Primary Colorectal Cancer Lesions Research Articles

Correlation Between DCAMKL-1 Protein Expression and K-ras Gene Mutation in Colorectal Cancer.

To investigate the correlation between doublecortin and CaM kinase-like-1 (DCAMKL-1) protein expression, K-ras gene mutation, and their impact on patient prognosis in colorectal cancer (CRC). Immunohistochemistry was used to detect the expression of DCAMKL-1 protein in 60 cases of colorectal adenoma, 82 cases of CRC (including 65 cases of lymph node metastasis) and paraffin-embedded paracancerous intestinal mucosal tissue. K-ras gene mutations in primary CRC lesions were detected using an amplification-refractory mutation system and fluorescent polymerase chain reaction. The relationship between DCAMKL-1 protein expression and K-ras gene mutations with the clinicopathological characteristics of patients with CRC was analyzed. Univariate Kaplan‒Meier survival analysis and multivariate Cox regression analysis were performed using follow-up data. The mutation rate of the K-ras gene in 82 cases of CRC was 48.8% (40/82). The positivity rate for the presence of DCAMKL-1 protein in CRC was 70.7% (58/82), significantly higher than that for colorectal adenomas (53.3%; 32/60) and paracancerous intestinal mucosa (0%; 0/82) (P<0.05). The positive expression rate for the presence of DCAMKL-1 protein in 65 patients with lymph node metastasis was higher in the primary lesions (69.2%; 45/65) than in the lymph node metastases (52.3%; 34/65) (χ2=12.087, P=0.001). The K-ras gene mutation status was positively correlated with DCAMKL-1 protein expression (r=0.252, P=0.022). In this study, a potential positive correlation between K-ras gene mutation and DCAMKL-1 protein expression was identified in CRC tissues. The assessment of K-ras gene mutation status and DCAMKL-1 protein expression holds promise for augmenting early diagnosis and prognosis evaluation in CRC. This approach may improve the overall prognosis and survival outcomes for CRC patients.

Diagnostic value of [68Ga]Ga-FAPI-04 in patients with colorectal cancer in comparison with [18F]F-FDG PET/CT.

This study aimed to compare the diagnostic performance of [68Ga]Ga-FAPI-04 PET/CT and [18F]F-FDG PET/CT in primary and metastatic colorectal cancer (CRC) lesions. This single-center preliminary clinical study (NCT04750772) was conducted at the Peking University Cancer Hospital & Institute and included 61 participants with CRC who underwent sequential evaluation through PET/CT with [18F]F-FDG and [68Ga]Ga-FAPI-04. Their PET/CT images were analysed to quantify the uptake of the two tracers in the form of maximum standardised uptake (SUVmax) values and target-to-background ratio (TBR), which were then compared using Wilcoxon's signed-rank test. The final changes in the tumour-node-metastasis (TNM) stage of all participants were recorded. Of all the participants, 21 were treatment naïve and 40 had been previously treated. In primary CRC lesions, the average TBRs of [68Ga]Ga-FAPI-04 and [18F]F-FDG were 13.3 ± 8.9 and 8.2 ± 6.5, respectively. The SUVmax of [68Ga]Ga-FAPI-04 in signet-ring/mucinous carcinomas (11.4 ± 4.9) was higher than that of [18F]F-FDG (7.9 ± 3.6) (P = 0.03). Both median SUVmax in peritoneal metastases and TBR in liver metastases of [68Ga]Ga-FAPI-04 were higher than those of [18F]F-FDG (5.2 vs. 3.8, P < 0.001; 3.7 vs. 1.9, P < 0.001, respectively). Compared with [18F]F-FDG PET/CT, clinical TNM staging based on [68Ga]Ga-FAPI-04 PET/CT led to upstaging and downstaging in 10 (16.4%) and 5 participants (8.2%), respectively. Therefore, the treatment options were changed in 13 participants (21.3%), including 9 with additional chemo/radiotherapy and/or surgery and others with avoidance or narrowed scope of surgery. [68Ga]Ga-FAPI-04 showed potential as a novel PET/CT tracer to detect lymph nodes and distant metastases, which improved CRC staging, thus prompting the optimisation or adjustment of treatment decisions.

(Pro)renin receptor promotes colorectal cancer progression through inhibiting the NEDD4L-mediated Wnt3 ubiquitination and modulating gut microbiota

BackgroundWe previously found that (pro)renin receptor ((P)RR) augments Wnt3 protein without affecting Wnt3 gene transcription in colorectal cancer (CRC) cells, thus contributes to CRC initiation. The present study aims to investigate whether (P)RR further promotes CRC progression following oncogenesis and the related mechanisms. Notably, we deeply elaborate how (P)RR affects Wnt3 protein level and the key enzyme that mediates this process.MethodsImmunohistochemistry, western blotting and immunofluorescence were performed to detect protein expression status. A kind of gastrointestinal epithelium-specific ATP6AP2 ((P)RR encoding gene) knock-in mice were generated using Crispr/Cas9 system.ResultsWe found that increased (P)RR expression in primary CRC lesions is positively associated with higher Wnt3 protein level and disease progression. Progressive CRC presents less colocalization of Wnt3 and an E3 ubiquitin ligase NEDD4L in primary lesions than non-progressive CRC. In colon cancer cells, (P)RR dramatically inhibits the NEDD4L-mediated Wnt3 protein ubiquitination. ATP6AP2 knock-in mice show more diminished Wnt3-NEDD4L colocalization in their gut epithelium in comparison to wildtype mice. They also have abnormal gut bacterial flora distribution. Especially, Lachnospiraceae_NK4A136 and Bacteroides genus, which are generally protective against CRC, are suppressed in guts of ATP6AP2 knock-in mice.ConclusionsCollectively, (P)RR promotes CRC progression through inhibiting the NEDD4L-mediated Wnt3 ubiquitination and modulating gut microbiota.5Fh7m3ebJHKpa3LjUnmMxGVideo

Hypoxia-Inducible Exosomes Facilitate Liver-Tropic Premetastatic Niche in Colorectal Cancer.

Liver metastasis is a frequent occurrence in patients with colorectal cancer (CRC), with 15%-25% of CRC patients having liver metastases at the time of initial diagnosis. Specifically, some regional-stage patients with mild symptoms (stage 1 or 2) will also advance to liver metastases rapidly, even if the CRC lesion in situ is resected in time. Nevertheless, the precisemechanism of liver metastasis isstill unclear. Fresh tumor tissues from patients with CRC, adjacent noncancerous tissues, and colorectal adenoma tissues were subjected to microarray analysis to identify differentially expressed microRNA. Exosomes from human serum and cell culture medium were separated, quantitated, and verified by transmission electronic microscopy and Zetasizer Nano. Luciferase reporter assay, real-time quantitative PCR, western blot, immunoprecipitation, chromatin and re-chromatin immunoprecipitation, migration and invasion assay, PDX mouse model, flow cytometry, immunohistochemistry, and immunofluorescence staining were employed to explore the regulation among CRC liver metastases, immunosuppression, and cell adhesion. In this study, we demonstrated that the hypoxic microenvironment in primary CRC lesions boosted exosome release, selectively initiated favorable premetastatic niche formation in the liver but not in other organs. Mechanistically, Kupffer cells (KCs) can phagocytose exosomes containing highly expressed miR-135a-5p from the blood circulation into the liver. Exosomal miR-135a-5p initiated the large tumor suppressor kinase 2-yes-associated protein-matrix metalloproteinase 7 axis to promote the occurrence of CRC liver metastasis, and cluster of differentiation 30-TNF receptor-associated factor 2-p65-mediated immunosuppression signaling also contributed to this process. Hypoxia-induced exosomal miR-135a-5p correlates with the development, clinical severity, and prognosis of CRC liver metastases through the premetastatic niche; and our findings revealed that miR-135a-5p might be a promising target in halting CRC liver metastases.

Overexpression of B7-H4 is associated with infiltrating immune cells and poor prognosis in metastatic colorectal cancer

Metastasis commonly occurs in colorectal cancer (CRC) patients and confers a poor prognosis. B7-H4, an immune checkpoint molecule, has been found to be expressed in numerous tumor tissues and play critical roles in tumor progression. However, B7-H4 expression and its prognostic significance in different metastases from CRC remain unclear. In the present study, we screened a novel mouse anti-human B7-H4 monoclonal antibody (mAb) which exhibited a higher degree of recognition and sensitivity than the commercial reagent in immunohistochemistry (IHC). Using this antibody, overall 110 metastatic and paired primary lesions of CRC were analyzed for their expression of B7-H4, CD8 and CD68. Our results showed that expression of B7-H4 and CD68 in metastastic lesions was significantly higher than that in matched primary lesions (P = 0.0016, P < 0.0001). We also found a significant increase of CD68-positive immune cell infiltration in the B7-H4 high expressing metastases (P = 0.041). Moreover, upregulated B7-H4 in metastatic lesions was correlated with poor prognosis of patients (P = 0.014), while in primary lesions, B7-H4 combined with CD8 was associated with the overall survival (OS) (P = 0.043). Further, B7-H4 expression in metastatic lesions was significantly correlated with hazard ratio (HR) both in univariate and multivariate analysis. Altogether, B7-H4 in metastatic lesions is promising to be a potential prognostic indicator of CRC, and may promote tumor progression and metastasis of this cancer.

A Distinctive microRNA (miRNA) Signature in the Blood of Colorectal Cancer (CRC) Patients at Surgery.

Background: Liquid biopsy (LB) provides an examination of the peripheral blood of cancer patients for circulating tumor cells, cell-free nucleic acids and microRNAs (miRNAs) and is an established tool of precision medicine. Unlike most previous LB studies that focused on advanced metastatic colorectal cancer (CRC), we assessed miRNA dysregulation in blood samples obtained on the day of surgery from patients with primary CRC lesions but no clinical evidence of extra-colonic diffusion. In this study, plasma preparation included miRNAs associated to exosomes, but excluded large macrovesicles from the preparation. Methods: The miRNA profile in plasma isolated from a cohort of 35 CRC patients at the day of surgery was analyzed by Next Generation Sequencing (NGS) and further confirmed by droplet digital RT-PCR (dd-RT-PCR). Results: A miR-141-3p/miR-221-3p/miR-222-3p upregulation signature previously described in advanced CRC did not discriminate the analyzed early-CRC cohort from six tumor-free donors (Tf-D). In contrast, NGS-based miRNome analysis of a training cohort of five CRC and three tumor-free donors identified a novel, distinct nine miRNA signature comprising five up-regulated and four down-regulated miRNAs, six of which could be confirmed in the full CRC and tumor-free donor validation dataset by dd-RT-PCR. Additionally, a KRAS (Kirsten Rat Sarcoma Viral Oncogene Homolog) mutant status was correlated with the plasma content of three identified miRNAs. Conclusions: When the data obtained were comparatively evaluated, at least one of the miRNAs belonging to the signature list was found to be dysregulated in 34/35 (97.1%) of our early-CRC plasma samples. The miRNA list provides diagnostic markers as well as possible molecular targets for protocols focusing on “microRNA therapeutics”.

Significance of RAS mutations in pulmonary metastases of patients with colorectal cancer.

RAS/BRAF mutations of colorectal cancer (CRC) play a crucial role in carcinogenesis and cancer progression and need to be considered for the therapeutic strategy choice. We used next-generation-sequencing (NGS) technology to assess RAS/BRAF mutation differences between primary CRC and corresponding pulmonary metastases (PMs). We examined the mutation statuses of the KRAS 12/13/61/146, NRAS 12/13/61/146, and BRAF 600 codons in genomic DNA from fresh-frozen or formalin-fixed paraffin-embedded tissues derived from 34 primary lesions and 52 corresponding PMs from 36 patients with CRC. We found RAS mutations in 76% (26/34) of primary CRC lesions and in 86% (31/36) of PMs. While 27% (7/26) of the primary CRC RAS mutations were heterogeneous, all the RAS mutations in PMs were homogeneous. Of the mutations in PMs, 71% (22/31) were KRAS G>A transitions, of which 82% (18/22) were KRAS G12D or G13D. The RAS mutation discordance between primary tumors and PMs was 12.1% (4/33). RAS mutations with the same genotyping were detected in all synchronous and metachronous PMs from 9 patients. We found no BRAF mutations in either primary or pulmonary tissues. Our NGS analysis suggests that RAS mutations of PM of patients with CRC are more common than initially thought. The presence of KRAS mutations in CRC specimens, especially G12D or G13D mutations, seems to promote PM formation.

Epidermal growth factor receptor-targeted molecular imaging of colorectal tumors: Detection and treatment evaluation of tumors in animal models.

To overcome the problem of overlooking colorectal tumors, a new and highly sensitive modality of colonoscopy is needed. Moreover, it is also important to establish a new modality to evaluate viable tumor volume in primary lesions of colorectal cancer (CRC) during chemotherapy. Therefore, we carried out molecular imaging of colorectal tumors targeting epidermal growth factor receptor (EGFR), which is highly expressed on tumor cells, for evaluating chemotherapeutic efficacy and for endoscopic detection of colorectal adenomas. We first attempted to image five CRC cell lines with various levels of EGFR expression using an Alexa Fluor‐labeled anti‐EGFR monoclonal antibody (AF‐EGFR‐Ab). A strong fluorescence signal was observed in the cells depending on the level of EGFR expression. When nude mice xenografted with LIM1215 CRC cells, which highly express EGFR, were i.v. injected with AF‐EGFR‐Ab, a strong fluorescence signal appeared in the tumor with a high signal to noise ratio, peaking at 48 hours after injection and then gradually decreasing, as shown using an IVIS Spectrum system. When the xenografted mice were treated with 5‐fluorouracil, fluorescence intensity in the tumor decreased in proportion to the viable tumor cell volume. Moreover, when the colorectum of azoxymethane‐treated rats was observed using a thin fluorescent endoscope with AF‐EGFR‐Ab, all 10 small colorectal adenomas (≤3 mm) were detected with a clear fluorescence signal. These preliminary results of animal experiments suggest that EGFR‐targeted fluorescent molecular imaging may be useful for quantitatively evaluating cell viability in CRC during chemotherapy, and also for detecting small adenomas using a fluorescent endoscope.

The Embryonic Stem Cell-Specific Transcription Factor ZFP57 Promotes Liver Metastasis of Colorectal Cancer

BackgroundThe embryonic stem cell-specific transcription factor, ZFP57, has been shown to play an important role in tumor formation. In this study, we examined if ZFP57 is involved in colorectal cancer metastasis. Materials and methodsFirst, we used colorectal cancer cell lines to perform in vivo metastatic experiments with nude mice. Next, we carried out immunohistochemical analysis of clinical specimens of colorectal cancers. ResultsIn liver metastatic experiments using human colorectal cancer HT29 and HCT116 cells, liver polymetastases occurred at high frequency in ZFP57-overexpressing HT29 and HCT116 cells, whereas both control cells only resulted in oligometastases. Next, we analyzed ZFP57 expression using clinical specimens. Liver metastasis-positive cases were more frequently associated with ZFP57 overexpression than negative cases in primary lesions of colorectal cancer, and the overexpression was particularly remarkable in tumor invasive lesions. Furthermore, ZFP57 overexpression was significantly correlated not only with liver metastasis but also with lymph node metastasis. In addition, the expression level of ZFP57 was significantly correlated with that of the metastasis-related gene NANOG. We also found that ZFP57 overexpression reduced the progression-free survival rate of patients with colorectal cancer. ConclusionsThis study demonstrated that ZFP57 plays an important role in the hematogenous metastasis of colorectal cancer, suggesting that it could be used as a novel treatment target.

Aberrant expression of long noncoding RNA SNHG15 correlates with liver metastasis and poor survival in colorectal cancer.

Long noncoding RNAs (lncRNAs) play a critical role in the initiation and progression of colorectal cancer (CRC), but little is known about the function of lncRNAs in the colorectal liver metastasis (CLM). This study was designed to identify specific lncRNAs correlating to liver metastasis of CRC, and to further assess their clinical value. Seventeen patients with primary CRC lesions, adjacent normal mucosa, and synchronous liver metastases lesions were divided into discovery set (six patients) and test set (11 patients). Transcriptome sequencing (RNAseq) was used to screen differential expression of lncRNAs in the discovery set. Based on bioinformatics data, quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was used to verify the target lncRNA in test set. The relationships between target lncRNA and clinical values were analysed in an expanded validation set of additional 91 patients. 23 upregulated and 14 downregulated lncRNAs were detected for distinguishing synchronous liver metastases, primary CRC lesions from adjacent normal mucosa in the RNAseq set. The expression levels of four lncRNAs in the 37 lncRNA signature were verified by qRT-PCR in the test set. Compared with the paired normal mucosa, high expression levels of lnc-small-nucleolar RNA host gene 15 (SNHG15) were detected not only in primary CRC lesions but also in liver metastases lesions in the test set. Furthermore, in the expanded validation set, high expression of lnc-SNHG15 was significantly associated with lymph-node metastasis and liver metastasis (p < 0.05), and patients displaying high lncRNA-SNHG15 expression exhibited a shorter median overall survival duration than those displaying low expression (30.7 vs. 35.2 months; p = 0.003). Multivariate analyses demonstrated that lncRNA-SNHG15 overexpression may serve as a poor prognostic biomarker for CRC patients (p = 0.049; Cox's regression: 2.731). Lnc-SNHG15 overexpression was significantly associated with CLM and high-expression of lnc-SNHG15 in CRC was an independent predictor of poor survival.

CDX2 expression is concordant between primary colorectal cancer lesions and corresponding liver metastases independent of chemotherapy: a single-center retrospective study in Japan.

ObjectiveLoss of caudal-type homeobox transcription factor 2 (CDX2) expression in colorectal cancers (CRCs) has recently been proposed as a promising predictive biomarker for not only prognosis but also response to chemotherapy. However, the relationship between alterations in CDX2 expression during cancer progression and response to chemotherapy remains unclear. We herein aimed to determine the concordance of CDX2 expression between primary CRCs and corresponding liver metastases, in association with chemotherapy.ResultsPrimary CRCs exhibited heterogeneous CDX2 expression. Seven of the 144 CRCs in the cohort (4.9%, 95% confidential interval, 2.0%–9.8%) were CDX2-negative. The concordance rate of the CDX2 expression status in patients who did not receive chemotherapy was 100% (P = 0.041), whereas the concordance rate among patients who received chemotherapy only after primary resection was 96.3% (P = 0.005). Moreover, the concordance rate in patients who received chemotherapy before both primary resection and liver metastasectomy was 100% (P < 0.001).ConclusionCDX2 expression status was highly concordant between primary CRCs and corresponding liver metastases, independent of chemotherapy, suggesting that the CDX2 expression status in CRCs was not affected by metastasis or chemotherapy.MethodsA total of 144 consecutive patients with CRC who were treated at a single center in Japan between 2006 and 2014 were included. Formalin-fixed paraffin-embedded whole sections of surgically resected primary CRCs and corresponding liver metastases were assessed for CDX2 expression by immunohistochemistry.

Down-Regulation of microRNA-132 is Associated with Poor Prognosis of Colorectal Cancer.

BackgroundGiven the role of microRNA in colorectal cancer (CRC) progression, we explored the association between microRNA (miRNA) expression and CRC-related prognosis.Methods Three types of tissue samples (primary CRC lesions without liver metastasis, primary lesions with liver metastasis, and liver metastatic tissues) were used for miRNA profiling to identify differentially expressed miRNA. Quantitative real-time PCR was used to examine miRNA expression in CRC cells and in tumor tissues.Results MiR-132 was significantly down-regulated in primary CRC tissues with liver metastasis and liver metastatic lesions compared to primary lesions without liver metastasis. Multivariate analysis for overall survival indicated that low miR-132 expression was an independent prognostic factor for CRC patients (overall survival P = 0.040, disease-free survival P = 0.015). Ectopic expression of miR-132 significantly inhibited cell proliferation and cell invasion. The luciferase reporter assay revealed that anoctamin 1 (ANO1) was a direct target of miR-132. Kaplan–Meier survival curves showed that high ANO1 expression was a significant prognostic factor for overall survival of patients with CRC (P = 0.0344).ConclusionsDown-regulation of miR-132 is associated with poor prognosis in CRC. ANO1 could be one of the crucial targets of miR-132 in CRC.Electronic supplementary materialThe online version of this article (doi:10.1245/s10434-016-5133-3) contains supplementary material, which is available to authorized users.

Dysregulation of over-expressed IL-32 in colorectal cancer induces metastasis.

BackgroundInterleukin (IL)-32 is a described intracellular pluripotent pro-inflammatory mediator, characterized by the signaling of NF-κB and STAT3.MethodsOur study investigated whether IL-32 expression has clinical significance in the metastases of colorectal cancer (CRC). A total of 70 CRC patients were enrolled, 47 cases of which were single CRC organic metastasis lesions while the rest of which were primary CRC lesions (T4NxM0). IL-32 expression was detected by immunohistochemistry, and the correlation between IL-32 expression and CRC metastases was analyzed.ResultsThe positive rates of IL-32 in the CRC organic metastasis group were more severe than those in the primary CRC group (P < 0.05). The positive rate of IL-32 in primary CRC with lymph node metastasis was more severe than that of IL-32 in primary CRC without lymph node metastasis (P < 0.05).ConclusionsThe level of IL-32 expression could influence the N grade of CRC. Thus, IL-32 expression may stimulate the organic metastasis and the lymph node metastasis of CRC.

Correlation between long-term aspirin use and F-fluorodeoxyglucose uptake in colorectal cancer measured by PET/CT.

PurposeThe aim of this study was to evaluate the relationship between long-term aspirin use with pretreatment 18 Fluorodeoxyglucose (FDG) uptake of primary lesions of Colorectal cancer (CRC) and evaluate their clinical significance.Materials and MethodsWe enrolled 84 patients with CRC who underwent 18F-FDG PET/CT scanning before surgery between 1st July 2008 and 1st March 2013 and followed up until 1st March 2014. Maximum standardized uptake value (SUVmax) of the primary tumor was measured by 18F-FDG PET/CT. The history of aspirin taken and other clinicopathogical factors were also obtained and their relationships were examined by Mann-Whitney or χ2 tests. Progression-free survival (PFS) was determined by standard Kaplan-Meier survival analysis. Cox proportional hazards regression was performed to determine whether history of aspirin taken, pretreatment SUVmax, age, gender, TNM stage, tumor sizes and differentiation influenced outcomes.ResultsCRC Patients with long-term history of aspirin use had lower SUVmax of primary lesions than control group (9.74±2.62 vs. 13.91±6.18) and showed a trend towards improved PFS after curative surgery. However, pretreatment of SUVmax showed no prognostic value in patients with CRC.ConclusionsLong-term aspirin use is associated with lower pretreatment SUVmax of CRC and is a promising prognostic factor for predicting PFS in patients with CRC.

Hepatic metastases of colorectal cancer are rather homogeneous but differ from primary lesions in terms of immune cell infiltration

The immune system plays an important role in shaping the clinical course of colorectal cancer (CRC). However, it is still unclear how the immune infiltrates of primary CRC lesions and distant metastases by immune effector cells are related to each other. To address this issue, we quantified CD3+, CD8+ and granzyme B+ lymphocytes in primary CRC samples and corresponding liver metastases. This analysis showed that the prognostic predictions that can be drawn from the infiltration of immune cells in primary CRCs and their metastases are heterogeneous. To investigate whether such heterogeneity would also be observed within CRC hepatic metastases, the density of the immune infiltrate and cytokine production were assessed in opposite sides of the same metastatic lesion. In addition, tumor-infiltrating lymphocytes were assessed in sequential sections of the same metastatic lesion, with a spacing of 30 μm. In summary, consistent cell counts and cytokine levels were detected within the same lesion. The study of a case of synchronous metastases, however, suggested that different metastatic lesions within the same patient may be heterogeneous, perhaps indicating a major impact for local causes on tumor infiltration by immune cells. In summary, our study demonstrates a consistent degree of heterogeneity between primary tumors and hepatic metastases but an excellent intra-lesional homogeneity. These findings may be of key importance for patient stratification and the development of personalized strategies against CRC.

Expression of the phosphorylated MEK5 protein is associated with TNM staging of colorectal cancer

BackgroundActivation of MEK5 in many cancers is associated with carcinogenesis through aberrant cell proliferation. In this study, we determined the level of phosphorylated MEK5 (pMEK5) expression in human colorectal cancer (CRC) tissues and correlated it with clinicopathologic data.MethodspMEK5 expression was examined by immunohistochemistry in a tissue microarray (TMA) containing 335 clinicopathologic characterized CRC cases and 80 cases of nontumor colorectal tissues. pMEK5 expression of 19 cases of primary CRC lesions and paired with normal mucosa was examined by Western blotting. The relationship between pMEK5 expression in CRC and clinicopathologic parameters, and the association of pMEK5 expression with CRC survival were analyzed respectively.ResultspMEK5 expression was significantly higher in CRC tissues (185 out of 335, 55.2%) than in normal tissues (6 out of 80, 7.5%; P < 0.001). Western blotting demonstrated that pMEK5 expression was upregulated in 12 of 19 CRC tissues (62.1%) compared to the corresponding adjacent nontumor colorectal tissues. Overexpression of pMEK5 in CRC tissues was significantly correlated to the depth of invasion (P = 0.001), lymph node metastasis (P < 0.001), distant metastasis (P < 0.001) and high preoperative CEA level (P < 0.001). Consistently, the pMEK5 level in CRC tissues was increased following stage progression of the disease (P < 0.001). Analysis of the survival curves showed a significantly worse 5-year disease-free (P = 0.002) and 5-year overall survival rate (P < 0.001) for patients whose tumors overexpressed pMEK5. However, in multivariate analysis, pMEK5 was not an independent prognostic factor for CRC (DFS: P = 0.139; OS: P = 0.071).ConclusionspMEK5 expression is correlated with the staging of CRC and its expression might be helpful to the TNM staging system of CRC.

Identification of chromosomal aberrations of metastatic potential in colorectal carcinoma

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.

Amphiregulin Is a Promising Prognostic Marker for Liver Metastases of Colorectal Cancer

Aberrant activation of epidermal growth factor receptors (EGFR/HER1) by ligand stimulation or heterodimerization with human epidermal growth factor 2 (HER2) is considered to play an important role in the development of colorectal carcinoma. Amphiregulin (AR) is a ligand of EGFR that might be related to the development and progression of gastrointestinal tumors. The aim of this study was to determine the AR, EGFR, and HER2 protein expression levels and to evaluate their prognostic relevance to the clinical course of colorectal cancer. The AR, EGFR, and HER2 protein levels in primary tumors of colorectal cancer (n = 106) were examined using immunohistochemistry. Metastatic sites in liver specimens (n = 16) were also analyzed in the same manner. Thirteen (81.6%) metastatic lesions of the liver stained positive for AR. Among the primary lesions of colorectal cancer, 58 (54.7%) stained positive for AR, 13 (12.3%) stained positive for EGFR, and 5 (4.7%) stained positive for HER2. When the relationships between each protein expression level and the clinicopathologic factors were examined, only the AR expression level was significantly related to liver metastasis (P = 0.0296). A multivariate analysis of liver metastasis proved that AR expression was an independent prognostic factor of liver metastasis from colorectal cancer (P = 0.0217). AR expression in primary lesions of colorectal cancer is an important predictive marker of liver metastasis.

PRL-3 Expression in Primary Colorectal Cancer

Purpose: Overexpression of the protein tyrosine phosphatase (PRL-3) is elevated in liver metastases derived from colorectal cancer. We examined PRL-3 expression in the primary lesion of colorectal cancer patients and investigated its relation to clinicopathological features. Methods: A total of 63 randomly selected patients who underwent surgical resection for colorectal cancer between May 2001 and June 2005 at our hospital were investigated. Formalin-fixed and paraffin-embedded specimens from colorectal cancer patients who underwent surgical resections for primary tumors were collected. The expression of PRL-3 was detected by immunohistochemistry and the relation with age, sex, primary tumor size, tumor cell differentiation, depth of invasion, microscopic lymph node metastases, vascular invasion, numbers of lymph node metastases, postoperative stage, and postoperative survival time were analyzed. Results: A total of 16 of the 63 colorectal cancer patients were detected with liver metastases during the follow-up periods. Liver resection was performed for those liver metastases patients. Five patients developed lung metastases after liver resection. PRL-3 expression was detected in 46 colorectal cancer patients. Fourteen patients with lymphatic invasion had positive expression of PRL-3 that was significant (P=0.042). The incidence of PRL-3 expression in the T stage was significant (P=0.019). Moreover, PRL-3 expression was closely associated with liver metastases (P=0.048). Conclusions: These results indicate that an investigation of PRL-3 expression in primary colorectal cancer lesions may contribute to the detection of occult liver metastases and to a differentiation between postoperative management strategies.

Expression of cyclooxygenase-2 in colorectal cancer and its clinical significance

To clarify the clinicopathologic significance of COX-2 expression in human colorectal cancer. A total of 128 surgically resected colorectal cancer specimens were immunohistochemically analyzed with the use of anti-COX-2, anti-VEGF and anti-MMP-2 antibodies. The relationship between the cyclooxygenase-2 expression in primary lesions of colorectal cancer and clinicopathologic parameters was evaluated by chi-square test. Among 128 cases of colorectal cancer, 87 (67.9%) were positive for cyclooxygenase-2. The expression of cyclooxygenase-2 was significantly correlated with the depth of invasion, stage of disease, and metastasis (lymph node and liver). Patients in T3-T4, stages III-IV and with metastasis had much higher expression of cyclooxygenase-2 than ones in T1-T2, stages I-II and without metastasis (P<0.05). Among 45 cases of colorectal cancer with lymph node metastasis, the COX-2- positive rate was 86.7% (39/45) for primary lesions and diffuse cytoplasmic staining for COX-2 protein was detected in cancer cells in 100% of metastatic lesions of the lymph nodes. VEGF expression was detected in 49 tumors (38.3%), and VEGF expression was closely correlated with COX-2 expression. The positive expression rate of VEGF (81.6%) in the cyclooxygenase-2-positive group was higher than that in the cyclooxygenase-2- negative group (18.4%, P<0.05). MMP-2 expression was detected in 88 tumors (68.8%), and MMP-2 expression was closely correlated with COX-2 expression. The positive expression rate of MMP-2 (79.6%) in the positive COX-2 group was higher than that in the negative COX-2 group (20.4%, P<0.05). Cyclooxygenase-2 may be associated with tumor progression by modulating the angiogenesis and cancer cell motility and invasive potential in colorectal cancer and it can be used as a possible biomarker.