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Background: Mutations in the TP53 tumor suppressor gene are well-established drivers of colorectal cancer (CRC) development. However, data on the prevalence of TP53 variants and their association with consensus molecular subtype (CMS) classification in patients with CRC from Rwanda are currently lacking. This study addressed this knowledge gap by investigating TP53 mutation status concerning CMS classification in a CRC cohort from Rwanda., Methods: Formalin-fixed paraffin-embedded (FFPE) tissue blocks were obtained from 51 patients with CRC at the University Teaching Hospital of Kigali, Rwanda. Exons 4 to 11 and their flanking intron-exon boundaries in the TP53 gene were sequenced using Sanger sequencing to identify potential variants. The recently established immunohistochemistry-based classifier was employed to determine the CMS of each tumor., Results: Sequencing analysis of cancerous tissue DNA revealed TP53 pathogenic variants in 23 of 51 (45.1%) patients from Rwanda. These variants were predominantly missense types (18/23, 78.3%). The most frequent were c.455dup (p.P153Afs*28), c.524G > A (p.R175H), and c.733G > A (p.G245S), each identified in three tumors. Trinucleotide sequence context analysis of the 23 mutations (20 of which were single-base substitutions) revealed a predominance of the [C > N] pattern among single-base substitutions (SBSs) (18/20; 90.0%), with C[C > T]G being the most frequent mutation (5/18, 27.8%). Furthermore, pyrimidine bases (C and T) were preferentially found at the 5' flanking position of the mutated cytosine (13/18; 72.2%). Analysis of CMS subtypes revealed the following distribution: CMS1 (microsatellite instability-immune) (6/51, 11.8%), CMS2 (canonical) (28/51, 54.9%), CMS3 (metabolic) (9/51, 17.6%), and CMS4 (mesenchymal) (8/51, 15.7%). Interestingly, the majority of TP53 variants were in the CMS2 subgroup (14/23; 60.1%)., Conclusion: Our findings indicate a high frequency of TP53 variants in CRC patients from Rwanda. Importantly, these variants are enriched in the CMS2 subtype. This study, representing the second investigation into molecular alterations in patients with CRC from Rwanda and the first to explore TP53 mutations and CMS classification, provides valuable insights into the molecular landscape of CRC in this understudied population., (© 2024. The Author(s).)

Mutations and inactivation of the TP53 gene are frequently observed in various types of malignancies. Precise knowledge of the genetic structure and detection of mutation hotspots are crucial, as these indicate a high probability of developing cancer. The aim of our study was to perform the bioinformatic detection of mutation hotspots in the TP53 gene in patients diagnosed with malignant colon neoplasms using self-developed software (version 1). We compared TP53 gene sequences from 50 healthy individuals with those from 50 patients diagnosed with colorectal carcinoma. Of the 50 samples from cancer patients, the most frequent mutations were observed in exons 5 and 8 (12 mutations per exon) and gene sequences of 12 samples, which differed from those of the 50 samples from healthy individuals. Based on our results, the distribution of mutations in the TP53 gene structure was not even across different exons. By comparing the gene sequences of healthy individuals with those of colon cancer samples, we conclude that structural changes occurring in similar gene regions are not associated with increases in susceptibility to malignancies in every case, namely, that the pathological mechanism is multifactorial.

Hypomagnesemia commonly occurs as a side effect of panitumumab treatment. In severe cases, temporary discontinuation or dose reduction of panitumumab may be necessary. Proton pump inhibitors (PPIs) are reportedly potential risk factors for hypomagnesemia. We conducted a multicenter study to assess the impact of PPIs on the risk of grade 3-4 hypomagnesemia in patients with metastatic colorectal cancer (mCRC) receiving panitumumab. We adjusted for potential bias using a propensity score-matched analysis and retrospectively reviewed the medical records of patients. Hypomagnesemia severity was graded according to the Common Terminology Criteria for Adverse Events, version 5.0. A total of 165 patients were enrolled in this study. The incidence of grade 3-4 hypomagnesemia was significantly higher in the PPI group than in the non-PPI group, both before (20.0% [30/60] vs. 8.0% [8/105], p = 0.026) and after propensity score matching (16.2% [6/37] vs. 0% [0/37], p = 0.025). In the propensity score-matched cohort, the risk of grade 3-4 hypomagnesemia was significantly higher in the PPI group (odds ratio, 2.19; 95% confidence interval, 1.69-2.84; p = 0.025). These findings suggest that concomitant use of PPIs significantly increases the risk of grade 3-4 hypomagnesemia in patients with mCRC receiving panitumumab. Therefore, close monitoring of these patients is imperative.

Although immunohistochemistry (IHC) for mismatch repair (MMR) proteins (MMR IHC) is used to identify DNA MMR status, universal screening of all patients with colorectal cancer (CRC) using a combination of both MMR IHC and genetic testing for the BRAFV600E mutation is limited in Japan. This study aimed to better understand the histopathological characteristics of CRCs, which exhibit both deficient mismatch repair (dMMR) and BRAFV600E mutation. MMR IHC of formalin-fixed paraffin-embedded tissues from tumor areas obtained from 651 patients with CRC who underwent surgical resection at Hamamatsu University Hospital (Hamamatsu, Japan) between August 2016 and March 2022 were used to evaluate MMR status, which was determined by staining for the expression of 4 MMR proteins (MLH1, MSH2, PMS2, and MSH6). All dMMR tumors were additionally evaluated for BRAFV600 mutation status via Sanger sequencing. Patient clinical characteristics (age, sex, tumor location, size, and tumor pathology) were then classified using their dMMR and BRAFV600 mutation statuses. Among the 651 patients with CRC, 58 carried tumors with dMMR, of which 52 were deficiency in MLH1 (dMLH1). Interestingly, all 16 medullary carcinomas that were analyzed showed characteristics corresponding to the presence of both dMLH1 and BRAFV600E mutation (P = .01). These results suggest that colorectal medullary carcinomas can be diagnosed based on their unique characteristics of harboring the BRAFV600E mutation and exhibiting dMLH1 expression., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2023 the Author(s). Published by Wolters Kluwer Health, Inc.)

Background/aim: Among colorectal cancer-associated intestinal microbiota, colibactin-producing (clb + ) bacteria are attracting attention. We aimed to clarify the interaction between clb + Escherichia coli and normal colorectal epithelial cells in vivo and in vitro., Materials and Methods: Five-week-old female Balb/c mice were divided in an untreated group, a group treated with clb + E. coli isolated from a Japanese patient with colorectal cancer (E. coli-50), and a group treated with non colibactin-producing E. coli (E. coli-50/ΔclbP). Mice were sacrificed at 18 weeks of treatment., Results: Treatment with clb + E. coli increased positivity for H2A histone family member X phosphorylated at Ser-139 (γH2AX) in epithelial cells of the luminal surface of the mouse rectum but this did not occur in the E. coli-50/ΔclbP and untreated groups. In an in vitro setting, the ratio of apoptotic cells was increased and cell counts were reduced by treatment with clb + E. coli more than in untreated cells and normal rat colorectal epithelial cells., Conclusion: E. coli-50 induced DNA damage in the mouse rectum, possibly by direct interaction between clb + E. coli and normal colorectal epithelial cells. Our findings imply that regulation of clb + E. coli infection may be a useful strategy for colorectal cancer control., (Copyright © 2022, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background: The Escherichia coli strain that is known to produce the genotoxic secondary metabolite colibactin is linked to colorectal oncogenesis. Therefore, understanding the properties of such colibactin-positive E. coli and the molecular mechanism of oncogenesis by colibactin may provide us with opportunities for early diagnosis or prevention of colorectal oncogenesis. While there have been major advances in the characterization of colibactin-positive E. coli and the toxin it produces, the infection route of the clb + strain remains poorly characterized., Results: We examined infants and their treatments during and post-birth periods to examine potential transmission of colibactin-positive E. coli to infants. Here, analysis of fecal samples of infants over the first month of birth for the presence of a colibactin biosynthetic gene revealed that the bacterium may be transmitted from mother to infant through intimate contacts, such as natural childbirth and breastfeeding, but not through food intake., Conclusions: Our finding suggests that transmission of colibactin-positive E. coli appears to be occurring at the very early stage of life of the newborn and hints at the possibility of developing early preventive measures against colorectal cancer., (© 2021. The Author(s).)

We investigated the relationship between colibactin-producing (clb + ) Escherichia coli and colorectal adenocarcinoma. In total, 729 E. coli colonies were isolated from tumor and surrounding non-tumor regions in resected specimens from 34 Japanese patients; 450 colonies were from the tumor regions and 279 from the non-tumor regions. clb + bacteria were found in tumor regions of 11 patients (11/34, 32.4%) and they were also detected in the non-tumor regions of 7 out of these 11 patients (7/34, 20.6%). The prevalence of clb + isolates was 72.7% (327/450) and 44.1% (123/279) in tumor and non-tumor regions, respectively. All the recovered clb + isolates belonged to the phylogenetic group B2 and were the most predominant type in tumor regions. Hemolytic (α-hemolysin-positive, hlyA + ) and non-hemolytic (α-hemolysin-negative, hlyA - ) clb + isolates were obtained from patient #19; however, the prevalence of hlyA + clb + isolates was significantly higher in tumor regions (35/43, 81.4%) than in non-tumor regions (3/19, 15.8%). Moreover, a significantly higher production of N-myristoyl-D-asparagine, a by-product of colibactin biosynthesis, was observed in hlyA + clb + isolates than in hlyA - clb + isolates. Our results suggest that hlyA + clb + E. coli may have a selective advantage in colorectal colonization and, consequently, might play a role in carcinogenesis. The presence of hlyA + clb + bacteria in healthy individuals is a potential risk marker of colorectal cancer.

While high-colibactin-producing Escherichia coli is thought to be associated with colorectal oncogenesis, this study is complicated part due to an inability to isolate colibactin adequately. Here, we created fluorescent probes activated by ClbP, the colibactin-maturing peptidase, to identify high-colibactin-producing strains. Our probe served as a valuable clinical diagnostic tool that allowed simple high-throughput diagnostic screening of clinical samples. Furthermore, the probe also allowed identification of high-colibactin producers that would help advance our understanding of colibactin biosynthesis.

Colibactin is a polyketide-peptide genotoxin produced by enteric bacteria such as E. coli, and is considered to contribute to the development of colorectal cancer. We previously isolated E. coli strains from Japanese colorectal cancer patients, and in the present study we investigated the genotoxic potency of the colibactin-producing (clb + ) E. coli strains that carry the polyketide synthases "pks" gene cluster (pks + ) and an isogenic clb - mutant in which the colibactin-producing ability is impaired. Measurement of phosphorylated histone H2AX indicated that DNA double strand breaks were induced in mammalian CHO AA8 cells infected with the clb + E. coli strains. Induction of DNA damage response (SOS response) by crude extract of the clb + strains was 1.7 times higher than that of the clb - E. coli in an umu assay with a Salmonella typhimurium TA1535/pSK1002 tester strain. Micronucleus test with CHO AA8 cells revealed that infection with the clb + strains induced genotoxicity, i.e., the frequencies of micronucleated cells infected with clb + strain were 4-6 times higher than with the clb - strain. Since the intestinal flora are affected by dietary habits that are strongly associated with ethnicity, these data may contribute to both risk evaluation and prevention of colorectal cancer in the Japanese population.

Background: Colorectal cancer (CRC) is the fourth most frequent cause of cancer deaths in the world. Novel biomarkers for the diagnosis, prognosis, and treatment of CRC are required to improve the clinical strategy., Methods: We applied shotgun proteomics using isobaric tags for relative and absolute quantitation (iTRAQ) to identify novel biomarkers of CRC, and then we detected leucine-rich PPR-motif-containing protein (LRPPRC) expression in 83 normal colorectal tissues and 133 CRC tissues by immunohistochemistry., Results: A total of 570 proteins were identified using iTRAQ. We validated the expression of LRPPRC protein by immunohistochemical analysis of the 77 proteins that showed expression changes in the cancer tissues >1.5-fold the levels in the normal tissues. The expression levels of LRPPRC were significantly higher in CRC tissues than those in normal colorectal tissues, and the expression levels were related with tumor differentiation and especially high in moderately differentiated CRC tissues., Conclusion: We identified a novel, differentially expressed protein, LRPPRC, which has the potential to serve as a molecular target for diagnosis and/or prognosis of CRC., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Spindle assembly abnormal protein 6 homolog (SASS6) plays an important role in the regulation of centriole duplication. To date, the genetic alteration of SASS6 has not been reported in human cancers. In the present study, we examined whether SASS6 expression is abnormally regulated in colorectal cancers (CRCs). Increased SASS6 mRNA and protein expression levels were observed in 49 (60.5%) of the 81 primary CRCs and 11 (57.9%) of the 19 primary CRCs, respectively. Moreover, the upregulation of SASS6 mRNA expression was statistically significant (P=0.0410). Next, using DLD-1 colon cancer cells inducibly expressing SASS6, SASS6 overexpression was shown to induce centrosome amplification, mitotic abnormalities such as chromosomal misalignment and lagging chromosome, and chromosomal numerical changes. Furthermore, SASS6 overexpression was associated with anaphase bridge formation, a type of mitotic structural abnormality, in primary CRCs (P<0.01). SASS6 upregulation in colon cancer was also revealed in the Cancer Genome Atlas (TCGA) data and was shown to be an independent predictor of poor survival (multivariate analysis: hazard ratio, 2.805; 95% confidence interval, 1.244‑7.512; P=0.0112). Finally, further analysis of the TCGA data demonstrated SASS6 upregulation in a modest manner in 8 of 11 cancer types other than colon cancer, and SASS6 upregulation was found to be associated with a poor survival outcome in patients with kidney renal cell carcinoma and lung adenocarcinoma. Our present findings revealed that the upregulation of SASS6 expression is involved in the pathogenesis of CRC and is associated with a poor prognosis among patients with colon cancer. They also suggest that SASS6 upregulation is a genetic abnormality relatively common in human cancer.

A CD44-SLC1A2 fusion has recently been discovered in a subset of primary gastric cancers, and an APIP-SLC1A2 fusion has been described in a colon cancer cell line (SNU-C1); however, whether such SLC1A2 fusions occur in primary colorectal cancer (CRC) and whether such fusions are specific for gastrointestinal cancers remain uncertain. In the present study, we examined 90 primary CRCs and 112 primary non-small cell lung cancers (NSCLCs) for CD44-SLC1A2 and APIP-SLC1A2 fusion transcripts using RT-PCR and subsequent sequencing analyses. Although the expression of both types of SLC1A2 fusion transcripts was not detected in any of the NSCLCs, the expression of CD44-SLC1A2, but not the APIP-SLC1A2 fusion transcript, was detected in one (1.1 %) CRC. The CD44-SLC1A2 fusion transcript was expressed in cancerous tissue but not in corresponding non-cancerous tissue, and the fusion occurred between exon 1 of CD44 and exon 2 of SLC1A2; it was expected that a slightly truncated but functional SLC1A2 protein would be produced under the CD44 promoter. A quantitative RT-PCR analysis revealed that SLC1A2 mRNA expression was upregulated in CRC containing SLC1A2 fusion transcripts, while it was downregulated in most other CRCs. The SLC1A2 fusion-positive carcinoma was located on the right-side of colon, was a mucinous adenocarcinoma, was immunohistochemically negative for MSH2 mismatch repair protein, and contained no APC or KRAS mutations. Together, these results suggest that the expression of SLC1A2 fusion transcripts is related to a subset of primary CRCs and may contribute to the elucidation of the characteristics of SLC1A2 fusion-positive CRCs in the future.

R-spondin (RSPO) gene fusions have recently been discovered in a subset of human colorectal cancer (CRC) in the U.S. population; however, whether the fusion is recurrent in CRC arising in patients from the other demographic areas and whether it is specific for CRC remain uncertain. In this study, we examined 75 primary CRCs and 121 primary lung cancers in the Japanese population for EIF3E-RSPO2 and PTPRK-RSPO3 fusion transcripts using RT-PCR and subsequent sequencing analyses. Although the expression of EIF3E-RSPO2 and PTPRK-RSPO3 was not detected in any of the lung carcinomas, RSPO fusions were detected in three (4%) of the 75 CRCs. Two CRCs contained EIF3E-RSPO2 fusion transcripts, and another CRC contained PTPRK-RSPO3 fusion transcripts. Interestingly, in one of the two EIF3E-RSPO2 fusion-positive CRCs, a novel fusion variant form of EIF3E-RSPO2 was identified: exon 1 of EIF3E was connected to exon 2 of RSPO2 by a 351-bp insertion. A quantitative RT-PCR analysis revealed that RSPO mRNA expression was upregulated in the three CRCs containing RSPO fusion transcripts, while it was downregulated in nearly all of the other CRCs. An immunohistochemical analysis and a mutational analysis revealed that the RSPO fusion-containing CRC had a CDX2 cell lineage, was positive for mismatch repair protein expression, and had the wild-type APC allele. Finally, the forced expression of RSPO fusion proteins were shown to endow colorectal cells with an increased growth ability. These results suggest that the expression of RSPO fusion transcripts is related to a subset of CRCs arising in the Japanese population.

We report a case of single-incision bilateral partial lung resection using the subxiphoid approach. This approach requires a 3-cm incision in the abdomen, making it aesthetically favorable. In addition, it does not cause postoperative intercostal neuropathy, and postoperative pain is minimal because the intercostal space is bypassed. Moreover, this technique enables exposure to both lungs through a single incision and has potential for widespread use if maneuverability can be increased by improving the instruments used., (Copyright © 2014 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)

The identification of cancer biomarkers is critical for target-linked cancer therapy. The overall level of phosphatidylcholine (PC) is elevated in colorectal cancer (CRC). To investigate which species of PC is overexpressed in colorectal cancer, an imaging mass spectrometry was performed using a panel of non-neoplastic mucosal and CRC tissues. In the present study, we identified a novel biomarker, PC(16:0/16:1), in CRC using imaging mass spectrometry. Specifically, elevated levels of PC(16:0/16:1) expression were observed in the more advanced stage of CRC. Our data further showed that PC(16:0/16:1) was specifically localized in the cancer region when examined using imaging mass spectrometry. Notably, because the ratio of PC(16:0/16:1) to lyso-PC(16:0) was higher in CRC, we postulated that lyso-PC acyltransferase (LPCAT) activity is elevated in CRC. In an in vitro analysis, we showed that LPCAT4 is involved in the deregulation of PC(16:0/16:1) in CRC. In an immunohistochemical analysis, LPCAT4 was shown to be overexpressed in CRC. These data indicate the potential usefulness of PC(16:0/16:1) for the clinical diagnosis of CRC and implicate LPCAT4 in the elevated expression of PC(16:0/16:1) in CRC., (© 2013 Japanese Cancer Association.)

Biallelic inactivating germline mutations in the base excision repair MUTYH (MYH) gene have been shown to predispose to MUTYH-associated polyposis (MAP), which is characterized by multiple colorectal adenomas and carcinomas. In this study, we successfully prepared highly homogeneous human MUTYH type 2 recombinant proteins and compared the DNA glycosylase activity of the wild-type protein and fourteen variant-type proteins on adenine mispaired with 8-hydroxyguanine, an oxidized form of guanine. The adenine DNA glycosylase activity of the p.I195V protein, p.G368D protein, p.M255V protein, and p.Y151C protein was 66.9%, 15.2%, 10.7%, and 4.5%, respectively, of that of the wild-type protein, and the glycosylase activity of the p.R154H, p.L360P, p.P377L, p.452delE, p.R69X, and p.Q310X proteins as well as of the p.D208N negative control form was extremely severely impaired. The glycosylase activity of the p.V47E, p.R281C, p.A345V, and p.S487F proteins, on the other hand, was almost the same as that of the wild-type protein. These results should be of great value in accurately diagnosing MAP and in fully understanding the mechanism by which MUTYH repairs DNA in which adenine is mispaired with 8-hydroxyguanine., (© 2010 Wiley-Liss, Inc.)

We previously reported that fecal cyclooxygenase 2 (COX-2) mRNA assay, detecting COX-2 mRNA in feces, is useful for identifying subjects with colorectal cancer (CRC). To further improve the sensitivity, we evaluated the usefulness of the combination of COX-2 mRNA and matrix metalloproteinase 7 (MMP-7) mRNA assays as a marker of CRC. The study cohort included 62 patients with CRC and 29 control patients without colorectal neoplasia. RNA was isolated from routinely collected fecal samples. The expression levels of COX-2 and MMP-7 mRNAs were determined by nested reverse transcription-PCR. PCR conditions were optimized where the specificity of fecal COX-2 and MMP-7 mRNA assay result in 100%. The sensitivity of each fecal assay was 87% [95% confidence interval (95% CI), 76-94%] and 65% (95% CI, 51-76%) for CRC, respectively. The sensitivity of fecal RNA test (either marker being positive) was high for CRC (90%; 95% CI, 80-96%). The sensitivity of the fecal RNA test was also high (93%; 95% CI, 80-98%) in patients with stage I or II who are often cured by surgical resection. The fecal RNA test using COX-2 and MMP-7 mRNAs improved the sensitivity to detect CRC without decreasing the specificity. These results suggest that the fecal RNA test would be a promising approach for CRC screening, although larger clinical investigations are indicated.

Background and Aims: Chromosomal instability (CIN) is recognised as a hallmark of cancer and is caused by a spindle assembly checkpoint disorder or chromosome mis-segregation during mitosis. Although the recent identification of human shugoshin (hSgo1), an important player in proper chromosome segregation, has suggested the involvement of hSgo1 in colorectal tumourigenesis, little is known about how it is involved. The aim of this study was to obtain information about the status of hSgo1 in human colorectal cancer., Method and Results: Among the 46 colorectal cancer cases, hSgo1 mRNA expression was decreased in the tumour tissue in comparison with the corresponding normal tissue (p = 0.032). Human Sgo1-downregulated tumours (tumour to normal mucosa ratio<0.5) had preferential location on the left side large bowel rather than on the right side (p = 0.012), and a higher variation of centromere numbers revealed by fluorescence in situ hybridisation (FISH). To assess the effects of hSgo1 downregulation, hSgo1 knockdown was performed by transfecting the diploid HCT116 cell line with a short hairpin RNA expression vector. hSgo1 knockdown cells proliferated slowly because of both G(2)/M arrest and apoptosis (p<0.001), and markers of CIN in the form of aneuploidy (p<0.001) and micronuclei (p<0.005) were later observed in hSgo1 knockdown cells. Increased centrosome amplification (p<0.05), the presence of binucleated cells and mitotic catastrophes were also noted in hSgo1 knockdown cells., Conclusions: These findings suggest that hSgo1-downregulated colorectal cancers have a clinicopathological character of CIN, and hSgo1 downregulation leads to CIN in colorectal cancer cells.

The Eph gene family has important roles in the developmental processes and may also be involved in the initiation, progression, and metastasis of certain types of cancers. In the present study, quantitative real-time reverse-transcriptase PCR was performed to detect the expression of EphA1 transcript in 5 colon cancer cell lines and 75 colorectal carcinomas. Immunohistochemical staining was used to check the expression of EphA1 protein in 20 colorectal adenomas and in 111 colorectal carcinomas specimens. EphA1 protein expression was not completely consistent with transcript expression. EphA1 protein was expressed in all adenomas and reduced in 54% colorectal cancers. Reduced expression of EphA1 protein occurred more often in male patients (P=0.028) and in patients with poor differentiation (P=0.027), greater depth of wall invasion (P=0.003), lymph node metastasis (P=0.034), and advanced tumor stage (P=0.003). Patients with reduced EphA1 expression had a poor overall survival (P=0.059). Reduced EphA1 expression in patients over 55 years or with rectal cancers and sigmoid colon cancers is associated with a poor overall survival (P=0.034 and 0.015, respectively). Our data indicate that the EphA1 may play different roles during the different stages of colorectal carcinoma progression.odern Pathology (2009) 22, 151-160; doi:10.1038/modpathol.2008.188; published online 14 November 2008.

The MUTYH gene encodes a DNA glycosylase that can initiate the base excision repair pathway and prevent G:C > T:A transversion by excising adenine mispaired with 8-hydroxyguanine. Biallelic germline mutations of MUTYH have been shown to predict familial and sporadic multiple colorectal adenomas and carcinomas, however, whether there is an association between single nucleotide polymorphisms (SNPs) of MUTYH and sporadic colorectal cancer (CRC) risk has remained unclear. In this study we investigated four MUTYH SNPs, IVS1+11C > T, IVS6+35G > A, IVS10-2A > G, and 972G > C (Gln324His), for an association with increased CRC risk in a population-based series of 685 CRC patients and 778 control subjects from Kyushu, Japan. A statistically significant association was demonstrated between IVS1+11T and increased CRC risk (odds ratio [OR]: 1.43; 95% confidence interval [CI]: 1.012-2.030; P = 0.042) and one of the five haplotypes based on the four SNPs, the IVS1+11T - IVS6+35G - IVS10-2A - 972C (TGAC) haplotype containing IVS1+11T, was demonstrated to be associated with increased CRC risk (OR, 1.43; 95% CI, 1.005-2.029; P = 0.046). Subsite-specific analysis showed that the TGAC haplotype was statistically significantly (P = 0.013) associated with an increased risk of distal colon, but not proximal colon or rectal cancer. Furthermore, IVS1+11C > T was found to be in complete linkage disequilibrium with -280G > A and 1389G > C (Thr463Thr). The results indicated that Japanese individuals with - 280A/IVS1+11T/1389C genotypes or the TGAC haplotype are susceptible to CRC.

Background: Over- or underexpression of certain Eph receptors has been associated with tumorigenesis of some types of cancer. EphB1 is a member of receptor tyrosine kinases of the EphB subfamily involved in the development, progress and prognosis of colorectal cancers. The expression levels of EphB1 in colon cancer cell lines and human colorectal carcinoma specimens were determined and association of EphB1 expression with clinicopathological parameters was analyzed., Methods: Quantitative real-time reverse transcription polymerase chain reaction and immunohistochemistry were used., Results: The EphB1 transcript is expressed in all colon cancer cell lines tested. However, there is marked variability in the expression of the EphB1 transcripts and proteins among colorectal carcinoma specimens. Reduced expression of EphB1 in colorectal cancers more often occurred in poorly differentiated and mucinous adenocarcinomas than in well- and moderately differentiated adenocarcinomas. Further, cancer cells with a low level of EphB1 protein showed more invasive power., Conclusion: Our data indicate that EphB1 may have roles in the pathogenesis and development of colorectal cancer., (Copyright 2008 S. Karger AG, Basel.)

Aim: To investigate associations between the Rsa I polymorphism of CYP2E1 and risk of colorectal cancer., Methods: A case-control study was conducted with 315 colorectal cancer cases (105 colon, 210 rectal) and 439 population-based controls in Jiangsu Province of China. Genomic DNA samples were assayed for restriction fragment length polymorphisms in CYP2E1 by PCR amplification followed by digestion with Rsa I. Information on smoking and alcohol drinking was collected using a questionnaire. Odds ratios (ORs) were estimated with an unconditional logistic model., Results: The proportional distribution of the CYP2E1 Rsa I c1/c1, c1/c2 and c2/c2 genotypes were 61.4%, 35.6% and 3.0% in controls, 60.6%, 33.7% and 5.8% in colon cancer cases, and 58.4%, 34.0% and 7.7% in rectal cancer cases, respectively. A significant difference was noted between controls and rectal cancer cases (P = 0.029), the c2/c2 genotype being associated with elevated OR (adjusted age, sex and status of the smoking and alcohol drinking) for rectal cancer (1.64, 95% CI, 1.12-2.41, vs c1 allele carriers), but not for colon cancer. In interaction analysis between the CYP2E1 Rsa I genotype and smoking and drinking habits, we found a significant cooperative action between the c2/c2 genotype and alcohol drinking in the sex-, age-adjusted ORs for both colon (4.74, 95% CI, 1.10-20.40) and rectal (5.75, 95% CI, 1.65-20.05) cancers. Among non-smokers, the CYP2E1 Rsa I c2/c2 genotype was also associated with elevated ORs in the two sites (1.95, 95% CI, 0.99-3.86 and 2.30, 95% CI, 1.32-3.99)., Conclusion: The results of the present study suggest that the CYP2E1 c2/c2 genotype increases susceptibility to rectal cancer and the gene-environmental interactions between the CYP2E1 polymorphism and smoking or alcohol drinking exist for colorectal neoplasia in general.

A significant reduction of EphA7 expression in human colorectal cancers was shown using semiquantitative reverse transcription-polymerase chain reaction analysis in 59 colorectal cancer tissues, compared to corresponding normal mucosas (P=0.008), and five colon cancer cell lines. To investigate the mechanism of EphA7 downregulation in colorectal cancer, we examined the methylation status of the 5'CpG island around the translation start site in five colon cancer cell lines using restriction enzymes, methylation-specific PCR, and bisulfite sequencing and found evidence of aberrant methylation. The expression of EphA7 in colon cancer cell lines was restored after treatment with 5-aza-2'-deoxycytidine. Analysis of methylation status in totally 75 tumors compared to clinicopathological parameters revealed that hypermethylation of colorectal cancers was more frequent in male than in female (P=0.0078), and in moderately differentiated than in well-differentiated adenocarcinomas (P=0.0361). There was a tendency that hypermethylation in rectal cancers was more frequent than in colon cancers (P=0.0816). Hypermethylation was also observed in colorectal adenomas. This is the first report describing the downregulation of an Eph family gene in a solid tumor via aberrant 5'CpG island methylation. It provides the evidence that EphA7 gene is involved in human colorectal carcinogenesis.

Background & Aims: Cyclooxygenase 2 (COX-2) is overexpressed frequently in aerodigestive tumors, especially in colorectal tumors. Therefore, it may be a suitable biomarker for colorectal cancer (CRC) screening. We performed a pilot study of whether detecting COX-2 expression in fecal RNA enables us to discriminate between patients with and without CRC., Methods: The study cohort included 29 patients with CRC, and 22 control patients without neoplastic disease of the colon or rectum. RNA was isolated from routinely collected stool samples using a modified method. The expression levels of carcinoembryonic antigen (CEA) and COX-2 were determined by nested reverse-transcription polymerase chain reaction (RT-PCR)., Results: The sensitivity and the specificity of fecal COX-2 assay for CRC were 90% (95% confidence interval [CI], 73%-98%) and 100% (95% CI, 85%-100%), respectively, whereas those of the fecal CEA assay for CRC were 100% (95% CI, 88%-100%) and 5% (95% CI, 2%-23%), respectively. COX-2 messenger RNA (mRNA) was detected in 3 of 4 patients with Dukes' stage A, 13 of 14 patients with Dukes' stage B, and 10 of 11 patients with Dukes' stage C or D. COX-2 mRNA was detected in 5 of 7 patients with proximal cancer and in 21 of 22 patients with distal cancer. The COX-2 assay was superior to the CEA assay for detecting CRC in terms of specificity, although both assays had high sensitivity., Conclusions: This fecal COX-2 assay had high sensitivity and high specificity for detecting CRC. These results suggest that it would be a promising approach for detecting CRC, although a larger study is necessary to assess the sensitivity and the specificity.

Evidence suggests that the erythropoietin-producing hepatocellular (EPH) receptor tyrosine kinases (RTKs) and their ephrin (EFN) ligands are involved in human carcinogenesis. Expression of two of them, EFNA1 ligand and its receptor, EPHA2, has been proposed to contribute to tumor-induced neovascularization. Colorectal cancers were examined for expressions of EPHA2 and its ligand EFNA1 by semi-quantitative RT-PCR, and double-immunostained for EPHA2 and CD34. Microvessels in the tumors were counted. Double-staining was also performed in 25 cases of adenoma with focal cancer for comparison. Trends of overexpression of both EPHA2 and EFNA1 was found in tumor tissue compared to the corresponding normal tissue in the same specimen [22/37 (59.5%) and 25/37 (67.5%), respectively; P = 0.100 for EPHA2 and P = 0.009 for EFNA1]. Overexpression of EPHA2 and EFNA1 was noted more frequently in the early stage than in the late stage [EPHA2, 15/21 (71.4%) vs. 7/16 (43.8%), P = 0.007; EFNA1, 15/21 (71.4%) vs. 10/16 (62.5%), P = 0.007]. Both EPHA2 and EFNA1 were more frequently overexpressed in smaller tumors (less than 5 cm) than in larger tumors [EPHA2, 15/21 (71.4%) vs. 7/16 (43.8%), P = 0.017; EFNA1, 16/21 (76.2%) vs. 8/16 (50%), P = 0.001]. Tumors less than 5 cm in diameter and in stages I and II were significantly more likely to overexpress EPHA2 and EFNA1 (P = 0.001 for EPHA2, P = 0.001 for EFNA1). Microvessel counts (MVCs) after immunostaining for CD34 were significantly correlated (r = 0.343, P = 0.037) with overexpression of EPHA2. EPHA2-expressing focal cancer also surrounded microvessels in adenomas with focal cancers. These findings suggest an involvement of EPHA2 in colon carcinogenesis, mainly in stages I and II, and probably through their effect on microvessel induction.

EphB2, a member of the Eph receptor protein-tyrosine kinase family, is overexpressed in several human gastrointestinal tumors. Furthermore, the EphB2 gene is localized at 1p35-p36.1, a frequently deleted region in colon and other cancers. So, despite its overexpression in some kind of tumors, we decided to study the possibility of involvement in the EphB2 gene (EPHB2) mutation in colon cancers, because some of the well known tumor suppressor genes (e.g. p53) is overexpressed (really accumulated) in tumors. Fifty colon tumor samples of matched with their respective normal tissues, were studied for mutation of the EPHB2. Analysis of the genomic structure of EphB2 and survey of all 16 exons revealed an infrequent polymorphism (intron 2) and mutation (intron 8). Another polymorphism in exon 6, localized at nucleotide 1359 (A-->G) was found to be rather frequent in Japanese and Chinese subjects, but very rare in Caucasians. Taking advantage of this polymorphism within EPHB2, we surveyed the loss of heterozygosity (LOH) status of this gene in Japanese colorectal tumors. Among the 50 samples analyzed, 24 were informative, and LOH was found in five of the15 (33.3%) informative rectal cancer cases. Mutation analysis covering all 16 exons in the remaining allele did not reveal any mutations. Thus, EPHB2 is not a classical tumor suppressor gene.

The insulin-like growth factor II receptor (IGFIIR) gene has been identified as a coding region target of microsatellite instability in human gastrointestinal (GI) tumors. IGFIIR normally has two growth-suppressive functions: it binds and stimulates the plasmin-mediated cleavage and activation of the latent transforming growth factor-beta1 (LTGF-beta1) complex, and it mediates the internalization and degradation of IGFII ligand, a mitogen. We used an immunohistochemical approach to determine whether IGFIIR mutation affected expression of these proteins in GI tumors. Four highly specific antibodies were used: LC(1-30), which recognizes the active form of TGF-beta1; anti-LTGF-beta1, which detects the LTGF-beta1 precursor protein; anti-IGFIIR; and anti-IGFII ligand. Twenty GI tumors either with (6 of 20) or without (14 of 20) known IGFIIR mutation were examined, along with matching normal tissues. Results were statistically significant in the following categories: (a) decreased active TGF-beta1 protein expression in IGFIIR-mutant tumor tissues versus matching normal tissues or IGFIIR-wild-type tumor tissues; (b) increased LTGF-beta1 protein expression in IGFIIR-mutant tumor tissues versus matching normal tissues or IGFIIR-wild-type tumor tissues; and (c) increased IGFII ligand protein expression in IGFIIR-mutant tumor tissues versus matching normal tissues or IGFIIR-wild-type tumor tissues. These data suggest that in genetically unstable GI tumors, mutation of a microsatellite within the coding region of IGFIIR functionally inactivates this gene, causing both diminished growth suppression (via decreased activation of TGF-beta1) and augmented growth stimulation (via decreased degradation of the IGFII ligand).

Genomic instability at simple repeated sequences has been observed in various types of human cancers and is considered an important mechanism in tumorigenesis. Alterations at microsatellite loci have been reported scattered throughout the genome. Recently, the transforming growth factor-beta receptor type II (TGF-beta RII) and the insulin-like growth factor II receptor (IGF-IIR) genes were shown to have inactivating mutations within coding microsatellite sequences. The demonstration of mutations in two growth regulatory genes supports the idea that other regulatory genes with repeat sequences may also be targets in tumours with defective mismatch repair. We examined genes involved in tumour suppression, cell adhesion and cell cycle regulation for mutations at small repeat sequences in replication error positive gastrointestinal cancers. Several polymorphisms were found which exhibited instability, but no other instability was present in the regions examined.

An 1194-nucleotide complementary DNA clone, FM1, encoding a human high-mobility group-1 protein (HMG-1) was isolated from a well-differentiated human gastric-carcinoma cell line complementary DNA library by a differential screening method. FM1 is similar to the published human HMG-1 in mature protein, with only 3 different codons at positions 11, 149, and 190. We analyzed 33 gastric and colorectal adenocarcinomas for expression of the FM1 gene. Northern-blot analysis revealed that all of the cancers expressed FM1 at a higher level than in corresponding non-cancerous mucosa, with 2 transcripts of approximately 1.4 and 2.4 kilobases. The FM1 expression level in the non-cancerous tissues increased with the depth of accompanying cancer invasion. Only 18.2% of well-differentiated cancers showed a higher expression level in corresponding non-cancerous tissues, whereas the expression in corresponding non-cancerous tissues was significantly higher in moderately (60%) and poorly differentiated (83.3%) cancers. In situ hybridization demonstrated the location of FM1 mRNA in well- and poorly differentiated gastric-cancer cells as well as in non-cancerous tissue adjacent to poorly differentiated gastric cancer, but no hybridization was detected in normal epithelial cells adjacent to well-differentiated gastric cancer. These findings may provide new information on HMG-1 mRNA expression in human gastrointestinal cancer and suggest a correlation between FM1 mRNA expression to the differentiation and the stage of human gastrointestinal adenocarcinomas.

Mutations within microsatellite sequences, consisting of additions or deletions of repeat units, are known as the replication/repair error positive (RER+) phenotype or micorsatellite instability (MI). Microsatellite instability has been demonstrated in hereditary and sporadic colorectal carcinomas and is usually observed in noncoding regions of genomic DNA. However, relatively few coding region targets of MI have been identified thus far. Using PCR, we amplified regions encompassing (A)8 and (C)8 microsatellite tracts within hMSH3 and hMSH6 from 31 RER+ sporadic colorectal tumors, 8 hereditary colon cancers, 23 RER+ gastric carcinomas, and 32 RER- gastric tumors. Mutations were found in 11 (36%) of 31 sporadic colon carcinomas, 4 (50%) of 8 hereditary colorectal cancers, and 5 (22%) of 23 RER+ gastric carcinomas, but in only 2 (6%) of 32 RER- gastric carcinomas. These frameshift mutations cause premature stop codons downstream that are predicted to abolish normal protein function. Our results and those of others suggest that DNA mismatch repair genes, such as hMSH3 and hMSH6, are targets for the mutagenic activity of upstream mismatch repair gene mutations and that this enhanced genomic instability may accelerate the accumulation of mutations in RER+ tumors.

Overexpression of the p53 protein was analyzed in colorectal adenomas, early cancers, advanced cancers and their hepatic metastasis by immunostaining using monoclonal antibody (DO-7). Positive staining for p53 was detected in 8 (22.9%) of 35 adenomas, 19 (82.6%) of 22 early cancers, and 59 (63.2%) of 95 advanced cancers. In adenomas, p53 immunoreactivity for severe dysplasia was higher than that for mild or moderate dysplasia (3/9.33.3% vs 2/16.12.5% or 3/10.33.3%), we found almost the same p53 immunoreactivity both in early cancers in adenoma (7/8.87.5%) and "de novo" cancers without an adenoma component (12/14. 85.7%). In 95 of advanced cancers, we found p53 positive cells in 33 (76.7%) of 43 cases with hepatic metastasis, against 26 (50.0%) of 52 cases without hepatic metastasis and surviving more than 5 years from the colorectal cancer resection. The former group had a significantly higher incidence of p53 overexpression (p < 0.05). In the 33 above mentioned cases, both primary and liver metastatic lesions were p53 positive, and the others were negative in both of them. In this series, it was suggested that p53 had important roles in the colorectal adenoma-carcinoma sequence from adenoma to advanced cancer and its metastatic potential, as well as in "de nove" carcinoma.

The prevalence of immunoreactive p53 and argyrophilic nucleolar organizer region (AgNOR) numbers were compared between colorectal cancers with (n=44) and without (n=51) hepatic metastasis for at least 5 years. At the same time, the distribution of p53-positive cells in primary, metastatic, and xenografted tumors from the same individuals were studied. Overall, p53 positivity was found more frequently in the cases with hepatic metastasis than in non-metastatic controls, regardless of the distribution pattern (P<0.05), whereas AgNOR counts were not different between the two groups. Significant heterogeneity in the distribution of p53 immunoreactivity was noted in both the primary and metastatic lesions. The intratumor distribution patterns of p53 immunoreactive cells in the primary (n=33), metastatic (n=33), and xenografted (n=7) tumors of the same individuals were consistent in the majority of cases. There were a few cases in which the p53 immunoreactive cells were more dominant in the metastatic tumor cells. Our observations suggest that p53 accumulation in colorectal cancer is associated with increased risk for hepatic metastasis, while cell proliferation as represented by AgNOR numbers is not. In addition, heterogeneity of abnormal p53 accumulation in the tumor is maintained during the course of metastasis and even after implantation in nude mouse. p53-Immunoreactive cells in the population of colorectal cancer cells do not necessarily have higher metastasizing potential.