society for the Promotion of Science) (No. 17H06639) (YA). No direct or indirect commercial, personal, academic, political, religious or ethical incentive is associated with publishing this article. © 2019 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetailsCited byAkiyama Y, Miyakawa J, O’Donnell M, Kreder K, Luo Y, Maeda D, Ushiku T, Kume H and Homma Y (2021) Overexpression of HIF1α in Hunner Lesions of Interstitial Cystitis: Pathophysiological ImplicationsJournal of Urology, VOL. 207, NO. 3, (635-646), Online publication date: 1-Mar-2022.Wein A (2020) Re: Update on the Pathophysiology of Interstitial Cystitis/Bladder Pain SyndromeJournal of Urology, VOL. 204, NO. 3, (625-625), Online publication date: 1-Sep-2020. Volume 202Issue 2August 2019Page: 290-300Supplementary Materials Advertisement Copyright & Permissions© 2019 by American Urological Association Education and Research, Inc.Keywordsurinary bladderinterstitialinflammationpathologiccystitisgene expressionneovascularizationAcknowledgmentKei Sakuma provided immunohistochemistry technical support.MetricsAuthor Information Yoshiyuki Akiyama Department of Urology, Graduate School of Medicine, University of Tokyo, Tokyo *Correspondence: Department of Urology, Graduate School of Medicine, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan telephone: +81-35-800-8753 [03-5800-8753]; FAX: +81-35-800-6435 [03-5800-6435]; E-mail Address: [email protected] More articles by this author Daichi Maeda Department of Clinical Genomics, Graduate School of Medicine, Osaka University, Osaka More articles by this author Hiroto Katoh Department of Genomic Pathology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo More articles by this author Teppei Morikawa Department of Diagnostic Pathology, NTT Medical Center Tokyo, Tokyo More articles by this author Aya Niimi Department of Urology, National Center for Global Health and Medicine, Tokyo More articles by this author Akira Nomiya Department of Urology, Graduate School of Medicine, University of Tokyo, Tokyo More articles by this author Yusuke Sato Department of Urology, Graduate School of Medicine, University of Tokyo, Tokyo More articles by this author Taketo Kawai Department of Urology, Graduate School of Medicine, University of Tokyo, Tokyo More articles by this author Akiteru Goto Department of Cellular and Organ Pathology, Graduate School of Medicine, Akita University, Akita More articles by this author Tetsuya Fujimura Department of Urology, Jichi Medical University, Tochigi, Japan More articles by this author Hiroshi Fukuhara Kyorin University School of Medicine, Tokyo More articles by this author Tohru Nakagawa Teikyo University School of Medicine, Tokyo More articles by this author Yasuhiko Igawa Department of Continence Medicine, Graduate School of Medicine, University of Tokyo, Tokyo More articles by this author Shumpei Ishikawa Department of Genomic Pathology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo More articles by this author Masashi Fukayama Department of Pathology, Graduate School of Medicine, University of Tokyo, Tokyo More articles by this author Haruki Kume Department of Urology, Graduate School of Medicine, University of Tokyo, Tokyo More articles by this author Yukio Homma Japanese Red Cross Medical Center, Tokyo More articles by this author Expand All The corresponding author certifies that, when applicable, a statement(s) has been included in the manuscript documenting institutional review board, ethics committee or ethical review board study approval; principles of Helsinki Declaration were followed in lieu of formal ethics committee approval; institutional animal care and use committee approval; all human subjects provided written informed consent with guarantees of confidentiality; IRB approved protocol number; animal approved project number. Supported by the Practical Research Project for Rare/Intractable Diseases of the Japan AMED (Agency for Medical Research and Development), the Japan Intractable Diseases Research Foundation, the Asahi Kasei Pharma Urological Academy and KAKENHI Grants-in-Aid from the PSPS (Japanese Society for the Promotion of Science) (No. 17H06639) (YA). No direct or indirect commercial, personal, academic, political, religious or ethical incentive is associated with publishing this article. Advertisement PDF downloadLoading ...

Renal cell carcinoma (RCC) is one of the most drug-refractory cancers. The aim of this study is to discover a novel therapeutic target molecule for clear cell RCC (CCRCC), which accounts for the majority of RCC.Gene expression profiles of 27 CCRCCs and 9 normal kidney tissues as well as 15 various adult normal tissues were examined by Affymetrix U133 Plus 2.0 arrays. Among the 34 genes specifically up-regulated in CCRCC, overexpression of Toll-like receptor 3 (TLR3) mRNA and its protein was validated by quantitative reverse transcription-PCR, immunoblot, and immunohistochemistry. The effects of TLR3 signaling on in vitro cell growth were examined.TLR3 gene was highly expressed in CCRCC, with only limited expression in a panel of normal tissues. On immunohistochemical analysis using a monoclonal antibody against TLR3, overexpression of TLR3 was observed in 139 of 189 (73.5%) cases of CCRCC as well as in lung metastatic CCRCC (6 of 8), whereas TLR3 expression was entirely absent in chromophobe RCC (0 of 8). Polyinosinic-polycytidilic acid, a TLR3 ligand, exerted a growth-inhibitory effect against RCC cells in a TLR3-dependent manner. Moreover, a combination of polyinosinic-polycytidilic acid and IFNalpha exerted a synergistic growth-inhibitory effect against Caki-1 RCC cells.This is the first report that TLR3 is overexpressed in CCRCC. These observations suggest that TLR3 pathway may represent a novel therapeutic target in CCRCC.

O6-methylguanine-DNA methyltransferase (MGMT) is a DNA repair enzyme. MGMT promoter hypermethylation and epigenetic silencing often occur as early events in carcinogenesis. However, prognostic significance of MGMT alterations in colorectal cancer remains uncertain. Utilizing a database of 855 colon and rectal cancers in two prospective cohort studies (the Nurses’ Health Study and the Health Professionals Follow-up Study), we detected MGMT promoter hypermethylation in 325 tumors (38%) by MethyLight and loss of MGMT expression in 37% (247/672) of tumors by immunohistochemistry. We assessed the CpG island methylator phenotype (CIMP) using eight methylation markers [CACNA1G, CDKN2A (p16), CRABP1, IGF2, MLH1, NEUROG1, RUNX3, and SOCS1], and LINE-1 (L1) hypomethylation, TP53 (p53), and microsatellite instability (MSI). MGMT hypermethylation was not associated with colorectal cancer–specific mortality in univariate or multivariate Cox regression analysis [adjusted hazard ratio (HR) = 1.03; 95% confidence interval (CI), 0.79–1.36] that adjusted for clinical and tumor features, including CIMP, MSI, and BRAF mutation. Similarly, MGMT loss was not associated with patient survival. MGMT loss was associated with G>A mutations in KRAS (p = 0.019) and PIK3CA (p = 0.0031). Despite a well-established role of MGMT aberrations in carcinogenesis, neither MGMT promoter methylation nor MGMT loss serves as a prognostic biomarker in colorectal cancer.

Ribonucleotide reductase M2 subunit is one of two subunits that constitute ribonucleotide reductase, the enzyme that catalyzes the conversion of ribonucleotide 5′-diphosphates into 2′-deoxyribonucleotides, which are required for DNA synthesis. This study was conducted to investigate the roles of ribonucleotide reductase M2 subunit in gastric cancer. The expression of ribonucleotide reductase M2 subunit protein was examined by immunohistochemistry. In normal gastric mucosa, ribonucleotide reductase M2 subunit expression was restricted to the neck regions of gastric pits and no expression was observed in the surface epithelium. Among 112 gastric cancer tissues, ribonucleotide reductase M2 subunit overexpression (≥10% cancer cells stained) was observed in 72 cases (64.3%). Ribonucleotide reductase M2 subunit overexpression was significantly associated with male sex (P = .015), presence of muscularis propria invasion (P = .020), presence of Epstein-Barr virus (P = .045), expression of survivin (P = .0014), and DNA methyltransferase 1 (P = .043), but not with age, histology, tumor size, lymph node metastasis or expression of phosphatase and tensin homolog, phosphorylated signal transducer, and activator of transcription 3 or p53. Suppression of ribonucleotide reductase M2 subunit synthesis, using small interfering RNA, inhibited the growth of 3 gastric cancer cell lines, MKN-1, MKN-7, and SNU-719. Our data suggest that ribonucleotide reductase M2 subunit overexpression could be associated with the gastric cancer progression and that suppression of its function is a potential therapeutic strategy in gastric cancer.

Fatty acid synthase (FASN) plays an important role in energy metabolism of fatty acids and is overexpressed in some colon cancers. We investigated whether associations between body mass index (BMI) and risk of colorectal cancer varied according to FASN expression. During follow-up of 109 051 women in the ongoing prospective Nurses’ Health Study, a total of 1351 incident colon and rectal cancers were diagnosed between 1986 and 2004. We constructed tissue microarrays of the available resected tumor samples (n = 536), and FASN expression was analyzed by immunohistochemistry. Hazard ratios (HRs) and 95% confidence intervals (CIs) were calculated using Cox proportional hazards regression models. All statistical tests were two-sided. High BMI was associated with an increased risk of FASN-negative (no or weak expression) colorectal cancer compared with normal BMI (high BMI [≥30 kg/m 2 ], ie, obese vs normal BMI [18.5–22.9 kg/m 2 ], HR = 2.25, 95% CI = 1.49 to 3.40, Ptrend < .001) but not with FASN-positive (moderate to strong expression) colorectal cancer. A statistically significant heterogeneity in colorectal cancer risks was observed between FASN-negative and FASN-positive tumors ( Pheterogeneity = .033). The age-adjusted incidence rates for FASN-positive and FASN-negative colorectal cancers were 10.9 and 7.1, respectively, per 100 000 person-years. This molecular pathological epidemiology study supports a role of energy metabolism in colorectal cancer pathogenesis.

Beyond known familial colorectal cancer (CRC) syndromes, the mechanisms underlying the elevated CRC risk associated with CRC family history remain largely unknown. A recent retrospective study suggests familial clustering of CRC with hypomethylation in long interspersed nucleotide element 1 (LINE-1). We tested the hypothesis that CRC family history might confer a higher risk of LINE-1 methylation-low CRC.Using the Nurses' Health Study and the Health Professionals Follow-up Study, we prospectively examined the association between CRC family history and the risk of rectal and colon cancer (N = 1224) according to tumor LINE-1 methylation level by duplication method Cox proportional hazards regression. We examined microsatellite instability (MSI) status to exclude the influence of Lynch syndrome. All statistical tests were two-sided.The association between CRC family history and non-MSI CRC risk differed statistically significantly by LINE-1 methylation level (P (heterogeneity) = .02). CRC family history was associated with a statistically significantly higher risk of LINE-1 methylation-low non-MSI cancer (multivariable hazard ratio [HR] = 1.68, 95% confidence interval [CI] = 1.19 to 2.38 for 1 vs 0 first-degree relatives with CRC; multivariable HR = 3.48, 95% CI = 1.59 to 7.6 for ≥2 vs 0 first-degree relatives with CRC; P (trend) < .001). In contrast, CRC family history was not statistically significantly associated with LINE-1 methylation-high non-MSI cancer (P (trend) = .35).This molecular pathological epidemiology study shows that CRC family history is associated with a higher risk of LINE-1 methylation-low CRC, suggesting previously unrecognized heritable predisposition to epigenetic alterations. Additional studies are needed to evaluate tumor LINE-1 methylation as a molecular biomarker for familial cancer risk assessment.

Colorectal mixed adenoneuroendocrine carcinoma (MANEC), which acts like an aggressive tumor, is a rare clinical manifestation on which only a limited amount of literature exists. Surgical resection by regional lymphadenectomy is considered as the only curative treatment for colorectal MANEC, and adjuvant chemotherapy or radiotherapy is recommended because of its high recurrence rate. Colorectal MANEC is frequently diagnosed at an advanced stage, when it is unresectable, and chemotherapy plays a central role in its treatment. Pathological confirmation of the target lesion component is critical for regimen selection. If the lesion comprises an adenocarcinomatous component, a regimen for colorectal adenocarcinoma should be administered. For lesions comprising mainly a neuroendocrine carcinomatous component, cisplatin combined with etoposide or irinotecan has proven to be clinically appropriate. Everolimus, a mechanistic target of rapamycin pathway inhibitor, also improves survival. Sunitinib malate, another molecular targeting agent, is effective for treating neuroendocrine carcinoma; however, the evidence on its effectiveness for treating gastrointestinal neuroendocrine carcinoma is insufficient.

Abstract An up-regulated CXCR3 pathway and affluent plasma cell infiltration are characteristic features of Hunner type interstitial cystitis (HIC). We further examined these two features using bladder biopsy samples taken from 27 patients with HIC and 15 patients with non-IC cystitis as a control. The number of CD3-positive T lymphocytes, CD20-positive B lymphocytes, CD138-positive plasma cells, and CXCR3-positive cells was quantified by digital image analysis. Double-immunofluorescence for CXCR3 and CD138 was used to detect CXCR3 expression in plasma cells. Correlations between CXCR3 positivity and lymphocytic and plasma cell numbers and clinical parameters were explored. The density of CXCR3-positive cells showed no significant differences between HIC and non-IC cystitis specimens. However, distribution of CXCR3-positivity in plasma cells indicated co-localization of CXCR3 with CD138 in HIC specimens, but not in non-IC cystitis specimens. The number of CXCR3-positive cells correlated with plasma cells in HIC specimens alone. Infiltration of CXCR3-positive cells was unrelated to clinical parameters of patients with HIC. These results suggest that infiltration of CXCR3-positive plasma cells is a characteristic feature of HIC. The CXCR3 pathway and specific immune responses may be involved in accumulation/retention of plasma cells and pathophysiology of the HIC bladder.

Objective Although evidence suggests an inverse association between calcium intake and the risk of colorectal cancer, the mechanisms remain unclear. The calcium-sensing receptor (CASR) is expressed abundantly in normal colonic epithelium and may influence carcinogenesis. We hypothesized that calcium intake might be associated with lower risk of CASR-positive, but not CASR-negative, colorectal cancer. Design We assessed tumour CASR protein expression using immunohistochemistry in 779 incident colon and rectal cancer cases that developed among 136 249 individuals in the Nurses’ Health Study and Health Professionals Follow-Up Study. Duplication method Cox proportional hazards regression analysis was used to assess associations of calcium intake with incidence of colorectal adenocarcinoma subtypes by CASR status. Results Total calcium intake was inversely associated with the risk of developing colorectal cancer (p trend =0.01, comparing ≥1200 vs &lt;600 mg/day: multivariable HR=0.75, 95% CI 0.60 to 0.95). For the same comparison, higher total calcium intake was associated with a lower risk of CASR-positive tumours (p trend =0.003, multivariable HR=0.67, 95% CI 0.51 to 0.86) but not with CASR-negative tumours (p trend =0.67, multivariable HR=1.15, 95% CI 0.75 to 1.78; p heterogeneity =0.06 between the CASR subtypes). The stronger inverse associations of calcium intake with CASR-positive but not CASR-negative tumours generally appeared consistent regardless of sex, tumour location and source of calcium. Conclusions Our molecular pathological epidemiology data suggest a causal relationship between higher calcium intake and lower colorectal cancer risk, and a potential role of CASR in mediating antineoplastic effect of calcium.

The present retrospective study aimed to examine the association between the expression of long non‑protein‑coding RNAs (lncRNAs) and clinical prognosis in the pretreatment formalin-fixed, paraffin-embedded (FFPE) tissue samples of cervical squamous cell carcinoma patients that underwent platinum‑based chemoradiation therapy. Between 2001 and 2013, 49 consecutive patients with squamous cell cervical carcinoma were selected for the present study (median follow‑up period, 44.1 months). The patients possessed an International Federation of Gynecology and Obstetrics stage of IB1/IIA1 (with pelvic lymph node metastasis), IB2 or IIA2‑IVA, and had been treated with definitive chemoradiation therapy. The pretreatment FFPE tumor biopsies of the patients obtained diagnosis were used for analysis. Total RNAs were extracted from the FFPE tumor tissues and reverse transcription‑quantitative polymerase chain reaction was performed to examine the expression level of lncRNAs. The expression level of X inactive‑specific transcript (XIST) demonstrated a significant association with the overall survival rate (P=0.014). The 4‑year overall survival rates were 87.1 and 54.4% in the high and low XIST expression groups, respectively. Since the expression of XIST is associated with the overall survival rate, this lncRNA has the potential to become a predictor for the prognosis of cervical squamous cell carcinoma patients that are treated with chemoradiation therapy. Additional studies are required to investigate the underlying mechanisms of XIST that are associated with prognosis.

Abstract Malignant melanoma is one of the untreatable cancers in which conventional therapeutic strategies, including chemotherapy, are hardly effective. Therefore, identification of novel therapeutic targets involved in melanoma progression is urgently needed for developing effective therapeutic methods. Overexpression of interleukin-13 receptor α2 (IL13Rα2) is observed in several cancer types including glioma and pancreatic cancer. Although IL13Rα2 is implicated in the progression of various types of cancer, its expression and roles in the malignant melanoma have not yet been elucidated. In the present study, we showed that IL13Rα2 was expressed in approximately 7.5% melanoma patients. While IL13Rα2 expression in human melanoma cells decreased their proliferation in vitro , it promoted in vivo tumour growth and angiogenesis in melanoma xenograft mouse model. We also found that the expression of amphiregulin, a member of the epidermal growth factor (EGF) family, was correlated with IL13Rα2 expression in cultured melanoma cells, xenograft tumour tissues and melanoma clinical samples. Furthermore, expression of amphiregulin promoted tumour growth, implicating causal relationship between the expression of IL13Rα2 and amphiregulin. These results suggest that IL13Rα2 enhances tumorigenicity by inducing angiogenesis in malignant melanoma, and serves as a potential therapeutic target of malignant melanoma.

Abstract Inactivation of the TP53 (p53) pathway by TP53 mutations is one of key steps in colorectal carcinogenesis. TP53 also plays an important role in cellular energy metabolism. We hypothesized that TP53‐altered tumor cells might behave aggressively independent of energy balance, while progression of TP53‐intact cells might depend on excess energy balance. Utilizing a database of 1,060 colon and rectal cancer patients in two prospective cohort studies, we evaluated TP53 expression by immunohistochemistry. Among 1,060 colorectal cancers, 457 (43%) tumors were positive for TP53. Cox proportional hazards model was used to compute mortality hazard ratio (HR), adjusting for clinical and tumoral features, including microsatellite instability, the CpG island methylator phenotype, LINE‐1 methylation, KRAS , BRAF and PIK3CA . TP53 positivity was not significantly associated with cancer‐specific survival in univariate analysis with HR of 1.16 [95% confidence interval (CI) = 0.92–1.45], which became significant after stage adjustment (multivariate HR = 1.30; 95% CI = 1.02–1.65). Notably, we found a possible modifying effect of patient's body mass index (BMI) on tumor TP53. In non‐obese patients (BMI &lt; 30 kg/m 2 ), TP53 positivity was associated with shorter cancer‐specific survival (multivariate HR = 1.53; 95% CI = 1.17‐2.00), while TP53 positivity was not significantly associated with survival among obese patients (BMI ≥30 kg/m 2 ). Effect of TP53 positivity on cancer‐specific survival significantly differed by BMI ( p interaction = 0.0051). The adverse effect of obesity on patient mortality was limited to TP53‐negative patients. These molecular pathological epidemiology data may support a dual role of TP53 alterations in cell‐cycle deregulation and cell autonomy with respect to energy balance status.

Abstract Renal clear cell carcinoma (RCCC) is the most frequent subpopulation of renal cell carcinoma and is derived from the proximal uriniferous tubules. We have previously reported that an actin‐binding protein, 4.1B/DAL‐1, is expressed in renal proximal tubules, whereas it is inactivated in 45% of RCCC by promoter methylation. In the lung and several epithelial tissues, 4.1B is shown to associate with a tumor suppressor protein, CADM1, belonging to the immunoglobulin‐superfamily cell adhesion molecules. Here, we demonstrate by immunohistochemistry that another member of the CADM‐family protein, CADM4, as well as 4.1B is expressed specifically in human proximal tubules, while CADM1 and 4.1N, another member of the 4.1 proteins, are expressed in the distal tubules. Immunoprecipitation analysis coupled with Western blotting revealed that CADM4 associated with 4.1B, while CADM1 associated with 4.1N in the lysate from normal human kidney, implicating that a cascade of CADM4 and 4.1B plays an important role in normal cell adhesion of the proximal tubules. On the other hand, CADM4 expression was lost or markedly reduced in 7 of 10 (70%) RCC cell lines and 28 of 40 (70%) surgically resected RCCC, including 10 of 16 (63%) tumors with T1a. CADM4 expression was more preferentially lost in RCCC with vascular infiltration ( p = 0.04), suggesting that loss of CADM4 is involved in tumor invasion. Finally, introduction of CADM4 into an RCC cell line, 786‐O, dramatically suppressed tumor formation in nude mice. These findings suggest that CADM4 is a novel tumor suppressor candidate in RCCC acting with its binding partner 4.1B.

Mononucleotide tracts in the coding regions of the TGFBR2 and BAX genes are commonly mutated in microsatellite instability-high (MSI-high) colon cancers. The receptor TGFBR2 plays an important role in the TGFB1 (transforming growth factor-β, TGF-β) signaling pathway, and BAX plays a key role in apoptosis. However, a role of TGFBR2 or BAX mononucleotide mutation in colorectal cancer as a prognostic biomarker remains uncertain.We utilized a database of 1072 rectal and colon cancers in two prospective cohort studies (the Nurses' Health Study and the Health Professionals Follow-up Study). Cox proportional hazards model was used to compute mortality hazard ratio (HR), adjusted for clinical, pathological and molecular features including the CpG island methylator phenotype (CIMP), LINE-1 methylation, and KRAS, BRAF and PIK3CA mutations. MSI-high was observed in 15% (162/1072) of all colorectal cancers. TGFBR2 and BAX mononucleotide mutations were detected in 74% (117/159) and 30% (48/158) of MSI-high tumors, respectively. In Kaplan-Meier analysis as well as univariate and multivariate Cox regression analyses, compared to microsatellite stable (MSS)/MSI-low cases, MSI-high cases were associated with superior colorectal cancer-specific survival [adjusted HR, 0.34; 95% confidence interval (CI), 0.20-0.57] regardless of TGFBR2 or BAX mutation status. Among MSI-high tumors, TGFBR2 mononucleotide mutation was associated with CIMP-high independent of other variables [multivariate odds ratio, 3.57; 95% CI, 1.66-7.66; p = 0.0011].TGFBR2 or BAX mononucleotide mutations are not associated with the patient survival outcome in MSI-high colorectal cancer. Our data do not support those mutations as prognostic biomarkers (beyond MSI) in colorectal carcinoma.

Patients with colorectal cancer (CRC) have high circulating levels of macrophage inhibitory cytokine-1 (MIC1 or growth differentiation factor 15), a marker of inflammation that might be involved in carcinogenesis. We analyzed blood samples collected from individuals before they were diagnosed with CRC to determine whether levels of MIC1 were associated with mortality.We collected data on survival of 618 participants diagnosed with CRC who provided prediagnosis blood specimens in 1990 (Nurses' Health Study) and 1994 (Health Professionals' Follow-up Study) and were followed through 2010. Levels of MIC1 were measured by enzyme-linked immunosorbent assay and then were categorized into quartiles based on the known distribution of MIC1 levels among previously matched individuals without CRC (controls) within each cohort. We then examined the association of MIC1 levels with overall and CRC-specific mortality using Cox proportional hazards models, with adjustments for mortality-associated risk factors and other plasma markers of inflammation. We also assessed the relationship between levels of MIC1 and levels of prostaglandin-endoperoxide synthase 2 expression (PTGS2 or cyclooxygenase-2), measured in 245 tumor samples by immunohistochemistry.Compared with participants in the lowest quartile for plasma level of MIC1, the multivariate hazard ratio for CRC-specific death for participants in the highest quartile of MIC1 level was 2.40 (95% confidence interval: 1.33-4.34; P for linear trend = .009). The association of MIC1 with survival varied with level of PTGS2 expression in tumor samples (Pinteraction = .04). For individuals with PTGS2-positive tumors, the hazard ratio for CRC-specific death among those with high levels of MIC1 (equal to or greater than the median) was 2.13 (95% confidence interval: 0.99-4.58) compared with participants with low levels of MIC1 (below the median). In individuals with PTGS2-negative CRC, a high level of MIC1 was not associated with an increased risk of CRC-specific death (multivariate hazard ratio = 0.61; 95% confidence interval: 0.13-2.93).Based on an analysis of blood and colorectal tumor samples from 2 large studies, high plasma levels of MIC1 (growth differentiation factor 15) before diagnosis of CRC are associated with greater CRC-specific mortality, particularly in individuals with PTGS2-positive tumors.

Higher levels of physical activity are associated with lower colorectal carcinoma incidence and mortality, perhaps through influencing energy balance, cellular prosta7 systemic inflammation. Although evidence suggests interactive effects of energetics, sedentary lifestyle, and tumor CTNNB1 (β-catenin) or CDKN1B (p27) status on colon cancer prognosis, interactive effects of physical activity and tumor PTGS2 (the official symbol for COX-2) status on clinical outcome remain unknown.Using molecular pathological epidemiology database of 605 stage I-III colon and rectal cancers in two prospective cohort studies (the Nurse's Health Study and the Health Professionals Follow-up Study), we examined patient survival according to postdiagnosis physical activity and tumor PTGS2 status (with 382 PTGS2-positive and 223 PTGS2-negative tumors by immunohistochemistry). Cox proportional hazards models were used to calculate colorectal cancer-specific mortality HR, adjusting for clinical and other tumor variables including microsatellite instability status.Among PTGS2-positive cases, compared with the least active first quartile, the multivariate HRs (95% confidence interval) were 0.30 (0.14-0.62) for the second, 0.38 (0.20-0.71) for the third, and 0.18 (0.08-0.41) for the fourth quartile of physical activity level (Ptrend = 0.0002). In contrast, among PTGS2-negative cases, physical activity level was not significantly associated with survival (Ptrend = 0.84; Pinteraction = 0.024, between physical activity and tumor PTGS2 status).Postdiagnosis physical activity is associated with better survival among patients with PTGS2-positive tumors but not among patients with PTGS2-negative tumors.Immunohistochemical PTGS2 expression in colorectal carcinoma may serve as a predictive biomarker in pathology practice, which may predict stronger benefit from exercise.

Analyses of associations between clinicopathologic outcomes and recurrent somatic mutations in clear cell renal cell carcinoma (ccRCC) have been limited to individual cohorts.To define clinicopathologic associations between specific mutations and ccRCC disease characteristics.DNA sequencing data were pooled from three collaborative genomic cohorts (n=754) and our institutional database (n=295). All patients had clinical data and identification of somatic mutations from their primary tumors.Analysis of gene mutations for associations with maximal tumor size (linear regression) and pathologic stage (logistic regression). Cancer-specific survival (CSS) and recurrence-free survival (RFS) were calculated using competing risks methods. Analyses were adjusted for cohort site, and results were adjusted for multiple testing (q value). Relevant genes were used in multivariable models that included confounding variables and the validated Mayo Clinic Stage, Size, Grade, and Necrosis (SSIGN) score.Association with tumor size was found for mutations in BAP1 (q=0.013). No mutations were found to be associated with stage after adjusted analysis. Mutations in BAP1 (q=0.004) and TP53 (q=0.001) were associated with decreased CSS in a multivariable model; only TP53 (q=0.005) remained significant when SSIGN score was included. SETD2 mutations (q=0.047) were associated with decreased RFS in multivariable models, including models with SSIGN score.In >1000 patients with ccRCC, pooled analysis and multivariable modeling demonstrated that three mutated genes have statistically significant associations with poor clinical outcomes. This included the more commonly mutated BAP1 and SETD2 and the less frequently mutated TP53. After adjustment for clinical confounders, mutations of TP53 and SETD2 were associated with decreased CSS and RFS, respectively.Using rigorous statistical methods, this study affirmed that certain mutations in clear cell renal cell carcinoma may portend inferior survival and an increased risk of recurrence.

Endothelial cells (ECs) are a key component of the tumor microenvironment. They have abnormal characteristics compared to the ECs in normal tissues. Here, we found a marked increase in lipocalin-type prostaglandin D synthase (L-PGDS) mRNA (Ptgds) expression in ECs isolated from mouse melanoma. Immunostaining of mouse melanoma revealed expression of L-PGDS protein in the ECs. In situ hybridization also showed L-PGDS (PTGDS) mRNA expression in the ECs of human melanoma and oral squamous cell carcinoma. In vitro experiments showed that stimulation with tumor cell-derived IL-1 and TNF-α increased L-PGDS mRNA expression and its product prostaglandin D2 (PGD2 ) in human normal ECs. We also investigated the contribution of L-PGDS-PGD2 to tumor growth and vascularization. Systemic or EC-specific deficiency of L-PGDS accelerated the growth of melanoma in mice, whereas treatment with an agonist of the PGD2 receptor, DP1 (BW245C, 0.1 mg/kg, injected intraperitoneally twice daily), attenuated it. Morphological and in vivo studies showed that endothelial L-PGDS deficiency resulted in functional changes of tumor ECs such as accelerated vascular hyperpermeability, angiogenesis, and endothelial-to-mesenchymal transition (EndMT) in tumors, which in turn reduced tumor cell apoptosis. These observations suggest that tumor cell-derived inflammatory cytokines increase L-PGDS expression and subsequent PGD2 production in the tumor ECs. This PGD2 acts as a negative regulator of the tumorigenic changes in tumor ECs. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

To identify factors that influence the total and negative lymph node counts in colorectal cancer resection specimens independent of pathologists and surgeons.We used multivariate negative binomial regression. Covariates included age, sex, body mass index, family history of colorectal carcinoma, year of diagnosis, hospital setting, tumor location, resected colorectal length (specimen length), tumor size, circumferential growth, TNM stage, lymphocytic reactions and other pathological features, and tumor molecular features (microsatellite instability, CpG island methylator phenotype, long interspersed nucleotide element 1 [LINE-1] methylation, and BRAF, KRAS, and PIK3CA mutations).Two US nationwide prospective cohort studies.Patients with rectal and colon cancer (N=918).The negative and total node counts (continuous).Specimen length, tumor size, ascending colon location, T3N0M0 stage, and year of diagnosis were positively associated with the negative node count (all P.002). Mutation of KRAS might also be positively associated with the negative node count (P=.03; borderline significance considering multiple hypothesis testing). Among node-negative (stages I and II) cases, specimen length, tumor size, and ascending colon location remained significantly associated with the node count (all P.002), and PIK3CA and KRAS mutations might also be positively associated (P=.03 and P=.049, respectively, with borderline significance).This molecular pathological epidemiology study shows that specimen length, tumor size, tumor location, TNM stage, and year of diagnosis are operator-independent predictors of the lymph node count. These crucial variables should be examined in any future evaluation of the adequacy of lymph node harvest and nodal staging when devising individualized treatment plans for patients with colorectal cancer.

Analysis of acetylation and tri-methylation of the same residue of histone molecules might identify a subset of hepatocellular carcinoma (HCC) with aggressive behavior. In the present study, we examined acetylation and tri-methylation of lysine 27 on histone H3 (H3K27ac and H3K27me3, respectively) because these two modifications are known to exhibit opposite effects (enhancing and silencing) on gene expression. Neoplastic and non-neoplastic tissues from 198 HCC cases were immunostained with specific monoclonal antibodies against H3K27ac and H3K27me3. The stained tissues were evaluated by an image analyzing program to generate histological scores (H-scores, range 0-300), which were determined by multiplying the percentage of positive-stained cells with the classified immunohistochemical marker intensity (0-3). HCC tissues showed significantly higher H3K27ac (156.7±86.8) and H3K27me3 H-scores (151.8±78.1) compared with the background liver (40.3±33.0 and 64.7±45.6, respectively) (both P<0.001). The cases with H-scores of high-H3K27ac/high-H3K27me3 (n = 54) showed significant correlation with poor differentiation of morphology (P<0.01) and p53-positive staining (P<0.05), and poor prognosis (P<0.01). Confocal microscopy revealed segregated intranuclear localization of both modifications in the individual cancer cells: H3K27ac localization in central euchromatin regions and H3K27me3 in peripheral heterochromatin regions. Concurrent acetylation and methylation at H3K27 occurs in HCC cells in association with p53 abnormalities. These findings demonstrate that image analyzer-assisted H-scores of H3K27ac and H3K27me3 identified an aggressive subgroup of HCC, and could serve as a prognostic marker for HCC.

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. 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. 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. 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.

Although genome-wide association studies (GWAS) have separately identified many genetic susceptibility loci for ulcerative colitis (UC), Crohn's disease (CD) and colorectal cancer (CRC), there has been no large-scale examination for pleiotropy, or shared genetic susceptibility, for these conditions. We used logistic regression modeling to examine the associations of 181 UC and CD susceptibility variants previously identified by GWAS with risk of CRC using data from the Genetics and Epidemiology of Colorectal Cancer Consortium and the Colon Cancer Family Registry. We also examined associations of significant variants with clinical and molecular characteristics in a subset of the studies. Among 11794 CRC cases and 14190 controls, rs11676348, the susceptibility single nucleotide polymorphism (SNP) for UC, was significantly associated with reduced risk of CRC (P = 7E-05). The multivariate-adjusted odds ratio of CRC with each copy of the T allele was 0.93 (95% CI 0.89-0.96). The association of the SNP with risk of CRC differed according to mucinous histological features (P heterogeneity = 0.008). In addition, the (T) allele was associated with lower risk of tumors with Crohn's-like reaction but not tumors without such immune infiltrate (P heterogeneity = 0.02) and microsatellite instability-high (MSI-high) but not microsatellite stable or MSI-low tumors (P heterogeneity = 0.03). The minor allele (T) in SNP rs11676348, located downstream from CXCR2 that has been implicated in CRC progression, is associated with a lower risk of CRC, particularly tumors with a mucinous component, Crohn's-like reaction and MSI-high. Our findings offer the promise of risk stratification of inflammatory bowel disease patients for complications such as CRC.

Recent studies suggest that CD204-positive (CD204+) M2-type tumor-associated macrophages (TAMs) are associated with the aggressive behavior of various cancers. However, the clinicopathologic significance of tumor-infiltrating CD204+ macrophages and their correlation with Epstein-Barr virus (EBV) in gastric cancer is unclear. Tissue microarrays were constructed from 119 surgically resected gastric cancer specimens (86 EBV-negative and 28 EBV-positive cases). After immunohistochemistry of CD204, the density of CD204+ cells was calculated using image analysis software, and associations between CD204 and clinicopathologic factors including patient survival were examined. High CD204+ cell density was significantly associated with several adverse prognostic factors, including older age (P = .008), advanced tumor depth (P < .001), lymph node metastasis (P < .001), presence of venous invasion (P < .001), and lymphatic invasion (P = .03). Low CD204+ cell density was significantly associated with EBV infection. Advanced tumor depth and presence of lymph node metastasis were significantly associated with high CD204+ cell density in both EBV-positive and EBV-negative subgroups. High CD204+ cell density was significantly associated with shorter cancer-specific survival (P = .0015). In conclusion, a high density of CD204+ TAMs was associated with the aggressive behavior and worse survival of gastric cancer. Low density of CD204+ TAMs was associated with infection of EBV, which may explain the favorable outcome of EBV-associated gastric carcinoma. Our results suggest that a specific immune microenvironment may be associated with the biological behavior of gastric cancer and that EBV-associated gastric carcinoma is distinctive from other gastric carcinomas in tumor immunity.

Background Recently, lymphocyte-to-monocyte ratio (LMR) has attracted attention as a new marker of the chronic systemic inflammatory response and has been associated with patient prognosis in those who underwent chemoradiotherapy (CRT) for several solid cancers. This study aimed to evaluate the association between LMR and the prognosis of patients with rectal cancer. Methods A total of 183 stage II-III rectal cancer patients who underwent preoperative CRT followed by surgical R0 resection were retrospectively reviewed. The LMR was calculated from pre- and post-CRT blood samples. To determine the optimal cutoff value for pre- and post-CRT LMR for predicting relapse-free survival (RFS) and overall survival (OS), a receiver operator characteristic curve was used. Cox's proportional hazard models were applied to identify risk factors for recurrence and overall mortality. Results Low LMR was observed in 54 patients (pre-CRT <4.0) and 29 patients (post-CRT <1.5). Although pre-CRT LMR correlated with tumor size and ypT stage, post-CRT LMR showed no correlation to any pathologic features. Median follow-up term was 66.3 months; the 5-year RFS and OS of all patients were 72.5% and 88.7%, respectively. We found that a low pre-CRT LMR was an independent risk factor for OS (hazard ratio, 2.83; 95% confidence interval 1.03-8.13; P = 0.043). Conclusions In rectal cancer patients who have undergone preoperative CRT, a low pre-CRT LMR is a poor prognostic factor for OS.

Glypican-1 (GPC1) protein in exosomes was recently identified as a biomarker for the early detection of pancreatic ductal adenocarcinoma (PDAC). Immunohistochemical analyses and in vitro assays were conducted to assess the usefulness of GPC1 as a PDAC biomarker, to reveal the biological role of GPC1 in pancreatic carcinogenesis, and to ascertain the regulation mechanism of GPC1. An aberrant overexpression of GPC1 protein which is usually absent in normal pancreatic duct, was a widespread marker across the full spectrum of human PDAC precursors, PDAC, and pancreatic cancerous stroma. In intraductal papillary-mucinous neoplasms (IPMNs), GPC1 tended to be positive in gastric-type IPMN. KRAS mutations were found in all GPC1-positive IPMN cases and in one-third of GPC1-negative IPMN cases. In pancreatic cell lines, GPC1 depletion caused remarkable inhibition of cell growth and migration, suggesting its oncogenic roles. GPC1 depletion upregulated the molecules associated with cell cycle arrest in pancreatic cell lines. Furthermore, KRAS and ecotropic viral integration site 1 (EVI1) oncoprotein upregulated GPC1 expression. In a clinical cohort, GPC1 overexpression was not correlated with pancreatic cancer prognosis. Taken together, these findings suggest the necessity of establishing a threshold of GPC1 value for detecting pancreatic malignancy because GPC1 is overexpressed even in low-grade PDAC precursors which do not always become malignant. Our study also reveals a new aspect of pancreatic carcinogenesis: KRAS and EVI1, two important molecules in early phases of pancreatic carcinogenesis, positively regulate GPC1 expression and likely promote pancreatic carcinogenesis.

Aims To evaluate the significance of mast cell infiltration in interstitial cystitis (IC) by comparison with equally inflamed controls using a digital quantification technique. Methods Bladder biopsy specimens from 31 patients with Hunner type IC and 38 patients with non‐Hunner type IC were analyzed. Bladder biopsy specimens from 37 patients without IC, including 19 non‐specific chronic cystitis (“non‐IC cystitis”) specimens and 18 non‐inflamed bladder (“normal bladder”) specimens, were used as controls. Mast cell tryptase‐, CD3‐, CD20‐, and CD138‐immunoreactive cells were quantified using digital image analysis software to evaluate both mast cell and lymphoplasmacytic cell densities. Mast cell and lymphoplasmacytic cell densities were counted independently in the entire lamina propria and detrusor areas and compared among the four groups. Results In the lamina propria, there were no significant differences in mast cell and lymphoplasmacytic cell densities between Hunner type IC and non‐IC cystitis or between non‐Hunner type IC and normal bladder specimens. In the detrusor, the mast cell densities were not significantly different among the four groups. Mast cell density was correlated with lymphoplasmacytic cell density, but not with clinical parameters. Conclusions Mast cell density is not significantly different between IC specimens and non‐IC control specimens with a similar degree of background inflammation. The intensity of mast cell infiltration generally correlated with that of lymphoplasmacytic cells. We conclude that mast cell count is of no value in the differential diagnosis between IC and other etiologies.

Intrahepatic cholangiocarcinoma (ICC) is an extremely aggressive carcinoma. Useful predictors for the patients' prognosis after surgery have not been fully established. From the University of Tokyo Hospital pathology archives, we reviewed 107 cases of ICC, 54 cases of perihilar cholangiocarcinoma, and 40 cases of extrahepatic cholangiocarcinoma (ECC); we also investigated the significance of tumor budding in ICC, in comparison with perihilar cholangiocarcinoma and ECC. The tumor-budding frequencies were different by tumor location: 40.2% (43/107) in ICC, 70.4% (38/54) in perihilar cholangiocarcinoma, and 60.0% (24/40) in ECC. Tumor budding in ICC was associated with many pathologic indicators associated with invasion, such as major vascular invasion (P=0.012) and Union for International Cancer Control stage (P=0.007). Univariate and multivariate Cox regression analyses revealed tumor budding as a powerful prognostic factor for both recurrence-free survival (RFS) and overall survival (OS) in ICC by univariate (RFS: hazard ratio [HR]: 2.666; 95% confidence interval [CI]: 1.517-4.683, OS: HR: 4.206; 95% CI: 2.447-7.230) and by multivariate analyses (RFS: HR: 3.038; 95% CI: 1.591-5.973, OS: HR: 4.547, 95% CI: 2.348-8.805). Tumor budding was also a significant prognostic factor of perihilar cholangiocarcinoma, but not of ECC. When ICC was divided into 2 subtypes, type 1 (hilar) and type 2 (peripheral), tumor budding was the strong prognostic factor in type 2 ICC, but not in type 1 ICC, suggesting that some differences in biological behavior exist between type 1 ICC and perihilar cholangiocarcinoma. Tumor budding is prognostically important in ICC, and its pathogenetic role in biliary tract carcinomas might be different by anatomic location.

Although histochemical staining has been believed to inhibit the DNA amplification reaction, no previous study has systematically evaluated the influence of histochemical staining on downstream molecular assays. To evaluate an influence of H&E staining on DNA testing, we isolated DNA from 10 unstained, 10 hematoxylin-stained, 10 eosin-stained, and 10 H&E-stained tissue sections (ie, 4 groups), from each of 5 colon cancers. Among the 4 groups, we did not observe any significant or appreciable difference in DNA fragmentation by agarose gel electrophoresis, in DNA amplification by real-time polymerase chain reaction (PCR), in microsatellite PCR fragment analyses, or in a PCR-pyrosequencing assay. As a proof-of-principle study, we successfully performed microsatellite instability analysis and sequencing of KRAS and BRAF on more than 1,300 colorectal cancers using DNA extracted from H&E-stained tissue sections. Our data provide no evidence for an interfering effect of H&E staining on DNA testing, suggesting that DNA from H&E-stained sections can be effectively used for routine DNA testing.

Pulmonary fibrosis (PF) is an intractable disorder with a poor prognosis. Lung macrophages have been reported to regulate both progression and remission of bleomycin-induced diffuse PF. However, it remains unclear how macrophages contribute to silica-induced progressive nodular PF and the associated tissue cell responses in vivo. We found that lack of monocyte-derived macrophages results in the formation of diffuse PF after silica instillation. We found that the proportion and the number of monocyte-derived macrophages were persistently higher in silica-induced progressive PF compared with bleomycin-induced PF. Surprisingly, in Ccr2(-/-) mice, in which monocyte-derived macrophage infiltration is impaired, silica administration induced diffuse PF with loose nodule formation and greater activation of tissue cells. In the diffuse lesions, the distribution of epithelial cells, distribution of myofibroblasts, and architecture of the basement membrane were disrupted. Consistent with the development of diffuse lesions, genes that were differentially expressed in CD45(-) tissue cells from the lung of wild-type and Ccr2(-/-) mice were highly enriched in human diffuse, progressive PF. In gene ontology network analyses, many of these genes were associated with tissue remodeling and included genes not previously associated with PF, such as Mmp14, Thbs2, and Fgfr4. Overall, these results indicate that monocyte-derived macrophages prevent transition from nodular to diffuse silica-induced PF, potentially by regulating tissue cell responses.

BACKGROUND: Nonoperative management for patients with rectal cancer who have achieved a clinical complete response after chemoradiotherapy is becoming increasingly important in recent years. However, the definition of and modality used for patients with clinical complete response differ greatly between institutions, and the role of endoscopic assessment as a nonoperative approach has not been fully investigated. OBJECTIVE: This study aimed to investigate the ability of endoscopic assessments to predict pathological regression of rectal cancer after chemoradiotherapy and the applicability of these assessments for the watchful waiting approach. DESIGN: This was a retrospective comparative study. SETTINGS: This study was conducted at a single referral hospital. PATIENTS: A total of 198 patients with rectal cancer underwent preoperative endoscopic assessments after chemoradiotherapy. Of them, 186 patients underwent radical surgery with lymph node dissection. MAIN OUTCOME MEASURES: The histopathological findings of resected tissues were compared with the preoperative endoscopic findings. Twelve patients refused radical surgery and chose watchful waiting; their outcomes were compared with the outcomes of patients who underwent radical surgery. RESULTS: The endoscopic criteria correlated well with tumor regression grading. The sensitivity and specificity for a pathological complete response were 65.0% to 87.1% and 39.1% to 78.3%. However, endoscopic assessment could not fully discriminate pathological complete responses, and the outcomes of patients who underwent watchful waiting were considerably poorer than the patients who underwent radical surgery. Eventually, 41.7% of the patients who underwent watchful waiting experienced uncontrollable local failure, and many of these occurrences were observed more than 3 years after chemoradiotherapy. LIMITATIONS: The number of the patients treated with the watchful waiting strategy was limited, and the selection was not randomized. CONCLUSIONS: Although endoscopic assessment after chemoradiotherapy correlated with pathological response, it is unsuitable for surveillance of patients treated via a nonoperative approach. Incorporation of a “watchful waiting” strategy without establishing proper surveillance protocols and salvage strategies might result in poor local control.

Tumor hypoxia is associated with a malignant phenotype of cancer cells and poor patient prognosis. To investigate the role of hypoxia in tumor progression, we studied the effects of hypoxia in the A549 lung adenocarcinoma cell line. First, we showed that hypoxic treatment decreased cell-cell adhesion and induced a scattering of cancer cells. Concomitant with these morphological changes, the motility of cancer cells was increased, as demonstrated by the Boyden chamber assay. Then, we used oligonucleotide array analyses to identify the genes causally related to the hypoxia-induced motile phenotype. The results showed that the expression of approximately 100 genes was induced more than 5-fold by hypoxia. These included (among others) epidermal growth factor receptor (EGFR), as well as other well-known hypoxia-induced genes, such as vascular endothelial growth factor. Immunohistochemical analyses of primary lung adenocarcinomas confirmed the induction of EGFR in tumor cells in the vicinity of necrotic areas, a histological indicator of tumor hypoxia. Remarkably, the EGFR inhibitor AG1478 (10 microM) completely blocked the increased cell motility induced by hypoxia. Thus, the present study demonstrates the importance of the EGFR pathway in the increased motility of cancer cells that occurs in a hypoxic tumor environment.

Epstein-Barr virus (EBV)-associated gastric carcinoma (GC) is a distinct subtype with characteristic clinicopathological features. To better characterize its cellular characteristics, 43 cases of EBV-associated GC, 68 cases of EBV-negative GC, and non-neoplastic gastric mucosa in adults and fetuses were examined immunohistochemically. We quantified the expression of the major tight-junction protein claudin (CLDN) -1, -3, -4, -7, and -18 together with gastric mucins (MUC5AC and MUC6), intestinal mucin (MUC2), and CD10. EBV-associated GC showed a high frequency of CLDN18 expression (84%) and a low frequency of CLDN3 expression (5%). This expression profile corresponded to that of normal gastric epithelium in adults and fetuses. Almost half of the EBV-associated GC cases demonstrated gastric mucin expression, whereas the other half lacked mucin or CD10 expression. In contrast, as demonstrated by the expression profiles of CLDN3 and CLDN18, EBV-negative GC comprised a heterogeneous group of four different CLDN phenotypes: gastric, intestinal, mixed, and an undifferentiated type with variable expression patterns of mucins. These results indicate that EBV-associated GC is considerably homogenous with regard to cellular differentiation and that it preserves well the nature of the cells of origin. EBV-associated GC may undergo distinct carcinogenic processes, which differ from those of EBV-negative GC.

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.

Abstract Experimental studies have shown that estrogen- or progesterone-activated signaling leads to growth inhibition effects on colon cancer cells through the upregulation of several cell-cycle regulators. However, epidemiologic studies evaluating hormone therapy use and colorectal cancer risk by the status of cell-cycle regulators are lacking. In this study, we used data from the prospective Nurses' Health Study to evaluate whether the association between hormone therapy use and colorectal cancer risk differs by the molecular pathologic status of microsatellite instability (MSI) and expression of cell-cycle–related tumor biomarkers, including CDKN1A (p21, CIP1), CDKN1B (p27, KIP1), and TP53 (p53) by immunohistochemistry. Duplication Cox regression analysis was used to determine an association between hormone therapy use, cancer risk, and specific tumor biomarkers in 581 incident colon and rectal cancer cases that occurred during 26 years of follow-up among 105,520 postmenopausal women. We found a difference between hormone therapy use and colorectal cancer risk according to CDKN1A expression (Pheterogeneity = 0.01). Current hormone therapy use was associated with a reduced risk for CDKN1A-nonexpressed [multivariate relative risk (RR), 0.61; 95% confidence interval (CI), 0.46–0.82] but not for CDKN1A-expressed (RR, 1.32; 95% CI, 0.76–2.31) tumors. The lower risk for CDKN1A-nonexpressed but not for CDKN1A-expressed cancers was also present among current users of estrogen-alone therapy. We found no significant difference in the relations between hormone therapy use and cancer risk according to MSI, CDKN1B, or TP53 status. Together, our molecular pathological epidemiology findings suggest a preventive effect of hormone therapy against colorectal carcinogenesis that depends, in part, on loss of cyclin-dependent kinase inhibitor CDKN1A. Cancer Res; 72(12); 3020–8. ©2012 AACR.

Abstract Fibroblasts are some of the major cells in tumour tissues that influence tumour progression and drug resistance. However, our understanding on fibroblast-mediated tumour malignancy remains incomplete. Munc18-1-interacting protein 3 (Mint3) is known as an activator of hypoxia-inducible factor-1 (HIF-1) even during normoxia in cancer cells, macrophages and fibroblasts. Although Mint3 promotes ATP production via glycolysis by activating HIF-1 in cancer cells and macrophages, the biological role of Mint3-mediated HIF-1 activation in fibroblasts remains unclear. To address this, we examined whether Mint3 in fibroblasts contributes to tumour growth. Mint3 depletion in mouse embryonic fibroblasts (MEFs) decreased tumour growth of co-injected human breast cancer cells, MDA-MB-231 and epidermoid carcinoma A431 cells in mice. In MEFs, Mint3 also promoted cancer cell proliferation in vitro in a cell–cell contact-dependent manner. Mint3-mediated cancer cell proliferation depended on HIF-1, and further gene expression analysis revealed that the cell adhesion molecule, L1 cell adhesion molecule (L1CAM), was induced by Mint3 and HIF-1 in fibroblasts. Mint3-mediated L1CAM expression in fibroblasts stimulated the ERK signalling pathway via integrin α5β1 in cancer cells, and promoted cancer cell proliferation in vitro and tumour growth. In cancer-associated fibroblasts (CAFs), knockdown of MT1-MMP, which promotes Mint3-mediated HIF-1 activation, or Mint3 decreased L1CAM expression. As MEFs, CAFs also promoted cancer cell proliferation in vitro , and tumour growth via Mint3 and L1CAM. In human breast cancer specimens, the number of fibroblasts expressing L1CAM, Mint3 and MT1-MMP was higher in cancer regions than in adjacent benign regions. In addition, more phospho-ERK1/2-positive cancer cells existed in the peripheral region surrounded by the stroma than in the central region of solid breast cancer nest. Thus, Mint3 in fibroblasts might be a good target for cancer therapy by regulating cancer cell-stromal cell communication.

Neuronal intranuclear inclusion disease (NIID) is a rare neurodegenerative disease with marked variety in its clinical manifestations. While characteristic neuroimaging and skin biopsy findings are important clues to the diagnosis, autopsy studies are still important for confirming the exact disease features. We herein report the case of a patient who received an antemortem diagnosis of familial NIID with dementia-dominant phenotype that was later confirmed by an autopsy. Our report is the first to document a case of autopsy-confirmed NIID involving both cognitive impairment and sensorimotor neuropathy.

Abstract Programmed cell death ligand‐1 ( PD ‐L1) is a ligand for programmed cell death‐1 ( PD ‐1) that negatively regulates T‐cell activation and plays a crucial role in suppressing anti‐tumor host immunity. Although PD ‐L1 is a promising immunotherapy target in various cancers, including urothelial carcinoma ( UC ), the prognostic significance of PD ‐L1 in UC is unclear. As platelets help protect tumor cells from immune elimination in the circulatory system, we hypothesized that tumor PD ‐L1 and circulating platelets might synergistically promote tumor metastasis, and that the prognostic significance of PD ‐L1 might vary according to platelet count. We immunohistochemically examined tumor PD ‐L1 expression in 271 patients with upper tract UC , which revealed PD ‐L1 positivity in 31 of 271 cases (11%). The associations of tumor PD ‐L1 expression with outcomes varied among patients with high or low platelet counts ( P interaction &lt; 0.004). Among patients with high platelet counts (N = 136), PD ‐L1 positivity (N = 15) was significantly associated with shorter metastasis‐free survival (univariate hazard ratio [ HR ]: 6.23, 95% confidence interval [ CI ]: 2.95‐13.1; multivariate HR : 2.68, 95% CI : 1.27‐5.64) and shorter overall survival (univariate HR : 4.92, 95% CI : 2.14‐11.3, multivariate HR : 2.78, 95% CI : 1.19‐6.51). In contrast, among patients with low platelet counts (N = 135), PD ‐L1 positivity (N = 16) was not significantly associated with these outcomes. Our results suggest that tumor PD ‐L1 expression and platelet count might interact and help regulate tumor progression. Although a larger prospective study is needed to validate our findings, this relationship is important to consider, as immunotherapies targeting the PD ‐1/ PD ‐L1 axis have gained significant attention as promising therapies for UC .

In colorectal cancer (CRC) care, treatment decisions depend on the efforts to estimate the metastatic potential of tumors. The liver is one of the most common metastatic sites of CRC and the prognosis of CRC patients often reflects metastases to distant sites. To identify chromosomal aberrations associated with liver metastasis, we performed allelic copy number analysis for CRC with or without synchronous liver metastasis using genotyping arrays. By allelic copy number analysis of CRC samples, we observed common aberrations in 14 chromosomal arms in two groups, that is, gains on 7p22.3-p11.2, 8q22.3-q24.3, 13q12.12-q34, and 20q11.22-q13.33 and loss of heterozygosity (LOH) on 4q12-q35.1, 5q11.2-q35.3, 8p23.3-p12, 15q11.2-q26.3, 17p13.3-p11.2, 17q11.2-q25.1, 18p11.32-p11.21, 18q11.2-q23, 20p13-p12.1, and 22q11.1-q13.32. We found that gains on 20p13-p12.1 and 20q11.21-q13.33 and LOH on 6q14.1-q25.1 were more frequent in CRC with liver metastasis. We also compared chromosomal aberrations in primary CRC lesions with those of the corresponding liver metastasis and found that the allelic genome imbalance status of a metastatic lesion is similar to that of the primary cancer, which suggests that chromosomal aberrations are largely maintained on hematogenous spread. Intriguingly, several chromosomal aberrations in CRC were found in the primary cancer but not in the corresponding liver metastasis, thus suggesting heterogeneity of cancer cells within solid tumors or the presence of events uniquely developed in primary tumors. Consequently, CRC with and without liver metastasis harbor similar chromosomal aberrations, and chromosomal aberration at 6q, 20p, and 20q may be involved in the process of liver metastasis of CRC.

It has been suggested that ubiquitin-conjugating enzyme E2C (UBE2C, also known as UBCH10) represents a promising cancer biomarker. However, the clinicopathological or prognostic significance as well as the functions of UBE2C in bladder cancer are largely unknown. To investigate the significance of UBE2C expression in bladder cancer, immunohistochemical analysis was performed using a tissue microarray. UBE2C positivity was observed in 51 of 82 (62%) bladder urothelial carcinoma cases treated with radical cystectomy. In contrast, UBE2C was negative in all of the non-neoplastic urothelium examined. UBE2C positivity was significantly associated with higher tumor stage (p=0.0061) and presence of lymphovascular invasion (p=0.0045). In addition, UBE2C positivity was significantly associated with shorter cancer-specific survival after cystectomy (log rank p=0.0017; multivariate hazard ratio, 2.49; 95% confidence interval, 1.09-5.71). Small interfering RNA-mediated suppression of UBE2C in UM-UC-3 bladder cancer cells inhibited cell proliferation in vitro. Taken together, our results suggest that UBE2C is a novel prognostic biomarker as well as a potential therapeutic target in bladder cancer.

Accumulating evidence suggests that post-diagnostic insulin levels may influence colorectal cancer (CRC) survival. Yet, no previous study has examined CRC survival in relation to a post-diagnostic diet rich in foods that increase post-prandial insulin levels. We hypothesized that glycemic and insulin scores (index or load; derived from food frequency questionnaire data) may be associated with survival from specific CRC subtypes sensitive to the insulin signaling pathway. We prospectively followed 1,160 CRC patients from the Nurses' Health Study (1980-2012) and Health Professionals Follow-Up Study (1986-2012), resulting in 266 CRC deaths in 10,235 person-years. CRC subtypes were defined by seven tumor biomarkers (KRAS, BRAF, PIK3CA mutations, and IRS1, IRS2, FASN and CTNNB1 expression) implicated in the insulin signaling pathway. For overall CRC and each subtype, hazard ratio (HR) and 95% confidence interval (95% CI) for an increase of one standard deviation in each of glycemic and insulin scores were estimated using time-dependent Cox proportional hazards model. We found that insulin scores, but not glycemic scores, were positively associated with CRC mortality (HR = 1.19, 95% CI = 1.02-1.38 for index; HR = 1.23, 95% CI = 1.04-1.47 for load). The significant positive associations appeared more pronounced among PIK3CA wild-type cases and FASN-negative cases, with HR ranging from 1.36 to 1.60 across insulin scores. However, we did not observe statistically significant interactions of insulin scores with PIK3CA, FASN, or any other tumor marker (p interaction > 0.12). While additional studies are needed for definitive evidence, a high-insulinogenic diet after CRC diagnosis may contribute to worse CRC survival.

Background/Aims: The therapeutic strategies for small rectal neuroendocrine tumors (NETs) have not been standardized. We examined the efficacy and safety of endoscopic submucosal resection with a ligation device (ESMR-L) and the long-term outcomes after endoscopic treatment. Methods: A total of 181 patients with rectal NETs &lt;10 mm who were treated between May 2002 and May 2017 were retrospectively enrolled. All the lesions had been resected using ESMR-L, and the follow-up strategies were determined according to the pathological examinations. The long-term outcomes after a 53-month follow-up period were also evaluated. Results: R0 resection was achieved in 180 cases (99.4%). Lymphovascular invasion was confirmed in 67 cases (37.0%), while a curative resection was achieved in 114 cases (63.0%). One perforation (0.6%) and 11 cases with delayed bleeding (6.1%) were observed. A multivariate logistic regression analysis revealed that a tumor size &gt; 5 mm (OR 2.06; 95% CI 1.04–4.08, p = 0.04) was a significant independent predictor of the presence of lymphovascular invasion. Of the 67 patients with non-curative resections, 11 patients underwent additional surgery; lymph node metastasis was confirmed in 2 cases (18.2%). No local or distant metastases were observed during the follow-up period in 77 patients with a curative resection, 9 patients who received additional surgery, and 50 patients with non-curative resections. Conclusion: ESMR-L is an easy, safe and effective treatment for rectal NETs &lt;10 mm in diameter, and the prognosis of patients seems to be good, despite a relatively high rate of lymphovascular invasion.

There are various mechanisms underlying the resistance of EGFR-mutant lung adenocarcinoma to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs). We herein report a case of pulmonary adenocarcinoma with EGFR mutation (exon 19 deletion and T790M) that acquired resistance to osimertinib treatment because of transformation into small-cell lung carcinoma (SCLC). A 67-year-old ex-smoking woman was diagnosed with left upper lobe adenocarcinoma of clinical stage IIIA (cT2bN2M0). She was treated with chemoradiotherapy (cisplatin and vinorelbine plus radiation), gefitinib, cisplatin, and pemetrexed followed by pemetrexed maintenance therapy and erlotinib. Since a sample extracted from the metastatic lung tumor taken obtained via a transbronchial lung biopsy was found to be positive for the T790M mutation at the time of disease progression during erlotinib treatment, she received osimertinib treatment for 15 months until progressive disease. She developed resistance to osimertinib due to the histologic transformation to SCLC. Although the standard chemotherapy of carboplatin and etoposide for SCLC was administered, she died due to metastatic liver failure.

Background Prostaglandin-endoperoxide synthase 2 (PTGS2, cyclooxygenase-2, COX-2)-prostaglandin E2 (PGE2) pathway promotes tumour progression. Considering evidence suggesting increased PGE2 synthesis by BRAF mutation in tumour cells, we hypothesised that the association of tumour PTGS2 (COX-2) expression with colorectal cancer mortality might be stronger in BRAF-mutated tumours than in BRAF-wild-type tumours. Methods Using 1708 patients, including 1200 stage I-IV colorectal carcinoma cases in the Nurses’ Health Study (NHS) and the Health Professionals Follow-up Study (HPFS) and 508 stage III colon cancer cases in a National Cancer Institute–sponsored randomised controlled trial of adjuvant therapy (CALGB/Alliance 89803), we evaluated tumour PTGS2 (COX-2) expression status using immunohistochemistry. We examined the prognostic association of PTGS2 (COX-2) expression in strata of BRAF mutation status by multivariable Cox proportional hazards regression models to adjust for potential confounders, including disease stage, tumour differentiation, microsatellite instability status and KRAS and PIK3CA mutations. Results In NHS and HPFS, the association of PTGS2 (COX-2) expression with colorectal cancer-specific survival differed by BRAF mutation status (Pinteraction = 0.0005); compared with PTGS2 (COX-2)-negative/low carcinomas, the multivariable-adjusted hazard ratios for PTGS2 (COX-2)-high carcinomas were 2.44 (95% confidence interval, 1.39–4.28) in BRAF-mutated cases and 0.82 (95% confidence interval, 0.65–1.04) in BRAF-wild-type cases. Differential prognostic associations of PTGS2 (COX-2) expression in strata of BRAF mutation status were similarly observed in CALGB/Alliance 89803 trial (Pinteraction = 0.03). Conclusions The association of tumour PTGS2 (COX-2) expression with colorectal cancer mortality is stronger in BRAF-mutated tumours than in BRAF-wild-type tumours, supporting interactive roles of PTGS2 (COX-2) expression and BRAF mutation statuses in prognostication of patients with colorectal cancer; ClinicalTrials.gov Identifier, NCT00003835.

Immune checkpoint inhibitor (ICI) is widely used and highly effective for some cancer patients but may result in disease progression in others. Hyperprogressive disease in particular is characterized by an acceleration of tumor growth during ICI therapy and has been reported in patients including those with urothelial carcinoma. Biomarkers predicting treatment efficacy are crucial to avoid tumor progression and unnecessary adverse effects. This study aims to clarify the predictors of disease progression for ICI treatment in patients with urothelial carcinoma. We analyzed the response pattern of 23 urothelial carcinomas treated with pembrolizumab and its association with pathological features and potential immunohistochemical markers including EGFR, MDM2, p53, p16, and programmed cell death ligand-1 (PD-L1) expression and CD8- and CD204-positive cell infiltration. During ICI therapy, 13 (57 %) patients showed progressive disease including 6 (26 %) with hyperprogressive disease. Notably, squamous differentiation combined with MAC387 expression was observed exclusively in cases with progressive disease (6 of 13, 46 %); it was not present in cases with stable disease or partial/complete response (0 of 10, p = 0.0019). All tumors with squamous differentiation showed positive staining for EGFR. Additionally, the loss of p16 expression occurred more frequently in cases with progressive disease (8 of 13, 62 %) than in other cases (3 of 10, 30 %), but this finding did not reach statistical significance. Squamous differentiation was also significantly associated with shorter overall survival. Based on our observations, squamous differentiation may be a novel biomarker for predicting disease progression in patients with urothelial carcinoma who receive pembrolizumab.

IgG4-related membranous nephropathy (MN) is often refractory to glucocorticoid (GC) therapy, and treatment remains unclear. We herein report a 67-year-old Japanese man with IgG4-related MN and tubulointerstitial nephritis. A post-gastroscopy antibody test revealed Helicobacter pylori infection. After eradication, his proteinuria decreased indefinitely. We started prednisolone (30 mg/day), long-term GCs, and immunosuppressant therapy. However, remission proved challenging to achieve, with persistent proteinuria present at 1.0-2.0 g/gCr. We performed multitarget therapy for refractory IgG4-related MN, achieving proteinuria remission (<0.3 g/gCr). Multitarget therapy with low-dose GCs can resolve refractory IgG4-related MN through remission induction of proteinuria and minimize the risks associated with GC therapy.

Patients with von Hippel-Lindau disease (vHL) are at risk of developing spatially and temporally multiple clear cell renal cell carcinomas (ccRCCs), which offers a valuable opportunity to analyze inter- and intra-tumor heterogeneity of genetic and immune profiles within the same patient. Here, we perform whole-exome and RNA sequencing, digital gene expression, and immunohistochemical analyses for 81 samples from 51 ccRCCs of 10 patients with vHL. Inherited ccRCCs are clonally independent and have less genomic alterations than sporadic ccRCCs. Hierarchical clustering of transcriptome profiles shows two clusters with distinct immune signatures: immune hot and cold clusters. Interestingly, not only samples from the same tumors but also different tumors from the same patients tend to show a similar immune signature, whereas samples from different patients frequently exhibit different signatures. Our findings reveal the genetic and immune landscape of inherited ccRCCs, demonstrating the relevance of host factors in shaping anti-tumor immunity.

Abstract Radical cystectomy is a gold-standard treatment for muscle-invasive bladder cancer. We recently introduced robot-assisted radical cystectomy (RARC) with perioperative enhanced recovery after surgery (ERAS). The medical records of patients with bladder cancer who underwent open radical cystectomy (ORC) or RARC/ERAS at NTT Medical Center Tokyo were retrospectively reviewed to compare the surgical outcomes, hospital stay, and medical costs between groups. Multidisciplinary full ERAS items were provided for the RARC/ERAS group. The median estimated blood losses in the ORC and RARC/ERAS groups were 650 and 100 mL, and the median operative times were 312 and 445 min, respectively. In addition, the median times to liquid food intake in these groups were 6 and 0 days, the median times to first flatus and first defecation were 2 and 1 day, and 3 and 1.5 days, respectively. The rates of postoperative ileus in the ORC and RARC/ERAS groups were 27.5% and 4.5%, and the median postoperative hospital stays was 26.5 and 12 days, respectively. Medical costs excluding surgery were significantly lower in the RARC/ERAS group. In conclusion, RARC/ERAS represents a safe treatment option for muscle-invasive bladder cancer with decreased perioperative complications and lower medical costs.

BackgroundDNA methylation accumulates in non-malignant gastric mucosa after exposure to pathogens. To elucidate how environmental, methylation, and lifestyle factors interplay to influence primary gastric neoplasia (GN) risk, we analyzed longitudinally monitored cohorts in Japan and Singapore.MethodsAsymptomatic subjects who underwent a gastric mucosal biopsy on the health check-up were enrolled. We analyzed the association between clinical factors and GN development using Cox hazard models. We further conducted comprehensive methylation analysis on selected tissues, including (i) mucosae from subjects developing GN later, (ii) mucosae from subjects not developing GN later, and (iii) GN tissues and surrounding mucosae. We also use the methylation data of mucosa collected in Singapore. The association between methylation and GN risk, as well as lifestyle and methylation, were analyzed.FindingsAmong 4234 subjects, GN was developed in 77 subjects. GN incidence was correlated with age, drinking, smoking, and Helicobacter pylori (HP) status. Accumulation of methylation in biopsied gastric mucosae was predictive of higher future GN risk and shorter duration to GN incidence. Whereas methylation levels were associated with HP positivity, lifestyle, and morphological alterations, DNA methylation remained an independent GN risk factor through multivariable analyses. Pro-carcinogenic epigenetic alterations initiated by HP exposure were amplified by unfavorable but modifiable lifestyle choices. Adding DNA methylation to the model with clinical factors improved the predictive ability for the GN risk.InterpretationThe integration of environmental, lifestyle, and epigenetic information can provide increased resolution in the stratification of primary GN risk.FundingThe funds are listed in Acknowledgements section.

Introduction Patients with hereditary leiomyomatosis and renal cell cancer (HLRCC) syndrome have high risks of uterine and cutaneous leiomyomas and renal cell carcinoma (RCC), which are caused by germline mutation of the fumarate hydratase (FH) gene. RCC lesions are mostly high‐grade tumors with a poor prognosis. Case presentation A 37‐year‐old man who had previously undergone treatment for a left RCC was referred to our hospital with a diagnosis of right RCC. Robot‐assisted partial nephrectomy was performed, and the pathological diagnosis revealed fumarate hydratase (FH)‐deficient RCC. The left RCC, which was originally diagnosed as mucinous tubular and spindle cell carcinoma, was reviewed and diagnosed as FH‐deficient RCC. The patient's father and uncle both died of RCC, and the father's tumor was also immunohistochemically proven to be FH‐deficient RCC. Conclusion HLRCC‐related RCC should be considered in a differential diagnosis of young patients with a family history of RCC.

Abstract A 51‐year‐old woman visited our hospital with the chief complaint of tarry stools. Contrast‐enhanced abdominal computed tomography revealed leakage of contrast medium into the lumen of the small intestine. Subsequently, a double‐balloon endoscopy was performed, which revealed a submucosal mass‐like lesion in the jejunum. Although hemostasis was attempted with clips, complete hemostasis was difficult to achieve, and angiographic embolization was performed. Nevertheless, the anemia progressed, and a small bowel resection was performed. Histopathological examination led to a diagnosis of a ruptured submucosal aneurysm of the small intestine. Endoscopic hemostasis is often difficult to achieve for submucosal aneurysms in the intestine. The submucosal tumor‐like finding observed on endoscopy in submucosal aneurysms is termed an “SMT‐like sign” and is considered an important finding to diagnose aneurysms.

A 25-year-old man was referred to our center for investigation of a gastric submucosal tumor and an ulcer that had developed on its oral side. Endoscopic ultrasonography findings suggested the presence of an ectopic pancreas, and treatment with an oral proton pump inhibitor was planned for the ulcer. Over the subsequent 3 years, the patient endured recurring epigastric pain and episodes of passing black stools. Emergency endoscopy revealed that the morphology of the gastric submucosal tumor had transformed into a pedunculated polyp-like morphology with a bleeding ulcer at the apex of the lesion. Endoscopic hemostasis using hemostatic forceps was performed. However, the patient continued to pass black stools. In light of the persistent symptoms and unique morphology of the lesion, endoscopic resection was attempted as a curative approach. The lesion was excised by hot snare polypectomy. Post-treatment, the patient exhibited no signs of recurrence, marking a successful resolution. Three months later, a gastroduodenal endoscopy showed that the excised site had undergone scar formation without recurrence of the lesion. This case holds significant clinical value as it demonstrates the efficacy of a minimally invasive treatment strategy in managing repeated bleeding ulcerations of an ectopic pancreas, ultimately achieving a complete cure.

A 57-year-old woman with no significant medical history was referred after a colonoscopy for abdominal distension, which revealed a tumor in the lower rectum. Pre-operative colonoscopy showed the tumor was 12 mm in size, located from the anorectal junction to beyond the dentate line, and was diagnosed as high-grade intramucosal neoplasia or shallow submucosal invasive cancer. Endoscopic submucosal dissection was performed, and the lesion was resected en bloc. Pathological examination revealed moderately differentiated tubular adenocarcinoma with tubulovillous adenoma. The stratified squamous epithelium adjacent to the anal side of the lesion showed pagetoid spread of atypical cells with positive horizontal margins. We referred her to a surgeon for radical treatment. The mucosa surrounding the endoscopic submucosal dissection scar was normal on narrow-band imaging magnification. We marked its oral side endoscopically as the resected boundary. Transanal local excision was performed. The horizontal margins were positive because atypical cells had spread into the stratified squamous epithelium of the anorectal side of the lesion. The patient was followed on an outpatient basis. Sixty days postoperatively, residual tumor growth was observed. The second local resection was performed after mapping biopsy. All resection margins were negative, there was no lymphovascular invasion. One year after surgery, no recurrence was observed. Regarding endoscopic findings, there are no reports of endoscopic findings of the rectal mucosa, or the squamous epithelium of the anus of pagetoid spread. Here, we report a review of perianal Paget's Disease that resulted in difficulties in borderline diagnosis of pagetoid spread, resulting in multiple therapeutic interventions.

A 52-year-old male patient was diagnosed with transverse colon cancer and synchronous stage IVA paraaortic lymph node (PALN) metastases (cT3N1bM1a of the lymph node).Six courses of mFOLFOX6 plus bevacizumab were administered as neoadjuvant chemotherapy.Computed tomography showed shrinkage of the primary tumor and PALN metastases.Extended right hemicolectomy, D3 lymph node dissection, and PALN dissection were performed.A pathologic examination indicated that the tumor had completely changed and comprised necrotic tissue with no viable cells.Therefore, it was considered that mFOLFOX6 plus bevacizumab resulted in a pathologic complete response.Postoperatively, six courses of mFOLFOX6 were administered.Six years postoperatively, the patient did not exhibit any signs of recurrence.There have been few reports of pathologic complete response after neoadjuvant therapy and resection for colon cancer with synchronous PALN metastases.This report describes a unique case involving a pathologic complete response with long-term survival after mFOLFOX6 plus bevacizumab and radical resection, including PALN dissection.Preoperative mFOLFOX6 plus bevacizumab followed by radical resection and adjuvant mFOLFOX6 therapy was safe and resulted in a good outcome.This regimen should be considered for advanced colon cancer with PALN metastases.