Clinico-pathological characteristics of CRC tumors in the study lot
1. Introduction
Colorectal carcinoma (CRC) is one of the most common human cancers. In 2008, 1.233.000 new CRC patients were diagnosed worldwide and about 608.000 deaths caused by colorectal cancer were estimated making it the fourth most common cause of death from cancer in the world. Five-year survival for CRC patients indicates a percent of 54.0% in Europe. Additionally, from the five-year survival, it was observed 74.0% of survival for patients with stage I, 66.5% for patients with stage IIA, 73.1% for patients with stage IIIA and only 5.7% for patients with stage IV disease (Stanczak, 2011). The success of colorectal cancer screening programs has resulted in an increasing number of biopsies of early neoplastic lesions with subtle histological features, making development of ancillary diagnostic testing for CRC essential. The incorporation of ancillary techniques, such as immunohistochemistry, cytochemical staining, electron microscopy, cytogenetic and, more recently, molecular testing, has made a significant impact in the diagnosis and management of solid tumors. Interpretation of hematoxylin-eosin stained slides by light microscopy remains the basic of anatomic pathology. However, an expanding menu of molecular assays continues to be implemented owing to their clinical utility in diagnosis, prognosis and risk assessment, therapy selection, as well as cancer screening and minimal residual disease detection. Carcinomas tend to carry multiple, complex, non-recurrent chromosomal and molecular aberrations, and they were not traditionally considered ideal candidates for molecular testing. However, this is changing with the discovery and implementation of new diagnostic, prognostic, and therapeutic molecular markers. Although single molecular biomarkers have proved useful, technical advances allowed performing the global genomic, epigenomic, or proteomic profiling of solid tumor malignancies. The research continues for more definitive molecular indicators that correlate with histological features and patient response to therapy and/ or survival.
Increasing understanding of cancer biology is beginning to explain the reasons for therapeutic failures. Signal transduction research have revealed that the receptors, enzymes and transcription factors that regulate cell fate are virtually all connected into an complex network of cross-regulatory interactions. The cell fate control system is not only interconnected but also highly redundant, such that if a gene or protein is disabled, another can perform a similar function (Rizzo, P, 2008). Key molecular mechanisms implicated in the genesis of CRC include chromosomal instability, DNA repair defects, and aberrant methylation. Chromosomal instability causes structural chromosomal anomalies, usually during DNA replication, with subsequent loss of tumor suppressor genes. DNA repair defects are caused by mutations in genes responsible for the repair of base-base DNA mismatches. These can be found as germline mutations or somatic methylation anomalies in acquired cases of CRC. A significant proportion of cases of CRC associated with mismatch repair anomalies occur on the right side of the colon and have a characteristic histological appearance. DNA repair defects can be detected indirectly by the associated epiphenomenon of microsatellite instability or unrepaired strand slippage within microsatellite regions.
Taking all these into account, we can conclude that study of colorectal carcinogenesis provides fundamental insights into the general mechanisms of cancer evolution. Now, it is believed that there are two patho-genetically distinct pathways for the development of colon cancer involving stepwise accumulation of multiple mutations. However, the genes involved and the mechanisms by which the mutations arisen are different.
The pathway, sometimes called the APC/ β-caterin pathway, is characterized by chromosomal instability that results in stepwise accumulation of mutations in a series of oncogenes and tumor suppressor genes. The molecular evolution of colon cancer along this pathway occurs through a series of morphologically identifiable stages. Initially, there is localized colon epithelial proliferation. This is followed by the formation of small adenomas that progressively enlarge, become more dysplastic, and ultimately develop into invasive cancers. This is referred to as the adenoma-carcinoma sequence. The genes that are correlated with this pathway are as follows:
Adenomatous Polyposis Coli (APC) -
β-Catenin is a member of the cadherin-based cell adhesive complex, which also acts as a transcription factor if the protein is translocated to the nucleus. When it is not bound to E-cadherin and participating in cell-to-cell adhesion, a cytoplasmic degradation complex (consisting of APC, Axin, GSK-3β, and β-catenin) leads to β-catenin phosphorylation and degradation. When
The E-cadherin gene (
Group IIA PLA2 is a 14-kDa enzyme found in a number of tissues and secretory products (Nevaleine TJ, 1993). The plasma concentration of the enzyme increases dramatically in severe infections and other diseases involving generalized inflammation and cancer (Ogawa M, 1991). In the gastrointestinal tract, expression of group IIA PLA2 has been localized in Paneth cells of the small intestine (Nevaleine TJ, 1995), metaplastic Paneth cells of gastric (Nevaleine TJ, 1995) and colonic mucosa (Haapamaki MM, 1999) as well as columnar epithelial cells of inflammeted colonic mucosa. Functional defects in PLA2 in tumor cells may interfere with the regulatory mechanisms of tumor growth. The
The EGFR is a member of the HER (human epidermal growth factor receptor) family, and includes HER1 (EGFR, ErbB-1), HER2 (ErbB-2), HER3 (ErbB-3), and HER4 (ErbB-4) (Boss JL, 1989). The natural ligands for EGFR include EGF, transforming growth factor (TGF), amphiregulin, heregulin, heparin-binding EGF, and cellulin. Ligand binding induces receptor dimerisation and subsequent auto-phosphorylation that activates critical pathways for cellular survival and proliferation such as PI3K/Akt, Stat, Src and MAPK. EGFR mediates signaling by activating the MAPK and PI3K signaling cascades (Jhawer M, 2008). EGFR modifications have been described in many cancers as a consequence of mutations or gene amplifications that induce protein over-expression, structural rearrangements and autocrine loops. EGFR abnormalities may have a relevant role in both carcinogenesis and clinical progression of CRC. EGFR is differentially expressed in normal, premalignant, and malignant tissues, and over-expression of EGFR has been documented in up to nearly 90% of cases of metastatic CRC (Boss JL, 1989; Arteaga CL, 2001). In addition, EGFR is over-expressed in a wide range of solid tumors and is involved in their growth and proliferation through various mechanisms. Given the documented role of EGFR in the development and progression of cancers, this receptor signaling pathway represents a rational target for drug development (Vokes EE, 2006; Lee JJ, 2007). Recent clinical data have shown that advanced colorectal cancer with tumor-promoting mutations of these pathways -- including activating mutations in KRAS, BRAF, and the p110 subunit of PI3K-- do not respond to anti-EGFR therapy.
The variability in clinical presentation, aggressiveness, and patterns of treatment failure suggests distinct genotypes and phenotypes identification, which can help future treatment strategies. A new concept called “personalized medicine” may be another beginning of a new era and it has been designed to offer every patient a suitable therapy. By this new approach, “Personalized medicine” can be defined as the tailoring of medical treatment to a specific subset of patients who are usually identified by genetic markers or other molecular profiling strategies. There is an increasing interest in this therapeutic strategy on the part of pharmaceutical and bio-pharmaceutical companies, consumers, and third party payers. Consequently, the level of clinical trial activity surrounding personalized medicines is intensifying as sponsors seek ways to target their therapies to patient populations that would most benefit from them. The aim of the present chapter is to elaborate an experimental model in order to improve the “personalized” therapeutically strategy, by evaluating some key gene expression involved into a crosstalk signaling, in colorectal cancer.
By our study design we have evaluated the comparative expression at proteic and genetic level of several key point proteins (
The idea of applying such a model to our studies was generated during the research that we conducted in our projects. We have noticed that between different proteins and genes is a very close relationship, which depends on the tumor type, cell grade and staging. Following a study of a large number of articles published in the international databases we observed that other researchers have drawn the same conclusion.
2. Results and discussion
2.1. Tissue samples and blood
Samples were obtained with the consent of 93 patients, consisting of histopatologically confirmed colorectal adenomas. Samples were obtained during colonoscopy with biopsy forceps, by harvesting at least four fragments from all the quadrants of the pathological tissue. The surgical intervention for CRC treatment included radical and palliative techniques (right or left hemicolectomy, segmentary colectomy, low anterior rectal resection–Dixon, Milles operation, Hartmann operation). All tumors were histologically (HP) examined by pathologist in order to: (a) confirm the diagnosis of adenocarcinoma, (b) confirm the presence of tumor and evaluate the percentage of tumor cells in these samples, and (c) carry out pathological staging. The complete HP diagnosis included: degree of differentiation (well/ moderate/ poor), vascular, neural and lymphatic invasion, status of the margins of resection (invaded/ noninvaded) and also TNM stadialisation. After surgical resection, tumor tissues were cut in small pieces, frozen immediately in liquid nitrogen and stored at - 800C until they were analyzed.
For the initial patients group, only 75 patients who had at least 75% tumor cells were taken in consideration for molecular biology analyses. To perform immunohistochemistry by immunofluorescence (IHF) analyses, five micrometers thick tissue serial sections were incubated with primary antibodies diluted in BSA (bovine serum albumin) in PBS (phosphate buffered saline). After washing with PBS, FITC-conjugated secondary antibodies (Invitrogen) were applied and then the samples were washed again. The protein expression was evaluated by fluorescent microscopy. In order to analyze the mutational status, DNA was extracted from patients’ venous blood (as control) and from tumours. DNA preparation was performed using the
2.2. Clinicopathological characteristics
The medical records of all 93 patients provided their birth date and sex, and the following parameters: tumor location, tumor size, lymph node metastases, pathological stage, vascular and neural invasion and tumoral differentiation grading.
Out of 93 cases, there were 40 womens and 53 mens. The mean age was 50 years. The majority had T3 tumors (31.8%); T2 tumors (25.80%) according to tumor stage of the TNM classification of colon and rectum neoplasm and 53 patients (57%) had lymph node involvement (N+). In the study lot, 17 cases (18.27%) presented metastasis at the time at CRC diagnosis. These were predominantly localized in the liver (12 cases, 70.58%) and rarely in the lungs (4 cases, 23.52%).
Regarding the histopathological type of colorectal tumors, the vast majority was adenocarcinomas (ADK) with different grades of differentiation. Most of the tumors (42 cases: 45.16%) were well differentiated (G1) while 33 cases (35.48 %) were moderately differentiated (G2) and 18 cases (19.35%) poor differentiated (G3) tumors. Beside typical adenocarcinoma another histopathological type of tumors was rare and was localized: i) to the right colon - especially mucinous ADK (5 cases from a total of 9 cases in the all study lot) and 1 adenosquamous carcinoma; ii) to the left colon - 2 cases of mucinous ADK and 1 case of “signet-ring” cell carcinoma; iii) to the rectum - 2 mucinous ADK, 1 squamocellular carcinoma and 1 case of anaplazic carcinoma. Patients characteristic is summarized in Table 1.
Our study has not taken into consideration the diet, because most of the patients do not know the food properties or they use food with pro-carcinogen potential. Regarding the diet, we consider that the patient instruction is extremely useful and has to be done by the surgeon doctor after the surgical treatment and then by the family doctor. This approach allows both secondary prophylaxis and control of possible relapses/ recidivists. A monitoring of the patients included in the study will shows the efficiency of medical control and the conscious of this mortal disease. In the studied lot of patients we have not registered cases with relapse, and we cannot predict their future behavior.
2.3. Immunohistochemical expression by immunofluoresce of the studied proteins
Because the interpretation of immunohistochemistry analyses remains the basic of anatomic pathology, in our study we first evaluated the protein expression of the key point proteins that were taken in our study. Unlike the normal histopathological analyses, our evaluation was based on protein fluorescent signal which, from our point of view, is more specific than classical immunohistochemistry.
The expression of α-SM (smooth muscle) was included in our study as a positive control to prove the method accuracy and it is used as a typical marker for myofibroblasts. It is one of the four muscle actin isoforms, a protein involved in supporting basic contractile apparatus in muscle cells. This expression can be found in vascular cells, intestinal muscularis mucosae and muscularis propria, and in the stromal tissue. In normal tissue, the immunofluorescence signal is strong (+3) around tumor crypts, in the vessel walls and stromal smooth muscle fibers. In the crypt epithelial cells the signal is absent (-). In CRC patients the α-SM expression decreases with increasing disease grade, and disappear in most of the advanced CRC, when the tissue is disorganized and a lot of tumor cells are present (Figure 1).
By labeling the APC C-terminus, there were observed changes of protein expression in tumor tissue compared with APC expression in normal tissues. In normal tissues, muscle tunic polyps analysis confirmed the expression of target protein in SM from blood vessels and fibers of the smooth muscle shell structure, where it is stored.
With few exceptions, the intensity of fluorescent signal given by the expression of APC is strong (3+), fluorescent signal obtained overlapping fluorescent signal of α-actin expression given by smooth muscle cells (Figure 2). Adenocarcinomas of the colorectal mucosa analysis revealed APC expression changes. During tumorigenesis process, the mucosa is invaded by stromal tissue, the crypts become large, elongate, their architecture is destroyed and the fluorescent signal intensity of epithelial cells (CE) decreases becoming weak (1+).
At the same time we observed an increase of its intensity in neoplastic infiltrated cells (CI). In the apical half of the fluorescent signal crypt, epithelial cells and infiltrated cells disappeared (-). The IHF expression pattern overlaps the APC sequential histopathological changes occurring in the colorectal carcinogenesis, in which β-catenin and APC play the role of so-called "Second Hit”.
In normal colorectal tissue, β-catenin expression appears on the membrane of epithelial cells. In tumor tissue, can occur either over-expression of β-catenin in the nucleus where it is translocated from the cytoplasm as a result of
We can observe how the fluorescent signal on the membrane of epithelial cells gradually decreases in intensity during the tumor progression, along with increased fluorescent signal by over-expression in cytoplasm (in 28 patients) and in the nucleus (in 5 patients).
Regarding E-cadherin expression, colorectal tumors showed a heterogeneous type of expression compared to the normal colorectal epithelium in which E-cadherin expression is present on the basolateral membrane to the whole length of the glandular crypts and on the intercellular membranes. An abnormal pattern of expression is observed on CRC tumor sections: i) a reduced expression (2+, 1+) at the membrane level was observed in 20% (15/ 75) of patients; ii) cytoplasmatic expression was observed in 37.33% (28/ 75) of patients and the expression is similar to that observed for β-catenin; iii) loss of expression (-) was observed in 12% (9/ 75) of patients. In 30.66% (23/ 75) of patients, the E-cadherin expression was similar with that observed in normal colon epithelium, in the cell membrane, with strong immunofluorescent signal (3+) and is co-localized with membrane β-catenin (Figure 4).
Comparative analyses of E-cadherin protein expression for CRC tumors with various histological differentiation grades (G1, G2, G3), showed an almost similar expression pattern for all G1, G2 and G3 tumor grades, although the majority of the well differentiated G1 tumors indicated strong membranous signal; the moderately differentiated tumors (G2) showed a heterogeneous membranous signal and some of the poorly differentiated tumors (G3) had no membranous expression for E-cadherin. In the case of lymph nodes analyses, there is a strong correlation between the presence of the lymph node invasion status and protein expression of E-cadherin. From a total number of 75 cases of CRC, we observed that patients with lymph node invasion N + (N1, N2, N3) have low or no expression of E-cadherin. Thus E-cadherin could be considered a biomarker that can help to determine the risk in patients with CRC, and a strong indicator of the lymph node status. In the group of N0 CRC tumors from 27 cases, only 77.77% (21/ 27) of patients presented E-cadherin membrane expression in different staining grades, scored as 0, 1+, 2+, while in the group of lymph node invasion N+ tumors (48 cases) only 35.41% (31/ 48) of patients were positive for membranous staining (0,1+, 2+).
In normal colon mucosa the sPLA2 type IIA enzyme was detected by a strong staining in muscularis mucosae in a large fraction of SM cells (recognized by α-SM actin antibody) and vascular SM cells (Figure 5). In lamina propria, the PLA2 type IIA enzyme was detected with a weaker staining (2+), surrounding the crypts (as determined by morphological and histological evaluation), and in vascular smooth muscle. These results show that PLA2 type IIA enzyme is expressed only in smooth muscle cells from normal colon mucosa. An abnormal pattern for PLA2 type IIA expression was observed in 27 of the 75 CRC cases (36.00%), which were examined. In muscularis externa and submucosa, the SM cells express PLA2 type IIA with a strong intensity (3+). The presence of PLA2 type IIA was not observed (-) in other types of cells.
Beginning with mucosa, the PLA2 type IIA expression started to be modified. Thus, near the submucosa, the immunofluorescence signal for PLA2 type IIA was observed in SM cells from lamina propria, but only around crypts, and with a weak signal comparative with the normal pattern (1+). As the crypts get longer with more ramifications, the number of SM cells that express PLA2 type IIA decrease, although we had a positive signal for α-SM actin from all the SM cells. In this area, PLA2 type IIA expression was found in epithelial cells, on the border of Lieberkühn crypts. The number of epithelial cells that express PLA2 type IIA increases during the crypts growing. The immunofluorescence signal is also stronger (3+) than fluorescent signal observed in SM cells. No immunoreaction for PLA2 (type II) was found in all 11 patients’ sections (14.66%) that were analyzed. This may suggest that the malignant cells lose their ability to express PLA2 type IIA, when invasive carcinoma develops in the adenoma.
We characterized the expression of BRCA1 in 75 sporadic colorectal carcinomas. It was found an increased BRCA1 expression in the apical cell pole of epithelial malignant cells and a significant increase in BRCA1 nuclear foci in tumor colorectal specimens in comparison with the corresponding normal tissues, in 10 cases out of 75 (13.33%). These increases in BRCA1 expression may be explained by the fact that colorectal tissue is subject to very active proliferation and differentiation. In 14 cases out 75 (18.66%) we observed the loss of BRCA1 expression (Figure 6).
The epidermal growth factor receptor (EGFR) expression had an abnormal pattern in 41.33% (31/ 75) of patients. Out of these, the signal intensity was weak (1+) in 22.58% (7/ 31) of patients and moderate (2+) in 32.25% (10/ 31) of patients. Moreover, in both cases EGFR expression was observed in cytoplasm of tumoral cells (Figure 7). Complete strong circumferential expression (3+) was found in 45.16% (14/ 31) of patients. Normal expression, like signal absence was observed in 58.67% (44/ 75) of patients. In our study (2+) and/ or (3+) were defined for those cases with EGFR expression in 50% or more tumoral cells on the section. By our study we observed that EGFR expression was significantly associated with higher rates of cell proliferation. EGFR activation and intracellular signal can be a result of its roles in transcription, up-regulation, degradation and gene amplification. Our results demonstrate that EGFR over-expression is correlated with higher tumor stage (III and IV) as compared with weaker EGFR expression. Due to the knowledge of EGFR expression in CRC, now it is possible to apply targeted therapy with cetuximab-EGFR monoclonal antibodies in the treatment algorithm of the CRC at the EGFR-positive patients identified by IHC examination. Also, the observed differentiated association between EGFR expression, ganglion EGFR status – N and tumor differentiation degree - G, could significantly assign to the EGFR the role of prognostic marker for disease recurrence. Determination of EGFR status may be used to identify cases of CRC, which could benefit from anti-EGFR therapies and on the other hand would have the potential to be a rigorous mean for monitoring efficacy of anti-EGFR therapy in CRC (Mendelsohn, 2003). Although EGFR remains a controversial prognostic factor, the association between EGFR over-expression and tumor stage may have an important role in the anti-EGFR therapy of patients with CRC.
2.4. Deletion/duplication evaluation for the interested genes (MLPA)
MLPA analysis detects large deletions or duplications in the gene. This is a semi quantitative reaction based on PCR identifying copy number variations and contributes for assessing predictive genetic markers giving an intra-individual variation spectrum of the genes included in this study. It is also a useful tool for the diagnosis of genetic diseases characterized by large genomic rearrangements. In order to perform the test on blood and tissue samples in the first step of our analyses we optimized the procedure for the specific genes. For each gene we optimized the range of DNA concentration in order to have a good signal and to obtain the most suitable mix of primers that we have to use. After protocol optimization we went through the technique and in each run we used three DNA samples from blood and tissue for each patient.
According to the microsatellites alteration assay we performed the MLPA analysis of
The interpretation of the results was made by the help of a specific soft that assesses the reaction products in accordance with their molecular weight and quantitative expression. The GeneMapper results were exported in Coffalyzer software for normalization and the relative probe signals were calculated by dividing each measured peak area by the sum of all peak areas of the sample. A value of 1.0 indicated the presence of two alleles, and values of 0.5 and 1.5 represented a heterozygous deletion or duplication at that locus, respectively.
The mutational analyses at
This patient showed two deletions, in blood and in the tumour, in the promoter 2 and mutation 1309 region, although the individual did not show microsatellite loci alteration. Another example is patient 31 who presents a large deletion in between exon 12 – exon 15 (Figure 9) and by immunohistochemistry we found
Regarding the
Mutational analyses at
2.5. Microsatellite instability correlation on APC , BRCA1 and PLA2G2A
During tumorigenesis, loss of wild-type alleles (inherited from the non-mutation-carrying parents) is frequently observed. Loss of heterozygosity (LOH) on tumor suppressor genes play a key role in colorectal cancer transformation, and LOH analysis of sporadic colorectal cancers could help discover unknown tumor suppressor genes (Ahmed B, 2011). For those patients who presented deletion/ duplication at the interested genes, in order to have a more accurate mutational analysis we decided to analyze the microsatellite instability. A panel of microsatellite markers, labeled with FAM, HEX, TET, were used to amplify DNA from normal and tumour tissues for LOH and MSI analyses of chromosomal loci specifics for
In order to analyze the polymorphic microsatellite markers, a PCR reaction was carried out for 10 ng DNA from normal and tumour tissue. The fluorescent specific-marker amplification PCR products were separated on ABI PRISM™ 310 Genetic Analyzer (Applied Biosystems). Resulted electrophoregrams were analyzed with GeneMapper ID v3.1 software for molecular size and peak heights. Data analysis was done with Sequencing DNA Analysis Software. The allelic imbalance can appear as loss of heterozygosity (LOH) or as microsatellite instability (MSI). LOH was determined using the following ratio: (T1:T2)/ (N1:N2), where 1 and 2 are the first and the second peaks of alleles identified in the tumour/ blood DNA samples from patients with colorectal cancer. When the ratio is lower than 0.67 or higher than 1.5, this is revealing the loss of one of the alleles (LOH). The presence of a novel allele in the tumour sample was interpreted as microsatellite instability (MSI).
In case of homozygosity, the two alleles are identical as dimension, and the corresponding picks are overlapped. Thus we cannot make distinction between the two alleles and their height.
Highly polymorphic markers were designed for AI analysis. The designed microsatellite markers for
At the microsatellite loci designed on chromosome 1, LOH/ MSI was observed in 28% (21/ 75) of patients and 68% (17/ 21) of these have had allelic imbalance at the D1S234 locus which covers the
On chromosome 5 LOH/ MSI was observed in 38.66% (29/ 75) of patients (Figure 17) and 51.72% (15/ 29) of these have had allelic imbalance at the D5S421 locus which overlap the
Microsatellites loci alterations corresponding to
By examining the allelic imbalance analyses for the three genes included in this study and for all the patients, we can conclude that instability variation was: a) 29.63% on the short arm of chromosome 1; b) 55.56% on the long arm of chromosome 5; c) 37.10% on the long arm of chromosome 17 (Figure 19, Table 2). Because MSI was observed only in 13 patients (14.81%) we suppose that this type of instability is no specific for sporadic colorectal cancer and appears to be a relatively specific pointer for HNPCC. As MSI is very rare in sporadic adenomas, routine screening of such lesions for MSI is not a high priority (Xue-Rong C, 2006). However, MSI analysis in adenomas is likely to be useful in the cases where clinical features or family history suggest hereditary predisposition (Jesus V, 2011). Consequently, these results can be associated with sporadic colon cancer and not with hereditary cancer, like in HNPCC.
By comparative analysis of all 15 microsatellite markers, we found that: a) 7/ 93 patients have instability on all three genes (7.52%); b) 20/ 93 patients on both
The frequencies of instability observed at
On chromosome 5q, in the region where
By comparing
3. Conclusions
In order to improve the “personalized” therapeutic strategy in CRC, by our study we have comparatively evaluated the protein and gene expression for several key point biomarkers (APC, PLA2G2A, CDH1, BRCA1, and EGFR). Our
We observed a close relationship in between different proteins and genes, which depends on the tumor type, cell grade and staging. For LOH/ MSI evaluation, our investigations were undertaken at the chromosomal regions where
Regarding the
Without making microsatellite instability analyze, at the
Acknowledgement
This work has been supported by the Government of Romania, through National Plan of Research II, grant no. 137/ and 42-158/ 2008. We are grateful to all our partners from Bucharest Emergency Clinical Hospital Bucharest, Romania and Department of Biochemistry and Molecular Biology from the University of Bucharest, for their excellent technical support.
References
- 1.
Aleksandra S. Rafal S. Lubomir B. Wojciech O. Marzena C. Wojciech K. Cezary S. Tadeusz P. Maciej W. Monika L. P. 2011 Prognostic Significance of Wnt-1, β-catenin and E-cadherin Expression in Advanced Colorectal Carcinoma, Pathol. Oncol. Res.,17 4 955 963 1219-4956 - 2.
Arteaga CL. 2001 The epidermal growth factor receptor: from mutant oncogene in nonhuman cancers to therapeutic target in human neoplasia. J Clin Oncol.,19 suppl),18 32S 40S 0073-2183 X - 3.
Avoranta T. Sundström J. Korkeila E. Syrjänen K. Pyrhönen S. Laine J. 2010 The expression and distribution of group IIA phospholipase A2 in human colorectal tumours, Virchows Arch.,457 6 659 667 0945-6317 - 4.
Bos JL. 1989 Ras oncogene in human cancer: a review. Cancer Res.,49 17 4682 4689 0008-5472 - 5.
Bryant D. M. Stow J. L. 2004 The ins and outs of E-cadherin trafficking. Trends Cell Biol.14 8 427 434 0962-8924 - 6.
Buhmeida A. Bendardaf R. Hilska M. Laine J. Collan Y. Laato M. Syrjänen K. Pyrhönen S. 2009 PLA2 (group IIA phospholipase A2) as a prognostic determinant in stage II colorectal carcinoma. Ann Oncol.,20 7 1230 1235 0923-7534 - 7.
Friedenson Bernard, 2004 BRCA1 and BRCA2 Founder Mutations and the Risk of Colorectal Cancer. Journal of the National Cancer Institute,96 15 1185 1186 0027-8874 - 8.
Goodwin M. Yap A. S. 2004 Classical cadherin adhesion molecules: coordinating cell adhesion, signaling and the cytoskeleton. J Mol Histol.35 8 839 844 1567-2379 - 9.
Haapamaki M. M. Gronroos J. M. Nurmi H. Alanen K. Kallajoki M. Nevalainen T. J. 1997 Gene expression of group II phospholipase A2 in intestine in ulcerative colitis. Gut,40 1 95 101 0017-5749 - 10.
Hardy R.G., Meltzer S.J. and Jankowski J.A. 2000 ABC of colorectal cancer: Molecular basis for risk factors. BMJ,321 7265 886 889 0959-8146 - 11.
Ishiguro Y. Ochiai M. Sugimura T. 1999 Strain differences of rats in the susceptibility to aberrant crypt foci formation by 2-amino-1-methyl-6phenylimidazo-[4,5-b]pyridine: no implication of Apc and Pla2g2a genetic polymorphisms in differential susceptibility. Carcinogenesis,20 1063 1068 - 12.
Jhawer M. Goel S. Wilson A. J. Montagna C. Ling Y. H. DS Byun Nasser. S. Arango D. Shin J. Klampfer L. Augenlicht L. H. Soler R. P. Mariadason J. M. 2008 PIK3CA mutation/PTEN expression status predicts response of colon cancer cells to the epidermal growth factor receptor inhibitor cetuximab. Cancer Res.,68 6 1953 1961 0008-5472 - 13.
Knudson A. G. (2001 2001 Two genetic hits (more or less) to cancer, Nat Rev Cancer.,1 2 157 162 - 14.
Sieber O. M. Tomlinson I. P. Lamlum H. 2000 The adenomatous polyposis coli (APC) tumour suppressor- genetics, function and disease. Molecular Medicine Today,6 12 462 469 1357-4310 - 15.
Lee J. J. Chu E. 2007 First-line use of anti-EGFR monoclonal antibodies in the treatment of metastatic colorectal cancer. Clin Colorectal Cancer,6 suppl 2),S42 S46 1533-0028 - 16.
Mendelsohn J.and. Baselga J. 2003 Status of epidermal growth factor receptor antagonist in the biology and treatment of cancer. J Clinical Oncology,21 2787 2799 0073-2183 X - 17.
Mihalcea A. Tica V. Georgescu S. E. Tesio C. Dinischiotu A. Condac E. Costache M. Ionica E. 2005 Allelic imbalance on chromosomes 1 and 5 in colorectal carcinoma. Plovdiv University Press,568 575 - 18.
Mihalcea . Chitu A. Stefan . Berlin I. Tica V. Costache M. Ionica E. 2009 The detection of mutations in the APC gene of Romanian patients with colorectal cancer through two independent techniques. Rom. Biotechnol. Lett.,14 5 4747 4755 1224-5984 - 19.
Mounier C. M. Wendum D. Greenspan E. Flejou J. Rosenberg D. W. Lambeau G. 2008 Distinct expression pattern of the full set of secreted phospholipases A2 in human colorectal adenocarcinomas: sPLA2-III as a biomarker candidate. Br J Cancer.,98 3 587 595 0007-0920 - 20.
Muhammad W. S. Edward C. 2010 Biology of Colorectal Cancer, Cancer J.,16 3 196 201 1528-9117 - 21.
Narayan S. Roy D. 2003 Role of APC and DNA mismatch repair genes in the development of colorectal cancers. Molecular cancer,2 1 1 15 1476-4598 - 22.
Nevalainen T. J. Gronroos J. M. Kallajoki M. 1995 Expression of group II phospholipase A2 in the human gastrointestinal tract. Lab Invest.,72 2 201 208 0023-6837 - 23.
Nevalainen T.J. and Haapanen T.J. 1993 Distribution of pancreatic (group I) and synovial type (group II) phospholipases A2 in human tissues. Inflammation,17 4 453 464 0360-3997 - 24.
Ogawa M. Yamashita S. Sakamoto K. Ikei S. 1991 Elevation of serum group II phospholipase A2 in patients with cancers of digestive organs. Res Commun Chem. Pathol Pharmacol.,74 2 241 244 0034-5164 - 25.
Porter D. E. Cohen B. B. Wallace M. R. Smyth E. Chetty U. Dixon M. J. Steel C. M. Carter D. C. 1994 Breast cancer incidence, penetrance and survival in probable carriers of BRCA1 gene mutations in families linked to BRCA1 on chromosome 17q12-21. Br J Surg,81 10 1512 1515 Online1365-2168 - 26.
Rizzo P. Osipo C. Foreman K. Golde T. Osborne B. Miele L. 2008 Rational targeting of Notch signaling in cancer. Oncogene,27 38 5124 5131 0950-9232 - 27.
Roukos D. 2010 Novel clinico-genome network modeling for 27 revolutionizing genotype-phenotype-based personalized cancer care. Expert Rev. Mol. Diagn.,10 1 33 48 1473-7159 - 28.
Samowitz W. S. Powers M. D. Spirio L. N. Nollet F. Frans van Roy. Slattery M. L. 1999 Catenin mutations are more frequent in small colorectal adenomas than in larger adenomas and invasive carcinomas. Cancer Res.,59 1442 1444 0008-5472 - 29.
Senda T. Shimomura A. Iizuka-Kogo A. 2005 Adenomatous polyposis coli (APC) tumor suppressor gene as a multifunctional gene. Anat Sci Int.,80 3 121 131 0022-7722 - 30.
Spano J. P. Lagorce C. Atlan D. Milano G. Domont J. Benamouzig R. Attar A. Benichou J. Martin A. Morere J. F. Raphael M. Penault-Llorca F. Breau J. L. Fagard R. Khayat D. Wind P. 2005 Impact of EGFR expression on colorectal cancer patient prognosis and survival. Annals of Oncology,16 1 102 108 0923-7534 - 31.
Spirio L. N. Kutchera W. Winstead M. V. Pearson B. Kaplan C. Robertson M. Lawrence E. Burt R. W. Tischfield J. A. Leppert M. F. Prescott S. M. White R. 1996 Three secretory phospholipase A(2) genes that map to human chromosome 1P35-36 are not mutated in individuals with attenuated adenomatous polyposis coli. Cancer Res.,56 5 955 958 0008-5472 - 32.
Stanczak A. Stec R. Bodnar L. Olszewski W. Cichowicz M. Kozlowski W. Szczylik C. Pietrucha T. Wieczorek M. Lamparska-Przybysz M. 2011 Prognostic Significance of Wnt-1, β-catenin and E-cadherin Expression in advanced colorectal carcinoma. Pathol Oncol Res.,17 4 955 963 1219-4956 - 33.
Thorstensen L. Ovist H. Heim S. Jan-Liefers G. Nesland J. M. Giercksky K. E. Löthe R. 2000 Evaluation of 1p losses in primary carcinomas, local recurrences and peripheral metastases from colorectal cancer patients. Neoplasia,2 6 514 522 1522-8002 - 34.
Valle J. Menendez M. Izquierdo A. Campos O. Velasco A. Feliubadalo L. Brunet J. Tornero E. Capella G. Darder E. Blanco I.and. Lazaro C. 2011 Identification of a new complex rearrangement affecting exon 20 of BRCA1, Breast Cancer Res Treat.,130 341 344 0167-6806 - 35.
Ng K. Zhu A. X. 2006 Anti Targeting the epidermal growth factor receptor in metastatic colorectal cancer. Critical Reviews in Oncology/Hematology,65 1 8 20 1040-8428 - 36.
Xue-Rong C. Wei-Zhong Z. Xing-Qiu L. Jin-Wei W. 2006 Genetic instability of BRCA1 gene at locus D17S855 is related to clinicopathological behaviors of gastric cancer from Chinese population, World J Gastroenterol.,12 26 1007-9327