Nanomagnetic levitation three-dimensional cultures of breast and colorectal cancers.

BACKGROUND: Innovative technologies for drug discovery and development, cancer models, stem cell research, tissue engineering, and drug testing in various cell-based platforms require an application similar to the in vivo system. MATERIALS AND METHODS: We developed for the first time nanomagnetically levitated three-dimensional (3-D) cultures of breast cancer (BC) and colorectal cancer (CRC) cells using carbon-encapsulated cobalt magnetic nanoparticles. BC and CRC xenografts grown in severe combined immunodeficient (SCID) mice were evaluated for N-cadherin and epidermal growth factor receptor expressions. These phenotypes were compared with two-dimensional and 3-D cultures grown in a gel matrix. RESULTS: The BC and CRC cells grown by magnetic levitation formed microtissues. The levitated cultures had high viability and were maintained in culture for long periods of time. It has been observed that N-cadherin and epidermal growth factor receptor activities were highly expressed in the levitated 3-D tumor spheres and xenografts of CRC and BC cells. CONCLUSIONS: Nanomagnetically levitated 3-D cultures tend to form stable microtissues of BC and CRC and maybe more feasible for a range of applications in drug discovery or regenerative medicine.

Cholesterol crystals induce mechanical trauma, inflammation, and neo-vascularization in solid cancers as in atherosclerosis.

Background and aims: Cancer and atherosclerosis share common risk factors and inflammatory pathways that promote their proliferation via vascular endothelial growth factor (VEGF). Because CCs cause mechanical injury and inflammation in atherosclerosis, we investigated their presence in solid cancers and their activation of IL-1ß, VEGF, CD44, and Ubiquityl-Histone H2B (Ub-H2B), that promote cancer growth. Methods: Tumor specimens from eleven different types of human cancers and atherosclerotic plaques were assessed for CCs, free cholesterol content and IL1-ß by microscopy, immunohistochemistry, and biochemical analysis. Breast and colon cancer cell lines were cultured with and without CCs to select for expression of VEGF, CD44, and Ub-H2B. Western blot and immunofluorescence were performed on cells to assess the effect of CCs on signaling pathways. Results: Cancers displayed higher CC content (+2.29 ± 0.74 vs +1.46 ± 0.84, p < 0.0001), distribution (5.06 ± 3.13 vs 2.86 ± 2.18, p < 0.001) and free cholesterol (3.63 ± 4.02 vs 1.52 ± 0.56 µg/mg, p < 0.01) than cancer free marginal tissues and similarly for atherosclerotic plaques and margins (+2.31 ± 0.51 vs +1.44 ± 0.79, p < 0.02; 14.0 ± 5.74 vs 8.14 ± 5.52, p < 0.03; 0.19 ± 0.14 vs 0.09 ± 0.04 µg/mg, p < 0.02) respectively. Cancers displayed significantly increased expression of IL1-ß compared to marginal tissues. CCs treated cancer cells had increased expression of VEGF, CD44, and Ub-H2B compared to control. By microscopy, CCs were found perforating cancer tumors similar to plaque rupture. Conclusions: These findings suggest that CCs can induce trauma and activate cytokines that enhance cancer growth as in atherosclerosis.

Prognostic significance of p53 codon 72 polymorphism differs with race in colorectal adenocarcinoma.

PURPOSE: Several studies have examined the prognostic value of the codon 72 polymorphism of the p53 gene in colorectal adenocarcinoma, but none have addressed patient race/ethnicity. Therefore, this study assessed the prognostic value of this polymorphism in African American and Caucasian colorectal adenocarcinoma patients separately. EXPERIMENTAL DESIGN: Colorectal adenocarcinomas from 137 African Americans and 236 non-Hispanic Caucasians were assessed for p53 mutations and genotyped for the codon 72 polymorphism. The phenotypes were correlated with p53 mutational status, clinicopathologic features, and patient survival using the chi(2) test and Kaplan-Meier and Cox regression models. RESULTS: The incidence of p53 mutations was similar in African American and Caucasian patients (50% versus 54%, respectively); however, the homozygous Pro72 allele frequency was higher in African Americans (17%) as compared with Caucasians (7%). In contrast, the homozygous Arg72 allele frequency was higher in Caucasians (36%) than in African Americans (19%). In African Americans but not Caucasians, the Pro/Pro phenotype significantly correlated with a higher incidence of missense p53 mutations and with nodal metastasis. African Americans, but not Caucasians, with the Pro/Pro phenotype had significantly higher mortality (log-rank P = 0.005 versus. P = 0.886) and risk of death due to colorectal adenocarcinoma (hazard ratio, 2.15; 95% confidence interval, 1.02-4.53 versus hazard ratio, 1.60; 95% confidence interval, 0.69-3.18) than those with the phenotype Arg/Arg or Arg/Pro. CONCLUSIONS: The higher frequency of the Pro/Pro phenotype of p53 in African American patients with colorectal adenocarcinoma is associated with an increased incidence of p53 mutations, with advanced tumor stage, and with short survival.

Clinical significance of a novel single nucleotide polymorphism in the 5' untranslated region of the Rabphillin-3A-Like gene in colorectal adenocarcinoma.

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.

p53 Nuclear accumulation and Bcl-2 expression in contiguous adenomatous components of colorectal adenocarcinomas predict aggressive tumor behavior.

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.

Functional consequence of the p53 codon 72 polymorphism in colorectal cancer.

BACKGROUND: The codon 72 polymorphism in p53 has been implicated in colorectal cancer (CRC) risk, prognosis and CRC health disparities. We examined the functional consequence of this polymorphism in CRC. EXPERIMENTAL DESIGN: Plasmids (pCMV6) that express different phenotypes of p53 [p53 wild type (wt) at codon 72 (R72wt), R72wt with mutation at codon 273 cysteine (R72273Cys), p53 mutation at codon 72 (P72wt) and P72wt with mutation at codon 273 (P72273Cys)] were constructed. The CRC cell line Caco2, which does not express p53 for in vitro studies, was used as host. CRC xenografts were established in severe combined immunodeficient (SCID) mice using established cell lines. CRC surgical specimens, corresponding normal colon, and tumor xenografts were sequenced for codon 72 polymorphism of p53. Proteins signaling mechanisms were evaluated to assess the functional consequence of P72 phenotype of p53. RESULTS: This study demonstrated a significantly increased survival of cells expressing P72wt, mutant phenotype, versus R72wt phenotype. WB analyses revealed that P72wt induced activation of p38 and RAF/MEK/ extracellular signal-regulated kinase (ERK) MAP kinases. Activation of CREB was found to be higher in tumors that exhibit P72 phenotype. Metastatic lesions of CRC expressed more phospho-CREB than non-metastatic lesions. The expression of P72wt promoted CRC metastasis. CONCLUSIONS: P72 contributes to the aggressiveness of CRC. Because P72 is over-expressed in CRC, specifically in African-American patients, this suggests a role for P72 in cancer health disparities. This work was supported by NIH/NCI Workforce Diversity Grant R21-CA171251 & U54CA118948.

Prognostic value of loss of heterozygosity and sub-cellular localization of SMAD4 varies with tumor stage in colorectal cancer.

BACKGROUND: Although loss of heterozygosity (LOH) at chromosome location 18q21 and decreased expression of SMAD4 in invasive colorectal cancers (CRCs) correlate with poor patient survival, the prognostic value of LOH at 18q21 and sub-cellular localization of SMAD4 have not been evaluated in relation to tumor stage. METHODS: Genomic DNA samples from 209 formalin-fixed, paraffin-embedded sporadic CRC tissues and their matching controls were analyzed for 18q21 LOH, and corresponding tissue sections were evaluated by immunohistochemistry for expression of SMAD4 and assessed for its sub-cellular localization (nuclear vs. cytoplasmic). In addition, 53 frozen CRCs and their matching control tissues were analyzed for their mutational status and mRNA expression of SMAD4. The phenotypic expression pattern and LOH status were evaluated for correlation with patient survival by the use of Kaplan-Meier and Cox regression models. RESULTS: LOH of 18q21 was detected in 61% of the informative cases. In 8% of the cases, missense point mutations were detected in Smad4. In CRCs, relative to controls, there was increased SMAD4 staining in the cytoplasm (74%) and decreased staining in the nuclei (37%). LOH of 18q21 and high cytoplasmic localization of SMAD4 were associated with shortened overall survival of Stage II patients, whereas low nuclear expression of SMAD4 was associated with worse survival, but only for patients with Stage III CRCs. CONCLUSIONS: LOH of 18q21 and high cytoplasmic localization of SMAD4 in Stage II CRCs and low nuclear SMAD4 in Stage III CRCs are predictors of shortened patient survival.

Association between Hepatitis C Virus Infection, p53 Phenotypes, and Gene Variants of Adenomatous Polyposis Coli in Hepatocellular Carcinomas.

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.

Nef-M1, a peptide antagonist of CXCR4, inhibits tumor angiogenesis and epithelial­to­mesenchymal transition in colon and breast cancers.

The Nef-M1 peptide competes effectively with the natural ligand of CXC chemokine receptor 4 (CXCR4), stromal cell-derived factor 1-alpha, to induce apoptosis and inhibit growth in colon cancer (CRC) and breast cancer (BC). Its role in tumor angiogenesis, and epithelial-to-mesenchymal transition (EMT) regulation, key steps involved in tumor growth and metastasis, are unknown. We evaluated the angioinhibitory effect of Nef-M1 peptide and examined its role in the inhibition of EMT in these cancers. Colon (HT29) and breast (MDA-MB231) cancer cells expressing CXCR4 were studied in vitro and in xenograft tumors propagated in severe combined immunodeficient mice. The mice were treated intraperitoneally with Nef-M1 or scrambled amino acid sequence of Nef-M1 (sNef-M1) peptide, a negative control, starting at the time of tumor implantation. Sections from tumors were evaluated for tumor angiogenesis, as measured by microvessel density (MVD) based on immunostaining of endothelial markers. In vitro tumor angiogenesis was assessed by treating human umbilical vein endothelial cells with conditioned media from the tumor cell lines. A BC cell line (MDA-MB 468) which does not express CXCR4 was used to study the actions of Nef-M1 peptide. Western blot and immunofluorescence analyses assessed the effect of Nef-M1 on tumor angiogenesis and EMT in both tumors and cancer cells. Metastatic lesions of CRC and BC expressed more CXCR4 than primary lesions. It was also found that tumors from mice treated with sNef-M1 had well established vascularity, while Nef-M1 treated tumors had very poor vascularization. Indeed, the mean MVD was lower in tumors from Nef-M1 treated mice than in sNef-M1 treated tumors. Nef-M1 treated tumor has poor morphology and loss of endothelial integrity. Although conditioned medium from CRC or BC cells supported HUVEC tube formation, the conditioned medium from Nef-M1 treated CRC or BC cells did not support tube formation. Western blot analyses revealed that Nef-M1 effectively suppressed the expression of VEGF-A in CRC and BC cells and tumors. This suggests that Nef-M1 treated CRC and BC cells are more consistent with E-cadherin signature, and thus appears more epithelial in nature. Our data indicate that Nef-M1 peptide inhibits tumor angiogenesis and the oncogenic EMT process. Targeting the chemokine receptor, CXCR4, mediated pathways using Nef-M1 may prove to be a novel therapeutic approach for CRC and BC.

Clinical Implications of Rabphillin-3A-Like Gene Alterations in Breast Cancer.

For the rabphillin-3A-like (RPH3AL) gene, a putative tumor suppressor, the clinical significance of genetic alterations in breast cancers was evaluated. DNA and RNA were extracted from formalin-fixed, paraffin-embedded (FFPE) cancers and matching normal tissues. DNA samples were assessed for loss of heterozygosity (LOH) at the 17p13.3 locus of RPH3AL and the 17p13.1 locus of the tumor suppressor, TP53. RPH3AL was sequenced, and single nucleotide polymorphisms (SNPs) were genotyped. RNA samples were evaluated for expression of RPH3AL, and FFPE tissues were profiled for its phenotypic expression. Alterations in RPH3AL were correlated with clinicopathological features, LOH of TP53, and patient survival. Of 121 cancers, 80 had LOH at one of the RPH3AL locus. LOH of RHP3AL was associated with nodal metastasis, advanced stage, large tumor size, and poor survival. Although ~50% were positive for LOH at the RPH3AL and TP53 loci, 19 of 105 exhibited LOH only at the RPH3AL locus. Of these, 12 were non-Hispanic Caucasians (Whites), 15 had large tumors, and 12 were older (>50 years). Patients exhibiting LOH at both loci had shorter survival than those without LOH at these loci (log-rank, P = 0.014). LOH at the TP53 locus alone was not associated with survival. Analyses of RPH3AL identified missense point mutations in 19 of 125 cases, a SNP (C>A) in the 5'untranslated region at -25 (5'UTR-25) in 26 of 104, and a SNP (G>T) in the intronic region at 43 bp downstream to exon-6 (intron-6-43) in 79 of 118. Genotype C/A or A/A of the SNP at 5'UTR-25 and genotype T/T of a SNP at intron-6-43 were predominantly in Whites. Low levels of RNA and protein expression of RPH3AL were present in cancers relative to normal tissues. Thus, genetic alterations in RPH3AL are associated with aggressive behavior of breast cancers and with short survival of patients.

Nef-M1, a CXCR4 Peptide Antagonist, Enhances Apoptosis and Inhibits Primary Tumor Growth and Metastasis in Breast Cancer.

Results from studies with animal models suggest that, in many cancers, CXCR4 is an important therapeutic target and that CXCR4 antagonists may be promising treatments for primary cancers and for metastases. The Nef protein effectively competes with CXCR4's natural ligand, SDF-1α, and induces apoptosis. As described in this report, the Nef-M1 peptide (Nef protein amino acids 50 - 60) inhibits primary tumor growth and metastasis of breast cancer (BC). Four BC cell lines (MDA-MB-231, MDA-MB-468, MCF 7, and DU4475) and primary human mammary epithelium (HME) cells were evaluated for their response to the Nef protein and to the Nef-M1 peptide. The presence of CXCR4 receptors in these cells was determined by RT-PCR, Western blot (WB), and immunohistochemical analyses. The apoptotic effect of Nef-M1 was assessed by terminal transferase dUTP nick-end labeling (TUNEL). WBs was used to assess caspase 3 activation. BC xenografts grown in SCID mice were evaluated for the presence of CXCR4 and for their metastatic potential. CXCR4 was presented in MDA-MB-231, MCF 7, and DU 4475 BC cells but not in MDA-MB-468 BC or HME cells. Cells expressing CXCR4 and treated with Nef-M1 peptide or the Nef protein had higher rates of apoptosis than untreated cells. Caspase-3 activation increased in MDA-MB 231 cells treated with the Nef protein, the Nef 41 - 60 peptide, or Nef-M1. Nef-M1, administered to mice starting at the time of xenograft implantation, inhibited growth of primary tumors and metastatic spread. Untreated mice developed diffuse intraperitoneal metastases. We conclude that, in BCs, Nef-M1, through interaction with CXCR4, inhibits primary tumor growth and metastasis by causing apoptosis.

Socioeconomic status, p53 abnormalities, and colorectal cancer.

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.

The prognostic value of microRNAs varies with patient race/ethnicity and stage of colorectal cancer.

PURPOSE: MicroRNAs (miRNA) have potential prognostic value for colorectal cancers; however, their value based on patient race/ethnicity and pathologic stage has not been determined. The goal was to ascertain the prognostic value of 5 miRNAs with increased expression in colorectal cancers of African American (black) and non-Hispanic Caucasian (white) patients. EXPERIMENTAL DESIGN: TaqMan quantitative real-time PCR was used to quantify expression of miR-20a, miR-21, miR-106a, miR-181b, and miR-203 in paired normal and tumor colorectal cancer archival tissues collected from 106 black and 239 white patients. The results were correlated with overall survival based on patient race/ethnicity and pathologic stage. Because decisions about adjuvant therapy are important for stage III colorectal cancers, and because miR-181b seemed to have prognostic value only for stage III black patients, we assessed its prognostic value in a separate cohort of 36 stage III colorectal cancers of blacks. RESULTS: All 5 miRNAs had higher expression in colorectal cancers (>1.0-fold) than in corresponding normal tissues. High expression of miR-203 was associated with poor survival of whites with stage IV colorectal cancers (HR = 3.00; 95% CI, 1.29-7.53), but in blacks it was an indicator of poor survival of patients with stages I and II colorectal cancers (HR = 5.63; 95% CI, 1.03-30.64). Increased miR-21 expression correlated with poor prognosis for white stage IV patients (HR = 2.50; 95% CI, 1.07-5.83). In both test and validation cohorts, high miR-181b expression correlated with poor survival of only black patients with stage III colorectal cancers (HR = 1.94; 95% CI, 1.03-3.67). CONCLUSION: These preliminary findings suggest that the prognostic value of miRNAs in colorectal cancers varies with patient race/ethnicity and stage of disease.

Dual-mode interaction between quercetin and DNA-damaging drugs in cancer cells.

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.

miRNAs are stable in colorectal cancer archival tissue blocks.

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.

Prognostic significance and gene expression profiles of p53 mutations in microsatellite-stable stage III colorectal adenocarcinomas.

Although the prognostic value of p53 abnormalities in Stage III microsatellite stable (MSS) colorectal cancers (CRCs) is known, the gene expression profiles specific to the p53 status in the MSS background are not known. Therefore, the current investigation has focused on identification and validation of the gene expression profiles associated with p53 mutant phenotypes in MSS Stage III CRCs. Genomic DNA extracted from 135 formalin-fixed paraffin-embedded tissues, was analyzed for microsatellite instability (MSI) and p53 mutations. Further, mRNA samples extracted from five p53-mutant and five p53-wild-type MSS-CRC snap-frozen tissues were profiled for differential gene expression by Affymetrix Human Genome U133 Plus 2.0 arrays. Differentially expressed genes were further validated by the high-throughput quantitative nuclease protection assay (qNPA), and confirmed by quantitative real-time polymerase chain reaction (qRT-PCR) and by immunohistochemistry (IHC). Survival rates were estimated by Kaplan-Meier and Cox regression analyses. A higher incidence of p53 mutations was found in MSS (58%) than in MSI (30%) phenotypes. Both univariate (log-rank, P = 0.025) and multivariate (hazard ratio, 2.52; 95% confidence interval, 1.25-5.08) analyses have demonstrated that patients with MSS-p53 mutant phenotypes had poor CRC-specific survival when compared to MSS-p53 wild-type phenotypes. Gene expression analyses identified 84 differentially expressed genes. Of 49 down-regulated genes, LPAR6, PDLIM3, and PLAT, and, of 35 up-regulated genes, TRIM29, FUT3, IQGAP3, and SLC6A8 were confirmed by qNPA, qRT-PCR, and IHC platforms. p53 mutations are associated with poor survival of patients with Stage III MSS CRCs and p53-mutant and wild-type phenotypes have distinct gene expression profiles that might be helpful in identifying aggressive subsets.

Evaluation of lymph node numbers for adequate staging of Stage II and III colon cancer.

BACKGROUND: Although evaluation of at least 12 lymph nodes (LNs) is recommended as the minimum number of nodes required for accurate staging of colon cancer patients, there is disagreement on what constitutes an adequate identification of such LNs. METHODS: To evaluate the minimum number of LNs for adequate staging of Stage II and III colon cancer, 490 patients were categorized into groups based on 1-6, 7-11, 12-19, and ≥ 20 LNs collected. RESULTS: For patients with Stage II or III disease, examination of 12 LNs was not significantly associated with recurrence or mortality. For Stage II (HR = 0.33; 95% CI, 0.12-0.91), but not for Stage III patients (HR = 1.59; 95% CI, 0.54-4.64), examination of ≥20 LNs was associated with a reduced risk of recurrence within 2 years. However, examination of ≥20 LNs had a 55% (Stage II, HR = 0.45; 95% CI, 0.23-0.87) and a 31% (Stage III, HR = 0.69; 95% CI, 0.38-1.26) decreased risk of mortality, respectively. For each six additional LNs examined from Stage III patients, there was a 19% increased probability of finding a positive LN (parameter estimate = 0.18510, p < 0.0001). For Stage II and III colon cancers, there was improved survival and a decreased risk of recurrence with an increased number of LNs examined, regardless of the cutoff-points. Examination of ≥7 or ≥12 LNs had similar outcomes, but there were significant outcome benefits at the ≥20 cutoff-point only for Stage II patients. For Stage III patients, examination of 6 additional LNs detected one additional positive LN. CONCLUSIONS: Thus, the 12 LN cut-off point cannot be supported as requisite in determining adequate staging of colon cancer based on current data. However, a minimum of 6 LNs should be examined for adequate staging of Stage II and III colon cancer patients.

miRNAs as biomarkers for management of patients with colorectal cancer.

miRNAs serve as micromanagers, negatively regulating gene expression. Since altered miRNA expression is implicated in the pathobiology of various cancers, including colorectal cancers (CRCs), these molecules serve as potential therapeutic targets. Manipulation of miRNAs may offer an alternative therapy for chemo- and radio-resistant CRCs. For CRC patients, miRNA expression patterns can be used for diagnosis, and to predict prognosis and efficacy of therapy. This article describes the methodological approaches for miRNA measurement, their function in the pathobiology of CRCs and their potential clinical utility.

Development of combination tapered fiber-optic biosensor dip probe for quantitative estimation of interleukin-6 in serum samples.

A combination tapered fiber-optic biosensor (CTFOB) dip probe for rapid and cost-effective quantification of proteins in serum samples has been developed. This device relies on diode laser excitation and a charged-coupled device spectrometer and functions on a technique of sandwich immunoassay. As a proof of principle, this technique was applied in a quantitative estimation of interleukin IL-6. The probes detected IL-6 at picomolar levels in serum samples obtained from a patient with lupus, an autoimmune disease, and a patient with lymphoma. The estimated concentration of IL-6 in the lupus sample was 5.9 ± 0.6 pM, and in the lymphoma sample, it was below the detection limit. These concentrations were verified by a procedure involving bead-based xMAP technology. A similar trend in the concentrations was observed. The specificity of the CTFOB dip probes was assessed by analysis with receiver operating characteristics. This analysis suggests that the dip probes can detect 5-pM or higher concentration of IL-6 in these samples with specificities of 100%. The results provide information for guiding further studies in the utilization of these probes to quantify other analytes in body fluids with high specificity and sensitivity.

Development and progression of colorectal neoplasia.

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).