Receptor-targeted quantum dots: fluorescent probes for brain tumor diagnosis

The intraoperative diagnosis of brain tumors and the timely evaluation of biomarkers that can guide therapy are hindered by the paucity of rapid adjunctive studies. This study evaluates the feasibility and specificity of using quantum dot-labeled antibodies for rapid visualization of epidermal growth factor receptor (EGFR) expression in human brain tumor cells and in surgical frozen section slides of glioma tissue. Streptavidin-coated quantum dots (QDs) were conjugated to anti-EGFR antibodies and incubated with target cultured tumor cells and tissues. The experiments were conducted first in human glioma tumor cell lines with elevated levels of EGFR expression (SKMG-3, U87) and then in frozen tissue sections of glioblastoma multiforme and of oligodendroglioma. The bioconjugated QDs used in the study were found to bind selectively to brain tumor cells expressing EGFR. QD complexed quickly to the cell membrane (less than 15 min), and binding was highly specific and depended on the expression level of EGFR on the cell membrane. Tissue experiments showed that only tumor specimens expressing EGFR were labeled in less than 30 min by QD complexes. These findings demonstrate that QD-labeled antibodies can provide a quick and accurate method for characterizing the presence or absence of a specific predictive biomarker.     

Fell-Muir Lecture: chondroitin sulphate glycosaminoglycans: fun for some and confusion for others

This review emphasizes the importance of glycobiology in nature and aims to highlight, simplify and summarize the multiple functions and structural complexities of the different oligosaccharide combinatorial domains that are found in chondroitin sulphate/dermatan sulphate (CS/DS) glycosaminoglycan (GAG) chains. For example, there are 1008 different pentasaccharide sequences possible within CS, DS or CS/DS hybrid GAG chains. These combinatorial possibilities provide numerous potential ligand-binding domains that are important for cell and extracellular matrix interactions as well as specific associations with cytokines, chemokines, morphogens and growth factors that regulate cellular differentiation and proliferation during tissue development, for example, morphogen gradient establishment. The review provides some details of the large and diverse number of different enzymes that are involved in CS/DS biosynthesis and attempts to explain how differences in their expression patterns in different cell types can lead to subtle but important differences in the GAG metabolism that influence cellular proliferation and differentiation in development as well as regeneration and repair in disease. Our laboratory was the first to generate and characterize monoclonal antibodies (mAb) that very specifically recognize different ‘native’ sulphation motif/epitopes in CS/DS GAG chains. These monoclonal antibodies have been used to identify very specific spatio-temporal expression patterns of CS/DS sulphation motifs that occur during tissue and organ development (in particular their association with stem/progenitor cell niches) and also their recapitulated expression in adult tissues with the onset of degenerative joint diseases. In summary, diversity in CS/DS sulphation motif expression is a very important necessity for animal life as we know it.     

Anti-CD3/anti-epidermal growth factor receptor-bispecific antibody retargeting of lymphocytes against human neoplastic keratinocytes in an autologous organotypic culture model.

Local cellular immune defects have been described in several tumors including human papillomavirus (HPV)-associated cervical cancer. This observation suggests the potential therapeutic benefit of immune manipulations that restore cellular immunity. Here, we evaluated the ability of bispecific monoclonal antibodies (bimAbs) to redirect T cells against keratinocytes transformed in vitro by HPV in an autologous three-dimensional culture model (organotypic cultures). The epidermal growth factor receptor (EGFR) was chosen as target for an anti-CD3/anti-EGFR bimAb because it is overexpressed in many malignant epithelial lesions and only weakly expressed in the basal layers of normal squamous epithelium. Interestingly, in organotypic cultures, the pattern of expression of EGFR was similar to that observed in vivo. The ability of T cells retargeted by CD3/EGFR bimAb to lyse HPV-transformed cell lines was confirmed in monolayer cultures. In autologous organotypic cultures, an increase in apoptotic HPV(+) keratinocytes and a significant decrease in the thickness of HPV(+) organotypic cultures were observed when activated lymphocytes and bimAbs were added to the cultures, whereas organotypic cultures of normal keratinocytes were not significantly affected. These data were similar to those obtained in the allogeneic model. These results suggest the potential usefulness of CD3-EGFR bimAb-retargeted lymphocytes in immunotherapeutic protocols for malignant epithelial lesions.     

Expression of epidermal growth factor receptor in human breast carcinoma and its correlation with morphological and biological features of tumour aggressiveness

A series of 54 patients presenting with primary breast cancer were investigated for tumour expression of epidermal growth factor receptor (EGFR) by the indirect three-step immunoperoxidase technique which used the monoclonal antibody EGFR1. The percentage of malignant cells positive for EGFR was determined and scored on a four-point (0-3) scale in each case. EGFR was demonstrated in 35 (64.8%) tumours. EGFR expression did not correlate (p greater than 0.05) with growth fraction immunohistochemically visualized by means of the monoclonal antibody Ki-67, tumour size, axillary lymph node status and malignancy grade. In contrast, a significant inverse relationship (p less than 0.05) was found between EGFR and estrogen receptor (ER) patterns. Expression of EGFR per se does not appear to be relevant to the biological behaviour of breast cancer as revealed through an evaluation of proliferative activity, pathological stage and histological differentiation. Recognition that EGFR is negatively related to ER supports the present evidence that the control of malignant cell growth and differentiation depends on complex regulatory mechanisms in which several extracellular messenger molecules, including hormones and peptide growth factors together with their specific cellular receptors, are involved and inextricably interwoven.     

Comparison of the Dako EGFR pharmDx kit and Zymed EGFR antibody for assessment of EGFR status in colorectal adenocarcinoma.

Immunohistochemistry is widely used to assess epidermal growth factor receptor (EGFR) expression on colorectal carcinomas to select patients for treatment with cetuximab, an anti-EGFR antibody. The data comparing different commercial EGFR antibodies is limited, and no cost comparisons have been made. We analyzed 65 advanced colorectal cancers from 36 patients using the EGFR pharmDx kit (DakoCytomation) and Clone 31G7 (Zymed Laboratories, Inc). EGFR expression was seen in 35 (53%) tumors (21 primary, 14 metastatic) with the Dako pharmDx kit. The Zymed antibody showed positive results in 41 (63%) tumors (25 primary, 16 metastatic). The cost per test was $40.00 with the pharmDx kit and $3.52 with the Zymed antibody. The Zymed antibody detects 10% more cases of colorectal cancer as EGFR positive, and is 10 times cheaper than the Dako pharmDx kit. There is little justification for the use of expensive kits for testing EGFR expression, when other available antibodies without the kit can give comparable or superior results.     

Isolation of tumor cell-specific single-chain Fv from immunized mice using phage-antibody libraries and the re-construction of whole antibodies from these antibody fragments

Enhanced expression of epidermal growth factor receptor (EGFR) occurs on a variety of malignant tissues thus making anti-EGFR antibodies possible agents for the diagnosis and therapy of human tumors. Standard hybridoma technology has been used successfully to isolate anti-EGFR antibodies from immunized mice and rats. This report demonstrates that phage-antibody libraries are an alternative, and more versatile, method for isolating antibodies from immunized mice. Anti-EGFR antibodies were isolated from phage-antibody libraries constructed not only from the spleen of an immunized mouse but also from the draining lymph node of an immunized mouse and from in vitro immunized mouse cells. Two of the single-chain Fv isolated from the phage-antibody libraries were engineered to create partially humanized whole antibody molecules.     

Molecular predictive markers of EGFR-targeted therapy in metastatic colorectal cancer

The monoclonal antibodies panitumumab and cetuximab that target the epidermal growth factor receptor are effective in approximately 10% and 20% of EGFR expressing, chemotherapy resistant metastatic colorectal cancer patients in monotherapy and in combination with chemotherapy, respectively. The evidence that EGFR expression by immunohistochemistry does not predict clinical outcome in EGFR targeted treatment has led to an intensive search for additional predicitive biomarkers. Oncogenic activation of signalling pathways downstream of the EGFR, such as mutation of KRAS, BRAF, or PIK3CA oncogenes, or inactivation of the PTEN tumor supressor gene is central to the progression of colorectal cancer. Tumor KRAS mutation, which may be present in 35%-45% of patients with colorectal cancer, is now recognized as an important predictive marker of resistance to cetuximab or panitumumab treatment and is also widely used in clinical practice. Among tumors carrying wild-type KRAS, mutations of BRAF or PIK3CA or loss of PTEN expression may be associated with resistance to the anti-EGFR monoclonal antibody treatment. On the other hand, EGFR ligands overexpression detected in tumor tissue is a promising positive predictive marker. There are also some initial observations that gene expression profiling could also contribute to clinical decision-making about the cetuximab and panitumumab treatments. These observations require further validation in prospective clinical trials before incorporation into clinical practice.     

Selective recognition of DNA antigenic determinants by murine monoclonal anti-DNA antibodies.

To assess the immune recognition of DNA in systemic lupus erythematosus, the antigenic specificity of monoclonal anti-DNA antibodies from autoimmune MRL-lpr/lpr mice was investigated Determinant specificity was assessed by ELISA in terms of binding to a panel of ssDNA antigens including calf thymus, human placenta, Escherichia coli, Clostridium perfringens, Micrococcus lysodeikticus, salmon testes, chicken blood and murine DNA. Among the monoclonal antibodies, a variety of binding patterns was observed, although for all antibodies tested murine DNA was among the most reactive antigens. Binding to other DNAs varied markedly, with some antibodies showing only low reactivity to certain antigens in the test panel. Similar results were obtained with sera of individual MRL-lpr/lpr mice. These results suggest that anti-DNA antibodies bind specific antigenic determinants variably expressed by DNAs of various species. Furthermore, the preferential binding to mouse DNA by some MRL-lpr/lpr antibodies may suggest a role of self-DNA in the in vivo selection of anti-DNA antibodies for expression.     

Patterns of expressions of transforming growth factor and epidermal growth factor receptor in squamous cell lesions of the urinary bladder.

AIM: To investigate the patterns of expression of transforming growth factor alpha (TGF-alpha) and epidermal growth factor receptor (EGFR) in squamous metaplasia and squamous cell carcinomas of the urinary bladder with and without schistosomiasis. METHODS: Immunohistochemical study of the expression of TGF-alpha and EGFR in squamous metaplasias (n = 12) and various grades of squamous cell carcinomas (n = 21) of the bladder with and without schistosomiasis. RESULTS: Focal cytoplasmic and membranous positivity for EGFR and TGF-alpha was seen in all cases of squamous metaplasia. The markers were diffusely coexpressed in a concordant pattern in areas of hyperplastic keratinising squamous metaplasia. A similar pattern of positivity was seen in verrucous carcinomas (n = 2) and well differentiated squamous carcinomas (n = 6). Progressive loss of differentiation was associated with increasing loss of EGFR staining while TGF-alpha staining was retained. Squamous cell carcinoma in situ (n = 2) showed focal positivity for TGF-alpha and EGFR. There were no differences in staining patterns between cases with and without schistosomiasis. CONCLUSIONS: The coexpression of TGF-alpha and EGFR by well differentiated squamous cell carcinomas and hyperplastic keratinising squamous metaplasia is consistent with the active regulatory role exerted by this autocrine loop. There is regional absence of expression of EGFR but not of TGF-alpha in squamous cell carcinomas of lesser differentiation, suggesting heterogeneity of such control in these tumours. The focal expression of the two markers in squamous cell carcinomas in situ indicates a possible second pathway of oncogenesis for less differentiated tumours. These observations may have important implications for the effectiveness of putative growth factor based treatments.     

The epidermal growth factor receptor in human pancreatic cancer.

The epidermal growth factor receptor (EGFR) and its ligands are thought to be important in the control of proliferation of many epithelial systems, including the exocrine pancreas. Abnormalities in expression of two of the known ligands of the EGFR, transforming growth factor alpha and epidermal growth factor, occur frequently in ductal adenocarcinoma of the human pancreas. We have examined an archival series of cases of pancreatic pathology for expression of the EGFR using the anti-EGFR antiserum 12E and found that there is almost ubiquitous overexpression of EGFR in pancreatic cancer and in chronic pancreatitis. Southern blot analysis showed no evidence of amplification or rearrangement of the EGFR gene. We conclude that an autocrine loop involving the EGFR system may be involved in the genesis of both neoplasia and reactive hyperplasia of pancreatic ductal epithelium.     

The influence of the adjuvant Quil A on the epitope specificity of meningococcal lipopolysaccharide anti-carbohydrate antibodies.

Rabbits were immunized with immunotype L3,7,9 phosphoethanolamine (PEA) group containing oligosaccharide-tetanus toxoid conjugates both with and without the addition of the adjuvant Quil A. The epitope specificity of the antibodies present in these antisera was analysed in an immunotype L2 and L3,7,9 specific inhibition ELISA using the homologous and heterologous lipopolysaccharide, oligosaccharide and partial dephosphorylated oligosaccharide as inhibitors. Two groups of antisera could be identified. In one group of antisera, at least two antibody populations are present, namely directed against the PEA group containing determinants on immunotype L3,7,9 lipopolysaccharide and against immunotype L2 specific epitopes in which no PEA group is present. In the second group of antisera, one but probably more antibody populations are detected with a similar specificity towards the conserved epitopes of both immunotypes. In general, immunization with the conjugates only resulted in the induction of antibodies against the PEA group containing epitopes on the L3,7,9 lipopolysaccharide (80%). Antibodies directed against the conserved epitopes of both immunotypes are mainly evoked with the conjugates in combination with the adjuvant Quil A (80%). Although these results suggest that the epitope specificity of the antibodies induced depends on the use of Quil A, the influence of genetic factors cannot be excluded. At the moment it is not known whether the differences in epitope specificities are reflected in biological function of these antibodies. However, the induction of antibodies with clearly different epitope specificities after immunization of different rabbits with the same antigen stresses the importance of this kind of analysis when developing a vaccine based on oligosaccharide-protein conjugates.     

Epidermal growth factor receptor regulates normal urothelial regeneration

Members of the epidermal growth factor (EGF) family and their receptors are involved in many cellular processes, including proliferation, migration, and differentiation. We have previously reported that these growth factors are expressed and have specific regulatory functions in an organ-like culture model of normal human urothelial cells. Here, we used this model to investigate the involvement of EGF receptor (EGFR) in human urothelial regeneration. Three 4-mm-diameter damaged areas were made in confluent normal human urothelial cell cultures with a biopsy punch. Regeneration was measured, on fixed stained cultures, with an image analyzer, at 4, 24, and 48 hours after injury. Cell proliferation was assessed by 5-bromo-2-deoxyuridine incorporation. To identify EGF family factors potentially involved in the healing process, we studied the effect of these factors on damaged confluent cultures and the level of expression of mRNAs extracted from these cultures. EGFR inhibition of the proliferation and migration of urothelial cells was tested with (1). a specific tyrosine kinase inhibitor (AG1478) and (2). a blocking anti-EGFR antibody (LA22). Exogenously added amphiregulin, EGF, transforming growth factor-alpha and heparin-binding EGF (HB-EGF) stimulated urothelial regeneration. The damaged areas were repaired by regrowth within 48 hours. Both AG1478 and LA22 inhibited the repair (by 50% and 30%, respectively), as well as proliferation and migration. This regeneration was accompanied by increased HB-EGF mRNA expression in cultures of cells from four of six subjects, but no corresponding change in EGFR protein level was observed. These results indicate that the EGFR signaling pathway is involved in urothelial regeneration. Our data support an autocrine role of HB-EGF in this process and suggest that the EGFR pathway is a potential therapeutic target for modulating urothelial cell proliferation.     

Cell surface oligosaccharide modulation during differentiation: VI. The effect of biomodulation on the senescent and neoplastic cell phenotype.

These investigations test the hypothesis developed previously, that there are biomolecules which control and integrate cellular differentiation. Our specific interest in cellular differentiation lies in the area of what we refer to as basal or primitive cellular differentiation mechanisms. These mechanisms are common to all cells, and are required for simple recognition and growth regulation. We have investigated two models, the IMR-90 human fetal lung fibroblast model as a representative of normal growth control, and the CG model, canine glioma cells, a transplantable growth transformed cell line. These two models represent normal, and aberrant cellular differentiation control. In previous studies we have shown that the arrangement of the cell surface oligosaccharide structure on these cell types are predictive of phenotypic transition. We have developed, and partially characterized a series of BIOMODULATORS (BM) which delay the onset of display of neoplastic cells. Three classes of BIOMODULATOR have been explored; (1) a large molecular weight natural product (25-35 kDa), PokeWeed Mitogen (PWM); (2) a small molecular weight natural product (500 Da) Cellular Activator and Differentiator (CAD) and a number of natural and synthetic analogs; and (3) an indolizidine alkaloid natural product, Swainsonine (Sw) which has a known cellular target (oligosaccharide biosynthesis). Preliminary data is presented which structurally links some of these BIOMODULATORS in terms of their effective stereochemistry. These BIOMODULATORS, when used before PDL 38, prevent the cell surface oligosaccharide display changes typical of morphological senescence and delay their onset to PDL 100 or more. These BIOMODULATORS also appear to have regulatory effects on the neoplastic cell models. This re-regulation results in increases in generation time and an increase in the ability of these cells to be recognized by cytotoxic lymphocytes. Proton NMR linewidth measurements of the fraction of 'bound' water associated with the cellular surface of treated and untreated cell populations showed induced physical changes in the cell surface related to the use of the BIOMODULATOR and correlated to the oligosaccharide display changes. These data were interpreted as indicating an increase in the organizational level of these cells. The data for normal and neoplastic cell populations are compared and contrasted in an effort to form the basis for an analytical approach to the control and integration of differentiation mechanisms.     

Inhibition of human brain malignant glioblastoma cells using carmustine-loaded catanionic solid lipid nanoparticles with surface anti-epithelial growth factor receptor

Innovated catanionic solid lipid nanoparticles (CASLNs) carrying carmustine (BCNU) (BCNU-CASLNs) were grafted with anti-epithelial growth factor receptor (EGFR) (anti-EGFR/BCNU-CASLNs) and applied to inhibiting the propagation of human brain malignant glioblastomas cells. U87MG cells were treated with anti-EGFR/BCNU-CASLNs and stained for the expression of EGFR. The minimal average diameter of BCNU-CASLNs and maximal entrapment efficiency of BCNU emerged when the concentration of catanionic surfactants was 1?mm. An increase in the weight percentage of cacao butter (CB) reduced the zeta potential, enhanced the viability of human brain microvasscular endothelial cells (HBMECs), and decreased the expression of tumor necrosis factor-α by HBMECs. The dissolution rate of BCNU and inhibition against the multiplication of U87MG cells using anti-EGFR/BCNU-CASLNs followed the order: 100% CB?>?0% CB?>?50% CB. Anti-EGFR/BCNU-CASLNs demonstrated the properties including an effective delivery to U87MG cells and antiproliferative efficacy against the growth of malignant brain tumors.     

Humoral immune response to epidermal growth factor receptor in lung cancer

The aim of this study was to explore the potential value of autoantibody to epidermal growth factor receptor (EGFR) in the diagnosis of lung cancer (LC) and its relation with EGFR mutations. Enzyme-linked immunosorbent assay (ELISA) was performed to detect the level of autoantibody to EGFR in sera from 254 LC patients and 222 normal controls (NCs). Besides, the mRNA and protein levels of EGFR were investigated in Gene Expression Profiling Interactive Analysis (GEPIA) and Human Protein Atlas (HPA) database, respectively. The level of autoantibody to EGFR (anti-EGFR) in LC even different types of LC was obviously higher than that in NC (P < 0.05). The area under the curve (AUC) of anti-EGFR was 0.695 (95% CI 0.645–0.742) when comparing LC patients with NC, while the AUC of carcinoembryonic antigen (CEA) was 0.681 (95% CI 0.629–0.730). Moreover, by integrating anti-EGFR with CEA to diagnose LC, the AUC was up to 0.784 (95% CI 0.737–0.826). However, the expression level of autoantibody to EGFR had no difference between LC patients with and without EGFR gene mutation (P > 0.05). EGFR mRNA expression level was obviously upregulated in squamous cell carcinoma (SCC) tissues compared with normal tissues (P < 0.05), but not in adenocarcinoma (ADC) (P > 0.05). The study confirmed that anti-EGFR could be a potential biomarker for LC diagnosis; additionally, it could improve the diagnostic value of CEA in clinical work.

     

Analysis of the expression of H, Lewis, X, Y and precursor blood group determinants in saliva and red cells using a panel of mouse monoclonal antibodies

Salivary glycoproteins from 33 normal individuals were analyzed with a panel of mouse monoclonal antibodies to H-1, H-2, Lea, Leb, X, Y and precursor blood group determinants. Samples from 19/33 individuals co-expressed Leb and Y-determinants (secretors) and 6/33 co-expressed Lea and X-determinants (non-secretors). Erythrocytes of these individuals were typed Le (a-b+) and Le (a + b-), respectively. In seven other salivas, only one specificity, either Lea, Leb, X or Y, was expressed and in one sample none of these determinants could be detected. Only one saliva sample expressed H-1 specificity and none expressed H-2 or type 1 precursor determinants. The absence of H-1 and H-2 structures in secretors and the resulting expression of difucosylated Leb and Y-structures is probably a tissue-specific trait of salivary gland secretions. The strict co-expression of Leb with Y and Lea with X supports the conclusion that only one 2-O-fucosyl-galactose transferase, which can fucosylate both type 1 and type 2 chains, exists in salivary glands. The finding that a number of individuals expressed neither X- nor Y-specificities was unexpected in view of previous work showing that the 3-O-fucosyl N-acetylglucosamine transferase involved in forming this structure is a ubiquitous enzyme. The individualistic expression of blood group phenotypes in tissues should be considered when the altered expression of blood groups in malignancy and other diseases is studied.     

High epidermal growth factor receptor amplification rate but low mutation frequency in Middle East lung cancer population

Epidermal growth factor receptor (EGFR) exon 18-21 mutations were shown to be highly predictive of response to gefitinib (Iressa) therapy in lung cancer. Studies on Western and Japanese lung cancers have indicated substantial differences in the EGFR mutation frequency between these populations. To investigate the prevalence of EGFR in another distinct ethnic group, EGFR alterations were studied in 47 consecutive non small cell lung cancers from Saudi Arabia by immunohistochemistry, fluorescence in situ hybridization, and DNA sequencing. Detectable EGFR expression was seen in 69.8% of 43 interpretable cancers. Epidermal growth factor receptor amplification, present in 15.3% of 39 analyzable cancers, was strongly associated with high levels of EGFR expression (P = .0047). Only 1 exon 18-21 mutation was seen among 34 lung cancers that could be successfully sequenced. It is concluded that EGFR exon 18-21 mutations are rare in Middle East patients with lung cancer and occur in a similar range as in Western patients. The remarkable high rate of EGFR gene amplifications could potentially facilitate studies on the predictive role of gene copy number changes for response to anti-EGFR therapies in Middle East patient sets.     

Cell surface oligosaccharide modulation during differentiation: V. Partial characterization of the regulated surface during substrate adhesion and spreading.

In previous studies with IMR-90 human fetal lung fibroblasts, it was shown that modulation of a small finite number of unique cell surface oligosaccharide structural specificities (as defined by reaction with specific lectins and monoclonal antibodies) define the cellular senescence phenotype. The cell surface oligosaccharide display can be characterized by assessing the epitope density and conformational arrangement of three or four individual carbohydrate specificities. Development of the senescence morphological phenotype was preceded by specific alterations in the cell surface oligosaccharide display. The senescence-dependent changes in these displays are primarily related to the binding affinity (the Kij of the Scatchard analyses) rather than the epitope density (the binding capacity, Rij of the Scatchard analyses). These alterations involve rearrangements within specific carbohydrate classes. In the course of these studies, the observation was made that low-density and contact-inhibited growth-retarded Phase II cells showed similar surface modulation of the oligosaccharide display. This suggests a broader significance for the date in growth regulation. These data suggest a structural/functional relationship between cellular senescence mechanisms and growth control in general. In this study we have investigated the possible role of cell surface oligosaccharide regulation in substrate interactions. Trypsinized cells were studied during substrate adherence, spreading and initiation of growth on control and poly-L-ornithine-treated polystyrene substrate. The cell surface oligosaccharide display was characterized using both biological assays and NMR-based measurements of the mobility of water at the cell surface. Results show that trypsinization does not remove or diminish the oligosaccharide display. The initial adherence of the cells to unmodified substrate is mediated by oligosaccharides. Cellular spreading results in specific changes in the display. Initiation of growth corresponds to further specific changes in the display. These data suggest a mechanistic connection between the cell surface oligosaccharide display and growth control of these fibroblasts.     

Molecular testing in colorectal cancer: diagnosis of Lynch syndrome and personalized cancer medicine

Currently, molecular testing in colorectal cancer (CRC) is aimed at detecting Lynch syndrome and predicting response to anti-epidermal growth factor receptor (EGFR) therapies. However, CRC is a complex disease, with at least 3 molecular pathways of carcinogenesis. The importance of the EGFR signaling pathway in colorectal carcinogenesis is underscored by the availability of anti-EGFR monoclonal antibodies for the treatment of some metastatic CRCs. Potentially, mutations in any of the genes in the EGFR signaling pathway may be associated with prognosis and may predict response to anti-EGFR or other targeted therapies. Although not currently the standard of care, molecular testing of CRCs is expanding to include mutational analysis of the genes in the EGFR pathway, in addition to more widely performed tests for identifying cancers with high microsatellite instability. Multiplex molecular prognostic panels for therapeutic decision making in stage II CRCs also represent expanding use of molecular testing for this common cancer.