Colorectal Adenocarcinoma Specimens Research Articles

Immunohistochemical detection of the BRAF V600E mutant protein in colorectal cancers in Taiwan is highly concordant with the molecular test.

The aims of this study were to investigate the incidence of BRAF mutations in colorectal cancers (CRCs) in Taiwan and the sensitivity and specificity of VE1 immunohistochemistry in detecting the BRAF(V) (600E) mutation. A total of 425 resected colorectal adenocarcinoma specimens were recruited into this study. Direct Sanger sequencing of exon 15 of the BRAF gene was performed for all cases. The incidence of BRAF mutation was 5.4% (23 of 425). Tissue microarrays were constructed for VE1 immunohistochemistry, and the staining intensity was scored as negative (0), weak (1+), moderate (2+) and strong (3+). In BRAF-mutated cases, two (8.7%) scored as 0, three (13.0%) as 1+, 13 (56.5%) as 2+ and five (21.7%) as 3+. Among 402 BRAF wild-type cases, five (1.2%) were scored as 1+, while the others were negative. The sensitivity and specificity of VE1 expression in detecting the BRAF mutation was 91.3% and 98.8%, respectively. Immunohistochemistry for VE1 antibody is a sensitive and specific marker for detection of BRAF mutations in CRCs. Incorporation of VE1 immunohistochemistry into Lynch syndrome screening protocol may be a reliable and cost-effective method.

Mutation analysis in colorectal adenocarcinoma specimens to targeted treatment.

e14639 Background: This study was conducted to assess whether tumors with mutations in KRAS codon 12 represent a less aggressive subtype compared to those with mutations in KRAS codon 13. Furthermo...

Enhanced miR-182 transcription is a predictor of poor overall survival in colorectal adenocarcinoma patients

Colorectal cancer is the second most frequent cause of cancer-related death in the developed world. Recent studies have tried to associate colorectal cancer with the aberrant expression of several microRNAs. The aim of the present study was the development of a highly sensitive quantitative real-time PCR which can be used to evaluate the miR-182 expression levels in colorectal adenocarcinoma and adjacent non-cancerous tissue specimens and associate them with several clinicopathological characteristics, aiming to examine the prognostic potential of miR-182. Total RNA was isolated from 116 malignant colorectal adenocarcinoma specimens and 60 paired non-cancerous tissues. Then, polyadenylation of 2 μg total RNA by poly(A) polymerase and reverse transcription with suitable oligo-dT-adapter followed. miR-182 levels were quantified by real-time PCR based on SYBR Green chemistry. The results were analyzed by the comparative quantification cycle method and by extensive biostatistical analysis. miR-182 was found to be significantly upregulated in colorectal adenocarcinoma specimens compared to their non-cancerous counterparts (p<0.001). miR-182 expression increases as the histological grade increases (p=0.013). miR-182 overexpression is associated with high depth of tumor invasion, positive regional lymph node status, and advanced TNM stage of patients. Therefore, miR-182 is an unfavorable prognostic marker in colorectal adenocarcinoma, predicting poor overall survival (p=0.007). Most importantly, miR-182 expression retained its unfavorable prognostic significance among patients with well- or moderately differentiated colorectal adenocarcinoma (p=0.006) and among metastasis-free patients (p=0.025). The increased levels of the oncogene-like miR-182 increase the risk for disease progression and predict poor overall survival for colorectal adenocarcinoma patients.

ROS1 and ALK Fusions in Colorectal Cancer, with Evidence of Intratumoral Heterogeneity for Molecular Drivers

Activated anaplastic lymphoma kinase (ALK) and ROS1 tyrosine kinases, through gene fusions, have been found in lung adenocarcinomas and are highly sensitive to selective kinase inhibitors. This study aimed at identifying the presence of these rearrangements in human colorectal adenocarcinoma specimens using a 4-target, 4-color break-apart FISH assay to simultaneously determine the genomic status of ALK and ROS1. Among the clinical colorectal cancer specimens analyzed, rearrangement-positive cases for both ALK and ROS1 were observed. The fusion partner for ALK was identified as EML4 and the fusion partner for one of the ROS1-positive cases was SLC34A2, the partner for the other ROS1-positive case remains to be identified. A small fraction of specimens presented duplicated or clustered copies of native ALK and ROS1. In addition, rearrangements were detected in samples that also harbored KRAS and BRAF mutations in two of the three cases. Interestingly, the ALK-positive specimen displayed marked intratumoral heterogeneity and rearrangement was also identified in regions of high-grade dysplasia. Despite the additional oncogenic events and tumor heterogeneity observed, elucidation of the first cases of ROS1 rearrangements and confirmation of ALK rearrangements support further evaluation of these genomic fusions as potential therapeutic targets in colorectal cancer. ROS1 and ALK fusions occur in colorectal cancer and may have substantial impact in therapy selection.

KRAS and BRAF Mutation Analysis in Colorectal Adenocarcinoma Specimens with a Low Percentage of Tumor Cells

Background and ObjectiveThe rapid development of molecular biology techniques allows for the introduction of real-time polymerase chain reaction (PCR) methods with a limit of mutation detection at 1 % in a background of wild-type DNA. Analysis of KRAS mutations in codons 12, 13, and 61, together with analysis of BRAF mutations in codon 600, are predictive biomarkers for anti-epidermal growth factor receptor (EGFR) treatment in colorectal cancer. Our aim was to compare PCR methods for KRAS mutations and BRAF mutation analysis using DNA isolated from tissue samples previously evaluated for presence of tumor cells using a quantitative scale and the percentage of tumor cells (PTC) scale. We addressed the question of whether a low number of tumor cells can be qualified for somatic mutation testing.ResultsOur study showed that PTC as low as 10 % was good enough to detect KRAS G12D, G13D, and Q61L mutations in formalin-fixed paraffin-embedded (FFPE) material. Furthermore, our results indicate that up to 20 % of colorectal cancer may carry mutations in the KRAS codon 61 and BRAF codon 600, which suggests the value of these mutation analyses because patients carrying them are unlikely to respond to cetuximab or panitumumab. A low level of KRAS somatic mutation detection has not been studied in depth in the context of clinical outcomes in patients; therefore, we compared new PCR methods, (KRBR-RT 50 Entrogen; ViennaLab StripAssay) and re-evaluated KRAS and BRAF status in patients with relapse after targeted therapy.ConclusionsThe importance of molecular results was confirmed by clinical observation of a patient with relapse who had qualified for targeted therapy with KRAS WT status (but was diagnosed by less sensitive single-stranded conformation polymorphisms method). Interestingly, during anti-EGFR treatment, it came to the selection of cells with KRAS G12C mutation which were present from the beginning in the tumor but at a low level (detected by PCR methods) only and led consequently to the metastasis. Taking into consideration the limit of detection, labor time, and assay cost, the real-time PCR method seems to be very promising especially for FFPE material with the PTC below 15 %.

Morphological distribution of μ chains and cd15 receptors in colorectal polyp and adenocarcinoma specimens

BackgroundWe have recently investigated the localisation of immunoglobulin-producing cells (IPCs) in inflamed intestinal tissue samples from patients with inflammatory bowel disease (IBD), and identified two main patterns of B lymphocyte infiltration: one characterised by the moderate strong stromal localisation of small B1 cell-like IgM+/CD79+/CD20-/CD21-/CD23-/CD5 ± IPCs, and the other by the peri-glandular localisation of IPCs with irregular nuclei that had surface markers specific for a B cell subset (IgM and CD79), but quantitative differences in their λ and κ chains. The same patients were also tested for CD15+ receptors, which were localised on inflammatory cell surfaces or in the crypts of the intestinal epithelium. CD15+ receptor distribution in inflamed tissues was limited to the cell structures. The aim of the study was to analyse variations in IPCs and CD15+ cell morphology or distribution in bowel biopsy specimens taken from patients with pre-malignant polyps or adenocarcinomas.MethodsIPCs were analysed by means of immunofluorescence using polyclonal goat anti-human μ chains. The pre-malignant polyp specimens were tested for B cell surface phenotype λ and κ chains, CD79, CD20, CD21 and CD23 using an immunoperoxidase method. CD15+ cells were evaluated using the immunoperoxidase method and monoclonal anti-CD15 IgM.ResultsThe study involved 14 patients (four with pre-malignant polyps and 10 with colorectal adenocarcinomas). The distribution of μ chains and CD15 markers varied in all of the biopsies, but delineated normal cell structures in the pre-malignant polyp specimens. B cell surface phenotype analysis of μ chain-positive cells identified a subset of CD79+/CD20-/CD21-/CD23- IPCs. The IPCs in certain areas showed the sporadic disintegration of inflammatory cell membranes or the accumulation of fluorescence in individual cells. IPC membrane disintegration was particularly marked in all of the adenocarcinoma samples, in which the CD15 markers also showed epithelial cell involvement. Furthermore, six of the ten adenocarcinoma samples had atypical and reorganised membranes that expressed an excess of both receptors and isolated small portions of tissue within the tumour.ConclusionThe findings of this preliminary morphological study suggest the presence of membrane disintegration and remodelling mechanisms in the tumours. The newly-formed membranes expressed high concentrations of inflammatory cell receptors that can confer adhesive properties.

Expression of GLI1 Correlates with Expression of Lymphangiogenesis Proteins, Vascular Endothelial Growth Factor C and Vascular Endothelial Growth Factor Receptor 3, in Colorectal Cancer

Aberrant activation of the hedgehog (Hh) signaling pathway is associated with tumorigenesis in various tissues. In colorectal cancer (CRC), evidence for Hh activation is inconsistent, and the relationship between the Hh signaling pathway and lymphangiogenesis has not been studied. The aim of this study was to determine the relationship between Hh signaling and lymphangiogenesis and the association of this relationship with lymph node metastasis in CRC. We investigated 189 patients who underwent curative surgical resection for CRC between 2002 and 2004 at Korea University Guro Hospital. Paraffin-embedded specimens of colorectal adenocarcinoma and adjacent normal mucosa were evaluated. Immunohistochemical staining for Sonic hedgehog (Shh), Gli1, vascular endothelial growth factor C (VEGFC), and VEGF receptor 3 (VEGFR3) was performed for each specimen. Tumor specimen showed significantly strong staining of Shh, Gli1, VEGFC, and VEGFR3 compared with a normal specimen. Shh expression was not associated with Gli1 expression. Gli1 expression correlated positively with VEGFC and VEGFR3 expression (P < 0.05 in both) but not with lymph node metastasis. Activation of the Hh signaling pathway associated with Gli1 promotes expression of lymphangiogenesis proteins, VEGFC and VEGFR3, in CRC. Further studies are necessary to determine the association of this relationship with lymph node metastasis in CRC.

Deficient mismatch repair phenotype is a prognostic factor for colorectal cancer in elderly patients

ObjectiveAbout 15% of colorectal adenocarcinomas have a deficient DNA mismatch repair phenotype. The frequency of deficient DNA mismatch repair tumours increases with age due to the hypermethylation of hMLH1 promoter. The study aimed to determine the prognostic value of deficient DNA mismatch repair phenotype in elderly patients. DesignMismatch repair phenotype was retrospectively determined by molecular analysis in consecutive resected colorectal adenocarcinoma specimens from patients over 75 years of age from 4 Oncology centres. Results231 patients (median age: 81, range: 75–100) were enrolled from 2005 to 2008. Mean prevalence of deficient DNA mismatch repair phenotype was 22.5%, and 36% for patients over 85 years. Deficient DNA mismatch repair status was significantly associated with older age, female sex, proximal colon primary and high grade tumour. For stage II tumours no deficient DNA mismatch repair tumours had a recurrence at end of follow-up compared to 17% for tumours with proficient phenotype. The proficient phenotype status was significantly associated with worse age-adjusted overall survival [HR 2.60; 95% CI 1.05–6.44; p=0.039]. For stage III tumours a trend for less recurrence was observed for deficient DNA mismatch repair phenotype (16%) compared to proficient phenotype (36%). Conclusiondeficient DNA mismatch repair phenotype is a prognostic factor in stage II colorectal tumour in elderly patients. Our results suggest that mismatch repair phenotype should be taken in consideration for adjuvant chemotherapy decision in elderly patients.

Concordance in KRAS and BRAF mutations in endoscopic biopsy samples and resection specimens of colorectal adenocarcinoma

BackgroundKRAS testing is mandatory if anti-EGFR therapy is considered in patients with metastatic colorectal cancer (CRC). In addition, BRAF mutations seem to be an important negative prognostic factor. The aim of this study is to establish the concordance of KRAS and BRAF mutational status in paired biopsy and resection specimens of primary CRC using several analytic methods. MethodsDNA was extracted from paraffin blocks of 126 CRC patients. KRAS codon 12/13 and BRAF V600E mutational status was assessed using high resolution melting (HRM), direct sequencing (DS) of the HRM polymerase chain reaction (PCR) product. In addition, the Therascreen Amplification Refractory Mutation System (ARMS)-Scorpion KRAS assay and BRAF pyrosequencing were employed; both assays claim to require less tumour cells in comparison with DS. ResultsKRAS and BRAF were found to be mutually exclusive. Mutation frequencies were 33.9% for KRAS, and for BRAF 19.0%, respectively. Concordance of KRAS mutational status between biopsy and resection specimens was 97.4% (ARMS), 98.4% (DS) and 99.2% (HRM), respectively. For BRAF concordance was 98.4% (Pyro, DS) and 99.2% (HRM). ConclusionsKRAS and BRAF mutational status of endoscopic biopsies and resection specimens of CRC showed a >95% concordance. Endoscopic biopsies can be confidently used for molecular analysis.

SAHA Inhibits the Growth of Colon Tumors by Decreasing Histone Deacetylase and the Expression of Cyclin D1 and Survivin

We studied the effects of suberoylanilide hydroxamic acid (SAHA), a histone deacetylase (HDAC) inhibitor, on colon cancer. The expression of HDACs in colorectal cancer specimens and the effects of SAHA on colon cancer cells and tumors of nude mice were assessed. Treatment with SAHA (3 μm) for 72 h induced downregulation of different subtypes of HDAC proteins and also induced acetylation of histone 3 and histone 4. SAHA significantly inhibited the expression of the oncogenic protein c-myc and also increased the expression of the p53 and Rb proteins. The immunohistochemical staining of HDACs, including HDAC1, HDAC2, HDAC3, and HDAC4, was significantly increased in colorectal adenocarcinoma specimens compared to healthy control tissues. In addition, murine studies showed that 100 mg/kg SAHA administered by intraperitoneal injection significantly induced tumor necrosis and inhibited the growth of colon tumors. Immunohistochemistry of the tumor tissues from nude mice revealed that SAHA inhibited the expression of different subtypes of histone deacetylase, the anti-apoptotic proteins cyclin D1, survivin, and also inhibited cell proliferative as determined by Ki67 expression. SAHA inhibited the growth of colon tumors by decreasing histone deacetylases and the expression of cyclin D1 and survivin in nude mice.

Correlation of polypoid colorectal adenocarcinoma with pre-existing adenomatous polyps and KRAS mutation

Cetuximab is an anti-epidermal growth factor receptor that helps effectively treat patients with advanced colorectal adenocarcinoma without KRAS activating mutations. KRAS mutations are associated with 16% to 50% of isolated villous adenomas and approximately 30% of colorectal cancer. Correlation between the gross and histological subset of colorectal adenocarcinoma with KRAS mutation is unknown. Archived surgical resection specimens of colorectal adenocarcinoma (n = 42) and villous adenoma (n = 9) were collected. The gross appearance and histopathological features of these lesions were thoroughly reviewed, including the presence of a pre-existing adenomatous polyp. DNA was extracted from formalin-fixed, paraffin-embedded tissue sections and then subjected to TaqMan real-time polymerase chain reaction to detect the seven most common KRAS mutations. KRAS mutations were found in 13 of 42 cases (31%) of colorectal adenocarcinoma and 7 of 9 cases (78%) of villous adenoma. All 13 cases of colorectal carcinoma with a KRAS mutation showed a gross polypoid configuration, compared to no KRAS mutation in the colorectal carcinomas with ulcerative configuration. In addition, 13 of 17 of these cases (76%) had histological features of adenocarcinoma with a persistent preexisting adenomatous polyp with villous architecture. In summary, grossly polypoid colorectal adenocarcinomas with a persistent pre-existing adenomatous polyp with villous architecture are strongly associated with KRAS mutations.

Abstract 5149: Intermedin stimulates cell growth, migration, and angiogenesis in human colorectal adenocarcinoma and hepatocellular carcinoma

Abstract Intermedin (IMD) functions as an angiogenic factor in a rat ischemic model and human endothelial cells. In our search for anti-angiogenic therapy for solid tumors, we analyzed the expression and possible role of IMD in tumor angiogenesis. IMD was expressed in human colorectal adenocarcinoma (CRC) and hepatocellular carcinoma (HCC). The IMD expression was significantly higher in stage I than stage 0 of both colon and liver tumors (stage I/0 ratio of mRNA level is 7.7 ± 0.4 and 3.2 ± 0.3, respectively). IMD was widely expressed in colon and liver tumor lines. Immunohistochemistry showed that the tumor regions were significantly more immunoreactive for IMD than adjacent benign regions of CRC and HCC specimens. Inhibition of IMD expression by RNA interference reduced cell proliferation by 29.7 ± 6.7% (n = 3, p = 0.047) and impaired cell invasion by 27.8 ± 3.6% (n = 3, p = 0.016) in a HCC cell line SK-Hep-1. Addition of IMD peptide for 5 minutes to serum starved SK-Hep-1 cells increased the phosphorylation level of p44 and p42 MAP Kinase (Erk1/2), suggesting that IMD regulates Sk-Hep-1 cell growth, at least partly, through activating Erk1/2 signaling pathway. Similarly, IMD knockdown in a CRC cell line HCT116 reduced cell growth by 35.0 ± 1.4 % (n = 3, p = 0.018). Conditioned medium from IMD siRNA-transfected SK-Hep-1 cells reduced endothelial cell tube formation by 31.6 ± 6.8%. Conversely, over-expression of IMD in CRC RKO cells increased cell growth and induced cell migration significantly. Furthermore, treatment with RKO/IMD conditioned medium increased endothelial cell tube formation by 14. 7 ± 0.8% (n=4, p = 0.049). Thus, IMD stimulated HCC and CRC cell growth, migration, and invasion, indicating a role for IMD in tumor angiogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5149. doi:10.1158/1538-7445.AM2011-5149

Lymph node harvest in colorectal adenocarcinoma specimens: the impact of improved fixation and examination procedures

A review of 1050 pathology reports from colorectal adenocarcinoma specimens examined at the Department of Pathology, Sørlandet sykehus HF, Kristiansand, Norway during the period 1995-2006 revealed a poor performance of most doctors concerning lymph node harvest. A mean of 8.1 nodes per specimen (range 12.3-2.1) and a mean proportion of 22.3% of specimens with ≥12 lymph nodes (range 47.1-0%) were found. A small pilot study was undertaken in 2007 to evaluate the effect of prolonged formalin fixation and the use of a special lymph node fixative [glacial acetic acid, ethanol, water and formaldehyde (GEWF) solution] with regard to the number of retrieved nodes. This showed that one extra day formalin fixation and the use of GEWF solution considerably enhanced the detection of lymph nodes, particularly those of smaller size. Based on these findings, our routines concerning handling of colorectal cancer specimens were changed during 2007. After this time all specimens have been fixed in a mixture of GEWF solution and formalin for at least 48 h and the doctors have been encouraged to find as many lymph nodes as possible. In cases revealing <12 nodes after microscopical examination, the specimens have been re-examined and searched for additional nodes. A review of lymph node retrieval in 423 cases of colorectal cancer during the period 2008-2010 showed that the mean number of nodes per specimen had increased to 16.8 (range 29.0-13.3) and the proportion of specimens with ≥12 nodes to 78.0% (range 96.8-63.6%). Thus, these changes of routines which were easy to implement without significant extra costs have considerably improved lymph node harvest at our department. The use of a special lymph node fixative (e.g. GEWF solution) is highly recommended not only for detection of nodes in colorectal specimens, but also for retrieval of lymph nodes embedded in fat tissue generally.

BDNF/TrkB Content and Interaction with Gastrin-Releasing Peptide Receptor Blockade in Colorectal Cancer

Objective: Neurotrophin and neuropeptide pathways are emerging targets in cancer. Here we show that brain-derived neurotrophic factor (BDNF) and its receptor, TrkB, are present in colorectal cancer and that BDNF levels are increased in tumors compared to nontumor tissue. In addition, we investigate the role of BDNF in influencing the response of colorectal cancer cells to inhibition of gastrin-releasing peptide receptors (GRPR). Methods: Fresh-frozen sporadic colorectal adenocarcinoma specimens and adjacent nonneoplastic tissue from 30 patients, as well as paraffin-embedded colorectal cancer samples from 21 patients, were used in this study. Cell proliferation and mRNA and protein levels were examined in HT-29 or SW620 cells treated with a GRPR antagonist, human recombinant BDNF (hrBDNF), a Trk antagonist K252a, or cetuximab. Results: Expression of BDNF and TrkB was detected in tumor samples and cell lines. BDNF levels were higher in tumor samples compared to nonneoplastic tissue. BDNF expression and secretion were increased by GRPR blockade in HT-29 cells through a mechanism dependent on epidermal growth factor receptors. Treatment with hrBDNF prevented the effect of GRPR blockade on cell proliferation, whereas a Trk inhibitor reduced proliferation. Conclusions: BDNF and TrkB are present in colorectal cancer and might contribute to resistance to GRPR antagonists.

The influence of surgeon and pathologist in obtaining high median lymph node yields after colorectal resection for adenocarcinoma

<h3>Introduction</h3> The number of lymph nodes evaluated in resected colorectal adenocarcinoma specimens is associated with patient survival and therefore used as a marker of quality in colon cancer care.1 The aim of this study was to assess whether individual surgeons and pathologists retrieving and analysing colorectal resection specimens at our centre differed in their lymph node yields (LNY). <h3>Methods</h3> Casenotes for all elective Right Hemicolectomies (RHC) and Anterior Resections (AR) performed at our centre for adenocarcinoma between 2008–2009 were identified using hospital coding and histology reports were assessed for LNYs. Total and positive LNYs were compared between surgeons and pathologists using GnuPSPP statistical software. <h3>Results</h3> Two dedicated colorectal surgeons and 5 pathologists produced 77 RHC and 125 AR (of which 62 were Total Mesorectal Excisions (TME)) specimens. Median total LNYs for each of our surgeons were: 22(16.5–28.6) and 23(17–31) p = 0.95 for RHC; 21(12–28) and 16(12.5–27) p = 0.37 for all ARs; and 19(9.5–25) and 15(12–24) p = 0.13 for TMEs. Positive LNYs from RHC (p = 0.5), AR (p = 0.80) and TME (p = 0.23) were also similar between the surgeons. Of the 5 pathologists the highest and lowest median total LNYs for RHC, AR and TME specimens were: 28(21–32) to 16.5(14.5–18.5); 28(21–35) to 14.5(10–19.72); and 25(16.5–34) to 9(8.5–16.5), respectively. Oneway ANOVA testing of the 5 pathologists total LNYs for RHC, AR and TME gave p values of 0.02, 0.00 and 0.01, respectively. The pathologist with the lowest LNY after TME had significantly more specimens (57%) treated with neoadjuvant radiotherapy than the other pathologists. Comparison of positive LNYs between pathologists showed no differences in yields with p = 0.05 for RHC, p = 0.80 for AR and p = 0.23 for TME. <h3>Conclusion</h3> Lymph node yields following colorectal resection at our centre are far higher than published yields from other centres.2 No difference was seen in the LNYs delivered by the 2 surgeons studied; however, there was a significant difference in total LNYs between our 5 pathologists; with some pathologists consistently producing huge yields. This did not, however, translate to a difference in positive LNYs produced by our pathologists although our study is underpowered to demonstrate equivalence.

Abstract 3358: Immunohistochemical features of stem cell transcription factors on colorectal carcinoma and adenoma in familial adenomatous polyposis

Abstract Background: The pathogenesis of colorectal cancer in familial adenomatous polyposis (FAP) has been studied genetically. Recently, intestinal stem cell (ISC) or cancer stem cell (CSC) is being focused on colorectal carcinogenesis. The aim of this study is to investigate the immunohistochemical features of stem cell transcription factors on colorectal adenocarcinomas, adenomataous polyps, and normal crypts in patients with FAP. Methods: Lgr5 as an ISC marker, CD133 and CD44 as CSC markers, and NANOG, OCT4, and SOX2 as embryonic stem cell transcription factors were evaluated immunohistochemically. Matched specimens of colorectal adenocarcinoma, adenomatous polyps, and adjacent normal crypts were obtained from individual FAP patients (n=4), to clarify the association between stepwise progression from normal crypt to adenomatous polyp to adenocarcinoma and expression of stem cell transcription factors. Results: Lgr5, NANOG, OCT4, and SOX2 were mainly located in cytoplasm of cancerous and adenomatous cells. CD133 showed cytoplasmic or intraluminal membranous staining in cancerous and adenomatous cells. Immunoreactive CD44 protein was present in cell membrane or cytoplasm of cancerous and adenomatous cells. Immunoreactive positivity and intensity was greater in colorectal adenocarcinomas than in adenomatous polyps, except for NANOG. Adjacent normal crypts showed negative or weak staining of all markers. CD133, CD44, Lgr5, OCT4, and SOX2 were strongly expressed on cancerous cells at the advancing front of colorectal adenocarcinoma. Conclusions: ISC or CSC may be associated with colorectal carcinogenesis of FAP. Stem cell transcription factors may be involved in tumor invasion of colorectal adenocarcinoma in FAP patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3358.

Computer‐assisted pathological immunohistochemistry scoring is more time‐effective than conventional scoring, but provides no analytical advantage

Interpretation of immunohistochemistry is primarily done through human visual scoring while computer-assisted scoring is relatively uncommon. This study aimed to examine (i) the level of agreement between human visual and computer-assisted pathological scoring of immunoreactivity expression in colorectal cancers, (ii) whether computer-assisted scoring affects the prognostic significance of biomarkers, and (iii) whether computer-assisted pathological scoring provides any time-saving or reproducibility advantages. Tissue microarray blocks were constructed from the primary colorectal adenocarcinoma specimens of 486 patients. Scoring of the six markers [cytokeratin (CK) 7, CK20, cyclooxygenase-2, Ki67, p27 and p53] was done independently by a qualified pathologist, a trained scientist and the Ariol SL-50 (Applied Imaging). Univariate analysis showed that human visual and computer-assisted scoring were strongly correlated (all kappa values >0.8). Both human visual and computer-assisted pathological scoring identified the same set of biomarkers with significant association with survival. Computer-assisted pathological scoring was shown to be a time-effective means of scoring larger numbers of slides (for high-throughput studies). Our results suggest that computer-assisted pathological scoring can be a viable alternative to pathologist scoring in a manner that is more practical and time-effective, but, interestingly, providing no analytical advantage.

TSPAN1 protein expression: A significant prognostic indicator for patients with colorectal adenocarcinoma

To determine if TSPAN1 overexpression is associated with clinicopathological and prognostic factors in human colorectal adenocarcinoma. Total RNA was extracted in 20 human adenocarcinoma tissues for TSPAN1 mRNA assay by RT-PCR. Eighty-eight specimens of human colorectal adenocarcinoma were surgically removed. TSPAN1 protein levels in cancer tissues were determined by immunohistochemistry using a polyclonal antibody against self-prepared TSPAN1. The correlation between TSPAN1 expression and the clinicopathological factors and the overall survival rate was analyzed by univariate and multivariate assay. TSPAN1 mRNA was detected in 90.0% (18/20) of cancerous tissues. The light density of TSPAN1 mRNA expression levels was 0.89 +/- 0.30 in adenocarcinoma by gel-image system. TSPAN1 protein expression was detected in 78.41% (69/88) and weakly expressed in 40% normal colorectal tissues. There were significant differences between colorectal adenocarcinoma and normal control epithelium (P < 0.05). TSPAN1 protein expression in colorectal cancerous tissue was significantly correlated with the histological grade, cell expression PCNA, lymph nodal metastasis and TNM staging of the disease. Patients with TSPAN1 protein overexpression had a significantly shorter survival period than that in patients with TSPAN1 protein negative or weak expression, respectively (P < 0.05). Furthermore, by multivariate analysis, TSPAN1 protein expression demonstrated an independent prognostic factor for human colorectal cancers (P < 0.05, relative risk 0.755; 95% confidence interval 0.302-1.208). The expression of TSPAN1 gene is increased in colorectal carcinoma, suggesting that TSPAN1 might serve as an independent prognostic factor for the colorectal adenocarcinoma patients.

Dysregulation of reg gene expression occurs early in gastrointestinal tumorigenesis and regulates anti-apoptotic genes

Expression of anti-apoptotic genes is frequently elevated in tumors, where they increase resistance to chemotherapeutic agents and predict poor patient outcomes. However, key cellular factors regulating anti-apoptotic genes in tumors remain unknown. Increased expression of the regenerating (Reg) genes is commonly observed in gastrointestinal (GI) malignancies including colorectal cancer (CRC). We therefore examined Reg gene expression and associated changes in anti-apoptotic genes in an animal model of GI tumorigenesis. Using real time RT-PCR, we measured expression of Reg genes in human colorectal adenocarcinoma specimens, colon adenocarcinoma cell lines and adenomas from multiple intestinal neoplasia (min) mice heterozygous for a germ-line mutation of the adenomatous polyposis coli (APC) gene. Expression of Reg genes is increased in human colorectal adenocarcinomas and in the intestine of APCmin/+ mice at 4 weeks of age, a time preceding the spontaneous second mutation in the APC gene. Individual Reg genes exhibited regional expression profiles across the GI tract in mice. Adenomas from 14-week old mice had significant increases in at least one member of the Reg gene family, most commonly Reg IV and an associated increase in expression of the anti-apoptotic gene, Bcl-2. Addition of exogenous recombinant human Reg IV to human colon adenocarcinoma cells significantly increased Bcl-2 and Bcl-xL expression and induced resistance to ionizing radiation. These results show that dysregulation of Reg genes occur early in tumorigenesis. Furthermore, increased expression of Reg genes, specifically Reg IV contribute to adenoma formation and lead to increased resistance to apoptotic cell death in CRC.

Prognostic Significance of p27kip1 Protein Expression and Spontaneous Apoptosis in Patients with Colorectal Adenocarcinomas

Tumor cell apoptosis, proliferation and p27 expression using an in situ apoptosis detection kit, anti-Ki67 antibody and anti-p27 antibody were investigated in 171 colorectal adenocarcinoma specimens, together with the assessment of mutated p53 expression. No apparent association was observed among p27 expression, mutated p53 accumulation, the Ki67 labeling index and the apoptotic index (AI). In the multivariate analysis using the median values as the cut-off points (46.8% for p27 expression and 0.89% for AI), p27 expression was identified as an independent and significant predictor for overall survival. When the present series of patients were dichotomized by these cut-off points to categorize the cases into 4 subgroups, the patients in the group with low p27 expression and a low AI had the poorest prognosis. The assessment of p27 expression and AI therefore may prove valuable in identifying patients with colorectal adenocarcinoma who may have a poor prognosis.