Genomic markers for decision making:What is preventing us from using markers?

The advent of novel genomic technologies that enable the evaluation of genomic alterations on a genome-wide scale has significantly altered the field of genomic marker research in solid tumors. Researchers have moved away from the traditional model of identifying a particular genomic alteration and evaluating the association between this finding and a clinical outcome measure to a new approach involving the identification and measurement of multiple genomic markers simultaneously within clinical studies. This in turn has presented additional challenges in considering the use of genomic markers in oncology, such as clinical study design, reproducibility and interpretation and reporting of results. This Review will explore these challenges, focusing on microarray-based gene-expression profiling, and highlights some common failings in study design that have impacted on the use of putative genomic markers in the clinic. Despite these rapid technological advances there is still a paucity of genomic markers in routine clinical use at present. A rational and focused approach to the evaluation and validation of genomic markers is needed, whereby analytically validated markers are investigated in clinical studies that are adequately powered and have pre-defined patient populations and study endpoints. Furthermore, novel adaptive clinical trial designs, incorporating putative genomic markers into prospective clinical trials, will enable the evaluation of these markers in a rigorous and timely fashion. Such approaches have the potential to facilitate the implementation of such markers into routine clinical practice and consequently enable the rational and tailored use of cancer therapies for individual patients. © 2010 Macmillan Publishers Limited. All rights reserved.

Prognostic and predictive markers in colorectal cancer - The role of new genomic technologies

Colorectal cancer (CRC) is one of the most frequently occurring malignancies worldwide, and the second leading cause of cancer related death in the Western World. Although early stage disease is curable by surgical resection alone, one half of patients with CRC will present with metastatic disease at some stage in the course of their disease. The most active drug in the treatment of CRC is 5-fluorouracil (5-FU) which is used in both the adjuvant and advanced settings. The use of adjuvant therapy is of proven benefit in Stage III CRC, however, its role in Stage II disease is less clear. There is therefore a need to identify those patients with early stage disease who will develop recurrent disease, and who would therefore benefit most from adjuvant treatment. In the advanced setting, the use of irinotecan and oxaliplatin in combination with 5-FU has proven beneficial, with yet further improvements in survival reported with the addition of new targeted agents such as bevacizamab. Despite this, a significant number of patients with advanced disease do not derive any benefit from the chemotherapy they receive, highlighting a need for the development of molecular or genomic markers predictive of response to these chemotherapeutic agents. This review will evaluate the recent advances in pharmacogenomics in CRC, in particular the development of predictive markers of response to chemotherapy. The successful identification of these markers of response will herald an era of personalised treatment, reducing treatment-related toxicity and improving outcome of patients with CRC. -cr 2007 Bentham Science Publishers Ltd.

Predictive Value of Depressive Symptoms for All-Cause Mortality: Findings From the PRIME Belfast Study Examining the Role of Inflammation and Cardiovascular Risk Markers

OBJECTIVES: To improve understanding about the potential underlying biological mechanisms in the link between depression and all-cause mortality and to investigate the role that inflammatory and other cardiovascular risk factors may play in the relationship between depressive symptoms and mortality.

METHODS: Depression and blood-based biological markers were assessed in the Belfast PRIME prospective cohort study (N = 2389 men, aged 50-59 years) in which participants were followed up for 18 years. Depression was measured using the 10-item Welsh Pure Depression Inventory. Inflammation markers (C-reactive protein [CRP], neopterin, interleukin [IL]-1 receptor antagonist [IL-1Ra], and IL-18) and cardiovascular-specific risk factors (N-terminal pro-b-type natriuretic peptide, midregion pro-atrial natriuretic peptide, midregion pro-adrenomedullin, C-terminal pro-endothelin-1 [CT-proET]) were obtained at baseline. We used Cox proportional hazards modeling to examine the association between depression and biological measures in relation to all-cause mortality and explore the mediating effects.

RESULTS: During follow-up, 418 participants died. Higher levels of depressive symptoms were associated with higher levels of CRP, IL-1Ra, and CT-proET. After adjustment for socioeconomic and life-style risk factors, depressive symptoms were significantly associated with all-cause mortality (hazard ratio = 1.10 per scale unit, 95% confidence interval = 1.04-1.16). This association was partly explained by CRP (7.3%) suggesting a minimal mediation effect. IL-1Ra, N-terminal pro-b-type natriuretic peptide, midregion pro-atrial natriuretic peptide, midregion pro-adrenomedullin, and CT-proET contributed marginally to the association between depression and subsequent mortality.

CONCLUSIONS: Inflammatory and cardiovascular risk markers are associated with depression and with increased mortality. However, depression and biological measures show additive effects rather than a pattern of meditation of biological factors in the association between depression and mortality.

Comprehensive molecular pathology analysis of small bowel adenocarcinoma reveals novel targets with potential for clinical utility

Small bowel accounts for only 0.5% of cancer cases in the US but incidence rates have been rising at 2.4% per year over the past decade. One-third of these are adenocarcinomas but little is known about their molecular pathology and no molecular markers are available for clinical use. Using a retrospective 28 patient matched normal-tumor cohort, next-generation sequencing, gene expression arrays and CpG methylation arrays were used for molecular profiling. Next-generation sequencing identified novel mutations in IDH1, CDH1, KIT, FGFR2, FLT3, NPM1, PTEN, MET, AKT1, RET, NOTCH1 and ERBB4. Array data revealed 17% of CpGs and 5% of RNA transcripts assayed to be differentially methylated and expressed respectively (p < 0.01). Merging gene expression and DNA methylation data revealed CHN2 as consistently hypermethylated and downregulated in this disease (Spearman -0.71, p < 0.001). Mutations in TP53 which were found in more than half of the cohort (15/28) and Kazald1 hypomethylation were both were indicative of poor survival (p = 0.03, HR = 3.2 and p = 0.01, HR = 4.9 respectively). By integrating high-throughput mutational, gene expression and DNA methylation data, this study reveals for the first time the distinct molecular profile of small bowel adenocarcinoma and highlights potential clinically exploitable markers.

EphA2 Expression Is a Key Driver of Migration and Invasion and a Poor Prognostic Marker in Colorectal Cancer

PURPOSE: EphA2, a member of the Eph receptor tyrosine kinases family, is an important regulator of tumor initiation, neovascularization, and metastasis in a wide range of epithelial and mesenchymal cancers; however, its role in colorectal cancer recurrence and progression is unclear.

EXPERIMENTAL DESIGN: EphA2 expression was determined by immunohistochemistry in stage II/III colorectal tumors (N = 338), and findings correlated with clinical outcome. The correlation between EphA2 expression and stem cell markers CD44 and Lgr5 was examined. The role of EphA2 in migration/invasion was assessed using a panel of KRAS wild-type (WT) and mutant (MT) parental and invasive colorectal cancer cell line models.

RESULTS: Colorectal tumors displayed significantly higher expression levels of EphA2 compared with matched normal tissue, which positively correlated with high CD44 and Lgr5 expression levels. Moreover, high EphA2 mRNA and protein expression were found to be associated with poor overall survival in stage II/III colorectal cancer tissues, in both univariate and multivariate analyses. Preclinically, we found that EphA2 was highly expressed in KRASMT colorectal cancer cells and that EphA2 levels are regulated by the KRAS-driven MAPK and RalGDS-RalA pathways. Moreover, EphA2 levels were elevated in several invasive daughter cell lines, and downregulation of EphA2 using RNAi or recombinant EFNA1 suppressed migration and invasion of KRASMT colorectal cancer cells.

CONCLUSIONS: These data show that EpHA2 is a poor prognostic marker in stage II/III colorectal cancer, which may be due to its ability to promote cell migration and invasion, providing support for the further investigation of EphA2 as a novel prognostic biomarker and therapeutic target. Clin Cancer Res; 22(1); 230-42. ©2015 AACR.

Expression of proteins associated with hypoxia and Wnt pathway activation is of prognostic significance in hepatocellular carcinoma

In the development and progression of hepatocellular carcinoma, tumor hypoxia plays an important role, as does activation of the Wnt pathway. The aim of this study was to characterize the expression and interrelationship between hypoxia and Wnt-pathway-associated proteins as prognostic factors for hepatocellular carcinoma. Expression of HIF-1α, CA-IX, E-cadherin, β-catenin, and Ki-67 was assessed by immunohistochemistry in 179 primary hepatocellular carcinoma cases. Univariate and multivariate analyses were performed to assess the relationship between the clinicopathological factors, protein expression, overall survival (OS), and recurrence-free survival (RFS). By univariate analysis, tumor stage, size, satellitosis, and vascular invasion were confirmed as prognostic factors for worse OS and RFS. High expression of HIF-1α, CA-IX, β-catenin, Ki-67, and E-cadherin was observed in 60, 15, 64, 8, and 64 % of tumors, respectively, and this was significantly associated with poor OS. CA-IX, HIF-1α, and E-cadherin were independent predictors of poor prognosis. We stratified 169 patients into four groups according to the expression level of hypoxia and Wnt pathway markers. The group with high expression of both hypoxia and Wnt-pathway-associated proteins showed worst OS. The poor survival of this group was also significant in patients with early stage disease and tumor size of less than 5 cm (p < 0.05). We identified a subgroup of hepatocellular carcinoma patients with high expression of both hypoxia and Wnt pathway proteins and found this predictive of poor survival. The therapeutic options for this group might need to be revisited.

PTEN deficiency promotes macrophage infiltration and hypersensitivity of prostate cancer to IAP antagonist/radiation combination therapy

PTEN loss is prognostic for patient relapse post-radiotherapy in prostate cancer (CaP). Infiltration of tumor-associated macrophages (TAMs) is associated with reduced disease-free survival following radical prostatectomy. However, the association between PTEN loss, TAM infiltration and radiotherapy response of CaP cells remains to be evaluated. Immunohistochemical and molecular analysis of surgically-resected Gleason 7 tumors confirmed that PTEN loss correlated with increased CXCL8 expression and macrophage infiltration. However PTEN status had no discernable correlation with expression of other inflammatory markers by CaP cells, including TNF-α. In vitro, exposure to conditioned media harvested from irradiated PTEN null CaP cells induced chemotaxis of macrophage-like THP-1 cells, a response partially attenuated by CXCL8 inhibition. Co-culture with THP-1 cells resulted in a modest reduction in the radio-sensitivity of DU145 cells. Cytokine profiling revealed constitutive secretion of TNF-α from CaP cells irrespective of PTEN status and IR-induced TNF-α secretion from THP-1 cells. THP-1-derived TNF-α increased NFκB pro-survival activity and elevated expression of anti-apoptotic proteins including cellular inhibitor of apoptosis protein-1 (cIAP-1) in CaP cells, which could be attenuated by pre-treatment with a TNF-α neutralizing antibody. Treatment with a novel IAP antagonist, AT-IAP, decreased basal and TNF-α-induced cIAP-1 expression in CaP cells, switched TNF-α signaling from pro-survival to pro-apoptotic and increased radiation sensitivity of CaP cells in co-culture with THP-1 cells. We conclude that targeting cIAP-1 can overcome apoptosis resistance of CaP cells and is an ideal approach to exploit high TNF-α signals within the TAM-rich microenvironment of PTEN-deficient CaP cells to enhance response to radiotherapy.

Tumor suppressor Ras-association domain family 1 isoform A is a novel regulator of cardiac hypertrophy

BACKGROUND: Ras signaling regulates a number of important processes in the heart, including cell growth and hypertrophy. Although it is known that defective Ras signaling is associated with Noonan, Costello, and other syndromes that are characterized by tumor formation and cardiac hypertrophy, little is known about factors that may control it. Here we investigate the role of Ras effector Ras-association domain family 1 isoform A (RASSF1A) in regulating myocardial hypertrophy.

METHODS AND RESULTS: A significant downregulation of RASSF1A expression was observed in hypertrophic mouse hearts, as well as in failing human hearts. To further investigate the role of RASSF1A in cardiac (patho)physiology, we used RASSF1A knock-out (RASSF1A(-)(/)(-)) mice and neonatal rat cardiomyocytes with adenoviral overexpression of RASSF1A. Ablation of RASSF1A in mice significantly enhanced the hypertrophic response to transverse aortic constriction (64.2% increase in heart weight/body weight ratio in RASSF1A(-)(/)(-) mice compared with 32.4% in wild type). Consistent with the in vivo data, overexpression of RASSF1A in cardiomyocytes markedly reduced the cellular hypertrophic response to phenylephrine stimulation. Analysis of molecular signaling events in isolated cardiomyocytes indicated that RASSF1A inhibited extracellular regulated kinase 1/2 activation, likely by blocking the binding of Raf1 to active Ras.

CONCLUSIONS: Our data establish RASSF1A as a novel inhibitor of cardiac hypertrophy by modulating the extracellular regulated kinase 1/2 pathway.

Novel functional interaction between the plasma membrane Ca2+ pump 4b and the proapoptotic tumor suppressor Ras-associated factor 1 (RASSF1)

Plasma membrane calmodulin-dependent calcium ATPases (PMCAs) are enzymatic systems implicated in the extrusion of calcium from the cell. We and others have previously identified molecular interactions between the cytoplasmic COOH-terminal end of PMCA and PDZ domain-containing proteins. These interactions suggested a new role for PMCA as a modulator of signal transduction pathways. The existence of other intracellular regions in the PMCA molecule prompted us to investigate the possible participation of other domains in interactions with different partner proteins. A two-hybrid screen of a human fetal heart cDNA library, using the region 652-840 of human PMCA4b (located in the catalytic, second intracellular loop) as bait, revealed a novel interaction between PMCA4b and the tumor suppressor RASSF1, a Ras effector protein involved in H-Ras-mediated apoptosis. Immunofluorescence co-localization, immunoprecipitation, and glutathione S-transferase pull-down experiments performed in mammalian cells provided further confirmation of the physical interaction between the two proteins. The interaction domain has been narrowed down to region 74-123 of RASSF1C (144-193 in RASSF1A) and 652-748 of human PMCA4b. The functionality of this interaction was demonstrated by the inhibition of the epidermal growth factor-dependent activation of the Erk pathway when PMCA4b and RASSF1 were co-expressed. This inhibition was abolished by blocking PMCA/RASSSF1 association with an excess of a green fluorescent protein fusion protein containing the region 50-123 of RASSF1C. This work describes a novel protein-protein interaction involving a domain of PMCA other than the COOH terminus. It suggests a function for PMCA4b as an organizer of macromolecular protein complexes, where PMCA4b could recruit diverse proteins through interaction with different domains. Furthermore, the functional association with RASSF1 indicates a role for PMCA4b in the modulation of Ras-mediated signaling.

IGF-Binding Protein 2 - Oncogene or Tumor Suppressor?

The role of insulin-like growth factor binding protein 2 (IGFBP2) in cancer is unclear. In general, IGFBP2 is considered to be oncogenic and its expression is often observed to be elevated in cancer. However, there are a number of conflicting reports in vitro and in vivo where IGFBP2 acts in a tumor suppressor manner. In this mini-review, we discuss the factors influencing the variation in IGFBP2 expression in cancer and our interpretation of these findings.

Low-grade malignant Triton tumor of the oral cavity:a case report

Malignant Triton tumor (MTT) is a malignant peripheral nerve sheath tumor showing rhabdomyoblastic differentiation. It is considered a high-grade neoplasm with poor outcome. This report describes an MTT appearing in the oral cavity. On histologic examination the encapsulated lesion was composed of interlacing fascicles of spindle cells and scattered, large, strap-like pleomorphic cells with abundant eosinophilic cytoplasm. No cross striations were seen. Examination of levels through the tissue showed a total of only 4 normal mitoses and no necrosis. Immunohistochemistry demonstrated diffuse S100 positivity in the spindle cells. The large pleomorphic cells were weakly positive for alpha-sarcomeric actin and myoglobin, although variably but strongly positive for desmin. Management involved a small en bloc resection of the maxilla. After 33 months there was no sign of recurrence or distant metastasis. It was concluded that low-grade variants of MTT occur that do not have an aggressive clinical course.

Multiple Markers of Inflammation in Crevicular Fluid During Experimental Gingivitis

Objective: To evaluate temporal changes in GCF levels of substance P, cathepsin G, interleukin 1 beta (IL-1&beta), neutrophil elastase and alpha1-antitrypsin (&alpha1AT) during development of and recovery from experimental gingivitis. Methods: Healthy human volunteers participated in a split-mouth study: experimental gingivitis was induced using a soft vinyl splint to cover test teeth during brushing over 21 days, after which normal brushing was resumed. Modified gingival index (MGI), gingival bleeding index (BI) and modified Quigley and Hein plaque index (PI) were assessed and 30-second GCF samples taken from 4 paired test and contra-lateral control sites in each subject at days 0, 7, 14, 21, 28 and 42. GCF volume was measured and site-specific quantification of one analyte per GCF sample was performed using radioimmunoassay (substance P), enzyme assay (cathepsin G) or ELISA (IL-1&beta, elastase, &alpha1AT). Site-specific data were analysed using analysis of repeated measurements and paired sample tests. Results: 56 subjects completed the study. All measurements at baseline (day 0) and at control sites throughout the study were low. Clinical indices and GCF volumes at the test sites increased from day 0, peaking at day 21 (difference between test and control for PI, BI, MGI and GCF all p<0.0001) and decreased again to control levels by day 28. Levels of four inflammatory markers showed a similar pattern, with significant differences between test and control apparent at 7 days (substance P p=0.0015; cathepsin G p=0.029; IL-1&beta p=0.026; elastase p=0.0129) and peaking at day 21 (substance P p=0.0023; cathepsin G, IL-1&beta and elastase all p<0.0001). Levels of &alpha1AT showed no apparent pattern over the course of the study. Conclusion: GCF levels of substance P, cathepsin G, IL-1&beta and neutrophil elastase have the potential to act as early markers of experimentally-induced gingival inflammation.

Validating the use of intrinsic markers in body feathers to identify inter‑individual differences in non‑breeding areas of northern fulmars

Many wildlife studies use chemical analyses to explore spatio-temporal variation in diet, migratory patterns and contaminant exposure. Intrinsic markers are particularly valuable for studying non-breeding marine predators, when direct methods of investigation are rarely feasible. However, any inferences regarding foraging ecology are dependent upon the time scale over which tissues such as feathers are formed. In this study, we validate the use of body feathers for studying non-breeding foraging patterns in a pelagic seabird, the northern fulmar. Analysis of carcasses of successfully breeding adult fulmars indicated that body feathers moulted between September and March, whereas analyses of carcasses and activity patterns suggested that wing feather and tail feather moult occurred during more restricted periods (September to October and September to January, respectively). By randomly sampling relevant body feathers, average values for individual birds were shown to be consistent. We also integrated chemical analyses of body feather with geolocation tracking data to demonstrate that analyses of δ13C and δ15N values successfully assigned 88 % of birds to one of two broad wintering regions used by breeding adult fulmars from a Scottish study colony. These data provide strong support for the use of body feathers as a tool for exploring non-breeding foraging patterns and diet in wide-ranging, pelagic seabirds.

Markers of vitamin D exposure and oesophageal cancer risk: a systematic review and meta-analysis.

Vitamin D has been associated with reduced risk of many cancers, but evidence for oesophageal cancer is mixed. To clarify the role of Vitamin D, we performed a systematic review and meta-analysis to evaluate the association of Vitamin D exposures and oesophageal neoplasia, including adenocarcinoma, squamous cell carcinoma (SCC), Barrett's oesophagus and squamous dysplasia. Ovid MEDLINE, EMBASE and Web of Science were searched from inception to September 2015. Fifteen publications in relation to circulating 25-hydroxyvitamin D (n=3), Vitamin D intake (n=4), UVB exposure (n=1), and genetic factors (n=7) were retrieved. Higher 25-OHD was associated with increased risk of cancer (adenocarcinoma or SCC, OR=1.39;95%CI:1.04-1.74), with the majority of participants coming from China. No association was observed between Vitamin D intake and risk of cancer overall (OR=1.03;0.65-1.42); however, a non-significantly increased risk for adenocarcinoma (OR=1.45;0.65-2.24) and non-significantly decreased risk for SCC (OR=0.80;0.48-1.12) were observed. One study reported a decreased risk of adenocarcinoma with higher UVB exposure. A decreased risk was found for VDR haplotype rs2238135(G)/rs1989969(T) carriers, OR=0.45;0.00-0.91, and a suggestive association was observed for rs2107301. No consistent associations were observed between Vitamin D exposures and occurrence of oesophageal lesions. Further adequately powered, well-designed studies are needed before conclusions can be made.