Prognostication in patients with chronic lymphocytic leukemia (CLL) is challenging due to heterogeneity in clinical course. We hypothesize that constitutional genetic variation affects disease progression and could aid prognostication. Pooling data from seven studies incorporating 842 cases identifies two genomic locations associated with time from diagnosis to treatment, including 10q26.13 (rs736456, hazard ratio (HR) = 1.78, 95% confidence interval (CI) = 1.47–2.15; P = 2.71 × 10 −9 ) and 6p (rs3778076, HR = 1.99, 95% CI = 1.55–2.55; P = 5.08 × 10 −8 ), which are particularly powerful prognostic markers in patients with early stage CLL otherwise characterized by low-risk features. Expression quantitative trait loci analysis identifies putative functional genes implicated in modulating B-cell receptor or innate immune responses, key pathways in CLL pathogenesis. In this work we identify rs736456 and rs3778076 as prognostic in CLL, demonstrating that disease progression is determined by constitutional genetic variation as well as known somatic drivers.

Advanced malignant pleural mesothelioma (MPM) has an extremely poor prognosis with limited chemotherapy options, therefore the identification of new therapeutic targets would aid in disease management. Arachidonic acid is metabolised by cyclooxygenase and lipoxygenase enzymes. The lipoxygenase isoenzymes 5-LOX and 12-LOX have been implicated in carcinogenesis. We aimed to examine 5-LOX and 12-LOX protein expression in a large retrospective series of mesothelioma samples. Further to this, the in vitro cytotoxic effects of lipoxygenase pathway inhibitors were investigated in mesothelioma cells. Archival samples from 83 patients with MPM were examined by immunohistochemistry for expression of the 5-LOX and 12-LOX proteins. The MTS assay was used to assess cell viability following 72 h treatment with the lipoxygenase pathway inhibitors baicalein, licofelone, MK-886 and zileuton in the MPM cell lines NCI-H2052, NCI-H2452 and MSTO-211H. Positive 12-LOX protein expression was recorded in 69/83 (83%) and positive 5-LOX expression was observed in 56/77 (73%) of MPM tissue samples. Co-expression of 5-LOX with 12-LOX was seen in 46/78 (58%) of MPM samples. Positive expression of 5-LOX, 12-LOX and COX-2 proteins was identified in the NCI-H2052, NCI-H2452 and MSTO-211H MPM cell lines. Baicalein (12-LOX and 15-LOX inhibitor) was effective in 3/3 MPM cell lines at low concentrations with an IC50 range of 9.6 μM to 20.7 μM. We have demonstrated that the 5-LOX and 12-LOX proteins are expressed in a significant proportion of MPM samples (73% and 83% respectively) and may represent novel therapeutic targets in this disease. We have demonstrated that the inhibition of the LOX pathway using baicalein may be effective as a novel treatment for MPM, however further human pharmacokinetic studies are required in order to establish whether the concentration used in vitro is clinically achievable.

Pathogenesis of chronic lymphocytic leukaemia (CLL) is contingent upon antigen receptor (BCR) expressed by malignant cells of this disease. Studies on somatic hypermutation of the antigen binding region, receptor expression levels and signal capacity have all linked BCR on CLL cells to disease prognosis. Our previous work showed that the src-family kinase Lck is a targetable mediator of BCR signalling in CLL cells, and that variance in Lck expression associated with ability of BCR to induce signal upon engagement. This latter finding makes Lck similar to ZAP70, another T-cell kinase whose aberrant expression in CLL cells also associates with BCR signalling capacity, but also different because ZAP70 is not easily pharmacologically targetable. Here we describe a robust method of measuring Lck expression in CLL cells using flow cytometry. However, unlike ZAP70 whose expression in CLL cells predicts prognosis, we find Lck expression and disease outcome in CLL are unrelated despite observations that its inhibition produces effects that biologically resemble the egress phenotype taken on by CLL cells treated with idelalisib. Taken together, our findings provide insight into the pathobiology of CLL to suggest a more complex relationship between expression of molecules within the BCR signalling pathway and disease outcome.

SummaryDefining the prognosis of individual cancer sufferers remains a significant clinical challenge. Here we assessed the ability of high‐resolution single telomere length analysis (STELA), combined with an experimentally derived definition of telomere dysfunction, to predict the clinical outcome of patients with chronic lymphocytic leukaemia (CLL). We defined the upper telomere length threshold at which telomere fusions occur and then used the mean of the telomere ‘fusogenic’ range as a prognostic tool. Patients with telomeres within the fusogenic range had a significantly shorter overall survival (P < 0·0001; Hazard ratio [HR] = 13·2, 95% confidence interval [CI] = 11·6–106·4) and this was preserved in early‐stage disease patients (P < 0·0001, HR=19·3, 95% CI = 17·8–802·5). Indeed, our assay allowed the accurate stratification of Binet stage A patients into those with indolent disease (91% survival at 10 years) and those with poor prognosis (13% survival at 10 years). Furthermore, patients with telomeres above the fusogenic mean showed superior prognosis regardless of their IGHV mutation status or cytogenetic risk group. In keeping with this finding, telomere dysfunction was the dominant variable in multivariate analysis. Taken together, this study provides compelling evidence for the use of high‐resolution telomere length analysis coupled with a definition of telomere dysfunction in the prognostic assessment of CLL.

Primary tamoxifen therapy has been widely used to treat elderly women with ER-positive breast cancer in the past. Aromatase inhibitors may be more beneficial than tamoxifen when used as primary endocrine therapy in elderly patients. We aimed to retrospectively evaluate a series of elderly women with ER-positive breast cancer treated with primary letrozole therapy as sole therapy with a minimum of 5 years follow up. To identify possible predictive biomarkers a pilot immunohistochemical analysis was performed to assess the expression of PR, HER2, EGFR, BCL2 and p53. A total of 45 women, aged more than 70 years with a diagnosis of ER-positive breast cancer that was treated with primary letrozole therapy were identified. A case note review was undertaken to obtain clinical information. Formalin fixed paraffin embedded tumour tissue from diagnostic core biopsies was available for all patients. Immunohistochemical analysis was performed to establish the protein expression status of p53, PR, HER2, EGFR and BCL2. The mean age of the 45 patients was 87 years (range 70-101). Clinical benefit was seen in 60% of the patients. Median progression free survival was 53 months (95% CI - 34-72) and the median time to progression was 43 months (95% CI - 22-64). BCL2 was expressed in 45/45 (100%); PR in 38/45 (84%); EGFR in 13/45 (28%); HER2 in 9/45 (20%) and p53 in 5/45 (11%) of tissue samples. Positive expression of p53 was associated with poor progression free survival (p = 0.03) in this pilot study. This study demonstrates that letrozole as sole treatment appears to be a suitable treatment option for elderly patients with ER-positive breast cancer who are not fit for, or decline, surgery. The analysis of p53 in a larger study is warranted in order to assess its role as a biomarker in this patient group.

Background: Neoadjuvant chemotherapy is a standard treatment for locally advanced breast cancer. However, chemoresistance can be a major obstacle in ER+ cancers. Using comparative proteomic approaches (antibody microarray/AbMA and 2D-PAGE with MALDI-TOF/TOF MS) to investigate a pilot series of breast cancer samples we recently identified 14-3-3 theta/tau, tBID and BcL-XL as putative biomarkers of response to neoadjuvant chemotherapy (Hodgkinson et al J Prot 2012, 75:1276–1283 and 75:2745–2752). Here we aimed to analyse further samples using the AbMA approach and to re-analyse the combined data.

We have previously shown that specific COX-2 inhibitors, including DuP 697, have anti-proliferative effects on mesothelioma cells and potentiate the cytotoxicity of pemetrexed. Here, we used a novel proteomic approach to explore the mechanism of action of this agent. COX-2-positive cell lines MSTO-211H (mesothelioma) and A549 (lung cancer) were exposed to DuP 697 for 72 h. Drug carrier only was added to control cells. Extracted proteins from treated and control cells were analysed using a comparative proteomic platform. Differentially expressed proteins, identified by the Panorama Xpress Profiler725 antibody microarray were submitted to Ingenuity Pathway Analysis. A total of 32 unique differentially expressed proteins were identified with a significant (>1.8-fold) difference in expression between treated and untreated cells in at least one cell line. Five molecules, BCL2L1 (Bcl-xL), BID, CHUK (IKK), FASLG and RAF1, were mapped to the Apoptosis Signaling pathway following Ingenuity Pathway Analysis. BCL2L1 (Bcl-xL) and BID were analysed using immuno-blotting and differential expression was confirmed. Proteomic (antibody microarray) analysis suggests that the mechanism of action of DuP 697 may be exerted via the induction of apoptosis. The antibody microarray platform can be utilised to explore the molecular mechanism of action of novel anticancer agents.

e22123 Background: Neoadjuvant chemotherapy is a standard treatment for locally advanced breast cancer however chemoresistance can be a major obstacle in ER+ cancers. Using comparative proteomic approaches (antibody microarray/AbMA and 2D-PAGE with MALDI-TOF/TOF MS) to investigate a pilot series of breast cancer samples we recently identified 14-3-3 theta/tau, tBID and BcL-XL as putative biomarkers of response to neoadjuvant chemotherapy (Hodgkinson et al. J Prot 2012, 75:1276-1283 and 75:2745-2752). Here we aimed to analyse further samples using the AbMA approach and to re-analyse the combined data. Methods: Samples from chemoresistant and chemosensitive breast cancers were selected following anthracycline-taxane chemotherapy and 4 experiments were performed using ductal ER+ tumours. Differential protein expression was compared between chemoresistant and chemosensitive samples using the Panorama XPRESS Profiler725 AbMA kit. The combined data from 9 AbMA assays and 3 2D-PAGE/MS experiments was then analysed using Ingenuity Pathway Analysis (IPA; Ingenuity Systems). Results: 89 differentially expressed proteins (DEPs) were seen in the 4 further AbMA experiments. Sixteen proteins (DR4, E2F6, ILK, MeCP2, MyD88, PI19INK4d, PRMT2, ID, FANCD2, FAKpTyr577, Pinin, Zyxin, 14-3-3 theta/tau, BcL-xL, Pancytokeratin and RIP) were found in at least 2/4 experiments. In the combined dataset (12 experiments from 2 proteomic platforms), 8 DEPs were seen in at least 3 experiments. These were 14-3-3 theta, 14-3-3 epsilon, 14-3-3 gamma, Bcl-xl, Bid, Phosphokinase B, Vimentin and FAK. 121 DEPs from the combined data were analysed using IPA; 12 DEPs were mapped onto the PI3K/AKT pathway. Conclusions: We have identified at least 8 proteins which could play a role in chemoresistance and clinical validation of these putative biomarkers is currently underway.

The kinin-kallikrein system (KKS) is an endogenous multiprotein cascade, the activation of which leads to triggering of the intrinsic coagulation pathway and enzymatic hydrolysis of kininogens with the consequent release of bradykinin-related peptides. This system plays a crucial role in inflammation, vasodilation, smooth muscle contraction, cardioprotection, vascular permeability, blood pressure control, coagulation and pain. In this review, we will outline the physiology and pathophysiology of the KKS and also highlight the association of this system with carcinogenesis and cancer progression.

CLL is an incurable disease with variable prognosis. The hyper reactivity of the B-cell receptor (BCR) to unknown antigen ligation plays a pivotal role in CLL-cell survival. We aimed to investigate the BCR signalling pathway using proteomics to identify novel proteins which may have clinical relevance in this disease.Three CLL samples were selected based upon BCR responsiveness, demonstrated by upregulation of phospho-ERK following in vitro stimulation. The differential expression of proteins, upon artificial stimulation of the BCR, was examined in these samples using two-dimensional gel electrophoresis in combination with mass spectrometry. Proteins of interest were subsequently examined using immunoblotting. Proteomic analysis revealed that kininogen, a critical protein of Irinin kallikrein system, was upregulated in all 3 clinical samples upon BCR stimulation. There are 2 forms of kininogen: HMWK and LMWK. The upregulation of LMWK upon BCR stimulation was confirmed by immunoblotting in all 3 of these samples. In a pilot series of 52 unselected CLL samples, 71% demonstrated basal LMWK expression. There was a trend towards shorter median survival in LMWK positive cases (147 months versus 253 months for LMWK negative cases; p = 0.125). Kininogen may be a novel therapeutic target in CLL and the possible association with prognosis warrants further investigation.Biological significanceWe have identified the upregulation of LMWK upon BCR stimulation of CLL samples. There is no previous published research to suggest a link between kininogen and normal B-cells or CLL cells. In 52 unselected CLL samples, 71% demonstrated basal LMWK expression. There was a trend towards shorter median survival in LMWK positive cases. The absence of LMWK protein expression on normal B-cells suggests that this could be a biomarker for CLL and further research should be undertaken. (C) 2013 Elsevier B.V. All rights reserved.

Background: Neo-adjuvant chemotherapy has become a standard treatment for locally advanced breast cancer as it allows disease down-staging and facilitates breast conservation surgery. However, resistance to chemotherapy can be a major obstacle in delivering an effective neo-adjuvant treatment in oestrogen receptor positive breast cancers. Using comparative proteomic approaches (antibody microarray and 2D gels with mass spectrometry) to investigate a pilot series of fresh tumour samples we have recently identified and validated 14-3-3 theta/tau, tBID and BcL-XL as putative biomarkers of response to anthracycline-taxane neo-adjuvant chemotherapy.

Background: Neoadjuvant chemotherapy is used to downstage locally advance breast cancer and allows increased rates of breast conserving surgery. Studies have shown Dynamic contrast enhanced- MRI (DCE-MRI) to provide a more accurate prediction of residual disease. However, therapy induced changes and presence of discontinuous foci from tumour fragmentation following chemotherapy can make prediction of response on DCE-MRI challenging.

Abstract Abstract 3904 CLL remains an incurable disease with standard therapy regimens. The hyper reactivity of the B cell Receptor (BCR) to unknown antigen ligation plays a pivotal role in B-cell survival. Recent clinical trials of BCR-targeted therapies prompted the need for a better understanding of the biology of BCR signaling. We previously employed proteomics to study protein expression changes associated with BCR ligation. Using 2-dimensional gel electrophoresis (2DE) with MALDI-TOF mass spectrometry (MS) we identified that Kininogen (KNG) was upregulated in 3/3 “poor prognosis” clinical samples upon BCR stimulation. KNG (a critical protein of Kinin-Kallikrein System) is the precursor for Kinins which act via the B1 and B2 receptors (B1R and B2R, respectively) and is known to play a critical role in cell migration, proliferation, vascular permeability, inflammation and intracellular Ca2+ influx. Consequently we hypothesized that there is a functional role for B1R and B2R, Kinins and their precursor KNG in CLL B-cell survival which may offer a potential therapeutic target. Following ethical approval, blood samples were collected from CLL patients. Time to first treatment, clinical stage, IgVh status, CD38 and ZAP-70 data were available. “Poor prognosis” samples were defined as unmutated IgVh status, high WBC and hyper-responsive BCR (p-ERK expression ≥ 2.0- fold increase upon stimulation). Immunoblotting was employed to confirm KNG upregulation and Tissue Kallikrein expression in CLL B-cells. A series of 59 CLL samples was screened for constituent KNG expression using immunoblotting. The presence of mRNA transcripts for KNG was assessed by PCR. An Automated One-stage Factor Assay on the Instrumentation Laboratory ACL-TOP analyser was used to determine the level of Plasma Kallikrein in CLL patients vs healthy controls. Fluorescence Activated Cell Sorting (FACS) was utilized for CLL B-cell enrichment according to CD20 status. Isolated CLL B-cells were subjected to immunodetection and flow cytometry for B1R and B2R cell surface expression identification. Proteomic analysis by 2DE/MALDI-TOF MS previously revealed upregulation of KNG after 5.5 hours of in vitro BCR stimulation in 3/3 clinical samples. Upregulation of KNG after stimulation was confirmed by immunoblotting in 5 samples, including all 3 which were previously analyzed using proteomics. There are 2 forms of KNG: HMWK and LMWK. The analysis of constitutive KNG by immunoblotting revealed positive expression in 72.9% (43/59) and 80% (28/35) for LMWK and HMWK, respectively. No statistical significance was found with preliminary clinical correlations, however, there was a trend towards shorter median survival in LMWK positive cases (152 months vs. 264 months for LMWK negative cases) (p=0.161). PCR confirmed the presence of KNG transcript in CLL samples. An automated one-stage factor assay revealed that 47.2% (26/55) CLL patients expressed elevated Plasma Prekallikrein (the zymogen form of Plasma Kallikrein) level. Tissue Kallikrein overexpression was confirmed in 14 CLL samples, using an anti-KLK 6 antibody. We investigated the presence of inducible B1R and constitutive B2R on CD20+ CLL B-cells and demonstrated that B1R and B2R are expressed by CLL B-cells, however, B1R expression was highly upregulated. We demonstrate for the first time that CLL B-cells express differing levels of KNG protein and that the expression can be significantly increased after BCR ligation. Consequently, high concentration of KNG is achieved in near proximity to B1R and B2R, identified on the surface of CLL B-cells. Therefore, we show that CLL B-cells comprise the complete system for the synthesis and liberation of Kinins which in turn mediate the activation of B1R and B2R via an autocrine loop. We suggest, that simultaneous and sustained activation of B1R and B2R, which is known to be advantageous for immediate triggering of ERK1/2 global phosphorylation and PLCγ2-dependent intracellular Ca2+ mobilization, play a functional role in the induction of pathologic signal transduction leading to B-cells survival and that Ca2+ influx-dependent transcriptional activation and MEK1/ERK1/2 pathway signaling may be dependent upon simultaneous complimentary stimuli input from BCR, B1R and B2R and possibly other disease-associated stimuli. The inhibition of Kinin receptor function may be a potential target for combined therapy in CLL. Disclosures: No relevant conflicts of interest to declare.

Background: Primary Tamoxifen therapy has been widely used to treat elderly women with ER-positive breast cancer in the past. Aromatase inhibitors may be more beneficial than Tamoxifen when used as primary endocrine therapy in elderly patients. We therefore aimed to retrospectively evaluate a series of elderly women with ER-positive breast cancer treated at our Breast Care Unit with primary Letrozole therapy with a minimum of 5 years follow-up.

Neoadjuvant chemotherapy is used to treat oestrogen receptor-positive breast cancer however chemo-resistance is a major obstacle in this molecular subtype. The ability to predict tumour response would allow chemotherapy administration to be directed towards patients who would most benefit, thus maximising treatment efficacy. We aimed to identify protein biomarkers associated with response to neoadjuvant chemotherapy, in a pilot study using comparative 2-DE MALDI TOF/TOF MS proteomic analysis of breast tumour samples. A total of 3 comparative proteomic experiments were performed, comparing protein expression between chemotherapy-sensitive and chemotherapy-resistant oestrogen receptor-positive invasive ductal carcinoma tissue samples. This identified a list of 132 unique proteins that were significantly differentially expressed (≥ 2 fold) in chemotherapy resistant samples, 57 of which were identified in at least two experiments. Ingenuity® Pathway Analysis was used to map the 57 DEPs onto canonical signalling pathways. We implicate several isoforms of 14-3-3 family proteins (theta/tau, gamma, epsilon, beta/alpha and zeta/delta), which have previously been associated with chemotherapy resistance in breast cancer. Extensive clinical validation is now required to fully assess the role of these proteins as putative markers of chemotherapy response in luminal breast cancer subtypes.

Breast conserving therapy is a currently accepted method for managing patients with early stage breast cancer. However, approximately 7% of patients may develop loco-regional tumour recurrence within 5 years. We previously reported that expression of the 26S proteasome may be associated with radio-resistance. Here we aimed to analyse the 26S proteasome in a pilot series of early breast cancers and correlate the findings with loco-regional recurrence. Fourteen patients with early breast cancer who developed loco-regional recurrence within 4 years of completing breast conserving therapy were selected according to strict criteria and compared with those from 14 patients who were disease-free at 10 years. Decreased expression of the 26S proteasome was significantly associated with radio-resistance, manifested as the development of a loco-regional recurrence within 4 years of breast conserving therapy (p=0.018). This small pilot study provides further suggestion that the 26S proteasome may be associated with response to radiotherapy.

Antibody microarrays are powerful new tools in the field of comparative proteomics. The success of the biomarker discovery pipeline relies on the quality of data generated in the discovery phase and careful selection of proteins for the verification phase. Recent meta-analyses found a number of repeatedly identified differentially expressed proteins (RIDEPs) from mass spectrometry-based proteomics research in a range of species. We aimed to assess RIDEPs based on antibody microarray data-sets. A total of 13 independent experiments encompassing a range of oncology-related research on human tissue, cells or cell lines from 5 distinct sample groups were performed utilising a commercial 725 antibody microarray platform (Panorama XPRESS Profiler725; Sigma-Aldrich). Analysis of all microarray slides was carried out by the same individual to reduce inter-observer variability. Fold changes of ≥1.8 were considered significant. A total of 13 RIDEPs were seen, each appearing in at least 4/13 (30%) antibody microarray analyses from at least 2 out of 5 experimental sample groups. The phenomenon of RIDEPs may exist in antibody microarray proteomics and we report a preliminary list of 13 RIDEPs from the XPRESS Profiler725 platform. This information will be useful when interpreting experimental data and considering which DEPs should be prioritised for verification.