Eldo Verghese

Combined analysis of eIF4E and 4E-binding protein expression predicts breast cancer survival and estimates eIF4E activity

Increased eukaryotic translation initiation factor 4E (eIF4E) expression occurs in many cancers, and makes fundamental contributions to carcinogenesis by stimulating the expression of cancer-related genes at post-transcriptional levels. This key role is highlighted by the facts that eIF4E levels can predict prognosis, and that eIF4E is an established therapeutic target. However, eIF4E activity is a complex function of expression levels and phosphorylation statuses of eIF4E and eIF4E-binding proteins (4E-BPs). Our hypothesis was that the combined analyses of these pathway components would allow insights into eIF4E activity and its influence on cancer. We have determined expression levels of eIF4E, 4E-BP1, 4E-BP2 and phosphorylated 4E-BP1 within 424 breast tumours, and have carried out analyses to combine these and relate the product to patient survival, in order to estimate eIF4E activity. We show that this analysis gives greater prognostic insights than that of eIF4E alone. We show that eIF4E and 4E-BP expression are positively associated, and that 4E-BP2 has a stronger influence on cancer behaviour than 4E-BP1. Finally, we examine eIF4E, estimated eIF4E activity, and phosphorylated 4E-BP1 as potential predictive biomarkers for eIF4E-targeted therapies, and show that each determines selection of different patient groups. We conclude that eIF4Es influence on cancer survival is modulated substantially by 4E-BPs, and that combined pathway analyses can estimate functional eIF4E.

Small is beautiful: microRNAs and breast cancer-where are we now?

MicroRNAs are a recently discovered class of small regulatory RNAs that influence the stability and translational efficiency of target mRNAs. They have been implicated in an increasing number of biological processes, including neoplasia. Recent studies have shown an involvement for these regulatory molecules in breast cancer. For example, miRNA profiling studies have identified microRNAs that are deregulated in breast cancer. Furthermore, functional studies have uncovered their roles in breast cancer as both tumour suppressor genes (eg miR‐335) and oncogenes (eg miR‐21). miRNAs deregulated in breast cancer influence the translational regulation of well‐established regulatory molecules, such as oestrogen receptor‐α, which is regulated by miR‐206, and novel cancer‐related molecules whose functions are not yet fully understood.. Here we present an overview of our current understanding of miRNA in breast cancer. Copyright

MiR‐26b is down‐regulated in carcinoma‐associated fibroblasts from ER‐positive breast cancers leading to enhanced cell migration and invasion

Carcinoma‐associated fibroblasts (CAFs) influence the behaviour of cancer cells but the roles of microRNAs in this interaction are unknown. We report microRNAs that are differentially expressed between breast normal fibroblasts and CAFs of oestrogen receptor‐positive cancers, and explore the influences of one of these, miR‐26b, on breast cancer biology. We identified differentially expressed microRNAs by expression profiling of clinical samples and a tissue culture model: miR‐26b was the most highly deregulated microRNA. Using qPCR, miR‐26b was confirmed as down‐regulated in fibroblasts from 15 of 18 further breast cancers. Next, we examined whether manipulation of miR‐26b expression changed breast fibroblast behaviour. Reduced miR‐26b expression caused fibroblast migration and invasion to increase by up to three‐fold in scratch‐closure and trans‐well assays. Furthermore, in co‐culture with MCF7 breast cancer epithelial cells, fibroblasts with reduced miR‐26b expression enhanced both MCF7 migration in trans‐well assays and MCF7 invasion from three‐dimensional spheroids by up to five‐fold. Mass spectrometry was used to identify expression changes associated with the reduction of miR‐26b expression in fibroblasts. Pathway analyses of differentially expressed proteins revealed that glycolysis/TCA cycle and cytoskeletal regulation by Rho GTPases are downstream of miR‐26b. In addition, three novel miR‐26b targets were identified (TNKS1BP1, CPSF7, COL12A1) and the expression of each in cancer stroma was shown to be significantly associated with breast cancer recurrence. MiR‐26b in breast CAFs is a potent regulator of cancer behaviour in oestrogen receptor‐positive cancers, and we have identified key genes and molecular pathways that act downstream of miR‐26b in CAFs.

A study of indefinite for dysplasia in Barrett’s oesophagus: reproducibility of diagnosis, clinical outcomes and predicting progression with AMACR (α-methylacyl-CoA-racemase)

Sonwalkar S A, Rotimi O, Scott N, Verghese E, Dixon M, Axon A T R A & Everett S M
(2010) Histopathology 56, 900–907
A study of indefinite for dysplasia in Barrett’s oesophagus: reproducibility of diagnosis, clinical outcomes and predicting progression with AMACR (α‐methylacyl‐CoA‐racemase)

Carcinoembryonic antigen is the preferred biomarker for in vivo colorectal cancer targeting

Background:Colorectal cancer-specific biomarkers have been used as molecular targets for fluorescent intra-operative imaging, targeted PET/MRI, and selective cytotoxic drug delivery yet the selection of biomarkers used is rarely evidence-based. We evaluated sensitivities and specificites of four of the most commonly used markers: carcinoembryonic antigen (CEA), tumour-associated glycoprotein-72 (TAG-72), folate receptor-α (FRα) and Epithelial growth factor receptor (EGFR).Methods:Marker expression was evaluated semi-quantitatively in matched mucosal and colorectal cancer tissues from 280 patients using immunohistochemistry (scores of 0–15). Matched positive and negative lymph nodes from 18 patients were also examined.Results:Markers were more highly expressed in tumour tissue than in matched normal tissue in 98.8%, 79.0%, 37.1% and 32.8% of cases for CEA, TAG-72, FRα and EGFR, respectively. Carcinoembryonic antigen showed the greatest differential expression, with tumours scoring a mean of 10.8 points higher than normal tissues (95% CI 10.31–11.21, P<0.001). Similarly, CEA showed the greatest differential expression between positive and negative lymph nodes. Receiver operating characteristic analyses showed CEA to have the best sensitivity (93.7%) and specificity (96.1%) for colorectal cancer detection.Conclusion:Carcinoembryonic antigen has the greatest potential to allow highly specific tumour imaging and drug delivery; future translational research should aim to exploit this.

Systematic review and meta‐analysis of cytokeratin 19‐based one‐step nucleic acid amplification versus histopathology for sentinel lymph node assessment in breast cancer

One‐step nucleic acid amplification (OSNA) is a new rapid assay for detecting breast cancer metastases during surgery, saving a second procedure for patients requiring an axillary clearance. Many centres in the UK and abroad have adopted OSNA in place of routine histopathology, despite no published meta‐analysis. The aim of this systematic review and meta‐analysis was to determine whether intraoperative OSNA for lymph node assessment is comparable to routine histopathology in the detection of clinically relevant metastases.

Neoadjuvant Chemotherapy Induces Expression Levels of Breast Cancer Resistance Protein That Predict Disease-Free Survival in Breast Cancer

Three main xenobiotic efflux pumps have been implicated in modulating breast cancer chemotherapy responses. These are P-glycoprotein (Pgp), Multidrug Resistance-associated Protein 1 (MRP1), and Breast Cancer Resistance Protein (BCRP). We investigated expression of these proteins in breast cancers before and after neoadjuvant chemotherapy (NAC) to determine whether their levels define response to NAC or subsequent survival. Formalin-fixed paraffin-embedded tissues were collected representing matched pairs of core biopsy (pre-NAC) and surgical specimen (post-NAC) from 45 patients with invasive ductal carcinomas. NAC regimes were anthracyclines +/− taxanes. Immunohistochemistry was performed for Pgp, MRP1 and BCRP and expression was quantified objectively using computer-aided scoring. Pgp and MRP1 were significantly up-regulated after exposure to NAC (Wilcoxon signed-rank p = 0.0024 and p<0.0001), while BCRP showed more variation in response to NAC, with frequent up- (59% of cases) and down-regulation (41%) contributing to a lack of significant difference overall. Pre-NAC expression of all markers, and post-NAC expression of Pgp and MRP1 did not correlate with NAC response or with disease-free survival (DFS). Post-NAC expression of BCRP did not correlate with NAC response, but correlated significantly with DFS (Log rank p = 0.007), with longer DFS in patients with low post-NAC BCRP expression. In multivariate Cox regression analyses, post-NAC BCRP expression levels proved to predict DFS independently of standard prognostic factors, with high expression associated with a hazard ratio of 4.04 (95% confidence interval 1.3–12.2; p = 0.013). We conclude that NAC-induced expression levels of BCRP predict survival after NAC for breast cancer, while Pgp and MRP1 expression have little predictive value.

Differential regulation of oestrogen receptor β isoforms by 5′ untranslated regions in cancer

Oestrogen receptors (ERs) are critical regulators of the behaviour of many cancers. Despite this, the roles and regulation of one of the two known ERs – ERβ– are poorly understood. This is partly because analyses have been confused by discrepancies between ERβ expression at mRNA and proteins levels, and because ERβ is expressed as several functionally distinct isoforms. We investigated human ERβ 5′ untranslated regions (UTRs) and their influences on ERβ expression and function. We demonstrate that two alternative ERβ 5′UTRs have potent and differential influences on expression acting at the level of translation. We show that their influences are modulated by cellular context and in carcinogenesis, and demonstrate the contributions of both upstream open reading frames and RNA secondary structure. These regulatory mechanisms offer explanations for the non‐concordance of ERβ mRNA and protein. Importantly, we also demonstrate that 5′UTRs allow the first reported mechanisms for differential regulation of the expression of the ERβ isoforms 1, 2 and 5, and thereby have critical influences on ERβ function.

Variation in pre-PCR processing of FFPE samples leads to discrepancies in BRAF and EGFR mutation detection: a diagnostic RING trial.

Aims Mutation detection accuracy has been described extensively; however, it is surprising that pre-PCR processing of formalin-fixed paraffin-embedded (FFPE) samples has not been systematically assessed in clinical context. We designed a RING trial to (i) investigate pre-PCR variability, (ii) correlate pre-PCR variation with EGFR/BRAF mutation testing accuracy and (iii) investigate causes for observed variation. Methods 13 molecular pathology laboratories were recruited. 104 blinded FFPE curls including engineered FFPE curls, cell-negative FFPE curls and control FFPE tissue samples were distributed to participants for pre-PCR processing and mutation detection. Follow-up analysis was performed to assess sample purity, DNA integrity and DNA quantitation. Results Rate of mutation detection failure was 11.9%. Of these failures, 80% were attributed to pre-PCR error. Significant differences in DNA yields across all samples were seen using analysis of variance (p<0.0001), and yield variation from engineered samples was not significant (p=0.3782). Two laboratories failed DNA extraction from samples that may be attributed to operator error. DNA extraction protocols themselves were not found to contribute significant variation. 10/13 labs reported yields averaging 235.8 ng (95% CI 90.7 to 380.9) from cell-negative samples, which was attributed to issues with spectrophotometry. DNA measurements using Qubit Fluorometry demonstrated a median fivefold overestimation of DNA quantity by Nanodrop Spectrophotometry. DNA integrity and PCR inhibition were factors not found to contribute significant variation. Conclusions In this study, we provide evidence demonstrating that variation in pre-PCR steps is prevalent and may detrimentally affect the patients ability to receive critical therapy. We provide recommendations for preanalytical workflow optimisation that may reduce errors in down-stream sequencing and for next-generation sequencing library generation.

Chemotherapy induces Notch1-dependent MRP1 up-regulation, inhibition of which sensitizes breast cancer cells to chemotherapy

BackgroundMulti-drug Resistance associated Protein-1 (MRP1) can export chemotherapeutics from cancer cells and is implicated in chemoresistance, particularly as is it known to be up-regulated by chemotherapeutics. Our aims in this study were to determine whether activation of Notch signalling is responsible for chemotherapy-induced MRP1 expression Notch in breast cancers, and whether this pathway can be manipulated with an inhibitor of Notch activity.MethodsMRP1 and Notch1 were investigated in 29 patients treated with neoadjuvant chemotherapy (NAC) for breast cancer, using immunohistochemistry on matched biopsy (pre-NAC) and surgical samples (post-NAC). Breast epithelial cell cultures (T47D, HB2) were treated with doxorubicin in the presence and absence of functional Notch1, and qPCR, siRNA, Western blots, ELISAs and flow-cytometry were used to establish interactions.ResultsIn clinical samples, Notch1 was activated by neoadjuvant chemotherapy (Wilcoxon signed-rank p < 0.0001) and this correlated with induction of MRP1 expression (rho = 0.6 p = 0.0008). In breast cell lines, doxorubicin induced MRP1 expression and function (non-linear regression p < 0.004). In the breast cancer line T47D, doxorubicin activated Notch1 and, critically, inhibition of Notch1 activation with the γ-secretase inhibitor DAPT abolished the doxorubicin-induced increase in MRP1 expression and function (t-test p < 0.05), resulting in enhanced cellular retention of doxorubicin and increased doxorubicin-induced apoptosis (t-test p = 0.0002). In HB2 cells, an immortal but non-cancer derived breast cell line, Notch1-independent MRP1 induction was noted and DAPT did not enhance doxorubicin-induced apoptosis.ConclusionsNotch inhibitors may have potential in sensitizing breast cancer cells to chemotherapeutics and therefore in tackling chemoresistance.