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Chemotherapeutic regimens containing camptothecin (CPT), 5-fluorouracil, and oxaliplatin are used to treat advanced colorectal cancer. We previously reported that an indole derivative, 3-(2-bromoethyl)indole (BEI-9), inhibited the proliferation of colon cancer cells and suppressed NF-κB activation. Here, we show that a combination of BEI-9 with either CPT or tumor necrosis factor alpha (TNFα) enhances cell death. Using colorectal cancer cells, we examined the activation of NF-κB by drugs, the potential of BEI-9 for inhibiting drug-induced NF-κB activation, and the enhancement of cell death by combination treatments. Cells were treated with the chemotherapeutic drugs alone or in combination with BEI-9. NF-κB activation, cell cycle profiles, DNA-damage response, markers of cell death signaling and targets of NF-κB were evaluated to determine the effects of single and co-treatments. The combination of BEI-9 with CPT or TNFα inhibited NF-κB activation and reduced the expression of NF-κB-responsive genes, Bcl-xL and COX2. Compared to CPT or BEI-9 alone, sequential treatment of the cells with CPT and BEI-9 significantly enhanced caspase activation and cell death. Co-treatment with TNFα and BEI-9 also caused more cytotoxicity than TNFα or BEI-9 alone. Combined BEI-9 and TNFα enhanced cell death through caspase activation and cleavage of the switch-protein, RIP1 kinase. BEI-9 reduced the expression of COX2 both alone and in combination with CPT or TNF. We postulate that BEI-9 enhances the effects of these drugs on cancer cells by turning off or redirecting NF-κB signaling. Therefore, the combination of BEI-9 with drugs that activate NF-κB needs to be evaluated for clinical applications.

This review provides updates on the efforts for the development of prognostic and predictive markers in colorectal cancer based on the race/ethnicity of patients. Since the clinical consequences of genetic and molecular alterations differ with patient race and ethnicity, the usefulness of these molecular alterations as biomarkers needs to be evaluated in different racial/ethnic groups. To accomplish personalized patient care, a combined analysis of multiple molecular alterations in DNA, RNA, microRNAs (miRNAs), metabolites, and proteins in a single test is required to assess disease status in a precise way. Therefore, a special emphasis is placed on issues related to utility of recently identified genetic and molecular alterations in genes, miRNAs, and various "-omes" (e.g., proteomes, kinomes, metabolomes, exomes, methylomes) as candidate molecular markers to determine cancer progression (disease recurrence/relapse and metastasis) and to assess the efficacy of therapy in colorectal cancer in relation to patient race and ethnicity. This review will be useful for oncologists, pathologists, and basic and translational researchers., Competing Interests: Conflict of Interest Upender Manne, Trafina Jadhav, Balananda-Dhurjati Kumar Putcha, Temesgen Samuel, Shivani Soni, Chandrakumar Shanmugam, and Esther A. Suswam declare that they have no conflict of interest.

Objective: To investigate the clinical value of p53 codon 72 single nucleotide polymorphisms (SNPs) and variants of adenomatous polyposis coli (APC) in hepatocellular carcinomas (HCCs)., Methods: DNA and RNA from 51 HCCs and their matching, uninvolved liver tissues were analyzed for p53 mutations, and the methylation and expression of APC variants were determined. Proliferation of each HCC was assessed by Ki67 immunohistochemistry. The results were correlated with the demographic and clinicopathologic features and patient survival., Results: Of 51 HCCs, 12% exhibited missense p53 mutations. SNP analysis of p53 codon 72 demonstrated the highest prevalence of the Arg/Arg (56%) phenotype, followed by Arg/Pro (33%) and Pro/Pro (11%). Four of five cases with the Pro/Pro phenotype were African Americans (AAs). All five cases with the Pro/Pro phenotype had hepatitis C virus (HCV) infections, a high Ki67 index, and lower median survival (15.5 months) compared to those with Arg/Arg or Arg/Pro phenotypes (32 months). The overall frequency of APC methylation was 31%, which was found predominantly in Caucasians. There was lower mRNA expression of APC variants-2 and -3 in both HCCs and corresponding adjacent, uninvolved liver tissues as compared to APC variant-1. The expression of APC variant-3, but not variants-1 and -2, was lower in HCCs relative to uninvolved tissues. Expression of all APC variants was lower in HCCs with APC methylation relative to HCCs without APC methylation, and low expression of APC variant-2 was associated with the Pro/Pro phenotype., Conclusions: These findings suggest that, for AA patients with HCCs, the p53 Pro/Pro phenotype and low expression of APC variant-2 are associated with aggressive tumor behavior, HCV infection, and poor clinical outcome.

Background: Low socioeconomic status (SES) has been associated with abnormal expression of p53 in breast cancer, but this relationship has not been evaluated for colorectal cancer (CRC). A cohort of CRC patients was evaluated to determine if SES is associated with abnormal p53 expression., Methods: The study population consisted of 249 patients who underwent curative or palliative resections for CRCs at the University of Alabama at Birmingham Hospital. Measures of SES and potential confounders were abstracted from medical records. Abnormal nuclear accumulation of p53 (p53(nac)) was measured in CRCs by immunohistochemistry. Logistic regression was used to assess the relationship between low SES and p53(nac)., Results: Over half (56.2%) of the patients exhibited p53(nac) in their CRCs. After adjustment, the odds ratio for p53(nac) was 1.28 (95% CI =0.55, 2.99) for Medicaid patients relative to those without Medicaid coverage. There was no association between the prevalence of p53(nac) and unemployment, private insurance coverage, or having Medicare due to disability., Conclusions: The odds of having p53(nac) were 1.28 times higher for patients with Medicaid coverage, although these findings were not statistically significant. The results of this pilot study, however, provide evidence of a molecular basis for the decreased survival of low SES patients with CRC.

Background: DNA-damaging drugs constitute standard chemotherapy regimen for advanced colorectal cancer. Here, the interactions between quercetin and 5-fluorouracil (5-FU), etoposide, and camptothecin were examined in cancer cells., Materials and Methods: HCT116 colorectal or PPC1 prostate cancer cells were treated with quercetin and the drugs. Clonogenicity assays, cell cycle profiles, and expressions of p53, p21, BAX, survivin and cyclin B1 proteins were used to examine the effects of the treatments., Results: Quercetin synergistically inhibited the clonogenicity of the wild-type cells, but inhibited the cell cycle effects of all the drugs tested. In p53-null cells, the combination of low dose 5-FU with up to 6 μM quercetin promoted clonogenic survival. Treatment of p53-wild-type cells with 50 μM quercetin reduced drug-induced up-regulation of p53, p21 and BAX. The combination of quercetin and the drugs also reduced the levels of cyclin B1 and survivin proteins., Conclusion: While high doses of quercetin synergize with DNA-damaging agents, the effect of drug combination with quercetin is influenced by the effective doses and the p53 status of the cells.

MicroRNAs (miRNAs) have prognostic and therapeutic value for colorectal cancers RCs). Although formalin-fixed paraffin-embedded (FFPE) tissues are available for biomarker studies, the stability of miRNAs in these tissues stored for long periods (more than 20 years) is unknown. The present effort involved analysis of 345 FFPE CRC tissues, stored for 6 to 28 years (1982-2004), for the expression of six miRNAs (miR-20a, miR-21, miR-106a, miR-181b, miR-203, and miR-324-5p) using TaqMan(r) microRNA assays and quantitative real-time PCR (qRT-PCR). Evaluation, by linear regression analysis, of miRNA expression among archived CRC tissues found similar levels of all six miRNAs in tissues stored over this period (correlation coefficients, R2, ranged from less than 0.0001-0.009; and t-test p-values were greater than or equal to 0.05). Thus, miRNAs are stable in FFPE tissues stored for long periods of time, and such samples can be used for discovery of biomarkers.

Objective: Since the anti-tumor activity of 5-fluorouracil (5-FU) is due to induction of apoptosis, we assessed the value of expression of key apoptotic molecules (Bax, Bcl-2 and p53) in predicting the efficacy of 5-FU therapy for colorectal adenocarcinomas (CRCs)., Methods: Archival tissues of CRCs from 56 patients who received a complete regimen of 5-FU-based chemotherapy after surgery, and 56 patients matched for age, gender, ethnicity, tumor stage, tumor location, and tumor differentiation who had undergone only surgery (without any pre- or post-surgery therapy), were evaluated for immunophenotypic expression of Bax, Bcl-2, and p53. Also, these CRCs were evaluated for Bax mutations. The predictive capacity or prognostic value of these markers was assessed by estimating overall survival., Results: The majority of low Bax expressing CRCs have exhibited mutations at the G (8) tract. There was no significant difference in overall survival rates between the categories of surgery alone and 5-FU-treated patients. However, a better survival was observed for patients who received chemotherapy when their CRCs had low Bax/Bcl2 ratio (HR, 1.55; 95% CI: 1.46-31.00). Patients who received surgery alone and whose CRCs lacked Bax expression had 5.33 times higher mortality than those with high Bax expression (95% CI: 1.78-15.94), when controlled for tumor stage and other confounders. Bcl-2 and nuclear p53 accumulation had no predictive value in either patient group., Conclusion: These findings are the first to demonstrate that high Bax expression is a good prognosticator for patients who underwent surgery alone, and that patient with low Bax/Bcl-2 expression ratio benefit from 5-FU-based adjuvant therapies.

Most discoveries of cancer biomarkers involve construction of a single model to determine predictions of survival.. 'Data-mining' techniques, such as artificial neural networks (ANNs), perform better than traditional methods, such as logistic regression. In this study, the quality of multiple predictive models built on a molecular data set for colorectal cancer (CRC) was evaluated. Predictive models (logistic regressions, ANNs, and decision trees) were compared, and the effect of techniques for variable selection on the predictive quality of these models was investigated. The Kolmogorov-Smirnoff (KS) statistic was used to compare the models. Overall, the logistic regression and ANN methods outperformed use of a decision tree. In some instances (e.g., for a model that included 'all variables without tumor stage' and use of a decision tree for variable selection), the ANN marginally outperformed logistic regression, although the difference between the accuracy of the KS statistic was minimal (0.80 versus 0.82). Regardless of the variable(s) and the methods for variable selection, all three predictive models identified survivors and non-survivors with the same level of statistical accuracy.

A rapid and cost-effective combination tapered fiber-optic biosensor (CTFOB) dip-probe was used for quantitative estimation of interleukin (IL)-6 in serum/plasma samples. Sandwich immunoassay was used as the detection technique. Probes could successfully detect presence of IL-6 in two serum samples, non-neoplastic autoimmune patient (lupus) sample and lymphoma patient sample. The estimated amount of IL-6 in lupus patient sample was 4.8 ± 0.9 pM and in lymphoma patient sample was 2 ± 1 pM. It is demonstrated that the developed CTFOB dip-probe is capable of quantitative estimation of proteins in serum/plasma samples with high specificity.

With the increasing use of individualized medical care (personalized medicine) in treating and managing patients with cancer, the utilization of biomarkers in selecting and tailoring such medical approaches also is increasing and becoming more important. Specifically, many therapies are effective against only a subgroup of a specific type of tumors and exposing patients with different non-responsive subgroups of the same tumor to ineffective therapies, not only exposes these patients needlessly to acute and chronic side effects of the therapy, but also adds to the costs of medical care. For example, the Oncotype Dx test for estrogen receptor positive tumors that are node negative has been used to identify low risk tumors for which surgery alone is an adequate therapy. Biomarkers may be used to aid in multiple aspects of medical care related to cancer, including early detection, diagnosis, risk assessment, as well as in predicting the aggressiveness of cancers (i.e., prognosis) and predicting the therapeutic efficacy of treatments (i.e., prediction). Biomarkers may be also used as surrogate endpoints to aid in evaluating therapies and preventive approaches. Types of biomarkers vary greatly and include histopathologic appearance, stage of the lesion, quantitative morphologic features, size of the lesion, metastatic pattern and extent of metastasis, as well as imaging and molecular features. The types of measurements of biomarkers also vary; for example, molecular features can be measured at the DNA, mRNA or protein levels as well as at regulatory levels (e.g., microRNA). The usefulness of each biomarker is limited by its sensitivity and specificity in fulfilling its role (e.g., in early detection) and the requirements of sensitivity and specificity to accomplish specific tasks are affected by multiple variables. For example, both very high specificity and sensitivity of a test are required to screen a population with a low prevalence of a specific tumor. The goal of this manuscript is to introduce the reader to how biomarkers may be used and the limitations on the uses of biomarkers in translational research.

A variety of genetic and molecular alterations underlie the development and progression of colorectal neoplasia (CRN). Most of these cancers arise sporadically due to multiple somatic mutations and genetic instability. Genetic instability includes chromosomal instability (CIN) and microsatellite instability (MSI), which is observed in most hereditary non-polyposis colon cancers (HNPCCs) and accounts for a small proportion of sporadic CRN. Although many biomarkers have been used in the diagnosis and prediction of the clinical outcomes of CRNs, no single marker has established value. New markers and genes associated with the development and progression of CRNs are being discovered at an accelerated rate. CRN is a heterogeneous disease, especially with respect to the anatomic location of the tumor, race/ethnicity differences, and genetic and dietary interactions that influence its development and progression and act as confounders. Hence, efforts related to biomarker discovery should focus on identification of individual differences based on tumor stage, tumor anatomic location, and race/ethnicity; on the discovery of molecules (genes, mRNA transcripts, and proteins) relevant to these differences; and on development of therapeutic approaches to target these molecules in developing personalized medicine. Such strategies have the potential of reducing the personal and socio-economic burden of CRNs. Here, we systematically review molecular and other pathologic features as they relate to the development, early detection, diagnosis, prognosis, progression, and prevention of CRNs, especially colorectal cancers (CRCs).

Invasive tumors (cancers or malignant lesions) typically develop in the setting in which there is the presence of putative non-invasive lesions and the development of these non-invasive lesions frequently precedes the development of cancers. For some organs, such as the oral cavity, cervix and skin, the respective putative pre-invasive lesions can be observed over time and documented to progress to invasive lesions. However, for less readily observable lesions, such as those of the prostate, the progression of the pre-invasive lesions, e.g., prostatic intraepithelial neoplasia (PIN) and prostatic proliferative inflammatory atrophy (PIA) to prostatic cancer are more difficult to document. Thus, for most organ systems, specific pre-invasive neoplastic lesions have been proposed based upon the apparent observations of one or more of the following: 1) microinvasive disease developing from a pre-invasive neoplastic lesion, 2) the general association of the pre-invasive lesion with invasive lesions, 3) the subsequent development of invasive lesions following diagnosis of the pre-invasive lesion, 4) correlations of the molecular features of the putative pre-invasive lesion with the matching invasive lesions, and 5) reductions in the rate of cancer following removal of the pre-invasive lesion. When there are mixtures of pre-invasive lesions with actual cancers in the same case, some of the above specific associations are more difficult to make. Several terms have been used to describe pre-invasive lesions, many of which are now less useful as our knowledge of these lesions increases. It is now commonly accepted that these lesions are a features of the spectrum of neoplastic development and most are accepted as ``neoplastic lesions'' with associated molecular features, even though they may be reversible even if they have mutations in suppressor genes (e.g., p53) or are associated with viral etiologies (e.g., cervical intraepithelial neoplasia). The overall term, "pre-invasive neoplasia", seems to best describe these putative pre-invasive lesions. Thus, terms such as incipient neoplasia should be abandoned. The term "intra-epithelial neoplasia" with an associated grade, which has been developed for pre-invasive neoplastic lesions of the cervix, i.e. cervical intraepithelial neoplasia (CIN), seems to be a terminology that adds consistency across epithelial organs. Thus, adoption of these terms for the additional organ sites of pancreas (PanIN) and prostate (PIN) seems accepted. Less descriptive terms such as the degrees of dysplasia of the oral cavity and bronchopulmonary system and actinic keratosis and Bowen's disease of the skin might be better designated as oral intraepithelial neoplasia (OIN), pulmonary intraepithelial neoplasia (PulIN) and dermal intraepithelial neoplasia (DIN). The etiology of pre-invasive neoplasia is the etiology of the matching cancers. Some obvious initiating factors include exposure to the whole range of ionizing and non-ionizing radiation, tobacco abuse and a broad range of other carcinogens (e.g., benzene). A frequent initiation factor is the setting of long standing continuing damage, inflammation and repair (LOCDIR) which leads to early molecular features associated with neoplasia after about one year. An excellent example of this is ulcerative colitis (UC) in which dysregulation of microsatellite repair enzymes have been documented one year following diagnosis of UC. While the nomenclature, description, diagnosis and etiology of pre-invasive neoplasia has advanced, approaches to therapy of such lesions have not progressed adequately even though it has been identified that, for example, removal of polyps periodically from the colorectum, DCIS from the breast, and high grade CIN from the cervix, results in a reduction in the development of cancers of the colorectum, breast, and cervix, respectively. With the development of more molecularly targeted therapy with fewer side effects, preventive therapies may be more successfully targeted to pre-invasive neoplastic lesions.

CXCR4 receptors have been implicated in tumorigenesis and proliferation, making it a potential target for colorectal cancer therapy. Expression of this chemokine receptor on cellular surfaces appears to promote metastasis by directly stimulating tumor cell migration and invasion. The receptor/ligand, CXCR4/SDF-1alpha, pair are critically important to angiogenesis and vascular remodeling which supports cancer proliferation. Our work has shown that a novel apoptotic peptide of HIV-1, Nef-M1, can act as a CXCR4 antagonist, inducing apoptosis in CXCR4 containing cells. Four colorectal tumor cell lines (HT-29, LS174t, SW480, WiDr), were evaluated for their response to Nef-M1 peptide via in vivo and in vitro. The presence of CXCR4 receptors on tumor cells was determined using immunohistochemical and RT-PCR analyses. Solid xenografts derived from tumor cell lines grown in SCID mice, were evaluated for the persistence of the receptor. Xenografts propagated in SCID mice from each of the four cell lines demonstrated high levels of receptor expression as well. The effects of Nef-M1 in vivo via splenic injected mice and subsequent hepatic metastasis also demonstrated dramatic reduction of primary tumor growth in the spleen and secondary invasion of the liver. We concluded that Nef-M1 peptide, through physical interaction(s) with CXCR4, drives apoptotic reduction in in vivo primary tumor growth and metastasis.

For subsets of colorectal adenocarcinoma (CRC) patients, nuclear accumulation of p53 (p53(nac)) and Bcl-2 expression are prognostic indicators. To understand their role in the progression of CRC we evaluated 90 CRCs and their contiguous adenomatous components (CAdCs) for immunohistochemical expression of these markers. In general, p53(nac) and Bcl-2 expression was significantly increased when comparing normal colonic epithelia to CAdCs and CRCs. Thirteen (14%) CAdCs that demonstrated p53(nac) continued to express p53(nac) in their contiguous CRCs. A similar trend was observed in Bcl-2 expression in that the majority of CAdCs expressing Bcl-2 continued to express it in their matching CRCs (39/44). Patients whose CAdCs and their contiguous CRCs demonstrate p53(nac) had shorter median survival (35.9 months) than those patients whose CAdCs and CRCs did not (80.56 months). However, patients whose CAdCs had p53(nac) and lacked Bcl-2 expression had the lowest median survival (15.74 months) when compared with patients whose CAdCs did not demonstrate p53(nac) but had increased expression of Bcl-2 (71.77 months). These findings suggest that in those adenomas that demonstrate p53(nac) but lack Bcl-2 expression, their contiguous CRCs are more likely to be aggressive as they progress.

The recently identified human ortholog of the Rabphillin-3A-Like (RPH3AL) gene, located at the 17p13.3 locus, has been assessed for its mutational status and clinical significance in colorectal adenocarcinoma (CRC). Prospectively collected 95 frozen CRCs and their matching benign colonic epithelial tissues were evaluated for mutations and mRNA expression. Since, we observed a higher incidence of a single nucleotide polymorphism (SNP) at the -25 position in the 5'untranslated region (5'UTR-25) of RPH3AL, we performed the genotyping analysis of this SNP in a retrospective CRC cohort (n=134) to assess their clinical importance. Univariate and multivariate outcome analyses were performed. The cDNA analysis has detected point mutations in 6 CRCs, coding region SNPs in 14 CRCs, and non-coding region SNPs in 38 CRCs. Combined analyses of both cohorts has demonstrated that the incidence of SNP at 5'UTR-25 was 41% (95 of 229), and its A/A genotype (9%, 20 of 229) was observed exclusively in non-Hispanic Caucasians, and 19 of these cases were diagnosed with nodal metastasis. Patients who exhibited homozygous for A or C alleles had a significantly decreased levels of mRNA expression, increased risk of CRC recurrence and mortality. Therefore, these findings have significant clinical implications in assessing the aggressiveness of CRC.

We compared the accuracy of 3 data-mining models, neural-network, decision-tree, and logistic-regression, in predicting the 5-year survival of patients with colorectal cancer. The database consisted of patient demographics, pathologic features, and levels of expression of 2 biomarkers (p53 and Bcl-2). All 3 methods demonstrated acceptable accuracy, from 64% to 70%. The neural-network model had the best specificity (80%) and accuracy (70%) but lowest sensitivity (59%). Both logistic-regression and decision-models demonstrated comparable sensitivity (72%).

With the availability of new technologies and the increased interest of medical practitioners to use molecular biomarkers in early detection and diagnosis, and in the prediction of therapeutic treatment efficacy and clinical outcomes, the academic and research institutions, as well as the pharmaceutical industry, have increased their efforts to develop novel molecular biomarkers for several human diseases, including cancer. The identification of molecular biomarkers also enables the development of a new generation of diagnostic products and to integrate diagnostics and therapeutics. This integrated approach will aid in 'individualizing' the medical practice. Here, we address issues related to the development of biomarkers, novel technological platforms used for drug development, future technologies and strategies for validating biomarkers for their clinical utility.

9-Cis-retinoic acid (aliretinoin) is a pan-retinoid receptor agonist and has been demonstrated in preclinical models to have potent chemoprevention effects. The purpose of this study was to determine the utility of using aliretinoin as a chemoprevention agent in cervical dysplasia. Patients with histological evidence of cervical intraepithelial neoplasia (CIN) 2/3 were randomized in a double-blind manner to receive high-dose aliretinoin (50 mg), low-dose of aliretinoin (25 mg), or placebo daily for 12 weeks. Compliance and side effects were monitored at various time points during therapy. At the completion of therapy, all of the patients underwent a loop procedure. Histology of pretreatment biopsies was compared with that of loop specimens. One-hundred and fourteen patients with CIN 2/3 were enrolled in the study. In the 112 patients evaluable for toxicity, headache was the most common clinical side effect and was experienced more frequently (74%) in the high-dose aliretinoin group. Eight patients withdrew from the study before completion of study medication because of unacceptable side effects. In the 104 patients evaluable for efficacy, there was no statistical difference in the rate of regression among the placebo (32%), the low-dose aliretinoin (32%), and the high-dose aliretinoin (36%) groups. (P = not significant; power 0.06). Aliretinoin at these dosages and this schedule does not appear to result in significant regression rates in CIN 2/3 patients when compared with placebo. Headache is encountered frequently and may thwart efforts to increase the dose or duration of aliretinoin in future cervical cancer chemoprevention studies. The rate of histological regression in biopsied CIN 2/3 patients is high even over a short time interval, and emphasizes the importance of having a placebo arm and an adequate sample size in cervical dysplasia chemoprevention studies.