Non-tumor Epithelium Research Articles

Overexpression of VSNL1 Enhances Cell Proliferation in Colorectal Carcinogenesis

Introduction: We have previously reported that overexpression of visinin-like protein 1 (VSNL1) is frequently observed in advanced colorectal adenocarcinomas and correlates with poorer prognosis. In this study, we determined the levels of VSNL1 expression in the earlier stages of colorectal tumors including adenomas and adenocarcinomas, and attempted to clarify the functional significance of VSNL1 overexpression in colorectal carcinogenesis. Methods: Levels of VSNL expression in colorectal tumor tissues were analyzed using immunohistochemistry. The effects of VSNL1 downregulation and overexpression on cell proliferation, resistance to apoptosis, and invasiveness were determined using two VSNL1-overexpressing colorectal cancer cell lines, CW-2 and HCT-116 and VSNL1 inducibly expressing SNU-C5, respectively. Gene expression signatures in VSNL1-downregulated CW-2 and HCT-116 were identified using transcriptome and gene set enrichment analyses. Results: VSNL1 expression was restricted to only a few crypt cells in the non-tumorous epithelium, whereas it became enhanced in adenomas and adenocarcinomas with the progression of tumorigenesis. Downregulation of VSNL1 in CW-2 and HCT-116 cells suppressed their proliferation through induction of apoptosis. Conversely, overexpression of VSNL1 in SNU-C5 cells enhanced resistance to anoikis. Transcriptome and gene set enrichment analyses revealed that downregulation of VSNL1 altered the expression level of the apoptosis-related gene set in CW-2 and HCT-116 cells. Conclusion: VSNL1 plays a role in both the development and progression of colorectal tumors by enhancing cell viability.

Stepwise activation of p63 and the MEK/ERK pathway induces the expression of ARL4C to promote oral squamous cell carcinoma cell proliferation.

Carcinogenesis is a multistep process wherein cells accumulate multiple genetic alterations and progress to a more malignant phenotype. It has been proposed that sequential accumulation of gene abnormalities in specific genes drives the transition from non-tumorous epithelia through a preneoplastic lesion/benign tumor to cancer. Histologically, oral squamous cell carcinoma (OSCC) progresses in multiple ordered steps that begin with mucosal epithelial cell hyperplasia, which is followed by dysplasia, carcinoma in situ and invasive carcinoma. It is therefore hypothesized that genetic alteration-mediated multistep carcinogenesis would be involved in the development of OSCC; however, the detailed molecular mechanisms are unknown. We clarified the comprehensive gene expression patterns and carried out an enrichment analysis using DNA microarray data from a pathological specimen of OSCC (including a non-tumor region, carcinoma in situ lesion and invasive carcinoma lesion). The expression of numerous genes and signal activation were altered in the development of OSCC. Among these, the p63 expression was increased and the MEK/ERK-MAPK pathway was activated in carcinoma in situ lesion and in invasive carcinoma lesion. Immunohistochemical analyses revealed that p63 was initially upregulated in carcinoma in situ and ERK was sequentially activated in invasive carcinoma lesions in OSCC specimens. ADP-ribosylation factor (ARF)-like 4c (ARL4C), the expression of which is reportedly induced by p63 and/or the MEK/ERK-MAPK pathway in OSCC cells, has been shown to promote tumorigenesis. Immunohistochemically, in OSCC specimens, ARL4C was more frequently detected in tumor lesions, especially in invasive carcinoma lesions, than in carcinoma in situ lesions. Additionally, ARL4C and phosphorylated ERK were frequently merged in invasive carcinoma lesions. Loss-of-function experiments using inhibitors and siRNAs revealed that p63 and MEK/ERK-MAPK cooperatively induce the expression of ARL4C and cell growth in OSCC cells. These results suggest that the stepwise activation of p63 and MEK/ERK-MAPK contributes to OSCC tumor cell growth through regulation of ARL4C expression.

Evidence for etiologic field changes in tongue distant from tumor in patients with squamous cell carcinoma of the oral tongue.

Oral cancer is a paradigm of Slaughter's concept of field cancerization, where tumors are thought to originate within an area of cells containing genetic alterations that predispose to cancer development. The field size is unclear but may represent a large area of tissue, and the origin of mutations is also unclear. Here, we analyzed whole exome and transcriptome features in contralateral tumor-distal tongue (i.e. distant from the tumor, not tumor-adjacent) and corresponding tumor tissues of 15 patients with squamous cell carcinoma of the oral tongue. The number of point mutations ranged from 41 to 237 in tumors and from one to 78 in tumor-distal samples. Tumor-distal samples showed mainly clock-like (associated with aging) or tobacco smoking mutational signatures. Tumors additionally showed mutations that associate with cytidine deaminase AID/APOBEC enzyme activities or a UV-like signature. Importantly, no point mutations were shared between a tumor and the matched tumor-distal sample in any patient. TP53 was the most frequently mutated gene in tumors (67%), whereas a TP53 mutation was detected in only one tumor-distal sample, and this mutation was not shared with the matched tumor. Arm-level copy number variation (CNV) was found in 12 tumors, with loss of chromosome (Chr) 8p or gain of 8q being the most frequent events. Two tumor-distal samples showed a gain of Chr8, which was associated with increased expression of Chr8-located genes in these samples, although gene ontology did not show a role for these genes in oncogenic processes. In situ hybridization revealed a mixed pattern of Chr8 gain and neutral copy number in both tumor cells and adjacent nontumor epithelium in one patient. We conclude that distant field cancerization exists but does not present as tumor-related mutational events. The data are compatible with etiologic field effects, rather than classical monoclonal field cancerization theory. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Spatiotemporal analysis of tumour-infiltrating immune cells in biliary carcinogenesis

BackgroundIntraductal papillary neoplasms (IPN) and biliary epithelial neoplasia (BilIN) are well‐defined precursor lesions of biliary tract carcinoma (BTC). The aim of this study was to provide a comprehensive characterisation of the inflammatory microenvironment in BTC precursor lesions.MethodsImmunohistochemistry was employed to assess tumour-infiltrating immune cells in tissue samples from patients, for whom precursor lesions were identified alongside invasive BTC. The spatiotemporal evolution of the immune microenvironment during IPN-associated carcinogenesis was comprehensively analysed using triplet sample sets of non-neoplastic epithelium, precursor lesion and invasive BTC. Immune-cell dynamics during IPN- and BilIN-associated carcinogenesis were subsequently compared.ResultsStromal CD3+ (P = 0.002), CD4+ (P = 0.007) and CD8+ (P < 0.001) T cells, CD20+ B cells (P = 0.008), MUM1+ plasma cells (P = 0.012) and CD163+ M2-like macrophages (P = 0.008) significantly decreased in IPN compared to non-tumorous biliary epithelium. Upon transition from IPN to invasive BTC, stromal CD68+ (P = 0.001) and CD163+ (P < 0.001) macrophages significantly increased. In contrast, BilIN-driven carcinogenesis was characterised by significant reduction of intraepithelial CD8+ T-lymphocytic infiltration from non-tumorous epithelium via BilIN (P = 0.008) to BTC (P = 0.004).ConclusionIPN and BilIN are immunologically distinct entities that undergo different immune-cell variations during biliary carcinogenesis. Intraepithelial CD8+ T-lymphocytic infiltration of biliary tissue decreased already at the IPN-precursor stage, whereas BilIN-associated carcinogenesis showed a slowly progressing reduction towards invasive carcinoma.

IFITM3 promotes malignant progression, cancer stemness and chemoresistance of gastric cancer by targeting MET/AKT/FOXO3/c-MYC axis

BackgroundTargeting the HGF/MET signaling pathway has been a viable therapeutic strategy for various cancer types due to hyperactivation of HGF/MET axis occurs frequently that leads to detrimental cancer progression and recurrence. Deciphering novel molecule mechanisms underlying complex HGF/MET signaling network is therefore critical to development of effective therapeutics for treating MET-dependent malignancies.ResultsUsing isobaric mass tag-based quantitative proteomics approach, we identified IFITM3, an interferon-induced transmembrane protein that was highly expressed in micro-dissected gastric cancer (GC) tumor regions relative to adjacent non-tumor epithelia. Analyses of GC clinical specimens revealed that expression IFITM3 was closely correlated to advanced pathological stages. IFITM3 has been reported as a PIP3 scaffold protein that promotes PI3K signaling. In present study, we unprecedentedly unraveled that IFITM3 associated with MET and AKT to facilitate HGF/MET mediated AKT signaling crosstalk in suppressing FOXO3, consequently leading to c-MYC mediated GC progression. In addition, gene ontology analyses of the clinical GC cohort revealed significant correlation between IFITM3-associated genes and targets of c-MYC, which is a crucial downstream effector of HGF/MET pathway in cancer progression. Moreover, we demonstrated ectopic expression of IFITM3 suppressed FOXO3 expression, consequently led to c-MYC induction to promote tumor growth, cell metastasis, cancer stemness as well as chemoresistance. Conversely, depletion of IFITM3 resulted in suppression of HGF triggered cellular growth and migration via inhibition of AKT/c-MYC signaling in GC.ConclusionsIn summary, our present study unveiled a novel regulatory mechanism for c-MYC-driven oncogenesis underlined by IFITM3-mediated signaling crosstalk between MET associated AKT signaling cascade.

Clinical significance and biomarker potential of MGMT protein measurement in colorectal cancer.

136 Background: O6-methylguanine DNA methyltransferase (MGMT) is the principal repair mechanism of DNA damage created by alkylating agents. Approximately 40% of colorectal cancers (CRC) exhibit MGMT promoter hypermethylation, expected to result in gene silencing. The role of MGMT as a biomarker is poorly understood, and quantitative measurement of MGMT protein has not been performed in CRC. We hypothesized that altered tumor MGMT would affect DNA damage response, modulate adaptive anti-tumor immune responses, and influence survival in CRC. Methods: We used multiplexed quantitative immunofluorescence (QIF) to study 4 clinically annotated retrospective patient cohorts treated from 2000 to 2017 at Yale, consisting of 400 formalin-fixed paraffin-embedded CRC cases represented in tissue microarray format. These included paired tumors and adjacent non-tumor colonic mucosa (n = 112 pairs in Cohort 1), paired primary CRC and lymph node metastases (n = 31 pairs in Cohort 2), and 2 cohorts with primary stage I-IV tumors (n = 250 in Cohort 3; n = 150 in Cohort 4). We established a QIF panel for simultaneous, localized measurement of DAPI (all cells), cytokeratin (CK; tumor epithelial cells), MGMT, γH2AX (DNA damage response), and CD8. The measurement of fluorescent signals in CK-positive tumor cells or CK-negative stromal cells was achieved using co-localization strategies and the AQUA QIF platform. Results: We identified lower MGMT protein levels in CRC cells than in non-tumor colonic epithelium (p &lt; 0.0001), and in lymph node metastases compared to paired primary CRC tumors (p = 0.0411). Using the visual detection threshold, tumor selective MGMT protein downregulation was identified in 20% of cases in Cohorts 3 and 4. Microsatellite instability-high (MSI-H) cases showed decreased tumoral MGMT protein compared to microsatellite stable (MSS) cases (p = 0.002). In Cohorts 3 and 4, low tumoral MGMT was consistently associated with increased CD8+ tumor infiltrating lymphocytes (p = 0.0472; p = 0.0002). The association between MGMT and γH2AX was inconsistent. The association between MGMT and CD8 was significant only in MSS cases (p = 0.0001) but not in MSI-H cases (p = 0.0979), supporting that the effect is not driven by MSI-H status. Tumor-selective MGMT downregulation evidenced by a low tumor-to-stroma ratio was associated with improved progression-free survival and overall survival in both Cohorts 3 and 4, but statistical significance was only seen in Cohort 3. Conclusions: MGMT protein downregulation occurs in 20% of CRCs and is associated with increased adaptive anti-tumor immune responses, mismatch repair (MMR) deficiency and better prognosis. MGMT deficiency may alter DNA repair in tumor cells and mediate the accumulation of antigenic mutations or neopeptides, independent from MMR status. Our results support a biomarker role of MGMT protein and suggest a role for immunotherapy combinations in MGMT deficient tumors.

Epithelial-specific ERBB3 deletion results in a genetic background-dependent increase in intestinal and colon polyps that is mediated by EGFR.

ERBB3 has gained attention as a potential therapeutic target to treat colorectal and other types of cancers. To confirm a previous study showing intestinal polyps are dependent upon ERBB3, we generated an intestinal epithelia-specific ERBB3 deletion in C57BL/6-ApcMin/+ mice. Contrary to the previous report showing a significant reduction in intestinal polyps with ablation of ERBB3 on a B6;129 mixed genetic background, we observed a significant increase in polyp number with ablation of ERBB3 on C57BL/6J compared to control littermates. We confirmed the genetic background dependency of ERBB3 by also analyzing polyp development on B6129 hybrid and B6;129 advanced intercross mixed genetic backgrounds, which showed that ERBB3 deficiency only reduced polyp number on the mixed background as previously reported. Increased polyp number with ablation of ERBB3 was also observed in C57BL/6J mice treated with azoxymethane showing the effect is model independent. Polyps forming in absence of ERBB3 were generally smaller than those forming in control mice, albeit the effect was greatest in genetic backgrounds with reduced polyp numbers. The mechanism for differential polyp number in the absence of ERBB3 was through altered proliferation. Backgrounds with increased polyp number with loss of ERBB3 showed an increase in cell proliferation even in non-tumor epithelia, while backgrounds showing reduced polyp number with loss of ERBB3 showed reduced cellular proliferation. Increase polyp number caused by loss of ERBB3 was mediated by increased epidermal growth factor receptor (EGFR) expression, which was confirmed by deletion of Egfr. Taken together, this study raises substantial implications on the use of ERBB3 inhibitors against colorectal cancer. The prediction is that some patients may have increased progression with ERBB3 inhibitor therapy, which is consistent with observations reported for ERBB3 inhibitor clinical trials.

A novel evaluation method for Ki-67 immunostaining in paraffin-embedded tissues.

The Ki-67 labeling index is traditionally used to investigate tumor aggressiveness. However, no diagnostic or prognostic value has been associated to the heterogeneous pattern of nuclear positivity. The aims of this study were to develop a classification for the patterns of Ki-67-positive nuclei; to search scientific evidence for the Ki-67 expression and location throughout the cell cycle; and to develop a protocol to apply the classification of patterns of Ki-67-positive nuclei in squamous epithelium with different proliferative activities. Based on empirical observation of paraffin sections submitted to immunohistochemistry for the determination of Ki-67 labeling index and literature review about Ki-67 expression, we created a classification of the patterns of nuclear positivity (NP1, NP2, NP3, NP4, and mitosis). A semi-automatic protocol was developed to identify and quantify the Ki-67 immunostaining patterns in target tissues. Two observers evaluated 7000 nuclei twice to test the intraobserver reliability, and six evaluated 1000 nuclei to the interobserver evaluation. The results showed that the immunohistochemical patterns of Ki-67 are similar in the tumoral and non-tumoral epithelium and were classified without difficulty. There was a high intraobserver reliability (Spearman correlation coefficient > 0.9) and moderate interobserver agreement (k = 0.523). Statistical analysis showed that non-malignant epithelial specimens presented a higher number of NP1 (geographic tongue = 83.8 ± 21.8; no lesion = 107.6 ± 52.7; and mild dysplasia = 86.6 ± 25.8) when compared to carcinoma in Situ (46.8 ± 34.8) and invasive carcinoma (72.6 ± 37.9). The statistical evaluation showed significant difference (p < 0.05). Thus, we propose a new way to evaluate Ki-67, where the pattern of its expression may be associated with the dynamics of the cell cycle. Future proof of this association will validate the use of the classification for its possible impact on cancer prognosis and guidance on personalized therapy.

Immunohistochemical analysis of epithelium adjacent to lip cancer: A meta‐analysis

To identify the immunohistochemical pattern of non-tumoral epithelium adjacent to lip cancer (ANTE) to unveil molecular alterations and potential biomarkers in lip cancer patients. A systematic search at MEDLINE, EMBASE and proceedings Web of Science, OpenGrey and WorldCat. The PICOS outline (Population: lip cancer patients; Intervention: immunostaining with biomarkers in surgical specimens; Comparisons: ANTE versus LC; Outcomes: primary-to identify the immunohistochemical pattern of peritumoral epithelium and secondary-to assess the immunoreactivity of LC epithelium; and Study design: cross-sectional, case-control and cohort studies). A total of 339 records were identified. Seven studies reporting on ANTE and LCs were included in the qualitative synthesis, and 4 of them (p53 immunostaining) were selected for meta-analysis. In the ANTE group, the pooled prevalence of p53 was 0.74 (95% CI: 0.66 - 0.82), while in the lip squamous cell carcinoma group this prevalence was0.83 (95% CI: 0.75 - 0.89). Similar immunoexpression was found in lip epithelium adjacent to lip carcinoma, even in epithelia with normal appearance or mild histological alterations. The role of biomarkers in the follow-up of actinic cheilitis patients deserves additional clinical assessment.

Healing potential of Caiman yacare (Daudin, 1802) visceral fat oil

Abstract Caiman yacare visceral fat oil was extracted, analyzed, and tested for healing potential. In vivo assays were performed by excisional wounds in rats and treated topically daily with Caiman oil for 10 days. The positive and negative controls were, respectively, with Dersani® (Essential Fatty Acids – EFA, reference substance) and saline solution (SS). The scattered area was measured daily, and at 3rd and 10th days the rats were euthanized and samples were processed for histological analyses. The cytotoxicity was evaluated by the MTT assay in non-tumor retinal pigment epithelium cells. The Caiman oil composition was 42.95 ± 1.03 % of saturated fatty acids and 43.74 ± 0.74 % of unsaturated ones. After 10 days of excisional wounds, the Caiman oil-treated animals present a larger scarred area than the negative control ones. Caiman oil and EFA treated animals present significantly more epidermal papillae than in SS-treated ones on day 10. Caiman oil treated animals, at 10th day, present a relative increased area of collagen fibers, as well as an elevated number of fibroblasts and monomorphonuclear cells in the wound region in comparison to both SS and EFA treated ones. The oil showed no significant cytotoxicity up to 500 μg/mL. Taken together, Caiman oil, extracted as a by-product of the C. yacare zootechnical disposal, demonstrated wound healing properties equal to the commercial available solution, subsidizing its ethnoknowledge.

Overexpression of JAG2 is related to poor outcomes in oral squamous cell carcinoma.

ObjectivesJAG2 is one of Notch ligands, which recently appear to exert various carcinogenesis. In the present study, we aimed to unveil the relation of JAG2 expression and clinicopathological features in oral squamous cell carcinoma (OSCC).Materials and MethodsWe examined JAG2 expression in OSCC plus adjacent nontumorous epithelia in eight patients. Ninety‐one OSCC tissue specimens were immunohistochemically stained with specific antibodies to JAG2. The immunoreactivities of JAG2 were correlated with clinicopathological factors, including the prognosis of patients. Chi‐square test, Kaplan–Meier survival, and Cox proportional hazard analysis were used to determine the statistical value of JAG2 expression in OSCC.Results JAG2 mRNA expression was much expressed in OSCC tissues compared with adjacent tissue specimens in five of eight patients. JAG2 immunoreactivity was found at invasion front in 31 of 91 OSCC. JAG2 immunoreactivity was significantly associated with age, less than 50 years old of patients (P = .048). Kaplan–Meier analysis demonstrated that the patients with JAG2 immunoreactvty have a short overall survival. With the Cox proportional hazard regression mode, the independent factors predictive of poor overall survival included JAG2 immunoreactivity (P < .05).ConclusionsThe present findings suggest that JAG2 overexpression, especially at the cancer invasion front, has potential prognostic value.

The expression and clinical significance of homeobox B7 and proliferation cell nuclear antigen in cholangiocarcinoma

Objective To investigate the expression of homeobox B7 (HOXB7) and proliferation cell nuclear antigen (Ki-67) in cholangiocarcinoma tissues and the clinical significances. Methods Immunohistochemical streptavid-in-peroxidase (SP) method was used to detect the expression of HOXB7 and Ki-67 in 70 cases of cholangiocarcinoma and 15 cases of non-tumor bile duct epithelium. The relationship between the expression of HOXB7 and Ki-67 and clinical progression and prognosis was analyzed. The correlation between the expression of HOXB7 and Ki-67 was analyzed by Spearman analysis method. Results The positive expression rate of HOXB7 and Ki-67 in cholangiocarcinoma tissue (57.1% and 71.4%) was significantly higher than that in non-tumor bile duct epithelium (20.0% and 26.7%; χ2=6.818, P=0.009; χ2=10.682, P=0.001). HOXB7 and Ki-67 expression was significantly correlated with clinical stage, tumor grade and lymph node metastasis. The positive expression rate of HOXB7 and Ki-67 was significantly higher in the stage Ⅲ+ Ⅳ group (χ2=4.073, P=0.044; χ2=4.488, P=0.034), in moderately or poorly-differentiated group than that in well-differentiated group (χ2=7.807, P=0.020; χ2=8.270, P=0.016) and in lymph node metastasis than that in no lymph node metastasis (χ2=6.076, P=0.014; χ2=4.667, P=0.031). A significant correlation was found between the expressions of HOXB7 and Ki-67 in cholangiocarcinoma (r=0.283, P=0.018). The post-operative mean survival time of patients was shortened with the increased expression levels of HOXB7 and Ki-67. Conclusion The abnormal expression of HOXB7 and Ki-67 may play an important role in the occurrence and development of cholangiocarcinoma. Key words: Cholangiocarcinoma; Homeobox B7; Proliferation cell nuclear antigen

Argininosuccinate synthetase 1 contributes to gastric cancer invasion and progression by modulating autophagy

Argininosuccinate synthetase 1 (ASS1) is a rate-limited enzyme in arginine biosynthesis. The oncogenic potential of ASS1 in terms of prognosis and cancer metastasis in arginine prototrophic gastric cancer (GC) remains unclear at present. We identify differentially expressed proteins in microdissected GC tumor cells relative to adjacent nontumor epithelia by isobaric mass tag for relative and absolute quantitation proteomics analysis. GC cells with stable expression or depletion of ASS1 were further analyzed to identify downstream molecules. We investigated their effects on chemoresistance and cell invasion in the presence or absence of arginine. ASS1 was highly expressed in GC and positively correlated with GC aggressiveness and poor outcome. Depletion of ASS1 led to inhibition of tumor growth and decreased cell invasion via induction of autophagy-lysosome machinery, resulting in degradation of active β-catenin, Snail, and Twist. Ectopic expression of ASS1 in GC cells reversed these effects and protected cancer cells from chemotherapy drug-induced apoptosis via activation of the AKT-mammalian target of rapamycin signaling pathway. ASS1 contributes to GC progression by enhancing aggressive potential resulting from active β-catenin, Snail, and Twist accumulation. Our results propose that ASS1 might contribute to GC metastasis and support its utility as a prognostic predictor of GC.-Tsai, C.-Y., Chi, H.-C., Chi, L.-M., Yang, H.-Y., Tsai, M.-M., Lee, K.-F., Huang, H.-W., Chou, L.-F., Cheng, A.-J., Yang, C.-W., Wang, C.-S., Lin, K.-H. Argininosuccinate synthetase 1 contributes to gastric cancer invasion and progression by modulating autophagy.

The Value of Diffusion-Weighted Imaging in Combination With Conventional Magnetic Resonance Imaging for Improving Tumor Detection for Early Cervical Carcinoma Treated With Fertility-Sparing Surgery.

This study aimed to investigate the value of diffusion-weighted imaging (DWI) in combination with conventional magnetic resonance imaging (MRI) for improving tumor detection in young patients treated with fertility-sparing surgery because of early cervical carcinoma. Fifty-four patients with stage Ia or Ib1 cervical carcinoma were enrolled into this study. Magnetic resonance examinations were performed for these patients using conventional MRI (including T1-weighted imaging, T2-weighted imaging, and dynamic contrast-enhanced MRI) and DWI. The apparent diffusion coefficient (ADC) values of cervical carcinoma were analyzed quantitatively and compared with that of adjacent epithelium. Sensitivity, positive predictive value, and accuracy of 2 sets of MRI sequences were calculated on the basis of histologic results, and the diagnostic ability of conventional MRI/DWI combinations was compared with that of conventional MRI. The mean ADC value from cervical carcinoma (mean, 786 × 10 mm/s ± 100) was significantly lower than that from adjacent epithelium (mean, 1352 × 10 mm/s ± 147) (P = 0.01). When the threshold ADC value set as 1010 × 10 mm/s, the sensitivity and specificity for differentiating cervical carcinoma from nontumor epithelium were 78.2% and 67.2%, respectively. The sensitivity and accuracy of conventional MRI for tumor detection were 76.0% and 70.4%, whereas the sensitivity and accuracy of conventional MRI/DWI combinations were 91.7% and 90.7%, respectively. Conventional MRI/DWI combinations revealed a positive predictive value of 97.8% and only 4 false-negative findings. The addition of DWI to conventional MRI considerably improves the sensitivity and accuracy of tumor detection in young patients treated with fertility-sparing surgery, which supports the inclusion quantitative analysis of ADC value in routine MRI protocol before fertility-sparing surgery.

Expression and clinical significance of annexin A1 and proliferating cell nuclear antigen in cholangiocarcinoma

Objective To investigate the expression of annexin A1 (ANXA1) and proliferating cell nuclear antigen (PCNA) in cholangiocarcinoma tissues and its clinical significance. Methods Immunohistochemical staining was used to detect the expression of ANXA1 and PCNA in 70 cases of cholangiocarcinoma and 15 cases of non-tumor bile duct epithelium. Results The expression of ANXA1 and PCNA in cholangiocarcinoma (54.3% and 77.1%) was significantly higher than that in non-tumor bile duct epithelium (13.3% and 33.3%; χ2=8.320, P=0.004; χ2=11.170, P=0.001). ANXA1 expression was significantly correlated with clinical stage and tumor grade. The positive rate of ANXA1 was significantly higher in the stage Ⅲ+ Ⅳ group and moderately or poorly-differentiated group than that in stage Ⅰ+ Ⅱ group and well-differentiated group (χ2=5.090, P=0.024; χ2=6.090, P=0.047). The PCNA expression was significantly correlated with clinical stage and tumor grade. The positive rate of PCNA was significantly higher in the stage Ⅲ+ Ⅳ group and moderately or poorly-differentiated group than that in stage Ⅰ+ Ⅱ group and well-differentiated group (χ2=13.010, P=0.001; χ2=6.660, P=0.017). A significant correlation was found between the expression of ANXA1 and PCNA in cholangiocarcinoma (r=0.250, P=0.036). Conclusion ANXA1 and PCNA may be important biomarkers in carcinogenesis and progression of cholangiocarcinoma. Key words: Cholangiocarcinoma; Annexin A1; Proliferation cell nuclear antigen

Abstract 5366: Mutation signature of TP53 gene in H. pylori-associated inflamed gastric mucosa during gastric carcinogenesis

Abstract Helicobacter pylori (H. pylori) infection and the resultant chronic gastric inflammation is an important etiologic factor for gastric cancer development. To explore the genetic basis of gastric cancer that develops in inflamed gastric mucosa, we investigated genetic aberrations that latently accumulate in non-tumorous gastric epithelium with H. pylori infection. Whole exome sequencing on gastric cancer, non-cancerous gastritis tissues and peripheral lymphocytes of 5 patients revealed that somatic mutations accumulated in various genes in inflamed gastric tissues. Additional deep sequencing analyses of selected tumor-related genes in 34 gastritis mucosa samples from patients with, or without, gastric cancer revealed non-synonymous low-abundance mutations in TP53 in 15 cases (44.1%) and ARID1A in 5 cases (14.7%). The mutation signature of whole exome sequences in gastric cancer genomes as well as low-abundance TP53 mutations latently accumulated in H. pylori-inflamed gastritis mucosa were predominantly biased to C:G&amp;gt;T:A transitions in the context of GpCpX motifs, the typical footprints of cytidine deamination triggered by activation-induced cytidine deaminase (AID). In vivo studies with a human TP53 knock-in mouse model revealed that constitutive AID expression in the gastric mucosa caused the emergence of human TP53 mutations at the amino acid positions identical to those detected in human gastric cancers. These findings provide novel evidence that somatic mutations latently accumulated in various genes in inflamed gastric mucosa with H. pylori infection, and cytidine deaminase activity might provide a putative genetic basis for the increased susceptibility to gastric carcinogenesis in patients with H. pylori infection. Citation Format: Takahiro Shimizu, Hiroyuki Marusawa, Tsutomu Chiba. Mutation signature of TP53 gene in H. pylori-associated inflamed gastric mucosa during gastric carcinogenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5366. doi:10.1158/1538-7445.AM2014-5366

CCR6, the sole receptor for the chemokine CCL20, promotes spontaneous intestinal tumorigenesis.

Interactions between the inflammatory chemokine CCL20 and its receptor CCR6 have been associated with colorectal cancer growth and metastasis, however, a causal role for CCL20 signaling through CCR6 in promoting intestinal carcinogenesis has not been demonstrated in vivo. In this study, we aimed to determine the role of CCL20-CCR6 interactions in spontaneous intestinal tumorigenesis. CCR6-deficient mice were crossed with mice heterozygous for a mutation in the adenomatous polyposis coli (APC) gene (APCMIN/+ mice) to generate APCMIN/+ mice with CCR6 knocked out (CCR6KO-APCMIN/+ mice). CCR6KO-APCMIN/+ mice had diminished spontaneous intestinal tumorigenesis. CCR6KO-APCMIN/+ also had normal sized spleens as compared to the enlarged spleens found in APCMIN/+ mice. Decreased macrophage infiltration into intestinal adenomas and non-tumor epithelium was observed in CCR6KO-APCMIN/+ as compared to APCMIN/+ mice. CCL20 signaling through CCR6 caused increased production of CCL20 by colorectal cancer cell lines. Furthermore, CCL20 had a direct mitogenic effect on colorectal cancer cells. Thus, interactions between CCL20 and CCR6 promote intestinal carcinogenesis. Our results suggest that the intestinal tumorigenesis driven by CCL20-CCR6 interactions may be driven by macrophage recruitment into the intestine as well as proliferation of neoplastic epithelial cells. This interaction could be targeted for the treatment or prevention of malignancy.

Expression of CXC chemokine recetper 4 and its clinical significance in cholangiocarcinoma

Objective To investigate the relationship between CXC chemokine recetper 4 (CXCR4) expression to clinicopathological factors and prognosis in cholangiocarcinoma.Methods The expression of CXCR4 in 82 cases of cholangiocarcinoma,7 cases of benign bile duct neoplasms and 12 cases of non-tumor bile duct epithelium was detected by immunohistochemical staining,and that in 30 cases of cholangiocarcinoma by reverse transcriptase-polymerase chain reaction (RT-PCR).Results The positive expression rate of CXCR4 in 82 cases of cholangiocarcinoma,7 cases of benign bile duct neoplasms,and 12 cases of non-tumor bile duct epithelium was 75.6％,28.6％ and 0,respectively.The expression of CXCR4 in cholangiocarcinoma was significantly higher than that in benign bile duct neoplasms and non-tumor bile duct epithelium (P ＜0.01).The positive rate of CXCR4 was slightly higher in the stage Ⅱ-Ⅳ cases than that in the stage Ⅰ cases (22.4％ vs.7.3％,P ＞0.05).RT-PCR results revealed that the expression of CXCR4 mRNA in stage Ⅱ and Ⅲ cases was higher than that in stage Ⅰ cases (0.43 ± 0.07 vs.0.25 ± 0.08,P ＜ 0.05).The expression of CXCR4 mRNA in the cases with lymph node metastasis was significantly higher than that without lymph node metastasis (0.44 ± 0.07 vs.0.23 ± 0.03,P ＜ 0.05).Conclusion CXCR4 may play a positive role in carcinogenesis,invasion and metastasis of cholangiocarcinoma. Key words: Cholangiocarcinoma; CXC chemokine recetper 4 ; Prognosis

Spatial distribution of cancer stem cells in head and neck squamous cell carcinomas

CD44 and aldehyde dehydrogenase 1 (ALDH1) are considered putative markers of highly tumorigenic cells (i.e., cancer stem-like cells) in head and neck squamous cell carcinomas. This small subset of cells is believed to be the primary responsible for tumor initiation and progression. The objectives of this study were (i) to evaluate the patterns of CD44 and ALDH1 expression in the tumor center and in the invasive front, as well as in adjacent non-tumor epithelium, and (ii) to correlate these findings with clinical parameters. The sample comprised 44 patients with primary head and neck squamous cell carcinomas. Hematoxylin and eosin (HE) staining was used for histopathological tumor grading and for morphological analysis of adjacent non-tumor epithelium. Semiquantitative analysis was performed in histological sections immunostained for CD44 and ALDH1. ALDH1 immunostaining in the invasive front showed positive association with tumor size, regional metastasis, tumor histopathological grading, and disease progression. Moreover, expression of this marker in both tumor invasive front and adjacent non-tumor epithelium was related with more aggressive tumors. CD44 immunostaining was heterogeneous in all areas evaluated and did not show association with clinical data. Collectively, these data suggest that ALDH1 immunostaining in the invasive front and in adjacent non-tumor epithelium may help identify tumors with a more aggressive behavior, potentially contributing to improving treatment customization and the monitoring of patients with head and neck cancer.

Asymmetrical proliferative pattern loss during malignant transformation of the oral mucosa

The aim of this study was to study the loss of asymmetrical proliferation in oral tumorigenesis. Samples: 92 oral squamous cell carcinomas (OSCC) with associated non-tumor epithelia (NTE). NTE and tumor were classified as distant from or close to the invasion point. Immunohistochemistry was performed using Mib-1 antibody. Ki-67 was assessed in basal, parabasal layer, medium and upper third, counting total and positive cells. Proliferative patterns were classified according to the ki-67 expression: 1 = expression in parabasal layers of well-differentiated tumor nest (WDTN); 2 = expression in parabasal and basal layers of WDTN; 3 = ki-67 expression in <20% cells in tumor tissue without WDTN; 4 = ki-67 expression in ≥20% of cells in tumor tissue without WDTN; and 5 = ki-67 expression exclusively found in basal layers of WDTN. Ki-67 expression was highest in parabasal layers of both close and distant NTE (39.7 ± 27.6 and 30.1 ± 20) and was also elevated in the close (43.4 ± 21.3) and distant (48.8 ± 21.9) tumor tissue samples. Close tumors largely corresponded to proliferation patterns 2 and 4, while distant tumors generally followed pattern 4. Of the 92 close NTE samples, 23 showed reduced basal proliferation with increased parabasal proliferation. Tumors derived from these epithelia followed patterns 2 (52%, 12/23 cases) or 4 (30.4%, 7/23 cases). Parabasal proliferation in distant NTE was significantly increased in patients with multiple vs. single tumors (36.7% vs. 25.4%; P = 0.032). The change from asymmetrical to symmetrical division appears to be an oncogenic mechanism in oral carcinogenesis.