p21 as a Predictor and Prognostic Indicator of Clinical Outcome in Rectal Cancer Patients.

The cell cycle plays a key and complex role in the development of human cancers. p21 is a potent cyclin-dependent kinase inhibitor (CDKI) involved in the promotion of cell cycle arrest and the regulation of cellular senescence. Altered p21 expression in rectal cancer cells may affect tumor cells' behavior and resistance to neoadjuvant and adjuvant therapy. Our study aimed to ascertain the relationship between the differential expression of p21 in rectal cancer and patient survival outcomes. Using tissue microarrays, 266 rectal cancer specimens were immunohistochemically stained for p21. The expression patterns were scored separately in cancer cells retrieved from the center and the periphery of the tumor; compared with clinicopathological data, tumor regression grade (TRG), disease-free, and overall survival. Negative p21 expression in tumor periphery cells was significantly associated with longer overall survival upon the univariate (p = 0.001) and multivariable analysis (p = 0.003, HR = 2.068). Negative p21 expression in tumor periphery cells was also associated with longer disease-free survival in the multivariable analysis (p = 0.040, HR = 1.769). Longer overall survival times also correlated with lower tumor grades (p= 0.011), the absence of vascular and perineural invasion (p = 0.001; p < 0.005), the absence of metastases (p < 0.005), and adjuvant treatment (p = 0.009). p21 expression is a potential predictive and prognostic biomarker for clinical outcomes in rectal cancer patients. Negative p21 expression in tumor periphery cells demonstrated significant association with longer overall survival and disease-free survival. Larger prospective studies are warranted to investigate the ability of p21 to identify rectal cancer patients who will benefit from neoadjuvant and adjuvant therapy.

Prognostic Significance of MRE11 Overexpression in Colorectal Cancer Patients.

Meiotic recombination 11 (MRE11) plays a critical role in the DNA damage response and maintenance of genome stability and is associated with the prognosis for numerous malignancies. Here, we explored the clinicopathological significance and prognostic value of MRE11 expression in colorectal cancer (CRC), a leading cause of cancer-related deaths worldwide. Samples from 408 patients who underwent surgery for colon and rectal cancer between 2006 and 2011, including a sub-cohort of 127 (31%) patients treated with adjuvant therapy, were analyzed. In Kaplan-Meier survival analyses, we found that high MRE11 expression in the tumor center (TC) was significantly associated with poor disease-free survival (DFS; p = 0.045) and overall survival (OS; p = 0.039). Intriguingly, high MRE11 expression in the TC was also significantly correlated with reduced DFS (p = 0.005) and OS (p = 0.010) in the subgroup with right-sided primary CRC. In multivariate analyses, high MRE11 expression (hazard ratio [HR] = 1.697, 95% confidence interval [CI]: 1.034-2.785; p = 0.036) and lymphovascular/perineural invasion (LVI/PNI; HR = 1.922, 95% CI 1.122-3.293; p = 0.017) showed significant association with worse OS in patients with right-sided tumors but not those with left-sided tumors. Moreover, in patients with right-sided tumors, high MRE11 was associated with worse OS for those with lymph node involvement (p = 0.006) and LVI/PNI (p = 0.049). Collectively, our results suggest that MRE11 may serve as an independent prognostic marker in those with right-sided severe CRC, with clinical value in the management of these patients.

Aberrant Expression of RAD52, Its Prognostic Impact in Rectal Cancer and Association with Poor Survival of Patients.

The DNA damage response enables cells to survive and maintain genome integrity. RAD52 is a DNA-binding protein involved in the homologous recombination in DNA repair, and is important for the maintenance of tumour genome integrity. We investigated possible correlations between RAD52 expression and cancer survival and response to preoperative radiotherapy. RAD52 expression was examined in tumour samples from 179 patients who underwent surgery for rectal cancer, including a sub-cohort of 40 patients who were treated with neoadjuvant therapy. A high score for RAD52 expression in the tumour centre was significantly associated with worse disease-free survival (DFS; p = 0.045). In contrast, reduced RAD52 expression in tumour centre samples from patients treated with neoadjuvant therapy (n = 40) significantly correlated with poor DFS (p = 0.025) and overall survival (OS; p = 0.048). Our results suggested that RAD52 may have clinical value as a prognostic marker of tumour response to neoadjuvant radiation and both disease-free status and overall survival in patients with rectal cancer.

Ibudilast sensitizes glioblastoma to temozolomide by targeting Macrophage Migration Inhibitory Factor (MIF).

Recurrence in patients with glioblastoma (GBM) is inevitable resulting in short survival times, even in patients with O-6-Methylguanine-DNA Methyltransferase (MGMT) methylation. Other pathways must be activated to escape from temozolomide (TMZ) treatment, however acquired resistance mechanisms to TMZ are not well understood. Herein, frozen tumors from 36 MGMT methylated patients grouped according to overall survival were extracted and proteins were profiled using surface-enhanced laser desorption/ionization (SELDI) with time-of flight (TOF) proteomics to identify low molecular weight proteins that associated with poor survival outcomes. Overexpression of macrophage migration inhibitory factor (MIF) was identified in human GBM specimens that were MGMT methylated but showed poor survival. This correlation was confirmed in an independent cohort of human GBM. MIF overexpression has been reported in several cancer types, including GBM. We repurposed ibudilast, a specific MIF inhibitor, and treated patient derived cell lines. Ibudilast showed modest anti-proliferative activity however, when combined with TMZ, significant synergism was observed, resulting in cell cycle arrest and apoptosis. In vivo, combined ibudilast and TMZ treatment of a patient derived xenograft (PDX) model resulted in significantly longer overall survival. Our findings have significant clinical implications for people with GBM. Since clinical trials involving ibudilast have shown no adverse side effects and the drug readily penetrates the blood brain barrier, treatment of GBM with this combination is clinically achievable.

MRN (MRE11-RAD50-NBS1) Complex in Human Cancer and Prognostic Implications in Colorectal Cancer.

The MRE11-RAD50-NBS1 (MRN) complex has been studied in multiple cancers. The identification of MRN complex mutations in mismatch repair (MMR)-defective cancers has sparked interest in its role in colorectal cancer (CRC). To date, there is evidence indicating a relationship of MRN expression with reduced progression-free survival, although the significance of the MRN complex in the clinical setting remains controversial. In this review, we present an overview of the function of the MRN complex, its role in cancer progression, and current evidence in colorectal cancer. The evidence indicates that the MRN complex has potential utilisation as a biomarker and as a putative treatment target to improve outcomes of colorectal cancer.

Overexpression of the MRE11-RAD50-NBS1 (MRN) complex in rectal cancer correlates with poor response to neoadjuvant radiotherapy and prognosis.

BACKGROUND: The MRE11/RAD50/NBS1 (MRN) complex plays an essential role in detecting and repairing double-stranded breaks, and thus the potential roles of MRE11, RAD50 and NBS1 proteins in the pathogenesis of various cancers is the subject of investigation. This study was aimed at assessing the three-protein panel of MRN complex subunits as a potential radiosensitivity marker and evaluating the prognostic and clinicopathological implications of MRN expression in rectal cancer. METHODS: Samples from 265 rectal cancer patients treated with surgery and adjuvant chemoradiotherapy, including samples from 55 patients who were treated with neoadjuvant radiotherapy between 2000 and 2011, were analyzed. Expression of MRN complex proteins in tissue samples was determined by immunohistochemistry. Univariate and multivariate analyses were carried out to identify clinicopathological characteristics that are associated with the MRN three-protein panel expression in rectal cancer samples. RESULTS: In Kaplan-Meier survival analyses, we found that high level expression of MRN complex proteins in postoperative samples was associated with poor disease-free (p = 0.021) and overall (P = 0.002) survival. Interestingly, high MRN expression also correlated with poor disease-free (P = 0.047) and overall (P = 0.024) survival in the neoadjuvant radiotherapy subgroup. In multivariate analysis, combined MRN expression (hazard ratio = 2.114, 95% confidence interval 1.096-4.078, P = 0.026) and perineural invasion (hazard ratio = 2.160, 95% confidence interval 1.209-3.859, P = 0.009) were significantly associated with a worse disease-free survival. CONCLUSIONS: Expression levels of MRN complex proteins significantly predict disease-free survival in rectal cancer patients, including those treated with neoadjuvant radiotherapy, and may have value in the management of these patients.

Early Postoperative Low Expression of RAD50 in Rectal Cancer Patients Associates with Disease-Free Survival.

BACKGROUND: Molecular biomarkers have the potential to predict response to the treatment of rectal cancer. In this study, we aimed to evaluate the prognostic and clinicopathological implication of RAD50 (DNA repair protein RAD50 homolog) expression in rectal cancer. METHODS: A total of 266 rectal cancer patients who underwent surgery and received chemo- and radiotherapy between 2000 and 2011 were involved in the study. Postoperative RAD50 expression was determined by immunohistochemistry in surgical samples (n = 266). RESULTS: Using Kaplan-Meier survival analysis, we found that low RAD50 expression in postoperative samples was associated with worse disease free survival (p = 0.001) and overall survival (p < 0.001) in early stage/low-grade tumors. In a comparison of patients with low vs. high RAD50 expression, we found that low levels of postoperative RAD50 expression in rectal cancer tissues were significantly associated with perineural invasion (p = 0.002). CONCLUSION: Expression of RAD50 in rectal cancer may serve as a prognostic biomarker for long-term survival of patients with perineural invasion-positive tumors and for potential use in early stage and low-grade rectal cancer assessment.

MRE11 and ATM Expression Levels Predict Rectal Cancer Survival and Their Association with Radiotherapy Response.

BACKGROUND: Aberrant expression of DNA repair proteins is associated with poor survival in cancer patients. We investigated the combined expression of MRE11 and ATM as a predictive marker of response to radiotherapy in rectal cancer. METHODS: MRE11 and ATM expression were examined in tumor samples from 262 rectal cancer patients who underwent surgery for rectal cancer, including a sub-cohort of 54 patients who were treated with neoadjuvant radiotherapy. The relationship between expression of the two-protein panel and tumor regression grade (TRG) was assessed by Mann-Whitney U test and receiver operating characteristics area under curve (ROC-AUC) analysis. The association between expression of the two-protein panel and clinicopathologic variables and survival was examined by Kaplan-Meier methods and Cox regression analysis. RESULTS: A high score for two-protein combined expression in the tumor center (TC) was significantly associated with worse disease-free survival (DFS) (P = 0.035) and overall survival (OS) (P = 0.003) in the whole cohort, and with DFS (P = 0.028) and OS (P = 0.024) in the neoadjuvant subgroup (n = 54). In multivariate analysis, the two-protein combination panel (HR = 2.178, 95% CI 1.115-4.256, P = 0.023) and perineural invasion (HR = 2.183, 95% CI 1.222-3.899, P = 0.008) were significantly associated with DFS. Using ROC-AUC analysis of good versus poor histological tumor response among patients treated preoperatively with radiotherapy, the average ROC-AUC was 0.745 for the combined panel, 0.618 for ATM alone, and 0.711 for MRE11 alone. CONCLUSIONS: The MRE11/ATM two-protein panel developed in this study may have clinical value as a predictive marker of tumor response to neoadjuvant radiotherapy, and a prognostic marker for disease-free and overall survival.

A novel truncated form of S100P predicts disease-free survival in patients with lymph node positive breast cancer.

The calcium-binding protein S100P is overexpressed in various cancers and may contribute to the oncogenic phenotype. This study used mass spectrometry to characterize a novel 9.2-kDa C-terminally truncated form of S100P (t-S100P), and to investigate its potential prognostic value in breast cancer. Univariate analysis demonstrated the association between breast tissue t-S100P levels (n = 148) and conventional pathological markers. Across all tumor samples, high t-S100P was strongly prognostic for poor disease-free survival (P = 0.005), its efficacy confined to lymph node-positive tumors (n = 74, P = 0.007). Matrix-assisted laser desorption/ionization imaging mass spectrometry confirmed differential t-S100P abundance between breast cancer and unaffected adjacent tissue. t-S100P was exclusively located in the cell nucleus of breast cancer tissue, and full-length S100P was essentially undetectable by mass spectrometry. We conclude that t-S100P is the predominant form of S100P in breast cancer tissue and is strongly prognostic for disease-free survival in women with lymph node-positive disease.

Novel serum protein biomarker panel revealed by mass spectrometry and its prognostic value in breast cancer.

INTRODUCTION: Serum profiling using proteomic techniques has great potential to detect biomarkers that might improve diagnosis and predict outcome for breast cancer patients (BC). This study used surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectrometry (MS) to identify differentially expressed proteins in sera from BC and healthy volunteers (HV), with the goal of developing a new prognostic biomarker panel. METHODS: Training set serum samples from 99 BC and 51 HV subjects were applied to four adsorptive chip surfaces (anion-exchange, cation-exchange, hydrophobic, and metal affinity) and analyzed by time-of-flight MS. For validation, 100 independent BC serum samples and 70 HV samples were analyzed similarly. Cluster analysis of protein spectra was performed to identify protein patterns related to BC and HV groups. Univariate and multivariate statistical analyses were used to develop a protein panel to distinguish breast cancer sera from healthy sera, and its prognostic potential was evaluated. RESULTS: From 51 protein peaks that were significantly up- or downregulated in BC patients by univariate analysis, binary logistic regression yielded five protein peaks that together classified BC and HV with a receiver operating characteristic (ROC) area-under-the-curve value of 0.961. Validation on an independent patient cohort confirmed the five-protein parameter (ROC value 0.939). The five-protein parameter showed positive association with large tumor size (P = 0.018) and lymph node involvement (P = 0.016). By matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS, immunoprecipitation and western blotting the proteins were identified as a fragment of apolipoprotein H (ApoH), ApoCI, complement C3a, transthyretin, and ApoAI. Kaplan-Meier analysis on 181 subjects after median follow-up of >5 years demonstrated that the panel significantly predicted disease-free survival (P = 0.005), its efficacy apparently greater in women with estrogen receptor (ER)-negative tumors (n = 50, P = 0.003) compared to ER-positive (n = 131, P = 0.161), although the influence of ER status needs to be confirmed after longer follow-up. CONCLUSIONS: Protein mass profiling by MS has revealed five serum proteins which, in combination, can distinguish between serum from women with breast cancer and healthy control subjects with high sensitivity and specificity. The five-protein panel significantly predicts recurrence-free survival in women with ER-negative tumors and may have value in the management of these patients.

Breast cancer biomarkers: proteomic discovery and translation to clinically relevant assays.

Although the molecular classification and prognostic assessment of breast tumors based on gene expression profiling is well established, a number of proteomic studies that propose potential breast cancer biomarkers has not yet led to any new diagnostic, prognostic or predictive test in wide clinical use. This review examines the current status of breast cancer biomarkers, discusses sample types (including plasma, tumor tissue, nipple aspirate and ductal lavage, as well as cell culture models) and different electrophoretic and mass spectrometry methods that have been widely used for the discovery of proteomic biomarkers in breast cancer, and also considers several approaches to biomarker validation. The pathway leading from the initial proteomic discovery and validation process to translation into a clinically useful test is also discussed.

Discovery of diagnostic biomarkers for pancreatic cancer in immunodepleted serum by SELDI-TOF MS.

MOTIVATION: Reports of serum pancreatic cancer (PC) biomarkers using SELDI-TOF MS have been inconsistent because different chip surfaces and interference with high-abundant proteins. This study examines the influence of these factors on the detection of discriminating diagnostic biomarkers. METHODS: Serum from fourteen from patients with PC, disease controls (DC, n = 14) and healthy volunteers (HV, n = 14) were evaluated by SELDI using H50, IMAC, Q10 and CM10 chips. A further evaluation was undertaken after depletion of seven high-abundant proteins using spin cartridges. RESULTS: More protein peaks were detected in whole serum than in depleted serum for IMAC, H50 and Q10 chips: 60 vs 39, 56 vs 48 and 69 vs 65, respectively, while the CM10 found less peaks in serum (27 vs 47 peaks). However, there were more differentially expressed peaks in the depleted serum samples for PC vs DC and PC vs HV samples using the H50, Q10 and CM10 ProteinChip arrays, whereas for IMAC arrays, more discriminating peaks were seen in non-depleted serum. The highly significant peaks observed on Q10, CM10 and H50 are consistent with the previous finding of ApoA-I (m/z 27,910-28000) and ApoA-II (m/z 8758 and 17,240). In addition, a number of new discriminating protein peaks were found on different ProteinChip arrays, notably peaks at m/z 4280 and 7763 on IMAC arrays. CONCLUSION: This study confirms the diagnostic value of ApoA-I&II and identifies further potential diagnostic biomarkers for pancreatic cancer when multiple chip surfaces are used with depletion of the most highly-abundant proteins.

Proteomic profiling of growth hormone-responsive proteins in human peripheral blood leukocytes.

CONTEXT: GH is a known modulator of the immune system, but the effect of exogenous GH administration on white blood cell proteins has not been investigated. Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) is a powerful platform for the study of GH effects on immune system proteins. OBJECTIVE: Our objective was to explore a novel approach for the detection of GH-responsive proteins in human leukocytes by proteomic analysis using SELDI-TOF MS. DESIGN: We conducted a randomized double-blind, placebo-controlled GH administration study of 8 wk treatment followed by 6 wk washout. Pre- and posttreatment samples from 30 subjects were used for biomarker discovery. SETTING: The study was performed at a clinical research facility. PARTICIPANTS: We studied 30 recreationally trained healthy athletes. INTERVENTION: Subjects received either recombinant human GH (2 mg/d sc; n = 22) or placebo (n = 8) for 8 wk. MAIN OUTCOME MEASURES: Proteomic profiles were determined using CM10 weak cation-exchange protein chips, and some GH-regulated proteins were purified and identified by mass spectrometry and/or immunoblotting. RESULTS: SELDI-TOF analysis revealed a number of GH-regulated peptides/proteins in the 3- to 22-kDa range that are either up- or down-regulated by GH. Several of these may be useful as biomarkers of GH action. The calcium-binding, proinflammatory calgranulins S100A8, S100A9, and S100A12 were all significantly down-regulated in response to GH treatment. CONCLUSION: This study illustrates the novel use of human leukocyte proteomic profiling by SELDI-TOF MS and reveals the negative regulation of proinflammatory S100 proteins by GH in human white blood cells.

Novel biomarkers of human growth hormone action from serum proteomic profiling using protein chip mass spectrometry.

CONTEXT: The detection of exogenous human GH (hGH) administration in athletes poses unique analytical problems, because its short circulating half-life provides only a brief opportunity to detect the administered hormone above endogenous levels. Measurement of novel GH-regulated serum protein biomarkers might provide an indirect method to detect exogenous GH. OBJECTIVE: The objective of this study was to identify new serum biomarkers of GH administration using proteomic profiling. DESIGN: Sera from a previously reported, double-blind, placebo-controlled GH administration trial were analyzed by protein chip mass spectrometry. SETTING: The study was performed at clinical research centers. SUBJECTS: Sixty healthy subjects, aged 18-40 yr, who were not elite athletes, were studied. INTERVENTIONS: Placebo or recombinant hGH treatment (0.1 or 0.2 IU/kg.d; 20 subjects/group) was administered for 4 wk, followed by an 8-wk washout period. MAIN OUTCOME MEASURES: Protein mass profiles were determined on immobilized Cu(2+) chips on d 0 and 21 of GH administration, and multivariate analysis was used to classify subjects into GH and placebo administration groups. RESULTS: When assessed by cross-validation, the classification performance of classifiers based on multivariate analysis of several GH-regulated peaks performed no better than classifiers based on the single best peak. This peak, a prominent biomarker of 15.1 kDa, was purified and identified as hemoglobin alpha-chain. The time course of the GH response of this biomarker is similar to that of other GH-dependent markers, such as IGF-I. CONCLUSION: This study demonstrates that protein mass profiling is an effective tool for the detection of GH administration and suggests that measurement of hemoglobin alpha-chain may have utility as a novel serum biomarker of GH action.