Staging of colorectal cancer using lipid biomarkers and machine learning.

INTRODUCTION: Colorectal cancer (CRC) is the third most commonly diagnosed cancer worldwide. Alteration in lipid metabolism and chemokine expression are considered hallmark characteristics of malignant progression and metastasis of CRC. Validated diagnostic and prognostic biomarkers are urgently needed to define molecular heterogeneous CRC clinical stages and subtypes, as liver dominant metastasis has poor survival outcomes. OBJECTIVES: The aim of this study was to integrate lipid changes, concentrations of chemokines, such as platelet factor 4 and interleukin 8, and gene marker status measured in plasma samples, with clinical features from patients at different CRC stages or who had progressed to stage-IV colorectal liver metastasis (CLM). METHODS: High-resolution liquid chromatography-mass spectrometry (HR-LC-MS) was used to determine the levels of candidate lipid biomarkers in each CRC patient's preoperative plasma samples and combined with chemokine, gene and clinical data. Machine learning models were then trained using known clinical outcomes to select biomarker combinations that best classify CRC stage and group. RESULTS: Bayesian neural net and multilinear regression-machine learning identified candidate biomarkers that classify CRC (stages I-III), CLM patients and control subjects (cancer-free or patients with polyps/diverticulitis), showing that integrating specific lipid signatures and chemokines (platelet factor-4 and interluken-8; IL-8) can improve prognostic accuracy. Gene marker status could contribute to disease prediction, but requires ubiquitous testing in clinical cohorts. CONCLUSION: Our findings demonstrate that correlating multiple disease related features with lipid changes could improve CRC prognosis. The identified signatures could be used as reference biomarkers to predict CRC prognosis and classify stages, and monitor therapeutic intervention.

High preoperative levels of circulating SFRP5 predict better prognosis in colorectal cancer patients.

The purpose of this research was to investigate the diagnostic and prognostic value of circulating SFRP5 (cSFRP5) in colorectal cancer (CRC). We evaluated preoperative cSFRP5 levels in CRC patients and controls (n = 208). We found significantly higher cSFRP5 levels in CRC patients compared with non-CRC controls (p < 0.001). Levels of cSFRP5 were significantly lower in CRC patients with either vascular invasion (p = 0.001) or liver metastasis (p = 0.016). High cSFRP5 levels were associated with longer disease-free survival in both univariate (p = 0.024) and multivariate (p = 0.015) analyses. Analysis of an independent tissue cohort from The Cancer Genome Atlas database revealed significantly lower SFRP5 RNA expression in CRC tumor tissue compared with adjacent normal mucosa (n = 590 vs 47; p < 0.0001). Our findings confirm the role of cSFRP5 as a physiologic tumor suppressor and demonstrate its potential diagnostic and prognostic value in CRC.

Combination of laser microdissection, 2D-DIGE and MALDI-TOF MS to identify protein biomarkers to predict colorectal cancer spread.

Biomarkers are urgently required to support current histological staging to provide additional accuracy in stratifying colorectal cancer (CRC) patients according to risk of spread to properly assign adjuvant chemotherapy after surgery. Chemotherapy is given to patients with stage III to reduce the risk of recurrence but is controversial in stage II patients. Up to 25% of stage II patients will relapse within 5 years after tumor removal and when this occurs cure is seldom possible. The aim of this study was to identify protein biomarkers to stratify risk of spread of CRC patients. Laser micro-dissection was used to isolate cancer cells from primary colorectal tumors of stage II patients which did or did not metastasize within 5 years after surgical resection. Protein expression differences between two groups of tumors were profiled by 2D-DIGE with saturation CyDye labeling and identified using MALDI-TOF mass spectrometry. Evaluation of protein candidates was conducted using tissue micro array (TMA) immunohistochemistry on 125 colorectal tumor tissue samples of different stages. A total of 55 differentially expressed proteins were identified. Ten protein biomarkers were chosen based on p value and ratio between non metastasized and metastazised groups and evaluated on 125 tissues using TMA immunohistochemistry. Expression of HLAB, protein 14-3-3ß, LTBP3, ADAMTS2, JAG2 and NME2 on tumour cells was significantly associated with clinical parameters related to tumour progression, invasion and metastasis. Kaplan-Meier survival curve showed strong expression of six proteins was associated with good CRC specific survival. Expression of HLAB, ADAMTS2, LTBP3, JAG2 and NME2 on tumour cells, was associated with tumour progression and invasion, metastasis and CRC specific survival may serve as potential biomarkers to stratify CRC patients into low and high risk of tumour metastasis. Combined methods of laser microdissection, 2D DIGE with saturation labelling and MALDI-TOF MS proved to be resourceful techniques capable of identifying protein biomarkers to predict risk of spread of CRC to liver.

Discordant frequencies of tissue-resident and circulating CD180-negative B cells in chronic rhinosinusitis.

BACKGROUND: The unconventional toll-like receptor (TLR) CD180 is implicated in chronic inflammatory diseases; however, its role in chronic rhinosinusitis (CRS) has yet to be investigated. Here we study the expression of CD180, its homologue TLR4 and myeloid differentiation factor 1 (MD1) on mucosal and systemic immune cell populations in relation to serum immunoglobulin G (IgG) levels. METHODS: A total of 70 patients were recruited to the study. Mucosal and peripheral blood samples were prospectively collected from CRS patients and non-CRS controls without evidence of sinus disease. The expression of TLR4, MD1, and CD180 was investigated using qualitative real-time polymerase chain reaction (qRT-PCR), immunohistochemistry, and flow cytometry. Serum IgG levels were determined using enzyme-linked immunosorbent assay (ELISA). RESULTS: CRS with nasal polyps (CRSwNP) patients had significantly increased messenger RNA (mRNA) expression of CD180 and MD1 compared to controls (5.54-fold and 2.1-fold, respectively, p < 0.01). B cells lacking CD180 were lower in CRSwNP tissue compared to CRS without nasal polyps (CRSsNP) and controls (21.07 ± 6.41 vs 41.61 ± 7.82 vs 40.06 ± 8.06; p < 0.01) but higher in blood (39.18 ± 8.3 vs 17.95 ± 7.82 and 12.49 ± 4.92; p ≤ 0.05). CONCLUSION: Changes in mucosal and peripheral CD180-expressing B cells were identified in CRSwNP patients compared to CRSsNP and controls. This suggests a role for these cells in the dysregulated immune response in these patients.

Soluble HLA-G is a differential prognostic marker in sequential colorectal cancer disease stages.

The expression of HLA-G by tumour cells is an established mechanism to escape recognition and immune mediated destruction, allowing tumour survival, growth and metastasis. However, the prognostic value of soluble HLA-G (sHLA-G) remains unknown. Mucinous carcinoma (MC) is a distinct form of colorectal cancer (CRC) found in 10 to 15% of patients, which has long been associated with poor response to treatment. To investigate the prognostic value of plasma sHLA-G levels in CRC patients, preoperative plasma sHLA-G levels were determined by ELISA in CRC patients (n = 133). In addition, the local expression of HLA-G in tumour biopsies was assessed using tissue microarray analysis (n = 255). Within the high 33rd percentile of sHLA-G levels (265-890 U/mL; n = 44) we observed higher frequency of MC patients (p = 0.012; Chi-square), and higher sHLA-G levels in patients with vascular invasion (p = 0.035; two-tailed t-test). Moreover, MC patients had significantly higher sHLA-G levels compared to those with adenocarcinoma not otherwise specified (p = 0.036; two-tailed t-test). Surprisingly, while stage II patients showed negative correlation between sHLA-G levels and liver metastasis free survival (LMFS) (p = 0.041; R = -0.321), in stage III patients high sHLA-G levels were associated with significantly longer LMFS (p = 0.002), and sHLA-G levels displayed positive correlation with LMFS (p = 0.006; R = 0.409). High HLA-G expression in tumours was associated with poor cancer specific overall survival in stage II to III (p = 0.01), and with shorter LMFS in stage II patients (p = 0.004). Our findings reveal that sHLA-G levels are associated with distinct progression patterns in consecutive disease stages, indicating a potential value as surrogate marker in the differential prognosis of CRC.

Overexpression of aminoacylase 1 is associated with colorectal cancer progression.

Aminoacylase 1 (ACY1) is a cytosolic enzyme responsible for amino acid deacylation during intracellular protein degradation. ACY1 has been implicated in a number of human tumor types. However, the exact role of ACY1 in tumor development remains elusive because it was found to be lost in small cell lung cancer and renal cell carcinoma but overexpressed in colorectal cancer (CRC). The present study aims to further clarify the relationship of ACY1 with CRC progression. Immunohistochemical staining was performed in tissue microarrays composed of 120 cases of CRC using a monoclonal anti-ACY1 antibody. Immunoreactivity was analyzed in association with patients' clinicopathologic parameters and survival time. The role of ACY1 in cell proliferation and apoptosis was assessed by silencing its expression in HCT116 cells using a small interfering RNA. Strong expression of ACY1 was found to be significantly associated with more advanced TNM stage, lymph node metastasis, positive vascular invasion, and shorter cancer-specific survival. ACY1 knockdown significantly inhibited cell proliferation and induced apoptosis. We concluded that ACY1 expression in CRC varies with stage and appears to play a role in cell proliferation and apoptosis. Further evaluation of ACY1 as a clinically useful prognostic marker and a potential drug target for CRC would seem worthwhile.

Cathepsin D Expression in Colorectal Cancer: From Proteomic Discovery through Validation Using Western Blotting, Immunohistochemistry, and Tissue Microarrays.

Despite recent advances in surgical techniques and therapeutic treatments, survival from colorectal cancer (CRC) remains disappointing with some 40-50% of newly diagnosed patients ultimately dying of metastatic disease. Current staging by light microscopy alone is not sufficiently predictive of prognosis and would benefit from additional support from biomarkers in order to stratify patients appropriately for adjuvant therapy. We have identified that cathepsin D expression was significantly greater in cells from invasive front (IF) area and liver metastasis (LM) than those from main tumour body (MTB). Cathepsin D expression was subsequently examined by immunohistochemistry in tissue microarrays from 119 patients with CRC. Strong expression in tumour cells at the IF did not correlate significantly with any clinico-pathological parameters examined or patient survival. However, cathepsin D expression in cells from the MTB was highly elevated in late stage CRC and showed significant correlation with subsequent distant metastasis and shorter cancer-specific survival. We also found that macrophages surrounding tumour cells stained strongly for cathepsin D but there was no significant correlation found between cathepsin D in macrophages at IF and MTB of CRC patient with the clinic-pathological parameters examined.

TUFM is a potential new prognostic indicator for colorectal carcinoma.

AIMS: Mitochondrial Tu translation elongation factor (TUFM) is a nuclear encoded protein that participates in mitochondrial polypeptide translation. TUFM has been reported to be over-expressed in many tumour types including colorectal carcinoma (CRC) by proteomics. The present study aims to examine the prognostic implication of TUFM in CRC. METHODS: Immunohistochemical staining was performed in tissue microarrays composed of 123 cases of CRC using a polyclonal anti-TUFM antibody. Immunoreactivity was quantified using Image-Pro plus software, and analysed in association with patients' clinicopathological parameters and survival time. RESULTS: The immunoreactivity of TUFM was negative in 25%, weak in 50% and strong in 25% of CRC cases. TUFM immunoreactivity had no significant association with the clinicopathological parameters examined including TNM stage and grade. However, strong TUFM expression significantly correlated with a higher 5-year recurrence rate (p = 0.024). Kaplan-Meier analysis revealed that patients with strong TUFM expression had significantly shorter cancer-specific survival than patients with negative TUFM (log-rank test, p = 0.038). In multivariate analysis, strong TUFM expression remained a stage-independent unfavourable prognostic indicator (p = 0.024). CONCLUSIONS: Increased expression of TUFM is a promising new prognostic indicator for CRC. Selective inhibition of TUFM in tumour cells may present a new avenue for the targeted therapy of this cancer.

Compatibility of toluidine blue with laser microdissection and saturation labeling DIGE.

Tissue fixation and staining protocols for laser microdissection are frequently not fully compatible with subsequent proteomic analysis. We compared the effect of three common histological stains (toluidine blue (TB), hemotoxylin, and hematoxylin and eosin (HE)) on tissue visualization, protein recovery, the saturation labeling reaction, and 2-D electrophoresis. TB provided the best visualization of colorectal tumor tissue during laser microdissection (LMD) and had a comparable effect on protein recovery and the saturation labeling reaction with hematoxylin, provided a modified 2-D clean-up protocol was used. Eosin inhibited both protein recovery and the saturation labeling reaction.

Naturally derived micelles for rapid in vitro screening of potential cholesterol-lowering bioactives.

A high plasma cholesterol level, especially low-density lipoprotein cholesterol, indicates increased risk of cardiovascular diseases. Plasma cholesterol levels are influenced by diet and cholesterol biosynthesis, uptake, and secretion. Cholesterol uptake involves solubilization into complex phospholipid spherical bodies termed micelles that facilitate the transport of lipids through the gut brush border membrane into enterocytes. In vitro assays reported to date to determine potential cholesterol-lowering effects of various compounds require artificial micelle preparations that are elaborate and time-consuming to prepare. The aims of this study were to compare the efficacy of artificially prepared micelles with naturally derived micelles from pig's bile and to test their ability to assess potential inhibitors of cholesterol uptake. The suitability of pig's bile-derived micelles was tested both at the level of the micelle and at cellular uptake using cultured Caco-2 cells. Known cholesterol uptake inhibitors at the micelle (green tea catechins) and at the Caco-2 cell (beta-lactoglobulin-derived peptide, IIAEK) were used as reference inhibitory compounds. It was concluded that pig's bile was a rapid, reproducible, convenient, and cost-effective source of micelles for cholesterol micelle solubility and cellular uptake assay systems and is suitable for screening purposes focused on identifying potential cholesterol-lowering agents.

Antitumor activity of extract of Zingiber aromaticum and its bioactive sesquiterpenoid zerumbone.

The anticancer properties of zerumbone (2,6,9 humulatriene-8-one, a sesquiterpenoid) from Zingiber aromaticum were compared with those of curcumin from Curcuma longa in an in vitro MTT tetrazolium salt assay using HT-29, CaCo-2, and MCF-7 cancer cells and in an azoxymethane (AOM)-induced animal model of colon cancer using aberrant crypt foci (ACFs) as a preneoplastic marker. The IC50 of zerumbone was approximately 10 mM and that of curcumin was 25 mM. Cell cycle arrest in HT-29 cells was observed at G0/G1 for 10 and 12.5 mM and G2/M for 25 mM after 24 h at concentrations of 10-25 mM of zerumbone, and a concentration-dependent increase in apoptosis (2-6% of viable cells) was observed after 48 h using the same concentration range. Male Sprague-Dawley rats were fed extracts in an AIN diet prepared from the equivalent of 4% by weight of dried rhizomes of Z. aromaticum and C. longa. ACFs were induced by two doses (15 mg/kg body weight) subcutaneously of AOM 1 wk apart, the rats were killed 10 wk later, and the ACFs were assessed in the colon. Total ACFs were significantly reduced by Z. aromaticum extract (down 21%, P < 0.05) relative to control, the effect being most evident with large ACFs (>3 aberrant crypts per focus). Similar reductions were observed with 4% C. longa extract in the diet (down 24%, P < 0.01) and with 2,000 ppm curcumin, the effect being particularly evident with large ACFs. The concentration of zerumbone in the Z. aromaticum extract diet was assayed at 300 ppm and of curcumin in the C. longa extract diet was also 300 ppm, i.e., the extract of C. longa was as effective at one-seventh the concentration of curcumin as the positive control. Zerumbone is effective as an anticancer agent, possibly by its apoptosis-inducing and antiproliferative influences. This latter possibility is currently being investigated.