Development, Validation, and Clinical Utility of a Six-gene Signature to Predict Aggressive Prostate Cancer.

BACKGROUND: Molecular signatures in prostate cancer (PCa) tissue can provide useful prognostic information to improve the understanding of a patient's risk of harbouring aggressive disease. OBJECTIVE: To develop and validate a gene signature that adds independent prognostic information to clinical parameters for better treatment decisions and patient management. DESIGN, SETTING, AND PARTICIPANTS: Expression of 14 genes was evaluated in radical prostatectomy (RP) tissue from an Irish cohort of PCa patients (n = 426). A six-gene molecular risk score (MRS) was identified with strong prognostic performance to predict adverse pathology (AP) at RP or biochemical recurrence (BCR). The MRS was combined with the Cancer of the Prostate Risk Assessment (CAPRA) score, to create a molecular and clinical risk score (MCRS), and validated in a Swedish cohort (n = 203). OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The primary AP outcome was assessed by the likelihood ratio statistics and area under the receiver operating characteristics curves (AUC) from logistic regression models. The secondary time to BCR outcome was assessed by likelihood ratio statistics and C-indexes from Cox proportional hazard regression models. RESULTS AND LIMITATIONS: The six-gene signature was significantly (p < 0.0001) prognostic and added significant prognostic value to clinicopathological features for AP and BCR outcomes. For both outcomes, both the MRS and the MCRS increased the AUC/C-index when added to European Association of Urology (EAU) and CAPRA scores. Limitations include the retrospective nature of this study. CONCLUSIONS: The six-gene signature has strong performance for the prediction of AP and BCR in an independent clinical validation study. MCRS improves prognostic evaluation and can optimise patient management after RP. PATIENT SUMMARY: We found that the expression panel of six genes can help predict whether a patient is likely to have a disease recurrence after radical prostatectomy surgery.

Prognostic value of the 6-gene OncoMasTR test in hormone receptor-positive HER2-negative early-stage breast cancer: Comparative analysis with standard clinicopathological factors.

AIM: The aim of the study was to assess the prognostic performance of a 6-gene molecular score (OncoMasTR Molecular Score [OMm]) and a composite risk score (OncoMasTR Risk Score [OM]) and to conduct a within-patient comparison against four routinely used molecular and clinicopathological risk assessment tools: Oncotype DX Recurrence Score, Ki67, Nottingham Prognostic Index and Clinical Risk Category, based on the modified Adjuvant! Online definition and three risk factors: patient age, tumour size and grade. METHODS: Biospecimens and clinicopathological information for 404 Irish women also previously enrolled in the Trial Assigning Individualized Options for Treatment [Rx] were provided by 11 participating hospitals, as the primary objective of an independent translational study. Gene expression measured via RT-qPCR was used to calculate OMm and OM. The prognostic value for distant recurrence-free survival (DRFS) and invasive disease-free survival (IDFS) was assessed using Cox proportional hazards models and Kaplan-Meier analysis. All statistical tests were two-sided ones. RESULTS: OMm and OM (both with likelihood ratio statistic [LRS] P < 0.001; C indexes = 0.84 and 0.85, respectively) were more prognostic for DRFS and provided significant additional prognostic information to all other assessment tools/factors assessed (all LRS P ≤ 0.002). In addition, the OM correctly classified more patients with distant recurrences (DRs) into the high-risk category than other risk classification tools. Similar results were observed for IDFS. DISCUSSION: Both OncoMasTR scores were significantly prognostic for DRFS and IDFS and provided additional prognostic information to the molecular and clinicopathological risk factors/tools assessed. OM was also the most accurate risk classification tool for identifying DR. A concise 6-gene signature with superior risk stratification was shown to increase prognosis reliability, which may help clinicians optimise treatment decisions.

Steroid Ligands, the Forgotten Triggers of Nuclear Receptor Action; Implications for Acquired Resistance to Endocrine Therapy.

PURPOSE: There is strong epidemiologic evidence indicating that estrogens may not be the sole steroid drivers of breast cancer. We hypothesize that abundant adrenal androgenic steroid precursors, acting via the androgen receptor (AR), promote an endocrine-resistant breast cancer phenotype. EXPERIMENTAL DESIGN: AR was evaluated in a primary breast cancer tissue microarray (n = 844). Androstenedione (4AD) levels were evaluated in serum samples (n = 42) from hormone receptor-positive, postmenopausal breast cancer. Levels of androgens, progesterone, and estradiol were quantified using LC/MS-MS in serum from age- and grade-matched recurrent and nonrecurrent patients (n = 6) before and after aromatase inhibitor (AI) therapy (>12 months). AR and estrogen receptor (ER) signaling pathway activities were analyzed in two independent AI-treated cohorts. RESULTS: AR protein expression was associated with favorable progression-free survival in the total population (Wilcoxon, P < 0.001). Pretherapy serum samples from breast cancer patients showed decreasing levels of 4AD with age only in the nonrecurrent group (P < 0.05). LC/MS-MS analysis of an AI-sensitive and AI-resistant cohort demonstrated the ability to detect altered levels of steroids in serum of patients before and after AI therapy. Transcriptional analysis showed an increased ratio of AR:ER signaling pathway activities in patients failing AI therapy (t test P < 0.05); furthermore, 4AD mediated gene changes associated with acquired AI resistance. CONCLUSIONS: This study highlights the importance of examining the therapeutic consequences of the steroid microenvironment and demonstrable receptor activation using indicative gene expression signatures.

Targeting the PI3K and MAPK pathways to improve response to HER2-targeted therapies in HER2-positive gastric cancer.

BACKGROUND: Aberrant PI3K signalling is implicated in trastuzumab resistance in HER2-positive gastric cancer (GC). The role of PI3K or MEK inhibitors in sensitising HER2-positive GCs to trastuzumab or in overcoming trastuzumab resistance is unclear. METHODS: Using mass spectrometry-based genotyping we analysed 105 hotspot, non-synonymous somatic mutations in PIK3CA and ERBB-family (EGFR, ERBB2, ERBB3 and ERBB4) genes in gastric tumour samples from 69 patients. A panel of gastric cell lines (N87, OE19, ESO26, SNU16, KATOIII) were profiled for anti-proliferative response to the PI3K inhibitor copanlisib and the MEK1/2 inhibitor refametinib alone and in combination with anti-HER2 therapies. RESULTS: Patients with HER2-positive GC had significantly poorer overall survival compared to HER2-negative patients (15.9 months vs. 35.7 months). Mutations in PIK3CA were only identified in HER2-negative tumours, while ERBB-family mutations were identified in HER2-positive and HER2-negative tumours. Copanlisib had anti-proliferative effects in 4/5 cell lines, with IC50s ranging from 23.4 (N87) to 93.8 nM (SNU16). All HER2-positive cell lines except SNU16 were sensitive to lapatinib (IC50s 0.04 µM-1.5 µM). OE19 cells were resistant to trastuzumab. The combination of lapatinib and copanlisib was synergistic in ESO-26 and OE-19 cells (ED50: 0.83 ± 0.19 and 0.88 ± 0.13, respectively) and additive in NCI-N87 cells (ED50:1.01 ± 0.55). The combination of copanlisib and trastuzumab significantly improved growth inhibition compared to either therapy alone in NCI-N87, ESO26 and OE19 cells (p < 0.05). CONCLUSIONS: PI3K or MEK inhibition alone or in combination with anti-HER2 therapy may represent an improved treatment strategy for some patients with HER2-positive GC, and warrants further investigation in a clinical trial setting.

Development of a Novel Weighted Ranking Method for Immunohistochemical Quantification of a Heterogeneously Expressed Protein in Gastro-Esophageal Cancers.

High expression of Junctional Adhesion Molecule-A (JAM-A) has been linked with poor prognosis in several cancers, including breast cancers overexpressing the human epidermal growth factor receptor-2 (HER2). Furthermore, JAM-A expression has been linked with regulating that of HER2, and associated with the development of resistance to HER2-targeted therapies in breast cancer patients. The purpose of this study was to establish a potential relationship between JAM-A and HER2 in HER2-overexpressing gastro-esophageal (GE) cancers. Interrogation of gene expression datasets revealed that high JAM-A mRNA expression was associated with poorer survival in HER2-positive gastric cancer patients. However, high intra-tumoral heterogeneity of JAM-A protein expression was noted upon immunohistochemical scoring of a GE cancer tissue microarray (TMA), precluding a simple confirmation of any relationship between JAM-A and HER2 at protein level. However, in a test-set of 25 full-face GE cancer tissue sections, a novel weighted ranking system proved effective in capturing JAM-A intra-tumoral heterogeneity and confirming statistically significant correlations between JAM-A/HER2 expression. Given the growing importance of immunohistochemistry in stratifying cancer patients for the receipt of new targeted therapies, this may sound a cautionary note against over-relying on cancer TMAs in biomarker discovery studies of heterogeneously expressed proteins. It also highlights a timely need to develop validated mechanisms of capturing intra-tumoral heterogeneity to aid in future biomarker/therapeutic target development for the benefit of cancer patients.

Genomic and Transcriptomic Characterisation of Response to Neoadjuvant Chemoradiotherapy in Locally Advanced Rectal Cancer.

Standard treatment for locally advanced rectal cancer (LARC) is neoadjuvant chemoradiotherapy (NACRT), followed by surgical resection. However, >70% of patients do not achieve a complete pathological response and have higher rates of relapse and death. There are no validated pre- or on-treatment factors that predict response to NACRT besides tumour stage and size. We characterised the response of 33 LARC patients to NACRT, collected tumour samples from patients prior to, during and after NACRT, and performed whole exome, transcriptome and high-depth targeted sequencing. The pre-treatment LARC genome was not predictive of response to NACRT. However, in line with the increasing recognition of microbial influence in cancer, RNA analysis of pre-treatment tumours suggested a greater abundance of Fusobacteria in intermediate and poor responders. In addition, we investigated tumour heterogeneity and evolution in response to NACRT. While matched pre-treatment, on-treatment and post-treatment tumours revealed minimal genome evolution overall, we identified cases in which microsatellite instability developed or was selected for during NACRT. Recent research has suggested a role for adaptive mutability to targeted therapy in colorectal cancer cells. We provide preliminary evidence of selection for mismatch repair deficiency in response to NACRT. Furthermore, pre-NACRT genomic landscapes do not predict treatment response but pre-NACRT microbiome characteristics may be informative.

Cleavage of the extracellular domain of junctional adhesion molecule-A is associated with resistance to anti-HER2 therapies in breast cancer settings.

BACKGROUND: Junctional adhesion molecule-A (JAM-A) is an adhesion molecule whose overexpression on breast tumor tissue has been associated with aggressive cancer phenotypes, including human epidermal growth factor receptor-2 (HER2)-positive disease. Since JAM-A has been described to regulate HER2 expression in breast cancer cells, we hypothesized that JAM-dependent stabilization of HER2 could participate in resistance to HER2-targeted therapies. METHODS: Using breast cancer cell line models resistant to anti-HER2 drugs, we investigated JAM-A expression and the effect of JAM-A silencing on biochemical/functional parameters. We also tested whether altered JAM-A expression/processing underpinned differences between drug-sensitive and -resistant cells and acted as a biomarker of patients who developed resistance to HER2-targeted therapies. RESULTS: Silencing JAM-A enhanced the anti-proliferative effects of anti-HER2 treatments in trastuzumab- and lapatinib-resistant breast cancer cells and further reduced HER2 protein expression and Akt phosphorylation in drug-treated cells. Increased epidermal growth factor receptor expression observed in drug-resistant models was normalized upon JAM-A silencing. JAM-A was highly expressed in all of a small cohort of HER2-positive patients whose disease recurred following anti-HER2 therapy. High JAM-A expression also correlated with metastatic disease at the time of diagnosis in another patient cohort resistant to trastuzumab therapy. Importantly, cleavage of JAM-A was increased in drug-resistant cell lines in conjunction with increased expression of ADAM-10 and -17 metalloproteases. Pharmacological inhibition or genetic silencing studies suggested a particular role for ADAM-10 in reducing JAM-A cleavage and partially re-sensitizing drug-resistant cells to the anti-proliferative effects of HER2-targeted drugs. Functionally, recombinant cleaved JAM-A enhanced breast cancer cell invasion in vitro and both invasion and proliferation in a semi-in vivo model. Finally, cleaved JAM-A was detectable in the serum of a small cohort of HER2-positive patients and correlated significantly with resistance to HER2-targeted therapy. CONCLUSIONS: Collectively, our data suggest a novel model whereby increased expression and cleavage of JAM-A drive tumorigenic behavior and act as a biomarker and potential therapeutic target for resistance to HER2-targeted therapies.

Stromal TRIM28-associated signaling pathway modulation within the colorectal cancer microenvironment.

BACKGROUND: Stromal gene expression patterns predict patient outcomes in colorectal cancer. TRIM28 is a transcriptional co-repressor that regulates an abundance of genes through the KRAB domain family of transcription factors. We have previously shown that stromal expression of TRIM28 is a marker of disease relapse and poor survival in colorectal cancer. Here, we perform differential epithelium-stroma proteomic network analyses to characterize signaling pathways associated with TRIM28 within the tumor microenvironment. METHODS: Reverse phase protein arrays were generated from laser capture micro-dissected carcinoma and stromal cells from fresh frozen colorectal cancer tissues. Phosphorylation and total protein levels were measured for 30 cancer-related signaling pathway endpoints. Strength and direction of associations between signaling endpoints were identified using Spearman's rank-order correlation analysis and compared to TRIM28 levels. Expression status of TRIM28 in tumor epithelium and stromal fibroblasts was assessed using IHC in formalin fixed tissue and the epithelium to stroma protein expression ratio method. RESULTS: We found distinct proteomic networks in the epithelial and stromal compartments which were linked to expression levels of TRIM28. Low levels of TRIM28 in tumor stroma (high epithelium: stroma ratio) were found in 10 out of 19 cases. Upon proteomic network analyses, these stromal high ratio cases revealed moderate signaling pathway similarity exemplified by 76 significant Spearman correlations (ρ ≥ 0.75, p ≤ 0.01). Furthermore, low levels of stromal TRIM28 correlated with elevated MDM2 levels in tumor epithelium (p = 0.01) and COX-2 levels in tumor stroma (p = 0.002). Low TRIM28 epithelium to stroma ratios were associated with elevated levels of caspases 3 and 7 in stroma (p = 0.041 and p = 0.036) and an increased signaling pathway similarity in stromal cells with 81 significant Spearman correlations (ρ ≥ 0.75, p ≤ 0.01). CONCLUSIONS: By dissecting TRIM28-associated pathways in stromal fibroblasts and epithelial tumor cells, we performed comprehensive proteomic analyses of molecular networks within the tumor microenvironment. We found modulation of several signaling pathways associated with TRIM28, which may be attributed to the pleiotropic properties of TRIM28 through its translational suppression of the family of KRAB domain transcription factors in tumor stromal compartments.

Impact of somatic PI3K pathway and ERBB family mutations on pathological complete response (pCR) in HER2-positive breast cancer patients who received neoadjuvant HER2-targeted therapies.

BACKGROUND: The Cancer Genome Atlas analysis revealed that somatic EGFR, receptor tyrosine-protein kinase erbB-2 (ERBB2), Erb-B2 receptor tyrosine kinase 3 (ERBB3) and Erb-B2 receptor tyrosine kinase 4 (ERBB4) gene mutations (ERBB family mutations) occur alone or co-occur with somatic mutations in the gene encoding the phosphatidylinositol 3-kinase (PI3K) catalytic subunit (PIK3CA) in 19% of human epidermal growth factor receptor 2 (HER2)-positive breast cancers. Because ERBB family mutations can activate the PI3K/AKT pathway and likely have similar canonical signalling effects to PI3K pathway mutations, we investigated their combined impact on response to neoadjuvant HER2-targeted therapies. METHODS: Baseline tumour biopsies were available from 74 patients with HER2-positive breast cancer who were enrolled in the phase II TCHL neoadjuvant study (ICORG 10-05) assessing TCH (docetaxel, carboplatin, trastuzumab) (n = 38) versus TCL (docetaxel, carboplatin, lapatinib) (n = 10) versus TCHL (docetaxel, carboplatin, trastuzumab, lapatinib) (n = 40), each for six cycles. Activating mutations in PIK3CA and ERBB family genes were identified using mass spectrometry-based genotyping. Phosphatase and tensin homolog (PTEN) expression was assessed by immunohistochemistry. RESULTS: PIK3CA and/or ERBB family mutations were detected in 23 (31.1%) tumour samples tested, whereas PTEN expression was low in 31.1% of cases tested. Mutation frequency was similar in each treatment arm (31.3% in TCH arm, 30% in TCL arm and 31.3% in TCHL arm) and was not influenced by oestrogen receptor (ER) status (27.6% in ER-negative patients, 33.3% in ER-positive patients) or progesterone receptor (PR) status (32.6% in PR-negative patients, 29% in PR-positive patients). There was no significant difference in pathological complete response (pCR) rates between 47 patients with wild-type (WT) tumours and 22 patients whose tumours carried mutations (in either PIK3CA or ERBB family genes) (42.5% vs. 54.5%; p = 0.439). Similarly, there was no significant difference in pCR rates between patients with PIK3CA/ERBB family mutated/PTEN-low (i.e., PI3K-activated) tumours and patients without PI3K activation (50% vs. 44%; p = 0.769). However, in the TCHL (but not the TCH) group, the pCR rate was higher for 9 patients with PIK3CA/ERBB family mutated tumours than for 20 patients with PIK3CA/ERBB family WT tumours (77.8% vs. 35%; p = 0.05). CONCLUSIONS: Our results indicate that patients who receive neoadjuvant TCHL and have PIK3CA/ERBB family mutated tumours may be more likely to have a pCR than patients with WT tumours. TRIAL REGISTRATION: ClinicalTrials.gov, NCT01485926 . Registered on 2 December 2011.

Analysis of the In Vivo Turnover of CD4+ T-Cell Subsets in Chronically SIV-Infected Sooty Mangabeys.

Aberrant turnover of memory CD4+ T-cells is central to Acquired Immunodeficiency Syndrome (AIDS) progression. Understanding the relationship between the turnover of CD4+ subsets and immunological homeostasis during simian immunodeficiency virus (SIV) infection in natural hosts may provide insight into mechanisms of immune regulation that may serve as models for therapeutic intervention in Human Immunodeficiency Virus (HIV)-infected persons. Sooty mangabeys (SMs) have naturally evolved with SIV to avoid AIDS progression while maintaining healthy peripheral CD4+ T-cell counts and thus represent a model by which therapeutic interventions for AIDS progression might be elucidated. To assess the relationship between the turnover of CD4+ subsets and immunological homeostasis during SIV infection in non-progressive hosts, we treated 6 SIV-uninfected and 9 SIV-infected SMs with 2'-bromo-5'-deoxyuridine (BrdU) for 14 days and longitudinally assessed CD4+ T-cell subset turnover by polychromatic flow cytometry. We observed that, in SIV-infected SMs, turnover of CD4+ T-cell naïve and central, transitional, and effector memory subsets is comparable to that in uninfected animals. Comparable turnover of CD4+ T-cell subsets irrespective of SIV-infection status likely contributes to the lack of aberrant immune activation and disease progression observed after infection in non-progressive hosts.

Protective role for caspase-11 during acute experimental murine colitis.

Activation of the noncanonical inflammasome, mediated by caspase-11, serves as an additional pathway for the production of the proinflammatory cytokines IL-1ß and IL-18. Noncanonical inflammasome activity occurs during host defense against Gram-negative bacteria and in models of acute septic shock. We propose that the noncanonical inflammasome is activated in mice during acute intestinal inflammation elicited by dextran sodium sulfate (DSS), a model of experimental colitis. We find that caspase-11(-/-) mice display enhanced susceptibility to DSS, because of impaired IL-18 production. The impaired IL-18 levels observed are shown to result in reduced intestinal epithelial cell proliferation and increased cell death. We also suggest that a novel type II IFN-dependent, type I IFN-TRIF-independent signaling pathway is required for in vivo caspase-11 production in intestinal epithelial cells during DSS colitis. Collectively, these data suggest that IFN-γ-mediated caspase-11 expression has a key role maintaining intestinal epithelial barrier integrity in vivo during experimentally induced acute colitis.

High CerS5 expression levels associate with reduced patient survival and transition from apoptotic to autophagy signalling pathways in colorectal cancer.

Ceramide synthase 5 is involved in the de novo synthesis of ceramide, a sphingolipid involved in cell death and proliferation. In this study, we investigated the role of ceramide synthase 5 in colorectal cancer by examining ceramide synthase 5 expression, clinico-pathological parameters and association with survival/death signalling pathways in cancer. Immunohistochemical analysis of CerS5 was performed on 102 colorectal cancer samples using tissue microarrays constructed from formalin-fixed and paraffin-embedded tissues. We found strong membranous ceramide synthase 5 staining in 57 of 102 (56%) colorectal cancers. A multivariate Cox regression analysis of ceramide synthase 5 expression adjusted for disease stage, differentiation and lymphovascular invasion revealed reduced 5-year overall survival (p = 0.001) and 5-year recurrence-free survival (p = 0.002), with hazard ratios of 4.712 and 4.322, respectively. The effect of ceramide synthase 5 expression on tumourigenic processes was further characterised by reverse phase protein array analysis. Reverse phase protein arrays were generated from laser capture microdissection-enriched carcinoma cells from 19 fresh-frozen colorectal cancer tissues. Measurements of phosphorylation and total levels of signalling proteins involved in apoptosis, autophagy and other cancer-related pathways revealed two distinct signalling networks; weak membranous ceramide synthase 5 intensity was associated with a proteomic network dominated by signalling proteins linked to apoptosis, whereas strong ceramide synthase 5 intensity was associated with a proteomic sub-network mostly composed of proteins linked to autophagy. In conclusion, high ceramide synthase 5 expression was found in colorectal cancer tissue and was associated with poorer patient outcomes. Our findings suggest that this may be mediated by a transition from apoptotic to autophagy signalling pathways in ceramide synthase 5 High expressing tumours, thus implicating ceramide synthase 5 in the progression of colorectal cancer.

The frequencies and clinical implications of mutations in 33 kinase-related genes in locally advanced rectal cancer: a pilot study.

BACKGROUND: Locally advanced rectal cancer (LARC: T3/4 and/or node-positive) is treated with preoperative/neoadjuvant chemoradiotherapy (CRT), but responses are not uniform. The phosphatidylinositol 3-kinase (PI3K), MAP kinase (MAPK), and related pathways are implicated in rectal cancer tumorigenesis. Here, we investigated the association between genetic mutations in these pathways and LARC clinical outcomes. METHODS: We genotyped 234 potentially clinically relevant nonsynonymous mutations in 33 PI3K and MAPK pathway-related genes, including PIK3CA, PIK3R1, AKT, STK11, KRAS, BRAF, MEK, CTNNB1, EGFR, MET, and NRAS, using the Sequenom platform. DNA samples were extracted from pretreatment LARC biopsy samples taken from 201 patients who were then treated with long-course neoadjuvant CRT followed by surgical resection. RESULTS: Sixty-two mutations were detected in 15 genes, with the highest frequencies occurring in KRAS (47 %), PIK3CA (14 %), STK11 (6.5 %), and CTNNB1 (6 %). Mutations were detected in BRAF, NRAS, AKT1, PIK3R1, EGFR, GNAS, MEK1, PDGFRA, ALK, and TNK2, but at frequencies of <5 %. As expected, a pathologic complete response (pCR) was associated with improved 5-year recurrence-free survival (RFS; hazard ratio, 0.074; 95 % CI 0.01-0.54; p = 0.001). Mutations in PI3K pathway-related genes (odds ratio, 5.146; 95 % CI 1.17-22.58; p = 0.030), but not MAPK pathway-related genes (p = 0.911), were associated with absence of pCR after neoadjuvant CRT. In contrast, in patients who did not achieve pCR, mutations in PI3K pathway-related genes were not associated with recurrence-free survival (p = 0.987). However, in these patients, codon 12 (G12D/G12 V/G12S) and 13 mutations in KRAS were associated with poor recurrence-free survival (hazard ratio, 1.579; 95 % confidence ratio, 1.00-2.48; p = 0.048). CONCLUSIONS: Mutations in kinase signaling pathways modulate treatment responsiveness and clinical outcomes in LARC and may constitute rational targets for novel therapies.

Immunological and virological analyses of rhesus macaques immunized with chimpanzee adenoviruses expressing the simian immunodeficiency virus Gag/Tat fusion protein and challenged intrarectally with repeated low doses of SIVmac.

Human adenovirus (AdHu)-based candidate AIDS vaccine can provide protection from simian immunodeficiency virus (SIV) transmission and disease progression. However, their potential use may be limited by widespread preexisting immunity to the vector. In contrast, preexisting immunity to chimpanzee adenoviruses (AdC) is relatively rare. In this study, we utilized two regimens of prime-boost immunizations with AdC serotype SAd-V23 (also called AdC6) and SAd-V24 (also called AdC7) expressing SIV Gag/Tat to test their immunogenicity and ability to protect rhesus macaques (RMs) from a repeated low-dose SIVmac239 challenge. Both AdC6 followed by AdC7 (AdC6/7) and AdC7 followed by AdC6 (AdC7/6) induced robust SIV Gag/Tat-specific T cell responses as measured by tetramer staining and functional assays. However, no significant protection from SIV transmission was observed in either AdC7/6- or AdC7/6-vaccinated RMs. Interestingly, in the RMs showing breakthrough infections, AdC7/6-SIV immunization was associated with a transient but significant (P = 0.035 at day 90 and P = 0.033 at day 120 postinfection) reduction in the setpoint viral load compared to unvaccinated controls. None of the measured immunological markers (i.e., number or functionality of SIV-specific CD8(+) and CD4(+) T cell responses and level of activated and/or CCR5(+) CD4(+) target cells) at the time of challenge correlated with protection from SIV transmission in the AdC-SIV-vaccinated RMs. The robust immunogenicity observed in all AdC-immunized RMs and the transient signal of protection from SIV replication exhibited by AdC7/6-vaccinated RMs even in the absence of any envelope immunogen suggest that AdC-based vectors may represent a promising platform for candidate AIDS vaccines.

Relationship between epithelial and stromal TRIM28 expression predicts survival in colorectal cancer patients.

BACKGROUND AND AIM: TRIM28 is a multi-domain nuclear protein with pleotropic effects in both normal and tumor cells. In this study, TRIM28 expression in epithelial and stromal tumor microenvironment and its prognostic role in colorectal cancer were investigated. METHODS: Immunohistological staining of TRIM28 was evaluated in tissue microarrays constructed from 137 colorectal cancer patients. The correlations of TRIM28 expression with clinicopathological features and p53 expression were studied. Kaplan-Meier analysis and Cox proportional hazard modeling were used to assess overall survival (OS) and recurrence-free survival (RFS). RESULTS: Strong epithelial TRIM28 expression was found in 42% of colorectal cancer tissues. TRIM28 expression correlated significantly with p53 expression in matched cases (P=0.0168, Spearman rank test). A high epithelial to stromal TRIM28 expression ratio was associated with shorter OS (P=0.033; log-rank test) and RFS (P=0.043; log-rank test). Multivariate analysis showed that the epithelial to stromal TRIM28 expression ratio was an independent predictor of OS (hazard ratio=2.136; 95% confidence interval 1.015-4.498, P=0.046) and RFS (hazard ratio=2.100; confidence interval 1.052-4.191, P=0.035). CONCLUSION: A high TRIM28 expression ratio between stromal and epithelial compartments in colorectal cancer tissue is an independent predictor of poor prognosis. The pathophysiological role of TRIM28 in carcinogenesis may be dependent on expression levels and cell type within the tumor microenvironment.

Giant right atrial myxoma: characterization with cardiac magnetic resonance imaging.

A 53-year-old woman presented to the emergency department with a 2-week history of dyspnoea and chest pain. Computed tomography pulmonary angiography was performed to exclude acute pulmonary embolism (PE). This demonstrated a large right atrial mass and no evidence of PE. Transthoracic echocardiography followed by cardiac magnetic resonance imaging confirmed a mobile right atrial mass. Surgical resection was then performed confirming a giant right atrial myxoma. We describe the typical clinical, radiologic, and pathologic features of right atrial myxoma.

N-acetylcysteine protects striated muscle in a model of compartment syndrome.

BACKGROUND: To avoid ischemic necrosis, compartment syndrome is a surgical emergency treated with decompression once identified. A potentially lethal, oxidant-driven reperfusion injury occurs after decompression. N-acetylcysteine is an antioxidant with the potential to attenuate the reperfusion injury. QUESTIONS/PURPOSES: We asked whether N-acetylcysteine could preserve striated muscle contractility and modify neutrophil infiltration and activation after simulated compartment syndrome release. MATERIALS AND METHODS: Fifty-seven rats were randomized to control, simulated compartment syndrome, and simulated compartment syndrome plus N-acetylcysteine groups. We isolated the rodent cremaster muscle on its neurovascular pedicle and placed it in a pressure chamber. Chamber pressure was elevated above critical closing pressure for 3 hours to simulate compartment syndrome. Experiments were concluded at three times: 1 hour, 24 hours, and 7 days after decompression of compartment syndrome. We assessed twitch and tetanic contractile function and tissue myeloperoxidase activity. Ten additional rats were randomized to control and N-acetylcysteine administration after which neutrophil respiratory burst activity was assessed. RESULTS: The simulated compartment syndrome decreased muscle contractility and increased muscle tissue myeloperoxidase activity compared with controls. Treatment with N-acetylcysteine preserved twitch and tetanic contractility. N-acetylcysteine did not alter neutrophil infiltration (myeloperoxidase activity) acutely but did reduce infiltration at 24 hours, even when given after decompression. N-acetylcysteine reduced neutrophil respiratory burst activity. CONCLUSION: N-acetylcysteine administration before or after simulated compartment syndrome preserved striated muscle contractility, apparently by attenuating neutrophil activation and the resultant oxidant injury. CLINICAL RELEVANCE: Our data suggest a potential role for N-acetylcysteine in the attenuation of muscle injury after release of compartment syndrome and possibly in the prophylaxis of compartment syndrome.

GLUT-1 expression and response to chemoradiotherapy in rectal cancer.

Preoperative chemoradiotherapy is used in locally advanced rectal cancer to reduce local recurrence and improve operability, however a proportion of tumors do not undergo significant regression. Identification of predictive markers of response to chemoradiotherapy would improve patient selection and may allow response modification by targeting of specific pathways. The aim of this study was to determine whether expression of glucose transporter-1 (GLUT-1) and p53 in pretreatment rectal cancer biopsies was predictive of tumor response to chemoradiotherapy. Immunohistochemical staining for GLUT-1 and p53 was performed on 69 pretreatment biopsies and compared to tumor response in the resected specimen as determined by the tumor regression grade (TRG) scoring system. GLUT-1 expression was significantly associated with reduced response to chemoradiotherapy and increasing GLUT expression correlated with poorer response (p=0.02). GLUT-1 negative tumors had a 70% probability of good response (TRG3/4) compared to a 31% probability of good response in GLUT-1 positive tumors. GLUT-1 may be a useful predictive marker of response to chemoradiotherapy in rectal cancer.

Quantitative cell signalling analysis reveals down-regulation of MAPK pathway activation in colorectal cancer.

Mitogen-activated protein kinases (MAPK) are considered to play significant roles in colonic carcinogenesis and kinase inhibitor therapy has been proposed as a potential tool in the treatment of this disease. Reverse-phase microarray assays using phospho-specific antibodies can directly measure levels of phosphorylated protein isoforms. In the current study, samples from 35 cases of untreated colorectal cancer colectomies were laser capture-microdissected to isolate epithelium and stroma from cancer as well as normal (i.e. uninvolved) mucosa. Lysates generated from these four tissue types were spotted onto reverse-phase protein microarrays and probed with a panel of antibodies to ERK, p-ERK, p38, p-p38, p-JNK, MEK and p-MEK. Whereas total protein levels were unchanged, or slightly elevated (p38, p = 0.0025) in cancers, activated isoforms, including p-ERK, p-p38 and p-JNK, were decreased two- to four-fold in cancers compared with uninvolved mucosa (p < 0.0023 in all cases except for p-JNK in epithelium, where decrement was non-significant). This was backed up by western blotting. Dukes' stage B and C cancers displayed lower p-ERK and p-p38 expression than Dukes' stage A cancers, although this was not statistically significant. It is concluded that MAPK activity may be down-regulated in colorectal cancer and that further exploration of inhibitory therapy in this system should be carefully evaluated if this finding is confirmed in larger series.

Cyclooxygenase-2 expression in chronic hepatitis C and the effect of interferon alpha treatment.

BACKGROUND AND AIM: Cyclooxygenase-2 (COX-2), a target of aspirin and other non-steroidal anti-inflammatory drugs (NSAIDs), is upregulated in chronic hepatitis B and may have a role in hepatocellular carcinoma. Little is known about the expression of COX-2 in chronic hepatitis C (HCV) infection. The aim the present study was to evaluate the extent of COX-2 expression in liver biopsies in patients with HCV infection and to determine the effect of treatment with interferon alpha (IFN). METHODS: Percutaneous liver biopsy specimens were retrieved. Following formalin fixation and paraffin embedding, the biopsies were histologically evaluated for inflammation and fibrosis. The extent of COX-2 expression was measured by the avidin biotin immunohistochemical technique using a monoclonal COX-2 antibody. The extent of COX-2 expression was graded according to the number of hepatocytes expressing COX-2. Data were analyzed using Student's t-test. RESULTS: Liver biopsies from 10 patients before and after treatment with IFN were obtained and compared with nine normal liver biopsies. All of the liver biopsies showed some COX-2 expression. COX-2 expression was confined to hepatocytes and bile duct epithelium and was not detected in vascular endothelium or inflammatory cells. The extent of COX-2 expression was greater in hepatitis C infected liver biopsies than in normal biopsies. Following treatment with IFN, there was a greater than threefold reduction in COX-2 expression (P < 0.01). This result was independent of the sustained virological response. CONCLUSION: In this small pilot study we have shown that COX-2 is overexpressed in liver biopsies infected with HCV and COX-2 expression is reduced following treatment with IFN.