Paraffin-embedded Colorectal Cancer Tissue Research Articles

High Prevalence of Chromosomal Rearrangements and LINE Retrotranspositions Detected in Formalin-Fixed, Paraffin-Embedded Colorectal Cancer Tissue

Structural variants (SVs) caused by chromosomal rearrangements in common fragile sites (CFS) or LINE retrotranspositions are highly prevalent in colorectal cancer (CRC). However, methodology for targeted detection of these SVs is lacking. We here report the use of formalin-fixed paraffin-embedded targeted locus capture (FFPE-TLC) sequencing as a novel technology for targeted detection of tumor-specific SVs. Analysis of 29 FFPE colorectal tumor and 8 matched normal samples revealed tumor-specific SVs in 24 patients (83%) with a median of 2 SVs per patient (range 1-21). 104 SVs were found in the CFS-associated genes MACROD2, PRKN, FHIT and WWOX in 18 patients (62%) and 39 SVs caused by three LINE transposable elements were found in 15 patients (52%). Tumor-specificity of SVs was independently verified by droplet digital PCR of tumor tissue DNA and their applicability as plasma circulating tumor DNA biomarkers was demonstrated. We conclude that FFPE-TLC sequencing enables the detection of tumor-specific SVs caused by chromosomal rearrangements and LINE retrotranspositions in FFPE tissue. Therefore, FFPE-TLC sequencing facilitates the investigation of the biological and clinical impact of SVs using FFPE material from (retrospective) cohorts of cancer patients and has potential clinical applicability to detect SV biomarkers in the routine molecular diagnostics setting.

PFKFB2 is a favorable prognostic biomarker for colorectal cancer by suppressing metastasis and tumor glycolysis.

This study was to investigate the biological effect of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 2 (PFKFB2) in colorectal cancer (CRC). PFKFB2 was selected by metabolism polymerase chain reaction (PCR) array from CRC cells under alkaline culture medium (pH 7.4) and acidic culture medium (pH 6.8). The expression of PFKFB2 mRNA and protein was detected by quantitative real-time PCR and immunohistochemistry in 70 paired fresh and 268 paired paraffin-embedded human CRC tissues, respectively, and then the prognostic value of PFKFB2 was investigated. The effects of PFKFB2 on CRC cells were also verified in vitro, which were through detecting the change of migration, invasion, sphere formation, proliferation, colony formation, and extracellular acidification rate of CRC cells after PFKFB2 knockdown in alkaline culture medium (pH 7.4) and overexpression in acidic culture medium (pH 6.8). PFKFB2 expression was downregulated in acidic culture medium (pH 6.8). In addition, we found PFKFB2 expression decreased in human CRC tissues compared with the adjacent normal tissues. Furthermore, the OS and DFS rate of CRC patients with low PFKFB2 expression was significantly shorter than those of patients with high PFKFB2 expression. Multivariate analysis indicated that low PFKFB2 expression was an independent prognostic factor for both OS and DFS in CRC patients. Moreover, the abilities of migration, invasion, spheroidizing ability, proliferation, and colony formation of CRC cells were significantly increased after depletion of PFKFB2 in alkaline culture medium (pH 7.4) and decreased after overexpression of PFKFB2 in acidic culture medium (pH 6.8) in vitro. Epithelial-mesenchymal transition (EMT) pathway was found and verified involved in the PFKFB2-mediated regulation of metastatic function in CRC cells. Further, glycolysis of CRC cells was significantly elevated after knockdown of PFKFB2 in alkaline culture medium (pH 7.4) and decreased after overexpression of PFKFB2 in acidic culture medium (pH 6.8). PFKFB2 expression is downregulated in CRC tissues and associated with worse survival for CRC patients. PFKFB2 could inhibit metastasis and the malignant progression of CRC cells by suppressing EMT and glycolysis.

High Wnt2 Expression Confers Poor Prognosis in Colorectal Cancer, and Represents a Novel Therapeutic Target in BRAF-Mutated Colorectal Cancer.

Background and Objectives: We aimed to investigate the role of Wnt2 expression in colorectal cancer (CRC) prognosis and evaluate its potential as a therapeutic target in BRAF-mutated CRC. Materials and Methods: Exactly 136 samples of formalin-fixed paraffin-embedded CRC tissue specimens were obtained from patients who underwent surgical resection for CRC. The gene mutation status of the samples was detected using fluorescence PCR. Wnt2 expression was detected using immunohistochemistry. Survival curves with high Wnt2 expression and BRAF mutations were compared using the Kaplan-Meier method. A nomogram was constructed to determine the estimated overall survival probability. We also predicted the 3-year and 5-year survival rates for patients with high Wnt2 expression and BRAF mutations. In total, 50 samples of BRAF-mutated CRC were collected and detected Wnt2 expression by immunohistochemistry. The Chi-squared test was used to analyze the association between Wnt2 expression and BRAF-mutated CRC. Results: High Wnt2 expression and BRAF mutations are associated with poor prognosis of CRC. Multivariate survival analyses indicated that high Wnt2 expression and BRAF mutations are significant independent predictors of CRC prognosis. Furthermore, high Wnt2 expression was significantly associated with BRAF-mutated CRC, and Wnt2 may be a potential therapeutic target for BRAF-mutated CRC. Conclusions: High Wnt2 expression confers poor prognosis in colorectal cancer and represents a novel therapeutic target in BRAF-mutated CRC.

Development of a Novel Anti-CD44 Variant 6 Monoclonal Antibody C44Mab-9 for Multiple Applications against Colorectal Carcinomas.

CD44 is a cell surface glycoprotein, and its isoforms are produced by the alternative splicing with the standard and variant exons. The CD44 variant exon-containing isoforms (CD44v) are overexpressed in carcinomas. CD44v6 is one of the CD44v, and its overexpression predicts poor prognosis in colorectal cancer (CRC) patients. CD44v6 plays critical roles in CRC adhesion, proliferation, stemness, invasiveness, and chemoresistance. Therefore, CD44v6 is a promising target for cancer diagnosis and therapy for CRC. In this study, we established anti-CD44 monoclonal antibodies (mAbs) by immunizing mice with CD44v3-10-overexpressed Chinese hamster ovary (CHO)-K1 cells. We then characterized them using enzyme-linked immunosorbent assay, flow cytometry, western blotting, and immunohistochemistry. One of the established clones (C44Mab-9; IgG1, kappa) reacted with a peptide of the variant 6-encoded region, indicating that C44Mab-9 recognizes CD44v6. Furthermore, C44Mab-9 reacted with CHO/CD44v3-10 cells or CRC cell lines (COLO201 and COLO205) by flow cytometry. The apparent dissociation constant (KD) of C44Mab-9 for CHO/CD44v3-10, COLO201, and COLO205 was 8.1 × 10-9 M, 1.7 × 10-8 M, and 2.3 × 10-8 M, respectively. C44Mab-9 detected the CD44v3-10 in western blotting, and partially stained the formalin-fixed paraffin-embedded CRC tissues in immunohistochemistry. Collectively, C44Mab-9 is useful for detecting CD44v6 in various applications.

Abstract 1960: Invasive potential of colorectal cancer through tumor budding in 3D

Abstract Background and Objectives: Tumor budding (TB) is defined as isolated single cancer cells or clusters of up to four cells located at the invasive tumor front of colorectal cancer (CRC) and other solid cancers. Despite the growing body of evidence establishing TB as a strong independent prognostic marker in CRC, further investigation is needed to elucidate the biological mechanisms behind TB and its invasive potential. Pseudobudding (PsB), in contrast to TB, is defined as single cancer cells or clusters of up to four cells that occur due to gland disruption and external influences such as inflammation. We hypothesize that the invasive potential of a tumor is therefore better reflected by the active process of TB rather than through the reactive process of PsB. By improved visualization of the invasive tumor front, we can characterize TB and PsB, elucidate their underlying biological processes, and improve future TB scoring accuracy in routine diagnostic practice. Methods: Cases containing TB and PsB were selected from the pathology archives. Thick sections (80-90µm) of formalin-fixed paraffin-embedded CRC tissue were taken using a microtome and stained using a free-floating immunostaining protocol for Pan-cytokeratin (epithelium) and DAPI (nuclei). Thick sections were imaged via confocal microscopy. Three-dimensional z-stacks were processed and analyzed using FIJI software. Tissue microarrays (TMAs) of regions with TB and PsB were created and RNA spatial transcriptomics was conducted on thin TMA sections (5µm) using the Nanostring GeoMx Digital Spatial Profiling Platform. Results: For sections where the invasive margin contained TB, both single cells and cell clusters appear isolated from the core tumor in 3D. These single cells and cell clusters exhibit migratory and invasive features associated with epithelial-mesenchymal transition (EMT), as evident by complete loss of cell-to-cell adhesions and shape shifting through elongation of the entire cell. In contrast, PsB is characterized by cellular debris surrounding a discontinuous epithelial lining. Undivided cells and cell clusters within the damaged area are observed as involuntarily disassociated with lagging remnants of cell-to-cell adhesions. This may be due to inflammatory cells disrupting the extracellular matrix and pulling the cells apart from one another. The identification of differing morphologies allows for further study of the RNA spatial transcriptome (of which the data will be presented during the meeting). Conclusion: Our findings show that TB and PsB are independent features within the invasive tumor margin which represent two separate biological processes. Considering the strong predictive capacity of TB as a marker of poor prognosis in CRC, TB scoring within routine diagnostic practice benefits from avoiding areas with a strong inflammatory reaction and PsB. Citation Format: Tariq Haddad, Luuk van den Dobbelsteen, Alessandro Lugli, Shannon van Lent-van Vliet, Femke Simmer, Inti Zlobec, Iris Nagtegaal. Invasive potential of colorectal cancer through tumor budding in 3D [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1960.

Evaluating the utility of tumour mutational signatures for identifying hereditary colorectal cancer and polyposis syndrome carriers

ObjectiveGermline pathogenic variants (PVs) in the DNA mismatch repair (MMR) genes and in the base excision repair gene MUTYH underlie hereditary colorectal cancer (CRC) and polyposis syndromes. We evaluated the...

Loss of CDX-2 expression is an independent poor prognostic biomarker in patients with early-stage deficient mismatch repair colorectal cancer.

To investigate the clinicopathological factors, molecular features, and prognostic implications associated with loss of Caudal-related homeobox transcription factor 2 (CDX-2) expression in colorectal cancer (CRC) patients. Immunohistochemistry for CDX-2 expression was performed on formalin-fixed, paraffin-embedded primary CRC tissue samples from 449 patients. Correlation between CDX-2 expression and clinicopathological and molecular characteristics was evaluated. Univariate and multivariate survival analyses were performed to determine the prognostic value of loss of CDX-2 expression. Of 449 patients, 84% were stage I-III. CDX-2-negative expression was identified in 18 of 441 (4.1%) patients. Loss of CDX-2 expression was more commonly found in patients with right-sided tumors rather than left-sided tumors (odds ratio [OR] = 3.57; p=0.009), deficient mismatch repair (dMMR) compared to proficient MMR (pMMR) (OR = 3.7; p=0.012), and BRAF mutation compared to BRAF wild type (OR = 8.06; p=0.002). Univariate analysis revealed that stage I-III CRC patients with loss of CDX-2 expression had significantly worse overall survival (OS) and disease-free survival (DFS) than those with positive CDX-2 expression (5-year OS = 33.3% vs. 74.6%, respectively; p<0.001, and 5-year DFS: 42.9% vs. 69.5%, respectively; p=0.004). Loss of CDX-2 expression remained significantly associated with worse OS compared to positive CDX-2 expression in multivariate analysis (hazard ratio [HR] = 2.4; 95% confidence interval [CI], 1.12-5.11; p=0.023). Loss of CDX-2 expression was found to be associated with right-sided tumor, dMMR status, and BRAF mutation. Moreover, loss of CDX-2 expression is a poor prognostic factor for OS in stage I-III, even among patients with dMMR tumors.

TNFRSF11B Remodel Immune Response in Colon Cancer Microenvironment: A Multi-Center Study

Background: Colorectal cancer is the lethal cancer in the world, due to the low tumor mutation burden and low proportion of tumor-infiltrating lymphocytes in the microenvironment of most patients, innovative immunotherapeutic approaches need to be identified. Methods: Using TCGA-COAD dataset (n = 514), we identified TNFRSF11B as a prognostic factor of colon cancer. Immunohistochemistry (IHC) dataset (n = 86), 290 single colorectal cancer cells (GSE81861) and 31 paired colon cancer transcriptional dataset were further applied to validate the function of TNFRSF11B, which was confirmed via fluorescence-activated cell sorting (FACS) analysis. Results: A risk scores system consisting of eight IRGs was constructed to predict the prognosis of colon cancer patients. Only TNFRSF11B correlated with TNM-stage, lymph node metastasis and prognosis. In our IHC dataset, High TNFRSF11B expression had a later TNM-stage, higher frequency of lymph node and lymphovascular invasion. Six genes (KRT18, ARPC5L, ACTG1, ARPC2, EZR, YWHAZ) related to pathogenic E. coli. infection were simultaneously increased with TNFRSF11B overexpression via GESA analysis. Finally, only activated memory CD4+ T cells (p =0.017) were significantly decreased in high TNFRSF11B expression group, which was supported by our FACS findings that TNFRSF11B downregulate the immune activity of central memory CD4 + T cells and effector memory CD4+ T cells (all p <0.001). Conclusion: TNFRSF11B, acts as a prognostic factor for colon cancer patient, could affect colon cancer immune response. TNFRSF11B was closely related with lymph node invasion and pathogenic E. coli. infection, which might negatively affect memory activated CD4+ T cell infiltration in colon cancer. Funding: This work was supported Joint Funds for the innovation of science and Technology, Fujian province (Grant number: 2018Y9203 to Jun-rong Zhang, 2018Y92042 to Xian-qiang Chen); Startup Fund for scientific research, Fujian Medical University (Grant number: 2019QH1016 to Ping Hou); Education Department of Fujian Province Young and middle-aged teacher education research project (Grant number: JAT190180 to Ping Hou, JT180181 to Jun-rong Zhang). Declaration of Interest: The authors declare that they have no competing interests. Ethical Approval: The validation of TNFRSF11B in paraffin-embedded colorectal cancer tissues were supported by general surgery of Fujian Medical University Union Hospital from January 2013 to December 2017. The study protocol was approved by the Institutional Review Board of Fujian Medical University Union Hospital, and all patients provided written informed consent.

Syntaxin 2 promotes colorectal cancer growth by increasing the secretion of exosomes.

Background: Colorectal cancer (CRC) is one of the most common cancers with high mortality worldwide. Uncontrolled growth is an important hallmark of CRC. However, the mechanisms are poorly understood. Methods: Syntaxin 2 (STX2) expression was analyzed in 160 cases of paraffin-embedded CRC tissue by immunohistochemistry, in 10 cases of fresh CRC tissue by western blot, and in 2 public databases. Gain- and loss-of-function analyses were used to investigate the biological function of STX2 in CRC growth. Exosomes isolation, characterization, Co-immunoprecipitation (Co-IP), flow cytometry and fluorescence were conducted to study the molecular mechanism of STX2 in CRC growth. Results: The expression of STX2 was obviously up-regulated in human CRC tissues. Overexpression of STX2 increased the growth of CRC cells in vitro and in vivo. Downregulation of STX2 repressed the growth of CRC. STX2 modulated exosomes secretion of CRC cells which might correlated with Rab8a expression. The secreted exosomes could be ingested by CRC cells, and ultimately promoted the growth of CRC by arresting the tumor cells at S phase. Conclusions: Our data provide evidence that STX2 promotes CRC growth by increasing exosomes secretion of CRC cells; And the modulation of STX2 in exosomes secretion correlates with Rab8a. Thus, our study identified a new mechanism of STX2 in CRC growth and may provide a possible strategy for CRC therapy.

Clinical and Prognostic Relevance of B7-H3 and Indicators of Glucose Metabolism in Colorectal Cancer.

ObjectiveThis study aimed to investigate the clinical and prognostic relevance of B7-H3 expression and indicators of glucose metabolism in patients with colorectal cancer (CRC).MethodsUsing immunohistochemistry, the expression of B7-H3 was detected in a total of 213 formalin-fixed paraffin-embedded CRC tissue specimens. Furthermore, levels of fasting blood glucose (FBG), lactic dehydrogenase (LDH), and fructosamine (FMN) as indicators of glucose metabolism were analyzed in CRC patients and stratified into high or low expression sub-groups based on Youden Index. The relationship between B7-H3, FBG, LDH, FMN expression, and clinicopathological characteristics were also evaluated to establish their prognostic significance in patients with CRC.ResultsB7-H3 was highly expressed in CRC tissue. The positive rates of B7-H3 expression was 63.8% (136/213). We found a linear correlation between B7-H3 and FBG in depth of tumor invasion (T3/4) (p = 0.037, r = 0.259), lymph node metastasis (N0) (p = 0.004, r = 0.259), and TNM stage (I/II) (p = 0.009, r = 0.242). High expression of FBG, LDH, FMN [hazard ratio (HR) = 1.916, 95% CI: 1.223–3.00, p = 0.005; HR = 1.801, 95% CI: 1.153–2.813, p = 0.010; HR = 2.154, 95% CI: 1.336–3.472, p = 0.002], respectively, was identified as a significant independent predictor of poor overall survival (OS). Although B7-H3 expression did not affect OS, CRC patients expressing both high B7-H3 and high FMN contributed to a significant decrease in OS (HR = 1.881, 95%CI: 1.059–3.339, p = 0.031). Moreover, with low expression of B7-H3, high expression of FBG, LDH and FMN were also recognized as predictors of inferior OS (HR = 3.393, 95% CI: 1.493-7.709, p = 0.004; HR = 7.107, 95% CI: 2.785–18.138, p = 0.000; HR = 2.800, 95% CI: 1.184–6.625, p = 0.019).ConclusionB7-H3 combined with FBG, LDH, or FMN, could reflect the clinical outcomes of patients with CRC.

Detection of the BRAF V600E Mutation in Colorectal Cancer by NIR Spectroscopy in Conjunction with Counter Propagation Artificial Neural Network.

This paper proposes a sensitive, sample preparation-free, rapid, and low-cost method for the detection of the B-rapidly accelerated fibrosarcoma (BRAF) gene mutation involving a substitution of valine to glutamic acid at codon 600 (V600E) in colorectal cancer (CRC) by near-infrared (NIR) spectroscopy in conjunction with counter propagation artificial neural network (CP-ANN). The NIR spectral data from 104 paraffin-embedded CRC tissue samples consisting of an equal number of the BRAF V600E mutant and wild-type ones calibrated and validated the CP-ANN model. As a result, the CP-ANN model had the classification accuracy of calibration (CAC) 98.0%, cross-validation (CACV) 95.0% and validation (CAV) 94.4%. When used to detect the BRAF V600E mutation in CRC, the model showed a diagnostic sensitivity of 100.0%, a diagnostic specificity of 87.5%, and a diagnostic accuracy of 93.8%. Moreover, this method was proven to distinguish the BRAF V600E mutant from the wild type based on intrinsic differences by using a total of 312 CRC tissue samples paraffin-embedded, deparaffinized, and stained. The novel method can be used for the auxiliary diagnosis of the BRAF V600E mutation in CRC. This work can expand the application of NIR spectroscopy in the auxiliary diagnosis of gene mutation in human cancer.

Overexpression of Arginase-1 is an indicator of poor prognosis in patients with colorectal cancer

AimArginase-1 (Arg-1) metabolizes l-arginine to l-ornithine and urea. It has been documented to have a role in various malignancies. However, the relationship between Arg-1 expression and clinicopathological characteristics of colorectal cancer (CRC) patients remains to be elucidated. The present study aimed to analyze the expression and prognostic value of Arg-1 in patients with CRC. Material and methodsThe mRNA and protein expressions of Arg-1 in fresh colorectal cancer tissue specimens and the corresponding noncancerous tissue specimens were examined by RT-qPCR (n = 24) and western blot analysis (n = 17). Arg-1 expression levels were determined in paraffin-embedded CRC tissue specimens (n = 236) by immunohistochemistry. The associations of Arg-1 expression and clinicopathological features and clinical prognosis in 236 CRC patients were analyzed. ResultsThe expression levels of Arg-1 were significantly higher in the CRC tissues compared with the matched noncancerous tissues, and elevated Arg-1 expression was remarkably associated with stage III-IV tumors (P = 0.007), lymph node metastasis (P = 0.019) and a plasma albumin concentration <35 g/l (P = 0.022). Kaplan-Meier analysis indicated that Arg-1 overexpression was associated with adverse prognoses for overall survival (OS) (P < 0.001) and disease-free survival (DFS) (P < 0.001) in all cases. Further analysis revealed that the patients with high Arg-1 expression had significantly shorter OS and DFS at the advanced stages (III + IV) (P = 0.032 for OS, and P = 0.012 for DFS) but not at the early stages (I + II) (P = 0.194 for OS, and P = 0.065 for DFS). Multivariate analysis revealed that Arg-1 overexpression was an independent prognostic factor for OS (P = 0.002) and DFS (P < 0.001) in patients with CRC. ConclusionThe data indicated that Arg-1 overexpression in CRC may be a marker that can discriminate subgroups of patients with a poor prognosis.

MALAT1 sponges miR-106b-5p to promote the invasion and metastasis of colorectal cancer via SLAIN2 enhanced microtubules mobility.

BackgroundThe low expression of miR93/25 (members of miR-106b~25 cluster) promoted the invasion and metastasis of colon cancer cells, which predicted poor survival. However, the role of miR-106b-5p, the member of miR-106b~25 cluster, in colorectal cancer (CRC) remains unclear.MethodsBioinformatics methods were used to predict the potential pairs of lncRNA-miRNA-mRNA. In situ hybridization and qPCR were used to evaluate the expression of MALAT1 and miR-106b-5p in the paraffin-embedded normal and CRC tissues. Kaplan–Meier analysis with the log-rank test was used for survival analyses. Immunohistochemistry staining was applied to investigate the expression of SLAIN2. Fluorescence recovery after photobleaching assay was applied to observe the microtubule (MT) mobility. In vitro and in vivo invasion and metastasis assays were used to explore the function of MALAT1/miR-106b-5p/SLAIN2 in the progression of CRC.FindingsmiR-106b-5p was identified as a suppressor in CRC. Functionally, ectopic or silencing the expression of miR-106b-5p inhibited or promoted the invasion and metastasis of CRC cells in vitro and in vivo. The long non-coding RNA MALAT1 regulated the miR-106b-5p expression and further mediated the mobility of SLAIN2-related MTs by functioning as a competing endogenous RNA in vitro and in vivo, which resulted in the progression of CRC. Clinically, low miR-106b-5p expression predicted poor survival of CRC patients, especially in combination with high MALAT1/ SLAIN2 expression.InterpretationmiR-106b-5p served as a suppressor in combination with MALAT1/miR-106b-5p/SLAIN2, which might be a group of potential prognostic biomarkers in the prognosis of CRC.FundThis work was supported by National Program Project for Precision Medicine in National Research and Development Plan of China (2016YFC0905300), National Natural Science Foundation of China (81572930), National Key Research and Development Program of the Ministry of Science and Technology of China (2016YFC0905303, 2016YFC1303200), Beijing Science and Technology Program (D17110002617004), Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences (2018PT32012), CAMS Innovation Fund for Medical Sciences (CIFMS) (2016-I2M-1-001), Incentive Fund for Academic Leaders of Oncology Hospital, Chinese Academy of Medical Sciences (RC2016003), and Beijing Hope Run Special Fund from Cancer Foundation of China (LC2017A19). The project of Shanghai Jiaotong Univversity (YG2017QN30).

Loss of claudin-1 expression induces epithelial-mesenchymal transition through nuclear factor-κB activation in colorectal cancer

ObjectiveThe aim of this study was to elucidate the clinicopathological significance and prognostic role of loss of claudin-1 in colorectal cancer (CRC). MethodsThe correlations between claudin-1 expression and clinicopathological characteristics, including survival rates, were assessed using immunohistochemistry on 260 archival, paraffin-embedded CRC tissues. In addition, the correlations between cludin-1 and nuclear factor-kappa B (NF-κB), epithelial-mesenchymal transition markers and tumor-infiltrating lymphocytes were investigated. ResultsClaudin-1 expression was markedly lost in 42.7% of the 260 CRCs analyzed. Loss of claudin-1 expression significantly correlated with larger tumor size, vascular invasion, higher pT stage, and high metastatic lymph node ratio. In addition, loss of claudin-1 expression significantly correlated with NF-κB activation (P < 0.001), high SNAI (P < 0.001), and low E-cadherin (P < 0.001) expressions. Patients with high immunoscores showed significantly lower rates of claudin-1 expression loss (P = 0.020). In detail, loss of claudin-1 expression were frequently found in CRCs low CD3- and CD8-positive lymphocytes. There were significant correlations between claudin-1 expression loss and poor overall and recurrence-free survivals (P < 0.001 and P < 0.001, respectively). ConclusionTaken together, our results suggest that the loss of claudin-1 expression significantly correlates with aggressive tumor behaviors, high SNAI expression, lower immunoscore, and poor prognoses.

SFRP2 Promoter Methylation Analysis in Tumor Tissue, Stool, and Plasma DNA of Patients with Colorectal Cancer

Noninvasive method for early detection of human cancers is promising way to provide more quality health service to the population. The serum and stool of colorectal cancer patients often harbor increased free DNA levels, which can potentially be used for cancer detection. Because SFRP2 (secreted frizzled related protein 2) is considered a Wnt inhibitor whose CpGs were frequently hypermethylated in several human cancers, we investigated the frequency of aberrant SFRP2 promoter methylation in primary tumors and serum or stool samples of colorectal cancer patients by MS-PCR. We detected methylation of SFRP2 in 111 of 117 (94.9%) paraffin-embedded colorectal cancer tissues. Fifty-nine of the 68 (86.8%) available serum samples from these cases carried detectable amounts of the methylated SFRP2 promoter. Sixty-one of 68 (89.7%) available stool samples from the same cases carried positive methylation in the promoter region of SFRP2 gene. In contrast, no methylated SFRP2 promoter DNA was detected in serum and stool samples from 60 healthy controls. Only 2 cases of 68 healthy controls showed methylated SFRP2 promoter DNA in their colorectal tissue. But high frequency of methylated SFRP2 gene promoter has no correlation with the clinic-pathologic features. We concluded that SFRP2 methylation analysis appeared to be a noninvasive tumor marker in serum and/or stool DNA

Loss of Yes-associated Protein Represents an Aggressive Subtype of Colorectal Cancer.

Background: Yes-associated protein (YAP) is a downstream effecter of Hippo signaling pathway, and has been linked to the initiation and development of colorectal cancer (CRC). However, the clinical significance of YAP in CRC remains controversial. This study was designed to investigate the clinical significance of YAP in CRC.Methods: We selected 206 eligible patients diagnosed with CRC from 2003 to 2007. Tissue microarray (TMA) blocks were made from 206 formalin-fixed paraffin-embedded CRC tissues and 158 corresponding normal colonic tissues. Using the TMA blocks, we performed immunohistochemical staining of YAP and assessed its expression status in different subcellular locations. The patients were divided into four groups according to the expression status of YAP in the cytoplasm and nucleus. Statistical analysis was performed to explore the correlation between YAP expression and clinicopathological features and overall survival (OS) in CRC patients.Results: Our results showed that both cytoplasmic YAP and nuclear YAP were overexpressed in CRC tissues compared to normal colonic tissues. Complete loss of YAP expression in CRC was significantly correlated with larger tumor size (p=0.023), proximal tumor location (p=0.038), higher tumor grade (p=0.022) and worse OS (p<0.001). Univariate and multivariate Cox regression analyses revealed that complete loss of YAP expression was an independent indicator of poor prognosis in CRC (p<0.001).Conclusions: Loss of YAP expression correlates with poor prognosis and may represent a subgroup with more aggressive biological features in CRC.

Rapid Somatic Mutation Testing in Colorectal Cancer by Use of a Fully Automated System and Single-Use Cartridge: A Comparison with Next-Generation Sequencing.

Molecular tests have been increasingly used in the management of various cancers as more targeted therapies are becoming available as treatment options. The Idylla™ system is a fully integrated, cartridge-based platform that provides automated sample processing (deparaffinization, tissue digestion, and DNA extraction) and real-time PCR-based mutation detection with all reagents included in a single-use cartridge. This retrospective study aimed at evaluating both the Idylla KRAS and NRAS-BRAF-EGFR492 Mutation Assay cartridges (research use only) against next-generation sequencing (NGS) by using colorectal cancer (CRC) tissue samples. Forty-four archived formalin-fixed paraffin-embedded (FFPE) CRC tissue samples previously analyzed by targeted NGS were tested on the Idylla system. Among these samples, 17 had a mutation in KRAS proto-oncogene, GTPase (KRAS), 5 in NRAS proto-oncogene, GTPase (NRAS), and 12 in B-Raf proto-oncogene, serine/threonine kinase (BRAF) as determined using the Ion AmpliSeq 50-gene Cancer Hotspot Panel v2. The remaining 10 samples were wild-type for KRAS, NRAS, and BRAF. Two 10-μm FFPE tissue sections were used for each Idylla run, 1 for the KRAS cartridge, and 1 for the NRAS-BRAF-EGFR492 cartridge. All cases met the Idylla minimum tumor content requirement for KRAS, NRAS, and BRAF (≥10%). Assay reproducibility was evaluated by testing commercial controls derived from human cell lines, which had an allelic frequency of 50% and were run in triplicate. The Idylla system successfully detected all mutations previously identified by NGS in KRAS (G12C, G12D, G12V, G13D, Q61K, Q61R, A146T), NRAS (G12V, G13R, Q61H), and BRAF (V600E). Compared with NGS, Idylla had a sensitivity of 100%. Analysis of the mutated commercial controls demonstrated agreement with the expected result for all samples and 100% reproducibility. The Idylla system produced results quickly with a turnaround time of approximately 2 h. The Idylla system offers reliable and sensitive testing of clinically actionable mutations in KRAS, NRAS, and BRAF directly from FFPE tissue sections.

Development of ultra-short PCR assay to reveal BRAF V600 mutation status in Thai colorectal cancer tissues.

The protein kinase BRAF is one of the key players in regulating cellular responses to extracellular signals. Somatic mutations of the BRAF gene, causing constitutive activation of BRAF, have been found in various types of human cancers such as malignant melanoma, and colorectal cancer. BRAF V600E and V600K, most commonly observed mutations in these cancers, may predict response to targeted therapies. Many techniques suffer from a lack of diagnostic sensitivity in mutation analysis in clinical samples with a low cancer cell percentage or poor-quality fragmented DNA. Here we present allele-specific real-time PCR assay for amplifying 35- to 45-base target sequences in BRAF gene. Forward primer designed for BRAF V600E detection is capable of recognizing both types of BRAF V600E mutation, i.e. V600E1 (c.1799T>A) and V600E2 (c.1799_1800delTGinsAA), as well as complex tandem mutation caused by nucleotide changes in codons 600 and 601. We utilized this assay to analyze Thai formalin-fixed paraffin-embedded tissues. Forty-eight percent of 178 Thai colorectal cancer tissues has KRAS mutation detected by highly sensitive commercial assays. Although these DNA samples contain low overall yield of amplifiable DNA, our newly-developed assay successfully revealed BRAF V600 mutations in 6 of 93 formalin-fixed paraffin-embedded colorectal cancer tissues which KRAS mutation was not detected. Ultra-short PCR assay with forward mutation-specific primers is potentially useful to detect BRAF V600 mutations in highly fragmented DNA specimens from cancer patients.

Aberrant expression of stress-induced phosphoprotein 1 in colorectal cancer and its clinicopathologic significance

Stress-induced phosphoprotein 1 (STIP1) is an adaptor protein that bridges HSP70 and HSP90 folding and a secretory protein that regulates malignant tumor progression. The aim of the present study was to demonstrate the clinicopathological significance and prognostic role of STIP1 in colorectal cancer (CRC). We used data from The Cancer Genome Atlas (TCGA) to analyze STIP1 expression in CRC and utilized 8 pairs of fresh-frozen tissue samples to investigate STIP1 expression in CRC tissues and adjacent normal tissues using quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot assays. We also used immunohistochemical staining to detect STIP1 expression in 144 formalin-fixed, paraffin-embedded (FFPE) CRC tissue samples and determine the clinical significance of STIP1 expression in CRC. The results of bioinformatics analysis, qRT-PCR, and Western blot showed that STIP1 expression was higher in CRC tissues than in adjacent normal tissues. High STIP1 expression was significantly correlated with advanced T stage (P = .01), N stage (P = .001), M stage (P < .001), and TNM stage (P < .001). Moreover, Kaplan-Meier analyses indicated that higher STIP1 expression predicted a worse prognosis in patients with CRC, and Cox regression analysis revealed that STIP1 was an independent prognostic factor for overall survival and disease-free survival in patients with CRC. In conclusion, our results suggest that STIP1 acts as an oncogene in CRC and can therefore serve as a biomarker for the prognosis of patients with CRC.

TPL2 expression is correlated with distant metastasis and poor prognosis in colorectal cancer

Tumor progression locus 2 (TPL2) is a mitogen-activated protein kinase (MAPK) kinase kinase (MAP3K). The present study aimed to elucidate the clinicopathological significance and prognostic role of TPL2 expression in colorectal cancer (CRC) through immunohistochemistry. In the present study, the correlations between TPL2 expression and clinicopathological parameters, including survival rate, were investigated using 262 archival paraffin-embedded CRC tissue samples. In addition, the correlation between TPL2 expression and tumor-infiltrating lymphocytes was evaluated using immunoscore. High TPL2 expression was found in 40.1% of the 262 CRCs analyzed. Patients with high TPL2 expression had frequent distant metastasis compared to patients with low TPL2 expression. However, there was no significant correlation between high TPL2 expression and other clinicopathological parameters. High TPL2 expression was significantly correlated with low immunoscore. In subgroup analysis based on distant metastasis, there was a significant correlation between high TPL2 expression and low immunoscore in only CRCs without distant metastasis. High TPL2 expression significantly correlated with poor overall survival. In both CRCs with and without distant metastasis, CRCs with high TPL2 expression showed worse prognosis compared to CRCs with low TPL2 expression (P = .034 and P = .010, respectively). Taken together, our results showed that high TPL2 expression was significantly correlated with distant metastasis and low immunoscore. In addition, TPL2 expression can be useful for predicting the prognosis of patients with CRC.