Eithne Costello

Circulating microRNAs as potential markers of human drug‐induced liver injury

New biomarkers of liver injury are required in the clinic and in preclinical pharmaceutical evaluation. Previous studies demonstrate that two liver‐enriched microRNAs (miR‐122 and miR‐192) are promising biomarkers of acetaminophen‐induced acute liver injury (APAP‐ALI) in mice. We have examined these molecules, for the first time, in humans with APAP poisoning. Serum miR‐122 and miR‐192 were substantially higher in APAP‐ALI patients, compared to healthy controls (median ΔΔCt [25th, 75th percentile]) (miR‐122: 1,265 [491, 4,270] versus 12.1 [7.0, 26.9], P < 0.0001; miR‐192: 6.9 [2.0, 29.2] versus 0.44 [0.30, 0.69], P < 0.0001). A heart‐enriched miR‐1 showed no difference between APAP‐ALI patients and controls, whereas miR‐218 (brain‐enriched) was slightly higher in the APAP‐ALI cohort (0.17 [0.07, 0.50] versus 0.07 [0.04, 0.12]; P = 0.01). In chronic kidney disease (CKD) patients, miR‐122 and ‐192 were modestly higher, compared to controls (miR‐122: 32.0 [21.1, 40.9] versus 12.1 [7.0, 26.9], P = 0.006; miR‐192: 1.2 [0.74, 1.9] versus 0.44 [0.30, 0.69], P = 0.005), but miR‐122 and ‐192 were substantially higher in APAP‐ALI patients than CKD patients (miR‐122: P < 0.0001; miR‐192: P < 0.0004). miR‐122 correlated with peak ALT levels in the APAP‐ALI cohort (Pearson R = 0.46, P = 0.0005), but not with prothrombin time. miR‐122 was also raised alongside peak ALT levels in a group of patients with non‐APAP ALI. Day 1 serum miR‐122 levels were almost 2‐fold higher in APAP‐ALI patients who satisfied Kings College Criteria (KCC), compared to those who did not satisfy KCC, although this did not reach statistical significance (P = 0.15). Conclusion: This work provides the first evidence for the potential use of miRNAs as biomarkers of human drug‐induced liver injury. (HEPATOLOGY 2011;)

Biology and management of pancreatic cancer

Pancreatic cancer continues to pose an enormous challenge to clinicians and cancer scientists. With a more affluent world the global incidence of pancreatic cancer is rising. For the first time significant advances are now being made into the management of the disease. There is a more sophisticated approach to palliative care and the centralisation of pancreatic cancer services is leading to greater tumour resection rates. Newer adjuvant modalities are also greatly increasing the 5 year survival rates. The molecular basis of pancreatic cancer is now better understood than ever before, leading to the development of new diagnostic approaches and the introduction of mechanistic based treatments. Technical advances in imaging and great improvements in conventional and molecular pathology have led to a deeper understanding of the pathological variables of the disease. This is now an important time for making big inroads into what still remains the most lethal of the common cancers. Pancreatic ductal adenocarcinoma remains one of the most difficult cancers to treat. It is the commonest cancer affecting the exocrine pancreas. In 2000, there were 217 000 new cases of pancreatic cancer and 213 000 deaths world wide and in Europe 60 139 new patients (10.4% of all digestive tract cancers) and 64 801 deaths.1 In 2002 there were 7152 new cases in the UK, with similar numbers in men and women.2 In the USA in 2006 there were 33 730 new cases and 32 300 deaths.3 Without active treatment, metastatic pancreatic cancer has a median survival of 3–5 months and 6–10 months for locally advanced disease, which increases to around 11–15 months with resectional surgery.4 The late presentation and aggressive tumour biology of this disease mean that only a minority (10–15%) of patients can undergo potentially curative surgery. Major advances in the past decade …

Pancreatic cancer hENT1 expression and survival from gemcitabine in patients from the ESPAC-3 trial

BACKGROUND Human equilibrative nucleoside transporter 1 (hENT1) levels in pancreatic adenocarcinoma may predict survival in patients who receive adjuvant gemcitabine after resection. METHODS Microarrays from 434 patients randomized to chemotherapy in the ESPAC-3 trial (plus controls from ESPAC-1/3) were stained with the 10D7G2 anti-hENT1 antibody. Patients were classified as having high hENT1 expression if the mean H score for their cores was above the overall median H score (48). High and low hENT1-expressing groups were compared using Kaplan-Meier curves, log-rank tests, and Cox proportional hazards models. All statistical tests were two-sided. RESULTS Three hundred eighty patients (87.6%) and 1808 cores were suitable and included in the final analysis. Median overall survival for gemcitabine-treated patients (n = 176) was 23.4 (95% confidence interval [CI] = 18.3 to 26.0) months vs 23.5 (95% CI = 19.8 to 27.3) months for 176 patients treated with 5-fluorouracil/folinic acid (χ(2) 1=0.24; P = .62). Median survival for patients treated with gemcitabine was 17.1 (95% CI = 14.3 to 23.8) months for those with low hENT1 expression vs 26.2 (95% CI = 21.2 to 31.4) months for those with high hENT1 expression (χ(2)₁= 9.87; P = .002). For the 5-fluorouracil group, median survival was 25.6 (95% CI = 20.1 to 27.9) and 21.9 (95% CI = 16.0 to 28.3) months for those with low and high hENT1 expression, respectively (χ(2)₁ = 0.83; P = .36). hENT1 levels were not predictive of survival for the 28 patients of the observation group (χ(2)₁ = 0.37; P = .54). Multivariable analysis confirmed hENT1 expression as a predictive marker in gemcitabine-treated (Wald χ(2) = 9.16; P = .003) but not 5-fluorouracil-treated (Wald χ(2) = 1.22; P = .27) patients. CONCLUSIONS Subject to prospective validation, gemcitabine should not be used for patients with low tumor hENT1 expression.

Molecular alterations in pancreatic carcinoma: expression profiling shows that dysregulated expression of S100 genes is highly prevalent

In order to expand our understanding of the molecular changes underlying the complex pathology of pancreatic malignancy, global gene expression profiling of pancreatic adenocarcinoma compared with normal pancreatic tissue was performed. Human cDNA arrays comprising 9932 elements were interrogated with fluorescence‐labelled normal and adenocarcinoma samples (nine tumours, three normal pancreata, and three cell lines). The data were analysed for differential gene expression, which was confirmed by serial analysis of gene expression (SAGE), digital differential display (DDD) analysis, and immunohistochemistry for selected cases. The array data were filtered to produce lists of a total of 75 genes significantly up‐regulated or down‐regulated in pancreatic adenocarcinoma. Two of those showing the highest differential were members of the S100 family of Ca‐binding proteins, namely S100P and S100A6, and therefore the S100 genes were studied in more detail. By immunohistochemical analysis of custom‐built, pancreas‐specific tissue arrays and commercially available, normal/cancer tissue arrays that included a wide variety of different tumour types, differential expression of S100P protein was found to be almost exclusive to pancreatic cancer. S100P could therefore represent a useful biomarker for pancreatic adenocarcinomas. Copyright

Analysis of gene expression in cancer cell lines identifies candidate markers for pancreatic tumorigenesis and metastasis

Pancreatic cancer is a highly aggressive type of malignancy and the prognosis for disease presenting typically at a late stage is extremely poor. A comprehensive understanding of its molecular genetics is required in order to develop new approaches to clinical management. To date, serial analysis of gene expression and more recently oligo/cDNA microarray technologies have been employed in order to identify genes involved in pancreatic neoplasia that can be developed as diagnostic markers and drug targets for this dismal disease. This study describes the expression profile obtained from 20 pancreatic cell lines using cDNA microarrays containing 9,932 human gene elements. Numerous genes were identified as being differentially expressed, some of which have been previously implicated in pancreatic adenocarcinoma (S100P, S100A4, prostate stem cell antigen, lipocalin 2, claudins 3 and 4, trefoil factors 1 and 2) as well as several novel genes. The differentially expressed genes identified are involved in a variety of cellular functions, including control of transcription, regulation of the cell cycle, proteolysis, cell adhesion and signaling. Validation of our array results was performed by exploring the SAGEmap database and by immunohistochemistry for a selection of 4 genes that have not previously been studied in pancreatic cancer: anterior gradient 2 homologue (Xenopus laevis), insulin‐like growth factor binding protein 3 and 4 and Forkhead box J1. Immunostaining was performed using pancreas‐specific tissue microarrays containing core biopsies from 305 clinical specimens. In addition, using statistical group comparison and hierarchical clustering, a selection of genes was identified that may be linked to the site of metastasis from which these cell lines were isolated.

Gemcitabine and capecitabine with or without telomerase peptide vaccine GV1001 in patients with locally advanced or metastatic pancreatic cancer (TeloVac): an open-label, randomised, phase 3 trial

BACKGROUND We aimed to assess the efficacy and safety of sequential or simultaneous telomerase vaccination (GV1001) in combination with chemotherapy in patients with locally advanced or metastatic pancreatic cancer. METHODS TeloVac was a three-group, open-label, randomised phase 3 trial. We recruited patients from 51 UK hospitals. Eligible patients were treatment naive, aged older than 18 years, with locally advanced or metastatic pancreatic ductal adenocarcinoma, and Eastern Cooperative Oncology Group performance status of 0-2. Patients were randomly assigned (1:1:1) to receive either chemotherapy alone, chemotherapy with sequential GV1001 (sequential chemoimmunotherapy), or chemotherapy with concurrent GV1001 (concurrent chemoimmunotherapy). Treatments were allocated with equal probability by means of computer-generated random permuted blocks of sizes 3 and 6 in equal proportion. Chemotherapy included six cycles of gemcitabine (1000 mg/m(2), 30 min intravenous infusion, at days 1, 8, and 15) and capecitabine (830 mg/m(2) orally twice daily for 21 days, repeated every 28 days). Sequential chemoimmunotherapy included two cycles of combination chemotherapy, then an intradermal lower abdominal injection of granulocyte-macrophage colony-stimulating factor (GM-CSF; 75 μg) and GV1001 (0·56 mg; days 1, 3, and 5, once on weeks 2-4, and six monthly thereafter). Concurrent chemoimmunotherapy included giving GV1001 from the start of chemotherapy with GM-CSF as an adjuvant. The primary endpoint was overall survival; analysis was by intention to treat. This study is registered as an International Standard Randomised Controlled Trial, number ISRCTN4382138. FINDINGS The first patient was randomly assigned to treatment on March 29, 2007, and the trial was terminated on March 27, 2011. Of 1572 patients screened, 1062 were randomly assigned to treatment (358 patients were allocated to the chemotherapy group, 350 to the sequential chemoimmunotherapy group, and 354 to the concurrent chemoimmunotherapy group). We recorded 772 deaths; the 290 patients still alive were followed up for a median of 6·0 months (IQR 2·4-12·2). Median overall survival was not significantly different in the chemotherapy group than in the sequential chemoimmunotherapy group (7·9 months [95% CI 7·1-8·8] vs 6·9 months [6·4-7·6]; hazard ratio [HR] 1·19, 98·25% CI 0·97-1·48, p=0·05), or in the concurrent chemoimmunotherapy group (8·4 months [95% CI 7·3-9·7], HR 1·05, 98·25% CI 0·85-1·29, p=0·64; overall log-rank of χ(2)2df=4·3; p=0·11). The commonest grade 3-4 toxic effects were neutropenia (68 [19%] patients in the chemotherapy group, 58 [17%] patients in the sequential chemoimmunotherapy group, and 79 [22%] patients in the concurrent chemoimmunotherapy group; fatigue (27 [8%] in the chemotherapy group, 35 [10%] in the sequential chemoimmunotherapy group, and 44 [12%] in the concurrent chemoimmunotherapy group); and pain (34 [9%] patients in the chemotherapy group, 39 [11%] in the sequential chemoimmunotherapy group, and 41 [12%] in the concurrent chemoimmunotherapy group). INTERPRETATION Adding GV1001 vaccination to chemotherapy did not improve overall survival. New strategies to enhance the immune response effect of telomerase vaccination during chemotherapy are required for clinical efficacy. FUNDING Cancer Research UK and KAEL-GemVax.

New biomarkers and targets in pancreatic cancer and their application to treatment.

Late diagnosis of pancreatic ductal adenocarcinoma (pancreatic cancer) and the limited response to current treatments results in an exceptionally poor prognosis. Advances in our understanding of the molecular events underpinning pancreatic cancer development and metastasis offer the hope of tangible benefits for patients. In-depth mutational analyses have shed light on the genetic abnormalities in pancreatic cancer, providing potential treatment targets. New biological studies in patients and in mouse models have advanced our knowledge of the timing of metastasis of pancreatic cancer, highlighting new directions for the way in which patients are treated. Furthermore, our increasing understanding of the molecular events in tumorigenesis is leading to the identification of biomarkers that enable us to predict response to treatment. A major drawback, however, is the general lack of an adequate systematic approach to advancing the use of biomarkers in cancer drug development, highlighted in a Cancer Biomarkers Collaborative consensus report. In this Review, we summarize the latest insights into the biology of pancreatic cancer, and their repercussions for treatment. We provide an overview of current treatments and, finally, we discuss novel therapeutic approaches, including the role of biomarkers in therapy for pancreatic cancer.

Nrf2 is overexpressed in pancreatic cancer: implications for cell proliferation and therapy

BackgroundNrf2 is a key transcriptional regulator of a battery of genes that facilitate phase II/III drug metabolism and defence against oxidative stress. Nrf2 is largely regulated by Keap1, which directs Nrf2 for proteasomal degradation. The Nrf2/Keap1 system is dysregulated in lung, head and neck, and breast cancers and this affects cellular proliferation and response to therapy. Here, we have investigated the integrity of the Nrf2/Keap1 system in pancreatic cancer.ResultsKeap1, Nrf2 and the Nrf2 target genes AKR1c1 and GCLC were detected in a panel of five pancreatic cancer cell lines. Mutation analysis of NRF2 exon 2 and KEAP1 exons 2-6 in these cell lines identified no mutations in NRF2 and only synonomous mutations in KEAP1. RNAi depletion of Nrf2 caused a decrease in the proliferation of Suit-2, MiaPaca-2 and FAMPAC cells and enhanced sensitivity to gemcitabine (Suit-2), 5-flurouracil (FAMPAC), cisplatin (Suit-2 and FAMPAC) and gamma radiation (Suit-2). The expression of Nrf2 and Keap1 was also analysed in pancreatic ductal adenocarcinomas (n = 66 and 57, respectively) and matching normal benign epithelium (n = 21 cases). Whilst no significant correlation was seen between the expression levels of Keap1 and Nrf2 in the tumors, interestingly, Nrf2 staining was significantly greater in the cytoplasm of tumors compared to benign ducts (P < 0.001).ConclusionsExpression of Nrf2 is up-regulated in pancreatic cancer cell lines and ductal adenocarcinomas. This may reflect a greater intrinsic capacity of these cells to respond to stress signals and resist chemotherapeutic interventions. Nrf2 also appears to support proliferation in certain pancreatic adenocarinomas. Therefore, strategies to pharmacologically manipulate the levels and/or activity of Nrf2 may have the potential to reduce pancreatic tumor growth, and increase sensitivity to therapeutics.

High Nuclear S100A6 (Calcyclin) Is Significantly Associated with Poor Survival in Pancreatic Cancer Patients

Recent studies have reported elevated levels of S100A6 in pancreatic ductal adenocarcinoma cells. Here, we describe a detailed analysis of S100A6 expression in benign (n = 32), malignant (n = 60), and premalignant pancreatic ductal cells [96 pancreatic intraepithelial neoplasias (PanIN) from 46 patients]. S100A6 staining was more intense in malignant cells than in benign cells (P = 0.0001). In malignant cells, staining was higher in the nucleus than in the cytoplasm (P = 0.003). Univariate analysis revealed a significant decrease in survival time for patients with high levels of nuclear (P = 0.01) but not cytoplasmic (P = 0.20) S100A6. No evidence was found for an association between nuclear S100A6 expression and other variables, including gender, age at surgery, tumor size or grade, nodal metastases, resection margin, vascular invasion, perineural invasion, p53 or Smad4 levels (both linked to survival in previous studies), or the p65 subunit of nuclear factor-kappaB (a potential regulator of S100A6). Although nodal metastases and resection margin involvement were also associated with poor survival (P = 0.06 in both cases), multivariate analysis suggests that nuclear S100A6 is a significant independent indicator of survival (P = 0.003). Whereas PanIN 1a lesions showed a general absence of S100A6 staining, there was a progressive increase in the proportion of positively stained PanINs with increasing PanIN grade. In particular, we observed an increase in the frequency and intensity of nuclear staining. Our results suggest that up-regulation of S100A6 is an early event in pancreatic cancer development and that elevated levels of nuclear S100A6 may affect clinical outcome.

Pancreatic cancer cells overexpress gelsolin family-capping proteins, which contribute to their cell motility

Background: Previously, proteomic methods were applied to characterise differentially expressed proteins in microdissected pancreatic ductal adenocarcinoma cells. Aims: To report that CapG and a related protein, gelsolin, which have established roles in cell motility, are overexpressed in metastatic pancreatic cancer; and to describe their pattern of expression in pancreatic cancer tissue and their effect on cell motility in pancreatic cancer cell lines. Methods: CapG was identified by mass spectrometry and immunoblotting. CapG and gelsolin expression was assessed by immunohistochemical analysis on a pancreatic cancer tissue microarray and correlated with clinical and pathological parameters. CapG and gelsolin levels were reduced using RNA interface in Suit-2, Panc-1 and MiaPaCa-2 cells. Cell motility was assessed using modified Boyden chamber or wound-healing assays. Results: Multiple isoforms of CapG were detected in pancreatic cancer tissue and cell lines. Immunohistochemical analysis of benign (n = 44 patients) and malignant (n = 69) pancreatic ductal cells showed significantly higher CapG staining intensity in nuclear (p<0.001) and cytoplasmic (p<0.001) compartments of malignant cells. Similarly, gelsolin immunostaining of benign (n = 24 patients) and malignant (n = 68 patients) pancreatic ductal cells showed higher expression in both compartments (both p<0.001). High nuclear CapG was associated with increased tumour size (p = 0.001). High nuclear gelsolin was associated with reduced survival (p = 0.01). Reduction of CapG or gelsolin expression in cell lines by RNAi was accompanied by significantly impaired motility. Conclusions: Up regulation of these actin-capping proteins in pancreatic cancer and their ability to modulate cell motility in vitro suggest their potentially important role in pancreatic cancer cell motility and consequently dissemination.