[Show abstract][Hide abstract] ABSTRACT: In locally advanced rectal cancer, neoadjuvant chemoradiotherapy is performed prior to surgery to downstage the tumour. Thirty to 40 % of patients do not respond. Defects in apoptotic machinery lead to therapy resistance; however, to date, no study quantitatively assessed whether B cell lymphoma 2 (BCL2)-dependent regulation of mitochondrial apoptosis, effector caspase activation downstream of mitochondria or a combination of both predicts patient responses. In a cohort of 20 rectal cancer patients, we performed protein profiling of tumour tissue and employed validated ordinary differential equation-based systems models of apoptosis signalling to calculate the ability of cancer cells to undergo apoptosis. Model outputs were compared to clinical responses. Systems modelling of BCL2-signalling predicted patients in the poor response group (p = 0.0049). Systems modelling also demonstrated that rectal cancers depended on BCL2 rather than B cell lymphoma-extra large (BCL(X)L) or myeloid cell leukemia 1 (MCL1) for survival, suggesting that poor responders may benefit from therapy with selective BCL2 antagonists. Dynamic modelling of effector caspase activation could not stratify patients with poor response and did not further improve predictive power. We deliver a powerful patient stratification tool identifying patients who will likely not benefit from neoadjuvant chemoradiotherapy and should be prioritised for surgical resection or treatment with BCL2 antagonists.
Journal of Molecular Medicine 11/2014; · 4.74 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Aim
In rectal cancer, not all tumours display a response to neoadjuvant treatment. An accurate predictor of response does not exist to guide patient-specific treatment. DNA methylation is a distinctive molecular pathway in colorectal carcinogenesis. Whether DNA methylation is altered by neoadjuvant treatment and is a potential response predictor is unknown. We aimed to determine whether DNA methylation is altered by neoadjuvant chemoradiotherapy (CRT) and to determine its role in predicting response to treatment.
Patients and Methods
Fifty-three (n=53) patients with locally advanced rectal cancers treated with neoadjuvant CRT followed by surgery were identified from the pathology databases of 2 tertiary referral centres over a 4-year period. Immunohistochemical staining of treatment specimens was carried out using the 5-Methylcytidine (Eurogentec, Seraing, Belgium) antibody. Quantitative analysis of staining was performed using an automated image analysis platform. The modified tumour regression grading system was used to assess tumour response to neoadjuvant therapy.
Seven (13%) patients showed complete pathological response while 46 (87%) patients were partial responders to neoadjuvant treatment. In 38 (72%) patients, significant reduction in methylation was observed in post-treatment resection specimens compared to pre-treatment specimens (171.5 vs 152.7, p=0.01); in 15 (28%) patients, methylation was increased. Pre-treatment methylation correlated significantly with tumour regression (p<0.001), T-stage (p=0.005), and was able to predict complete and partial pathological responders (p=0.01).
Neoadjuvant CRT appears to alter the rectal cancer epigenome. The significant correlation between pre-treatment DNA methylation with tumour response suggests a potential role for methylation as a biomarker of response.
European Journal of Surgical Oncology 11/2014; · 2.89 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Ceramide synthase 5 (CerS5) 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 CerS5 in colorectal cancer (CRC) by examining CerS5 expression, clinico-pathological parameters and association with survival/death signalling pathways in cancer. CerS5 immunohistochemical analysis was performed on 102 CRC samples using tissue microarrays constructed from formalin-fixed and paraffin-embedded (FFPE) tissues. We found strong CerS5 membranous staining in 57 of 102 (56%) CRCs. A multivariate Cox regression analysis of membranous CerS5 IHC intensity adjusted for TNM stage, differentiation and lymphovascular invasion revealed reduced 5-year overall survival (OS) (p = 0.001) and 5-year recurrence-free survival (RFS) (p = 0.002), with hazard ratios of 4.712 and 4.322, respectively. The effect of CerS5 expression on tumorigenic processes was further characterised by reverse phase protein array (RPPA) analysis. RPPAs were generated from laser capture microdissection enriched carcinoma cells from 19 fresh-frozen CRC tissues. Measurements of phosphorylation and total levels of signalling proteins involved in apoptosis, autophagy and other cancer related pathways revealed 2 distinct signalling networks; Weak membranous CerS5 IHC intensity was associated with a proteomic network dominated by signalling proteins linked to apoptosis, whereas strong Cer5S IHC intensity was associated with a proteomic sub-network mostly composed of proteins linked to autophagy. In conclusion, high CerS5 expression was found in CRC 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 CerS5 high expressing tumours, thus implicating CerS5 in the progression of colorectal cancer.
The Journal of Pathology: Clinical Research. 09/2014;
[Show abstract][Hide abstract] ABSTRACT: Ovarian carcinoma (OC) is the most lethal of the gynaecological malignancies, often presenting at an advanced stage. Treatment is hampered by high levels of drug resistance. The taxanes are microtubule stabilising agents, used as first-line agents in the treatment of OC that exert their apoptotic effects through the Spindle Assembly Checkpoint (SAC). BUB1-Related protein kinase (BUBR1) and Mitotic-Arrest Deficient-2 (MAD2), essential SAC components, play a key role in response to taxanes. BUBR1, MAD2 and Ki-67 were assessed on an OC tissue microarray (TMA) platform representing 72 OC tumours of varying histological subtypes. Sixty-one of these patients received paclitaxel and platinum agents combined; 11 received platinum alone. Overall survival (OS) was available for all 72 patients, whereas recurrence-free survival (RFS) was available for 66 patients. Increased BUBR1 expression was seen in serous carcinomas, compared with other histologies (p=0.03). Increased BUBR1 was significantly associated with tumours of advanced stage (p=0.05). Increased MAD2 and BUBR1 expression also correlated with increased cellular proliferation (p<0.0002 and p=0.02, respectively). Reduced MAD2 nuclear intensity was associated with a shorter RFS (p=0.03), in ovarian tumours of differing histological subtype (n=66). In this subgroup, for those women who received paclitaxel and platinum agents combined (n=57), reduced MAD2 intensity also identified women with a shorter RFS (p<0.007). For the entire cohort of patients, irrespective of histological subtype or treatment, MAD2 nuclear intensity retained independent significance in a multivariate model, with tumours showing reduced nuclear MAD2 intensity identifying patients with a poorer RFS (p=0.05).
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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).
Mutations in kinase signaling pathways modulate treatment responsiveness and clinical outcomes in LARC and may constitute rational targets for novel therapies.
Annals of Surgical Oncology 04/2014; · 3.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Thromboxane synthase (TXS) metabolizes prostaglandin H2 into thromboxanes, which are biologically active on cancer cells. TXS over-expression has been reported in a range of cancers, and associated with angiogenesis and poor outcome. TXS has been identified as a potential therapeutic target in NSCLC. This study examines a link between TXS expression, angiogenesis and survival in NSCLC.
TXS and VEGF metabolite levels were measured in NSCLC serum samples (n=46) by EIA. TXB2 levels were correlated with VEGF. A 204-patient TMA was stained for TXS, VEGF, and CD-31 expression. Expression was correlated with a range of clinical parameters, including overall survival. TXS expression was correlated with VEGF and CD-31. Stable TXS clones were generated and the effect of overexpression on tumour growth and angiogenesis markers examined in-vitro and in-vivo (xenograft mouse model).
Serum TXB2 levels were correlated with VEGF (p<0.05). TXS and VEGF were expressed to a varying degree in NSCLC tissue. TXS was associated with VEGF (p<0.0001) and microvessel density (CD-31; p<0.05). TXS and VEGF expression levels were higher in adenocarcinoma (p<0.0001) and female patients (p<0.05). Stable overexpression of TXS increased VEGF secretion in-vitro. While no significant association with patient survival was observed for either TXS or VEGF in our patient cohort, TXS overexpression significantly (p<0.05) increased tumour growth in-vivo. TXS overexpression was also associated with higher levels of VEGF, microvessel density and reduced apoptosis in xenograft tumours.
TXS promotes tumour growth in-vivo in NSCLC, an effect which is at least partly mediated through increased tumour angiogenesis.
Biochimica et Biophysica Acta 01/2014; · 4.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Non-small cell lung carcinoma remains by far the leading cause of cancer-related deaths worldwide. Overexpression of FLIP, which blocks the extrinsic apoptotic pathway by inhibiting caspase-8 activation, has been identified in various cancers. We investigated FLIP and procaspase-8 expression in NSCLC and the effect of HDAC inhibitors on FLIP expression, activation of caspase-8 and drug resistance in NSCLC and normal lung cell line models. Immunohistochemical analysis of cytoplasmic and nuclear FLIP and procaspase-8 protein expression was carried out using a novel digital pathology approach. Both FLIP and procaspase-8 were found to be significantly overexpressed in tumours, and importantly, high cytoplasmic expression of FLIP significantly correlated with shorter overall survival. Treatment with HDAC inhibitors targeting HDAC1-3 downregulated FLIP expression predominantly via post-transcriptional mechanisms, and this resulted in death receptor- and caspase-8-dependent apoptosis in NSCLC cells, but not normal lung cells. In addition, HDAC inhibitors synergized with TRAIL and cisplatin in NSCLC cells in a FLIP- and caspase-8-dependent manner. Thus, FLIP and procaspase-8 are overexpressed in NSCLC, and high cytoplasmic FLIP expression is indicative of poor prognosis. Targeting high FLIP expression using HDAC1-3 selective inhibitors such as entinostat to exploit high procaspase-8 expression in NSCLC has promising therapeutic potential, particularly when used in combination with TRAIL receptor-targeted agents.
Cell Death & Disease 12/2013; 4:e951. · 5.18 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Recent experimental and biomarker evidence indicates that the epidermal growth factor receptor (EGFR) and insulin-like growth factor receptor 1 (IGF1R) interact in the pathogenesis of malignant epithelial tumors, including lung cancer. This study examines the expression of both receptors and their prognostic significance in surgically resected non-small-cell lung cancer (NSCLC).
EGFR and IGF1R expression were evaluated in 184 patients with NSCLC (83 squamous cell carcinomas [SCCs], 83 adenocarcinomas [ADCs], and 18 other types) using immunohistochemical (IHC) analysis. Expression of both receptors was examined in matched fresh frozen normal and tumor tissues from 40 patients with NSCLC (20 SCCs and 20 ADCs) by Western blot analysis.
High EGFR expression was detected in 51% of patients, and SCCs had higher EGFR expression than did non-SCCs (57.4% vs. 42.5%; P = .028). High IGF1R expression was observed in 53.8% of patients, with SCC having higher expression than non-SCC (62.6% vs. 37.3%; P = .0004). A significant association was shown between EGFR and IGF1R protein overexpression (P < .005). Patients with high expression of both receptors had a poorer overall survival (OS) (P = .04). Higher EGFR and IGF1R expression was detected in resected tumors relative to matched normal tissues (P = .0004 and P = .0009), with SCC having higher expression levels than ADC.
Our findings indicate a close interrelationship between EGFR and IGF1R. Coexpression of both receptors correlates with poor survival. This subset of patients may benefit from treatments cotargeting EGFR and IGF1R.
Clinical Lung Cancer 11/2013; · 2.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Triple negative breast cancer (TNBC) is responsible for a disproportionate number of breast cancer (BC) deaths due to intrinsic aggressiveness and lack of treatment options, especially targeted therapies. Thus, there's urgent requirement for the development of better targeted treatments for TNBC. Molecular alteration of AKT-3 was previously reported in estrogen receptor positive BC (ER+BC). AKT-3 has also been suggested to play a role in hormone unresponsive BC. The aim of this study was to investigate molecular alterations of AKT-3 in TNBC and associated survival analysis and compare these findings with the incidence of AKT-3 molecular alterations in ER+BC.
Our study revealed AKT-3 amplification and deletions in 11% (9/82) and 13%(11/82) of TNBC, respectively. In contrast, 1%(2/209) ER+BCs were found to have AKT-3 amplifications and deletions. A higher prevalence of AKT-3 copy number gains was observed in TNBC (26%(21/82)) compared with ER+BC (9%(19/209)). AKT-3 gene amplification together with positive Akt-3 protein expression was negatively associated with recurrence-free survival in TNBC. Furthermore, negative association between high AKT-3 gene copy number and recurrence-free survival was observed.
AKT-3 amplification could represent a potentially relevant oncogenic event in a subset of TNBC, which may in turn select cells sensitive to Akt-3 inhibitors. This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: To date, there is no uniform consensus whether tumour regression grade (TRG) is predictive of outcome in rectal cancer. Furthermore, the lack of standardization of TRG grading is a major source of variability in published studies. The aim of this study was to evaluate the prognostic impact of TRG in a cohort of patients with locally advanced rectal cancer treated with neoadjuvant chemoradiation therapy (CRT). In addition to Mandard TRG, we utilized four TRG systems modified from the Mandard TRG system and applied them to the cohort to assess which TRG system is most informative.
153 patients with a T3/T4 and/or a node positive rectal cancer underwent neoadjuvant 5-fluorouracil-based CRT followed by surgical resection.
Thirty-six (23.5%) patients achieving complete pathologic response (ypCR) had a 5-year disease-free survival (DFS) of 100% vs. 74% for 117 (76.5%) patients without ypCR (P=0.003). The Royal College of Pathologists (RCPath) TRG best condenses the Mandard 5-point TRG by stratifying patients into three groups with distinct 5-year DFS rates of 100%, 86% and 67%, respectively (P=0.001). In multivariate analysis, pathological nodal status and circumferential resection margin (CRM) status, but not TRG, remained significant predictors of DFS (P=0.002, 0.035, 0.310, respectively).
Our findings support the notion that ypCR status, nodal status after neoadjuvant CRT and CRM status but not TRG are predictors of long-term survival in patients with locally advanced rectal cancer. This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: Genetic testing of an Irish kindred identified an exonic nucleotide substitution c.1664T>C (p.Leu555Pro) in the MLH1 mismatch repair (MMR) gene. This previously unreported variant is classified as a "variant of uncertain significance" (VUS). Immunohistochemical (IHC) analysis and microsatellite instability (MSI) studies, genetic testing, a literature and online MMR mutation database review, in silico phenotype prediction tools, and an in vitro MMR activity assay were used to study the clinical significance of this variant. The MLH1 c.1664T>C (p.Leu555Pro) VUS co-segregated with three cases of classic Lynch syndrome-associated malignancies over two generations, with consistent loss of MLH1 and PMS2 protein expression on IHC, and evidence of the MSI-High mutator phenotype. The leucine at position 555 is well conserved across a number of species, and this novel variant has not been reported as a normal polymorphism in the general population. In silico and in vitro analyses suggest that this variant may have a deleterious effect on the MLH1 protein and abrogate MMR activity. Evidence from clinical, histological, immunohistochemical, and molecular genetic data suggests that MLH1 c.1664T>C (p.Leu555Pro) is likely to be the pathogenic cause of Lynch syndrome in this family.
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND AND AIM: TRIM28 is a multi-domain nuclear protein with pleotropic effects in both normal and tumor cells. In this study, we investigate TRIM28 expression in epithelial and stromal tumor microenvironment and its prognostic role in colorectal cancer. METHODS: Immunohistological staining of TRIM28 was evaluated in tissue microarrays constructed from 137 colorectal cancer patients. The correlations of TRIM28 expression with clinicopathologic 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 (HR) = 2.136; 95% confidence interval (CI) 1.015-4.498, P = 0.046) and RFS (HR = 2.100; CI 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.
Journal of Gastroenterology and Hepatology 02/2013; · 3.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Biobank Ireland Trust (BIT) was established in 2004 to promote and develop an Irish biobank network to benefit patients, researchers, industry, and the economy. The network commenced in 2008 with two hospital biobanks and currently consists of biobanks in the four main cancer hospitals in Ireland. The St. James's Hospital (SJH) Biobank coordinates the network. Procedures, based on ISBER and NCI guidelines, are standardized across the network. Policies and documents-Patient Consent Policy, Patient Information Sheet, Biobank Consent Form, Sample and Data Access Policy (SAP), and Sample Application Form have been agreed upon (after robust discussion) for use in each hospital. An optimum sequence for document preparation and submission for review is outlined. Once consensus is reached among the participating biobanks, the SJH biobank liaises with the Research and Ethics Committees, the Office of the Data Protection Commissioner, The National Cancer Registry (NCR), patient advocate groups, researchers, and other stakeholders. The NCR provides de-identified data from its database for researchers via unique biobank codes. ELSI issues discussed include the introduction of prospective consent across the network and the return of significant research results to patients. Only 4 of 363 patients opted to be re-contacted and re-consented on each occasion that their samples are included in a new project. It was decided, after multidisciplinary discussion, that results will not be returned to patients. The SAP is modeled on those of several international networks. Biobank Ireland is affiliated with international biobanking groups-Marble Arch International Working Group, ISBER, and ESBB. The Irish government continues to deliberate on how to fund and implement biobanking nationally. Meanwhile BIT uses every opportunity to promote awareness of the benefits of biobanking in events and in the media.
Biopreservation and Biobanking 02/2013; 11(1):3-11. · 1.50 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Enteric neural dysfunction leads to increased mucous production and dysmotility in inflammatory bowel disease (IBD). Prior studies have shown that tissue eosinophilia is related to disease activity. We hypothesized that interactions between eosinophils and nerves contribute to neural dysfunction in IBD. Tissue from patients with intractable IBD, endoscopic biopsies from patients with steroid responsive IBD, both when active and quiescent, and control tissue were studied. Immunohistochemical studies showed that eosinophils localize to nerves in the mucosal layer of patients with Crohn's disease (CD) (p<0.001) and ulcerative colitis (UC), (p<0.01). Eosinophils localized to substance P and choline acetyltransferase (ChAT) immunostained nerves. Real time PCR of laser capture micro-dissected enteric ganglia demonstrated Intercellular Adhesion Molecule 1 (ICAM-1) mRNA was increased 7-fold in UC (n = 4), (p = 0.03), and 10-fold in CD (n = 3), (p = 0.05). Compared with controls, eotaxin-3 (CCL-26) mRNA was increased 9-fold in UC (p = 0.04) and 15-fold in CD (p = 0.06). Eosinophil numbers correlated with disease activity, while deposition of major basic protein (MBP) and eosinophil Transforming Growth Factor β -1 (TGFβ-1) expression were seen in therapeutically responsive disease. These data indicate a significant localization of eosinophils to nerves in IBD, mediated through neurally expressed ICAM-1 and eotaxin-3. This cell/neural interaction may influence the function of nerves and contribute to symptoms in IBD.
PLoS ONE 01/2013; 8(5):e64216. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Aberrant activation of Wnts is common in human cancers, including prostate. Hypermethylation associated transcriptional silencing of Wnt antagonist genes SFRPs (Secreted Frizzled-Related Proteins) is a frequent oncogenic event. The significance of this is not known in prostate cancer. The objectives of our study were to (i) profile Wnt signaling related gene expression and (ii) investigate methylation of Wnt antagonist genes in prostate cancer. Using TaqMan Low Density Arrays, we identified 15 Wnt signaling related genes with significantly altered expression in prostate cancer; the majority of which were upregulated in tumors. Notably, histologically benign tissue from men with prostate cancer appeared more similar to tumor (r = 0.76) than to benign prostatic hyperplasia (BPH; r = 0.57, p < 0.001). Overall, the expression profile was highly similar between tumors of high (≥ 7) and low (≤ 6) Gleason scores. Pharmacological demethylation of PC-3 cells with 5-Aza-CdR reactivated 39 genes (≥ 2-fold); 40% of which inhibit Wnt signaling. Methylation frequencies in prostate cancer were 10% (2/20) (SFRP1), 64.86% (48/74) (SFRP2), 0% (0/20) (SFRP4) and 60% (12/20) (SFRP5). SFRP2 methylation was detected at significantly lower frequencies in high-grade prostatic intraepithelial neoplasia (HGPIN; 30%, (6/20), p = 0.0096), tumor adjacent benign areas (8.82%, (7/69), p < 0.0001) and BPH (11.43% (4/35), p < 0.0001). The quantitative level of SFRP2 methylation (normalized index of methylation) was also significantly higher in tumors (116) than in the other samples (HGPIN = 7.45, HB = 0.47, and BPH = 0.12). We show that SFRP2 hypermethylation is a common event in prostate cancer. SFRP2 methylation in combination with other epigenetic markers may be a useful biomarker of prostate cancer.
International Journal of Cancer 08/2012; · 6.20 Impact Factor