Showing papers in "Cancer Medicine in 2012"
TL;DR: Increased Snail expression induces EMT and the CSC‐like phenotype in CRC cells, which enhance cancer cell invasion and chemoresistance, and is a potential therapeutic target in metastatic CRC.
Abstract: Epithelial-mesenchymal transition (EMT) is a critical process providing tumor cells with the ability to migrate and escape from the primary tumor and metastasize to distant sites. Recently, EMT was shown to be associated with the cancer stem cell (CSC) phenotype in breast cancer. Snail is a transcription factor that mediates EMT in a number of tumor types, including colorectal cancer (CRC). Our study was done to determine the role of Snail in mediating EMT and CSC function in CRC. Human CRC specimens were stained for Snail expression, and human CRC cell lines were transduced with a retroviral Snail construct or vector control. Cell proliferation and chemosensitivity to oxaliplatin of the infected cells were determined by the MTT (colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Migration and invasion were determined in vitro using modified Boyden chamber assays. EMT and putative CSC markers were analyzed using Western blotting. Intravenous injection of tumor cells was done to evaluate their metastatic potential in mice. Snail was overexpressed in human CRC surgical specimens. This overexpression induced EMT and a CSC-like phenotype in human CRC cells and enhanced cell migration and invasion (P < 0.002 vs. control). Snail overexpression also led to an increase in metastasis formation in vivo (P < 0.002 vs. control). Furthermore, the Snail-overexpressing CRC cells were more chemoresistant to oxaliplatin than control cells. Increased Snail expression induces EMT and the CSC-like phenotype in CRC cells, which enhance cancer cell invasion and chemoresistance. Thus, Snail is a potential therapeutic target in metastatic CRC.
207 citations
TL;DR: The concept that nisin decreases HNSCC tumorigenesis in vitro and in vivo by inducing increased cell apoptosis and decreased cell proliferation is supported; effects that are mediated by activation of CHAC1, increased calcium influxes, and induction of cell cycle arrest.
Abstract: Nisin, a bacteriocin and commonly used food preservative, may serve as a novel potential therapeutic for treating head and neck squamous cell carcinoma (HNSCC), as it induces preferential apoptosis, cell cycle arrest, and reduces cell proliferation in HNSCC cells, compared with primary keratinocytes. Nisin also reduces HNSCC tumorigenesis in vivo. Mechanistically, nisin exerts these effects on HNSCC, in part, through CHAC1, a proapoptotic cation transport regulator, and through a concomitant CHAC1-independent influx of extracellular calcium. In addition, although CHAC1 is known as an apoptotic mediator, its effects on cancer cell apoptosis have not been examined. Our studies are the first to report CHAC1's new role in promoting cancer cell apoptosis under nisin treatment. These data support the concept that nisin decreases HNSCC tumorigenesis in vitro and in vivo by inducing increased cell apoptosis and decreased cell proliferation; effects that are mediated by activation of CHAC1, increased calcium influxes, and induction of cell cycle arrest. These findings support the use of nisin as a potentially novel therapeutic for HNSCC, and as nisin is safe for human consumption and currently used in food preservation, its translation into a clinical setting may be facilitated.
186 citations
TL;DR: Persistent disparities in colorectal cancer screening affect rural populations and future research to reduce this disparity should focus on screening methods that are acceptable and feasible in rural areas.
Abstract: Despite the existence of effective screening, colorectal cancer remains the second leading cause of cancer death in the United States. Identification of disparities in colorectal cancer screening will allow for targeted interventions to achieve national goals for screening. The objective of this study was to contrast colorectal cancer screening rates in urban and rural populations in the United States. The study design comprised a cross-sectional study in the United States 1998-2005. Behavioral Risk Factor Surveillance System data from 1998 to 2005 were the method and data source. The primary outcome was self-report up-to-date colorectal cancer screening (fecal occult blood test in last 12 months, flexible sigmoidoscopy in last 5 years, or colonoscopy in last 10 years). Geographic location (urban vs. rural) was used as independent variable. Multivariate analysis controlled for demographic and health characteristics of respondents. After adjustment for demographic and health characteristics, rural residents had lower colorectal cancer screening rates (48%; 95% CI 48, 49%) as compared with urban residents (54%, 95% CI 53, 55%). Remote rural residents had the lowest screening rates overall (45%, 95% CI 43, 46%). From 1998 to 2005, rates of screening by colonoscopy or flexible sigmoidoscopy increased in both urban and rural populations. During the same time, rates of screening by fecal occult blood test decreased in urban populations and increased in rural populations. Persistent disparities in colorectal cancer screening affect rural populations. The types of screening tests used for colorectal cancer screening are different in rural and urban areas. Future research to reduce this disparity should focus on screening methods that are acceptable and feasible in rural areas.
130 citations
TL;DR: The development of DNA methylation‐ and hydroxymethylation‐based biomarkers is an exciting and rapidly evolving area of research that holds promise for potential applications in diverse clinical settings.
Abstract: DNA methylation, consisting of the addition of a methyl group at the fifth-position of cytosine in a CpG dinucleotide, is one of the most well-studied epigenetic mechanisms in mammals with important functions in normal and disease biology. Disease-specific aberrant DNA methylation is a well-recognized hallmark of many complex diseases. Accordingly, various studies have focused on characterizing unique DNA methylation marks associated with distinct stages of disease development as they may serve as useful biomarkers for diagnosis, prognosis, prediction of response to therapy, or disease monitoring. Recently, novel CpG dinucleotide modifications with potential regulatory roles such as 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine have been described. These potential epigenetic marks cannot be distinguished from 5-methylcytosine by many current strategies and may potentially compromise assessment and interpretation of methylation data. A large number of strategies have been described for the discovery and validation of DNA methylation-based biomarkers, each with its own advantages and limitations. These strategies can be classified into three main categories: restriction enzyme digestion, affinity-based analysis, and bisulfite modification. In general, candidate biomarkers are discovered using large-scale, genome-wide, methylation sequencing, and/or microarray-based profiling strategies. Following discovery, biomarker performance is validated in large independent cohorts using highly targeted locus-specific assays. There are still many challenges to the effective implementation of DNA methylation-based biomarkers. Emerging innovative methylation and hydroxymethylation detection strategies are focused on addressing these gaps in the field of epigenetics. The development of DNA methylation- and hydroxymethylation-based biomarkers is an exciting and rapidly evolving area of research that holds promise for potential applications in diverse clinical settings.
113 citations
TL;DR: The prevalence of HPV and/or p16 is much lower in OOSCC compared to earlier reports including all OSCC, or tonsillar‐ and base of tongue cancer alone and HPV and p16 had no impact on clinical outcome in OSCC in this study.
Abstract: Today, most oropharyngeal squamous cell carcinoma (OSCC) is human papillomavirus (HPV) positive and HPV alone or in combination with p16 is reported to be a favorable prognostic factor for OSCC. Patients with tumors at other OSCC sites (OOSCC) are often included in the same treatment and study protocols as patients with tonsillar- and base of tongue SCC, even though the prevalence and clinical significance of HPV infection in OOSCC is unknown. Since tonsillar and base of tongue SSC cover roughly 90% of all OSCC, there is an obvious risk that there may be a misinterpretation of the results for OOSCC. Herein, we therefore study the prevalence of HPV and p16 and their impact on survival in OOSCC. A total of 69 patients were included in the study, and 61 were included in the survival analysis. HPV and p16 were present in only 17% (12/69) and 25% (17/69) of the OOSCC cases, respectively, while the majority 69% (48/69) was both HPV and p16 negative. Neither HPV nor p16 had predictive value for clinical outcome in OOSCC in this study. In conclusion, the prevalence of HPV and/or p16 is much lower in OOSCC compared to earlier reports including all OSCC, or tonsillar- and base of tongue cancer alone and HPV and p16 had no impact on clinical outcome in OSCC in this study. Our data highlight the diversity of head neck cancer sub-sites and the importance of taking OSCC sub-sites in consideration in future clinical trials and treatment.
79 citations
TL;DR: The data suggest that the mutations in miR‐142 probably lead to a loss rather than a gain of function, which is the first report describing mutations of a miRNA gene in a large percentage of a distinct lymphoma subtype.
Abstract: MicroRNAs (miRNAs) are short 18-23 nucleotide long noncoding RNAs that posttranscriptionally regulate gene expression by binding to mRNA. Our previous miRNA profiling of diffuse large B-cell lymphoma (DLBCL) revealed a mutation in the seed sequence of miR-142-3p. Further analysis now showed that miR-142 was mutated in 11 (19.64%) of the 56 DLBCL cases. Of these, one case had a mutation in both alleles, with the remainder being heterozygous. Four mutations were found in the mature miR-142-5p, four in the mature miR-142-3p, and three mutations affected the miR-142 precursor. Two mutations in the seed sequence redirected miR-142-3p to the mRNA of the transcriptional repressor ZEB2 and one of them also targeted the ZEB1 mRNA. However, the other mutations in the mature miR-142-3p did not influence either the ZEB1 or ZEB2 3' untranslated region (3' UTR). On the other hand, the mutations affecting the seed sequence of miR-142-3p resulted in a loss of responsiveness in the 3' UTR of the known miR-142-3p targets RAC1 and ADCY9. In contrast to the mouse p300 gene, the human p300 gene was not found to be a target for miR-142-5p. In one case with a mutation of the precursor, we observed aberrant processing of the miR-142-5p. Our data suggest that the mutations in miR-142 probably lead to a loss rather than a gain of function. This is the first report describing mutations of a miRNA gene in a large percentage of a distinct lymphoma subtype.
78 citations
TL;DR: Examining the expression profiles of circulating miRNAs in the serum of patients with high‐risk oral lesions and exploring their utility as potential oral cancer biomarkers suggested utility as noninvasive biomarkers for detection of oral cancer or high‐grade lesions.
Abstract: Oral cancer is one of the most commonly diagnosed cancers worldwide. Disease is often diagnosed at later stages, which is associated with a poor 5-year survival rate and a high rate of local recurrence. MicroRNAs (miRNAs), a group of small, noncoding RNAs, can be isolated from blood serum samples and have demonstrated utility as biomarkers in multiple cancer types. The aim of this study was to examine the expression profiles of circulating miRNAs in the serum of patients with high-risk oral lesions (HRLs; oral cancer or carcinoma in situ) and to explore their utility as potential oral cancer biomarkers. Global serum miRNA profiles were generated using quantitative PCR method from 1) patients diagnosed with HRLs and undergoing intent-to-cure surgical treatment (N = 30) and 2) a demographically matched, noncancer control group (N = 26). We next honed our list of serum miRNAs associated with disease by reducing the effects of interpatient variability; we compared serum miRNA profiles from samples taken both before and after tumor resections (N = 10). Based on these analyses, fifteen miRNAs were significantly upregulated and five were significantly downregulated based on presence of disease (minimum fold-change >2 in at least 50% of samples, P 0.8, suggesting utility as noninvasive biomarkers for detection of oral cancer or high-grade lesions. Combining these serum miRNA profiles with other screening techniques could greatly improve the sensitivity in oral cancer detection.
78 citations
TL;DR: In vitro studies showed cancer‐derived exosomes, epithelial–mesenchymal transition process, fibroblast‐to‐CAF‐like cell transdifferentiation, and reciprocal interrelations between different cytokines suggesting the presence of molecular crosstalk between cancer cells and TME components.
Abstract: We characterized tumor microenvironment (TME) components of mobile tongue (MT) cancer patients in terms of overall inflammatory infiltrate, focusing on the protumorigenic/anti-inflammatory phenotypes and on cancer-associated fibroblasts (CAFs) in order to determine their interrelations and associations with clinical outcomes In addition, by culturing tongue carcinoma cells (HSC-3) on a three-dimensional myoma organotypic model that mimics TME, we attempted to investigate the possible existence of a molecular crosstalk between cancer cells and TME components Analysis of 64 cases of MT cancer patients revealed that the overall density of the inflammatory infiltrate was inversely correlated to the density of CAFs (P = 001), but that the cumulative density of the protumorigenic/anti-inflammatory phenotypes, including regulatory T cells (Tregs, Foxp3+), tumor-associated macrophages (TAM2, CD163+), and potentially Tregs-inducing immune cells (CD80+), was directly correlated with the density of CAFs (P = 001) The hazard ratio (HR) for recurrence in a TME rich in CD163+ Foxp3+ CD80+ was 29 (95% CI 103-86, P = 0043 compared with low in CD163+ Foxp3+ CD80+) The HR for recurrence in a TME rich in CAFs was 41 (95% confidence interval [CI] 13-128, P = 0012 compared with low in CAFs) In vitro studies showed cancer-derived exosomes, epithelial-mesenchymal transition process, fibroblast-to-CAF-like cell transdifferentiation, and reciprocal interrelations between different cytokines suggesting the presence of molecular crosstalk between cancer cells and TME components Collectively, these results highlighted the emerging need of new therapies targeting this crosstalk between the cancer cells and TME components in MT cancer
66 citations
TL;DR: It is concluded that several genes and pathways, particularly those associated with macronutrient and steroid hormone metabolism, are associated with the anticancer effects of CR, and that reduced IGF‐1 levels can account, at least in part, for many of the effects ofCR on gene expression and mammary tumor burden.
Abstract: The prevalence of obesity, an established risk and progression factor for breast and many other cancer types, remains very high in the United States and throughout the world. Calorie restriction (CR), a reduced-calorie dietary regimen typically involving a 20-40% reduction in calorie consumption, prevents or reverses obesity, and inhibits mammary and other types of cancer in multiple tumor model systems. Unfortunately, the mechanisms underlying the tumor inhibitory effects of CR are poorly understood, and a better understanding of these mechanisms may lead to new intervention targets and strategies for preventing or controlling cancer. We have previously shown that the anticancer effects of CR are associated with decreased systemic levels of insulin-like growth factor-1 (IGF-1), the primary source of which is liver. We have also reported that CR strongly suppresses tumor development and growth in multiple mammary cancer models. To identify CR-responsive genes and pathways, and to further characterize the role of IGF-1 as a mediator of the anticancer effects of CR, we assessed hepatic and mammary gland gene expression, hormone levels and growth of orthotopically transplanted mammary tumors in control and CR mice with and without exogenous IGF-1. C57BL/6 mice were fed either control AIN-76A diet ad libitum (AL), subjected to 20%, 30%, or 40% CR plus placebo timed-release pellets, or subjected to 30% or 40% CR plus timed-release pellets delivering murine IGF-1 (mIGF-1, 20 μg/day). Compared with AL-fed controls, body weights were decreased 14.3% in the 20% CR group, 18.5% in the 30% CR group, and 38% in the 40% CR group; IGF-1 infusion had no effect on body weight. Hepatic transcriptome analyses indicated that compared with 20% CR, 30% CR significantly modulated more than twice the number of genes and 40% CR more than seven times the number of genes. Many of the genes specific to the 40% CR regimen were hepatic stress-related and/or DNA damage-related genes. Exogenous IGF-1 rescued the hepatic expression of several metabolic genes and pathways affected by CR. Exogenous IGF-1 also rescued the expression of several metabolism- and cancer-related genes affected by CR in the mammary gland. Furthermore, exogenous IGF-1 partially reversed the mammary tumor inhibitory effects of 30% CR. We conclude that several genes and pathways, particularly those associated with macronutrient and steroid hormone metabolism, are associated with the anticancer effects of CR, and that reduced IGF-1 levels can account, at least in part, for many of the effects of CR on gene expression and mammary tumor burden.
53 citations
TL;DR: Increased Id1 and Id3 expression attenuates all three cyclin‐dependent kinase inhibitors resulting in a more aggressive PCa phenotype and both these proteins are noncompensatory at least in PCa progression.
Abstract: As transcriptional regulators of basic helix–oop–helix (bHLH) transcription and non-bHLH factors, the inhibitor of differentiation (Id1, Id2, Id3, and Id4) proteins play a critical role in coordinated regulation of cell growth, differentiation, tumorigenesis, and angiogenesis. Id1 regulates prostate cancer (PCa) cell proliferation, apoptosis, and androgen independence, but its clinical significance in PCa remains controversial. Moreover, there is lack of evidence on the expression of Id2 and Id3 in PCa progression. In this study we investigated the expression of Id2 and Id3 and reevaluated the expression of Id1 in PCa. We show that increased Id1 and Id3 protein expression is strongly associated with increasing grade of PCa. At the molecular level, we report that silencing either Id1 or Id3 attenuates cell cycle. Although structurally and mechanistically similar, our results show that both these proteins are noncompensatory at least in PCa progression. Moreover, through gene silencing approaches we show that Id1 and Id3 primarily attenuates CDKN1A (p21) and CDKN1B (p27), respectively. We also demonstrate that silencing Id3 alone significantly attenuates proliferation of PCa cells as compared with Id1. We propose that increased Id1 and Id3 expression attenuates all three cyclin-dependent kinase inhibitors (CDKN2B, -1A, and -1B) resulting in a more aggressive PCa phenotype.
51 citations
TL;DR: Results suggest that equilibrium of dimerization among the ERBB proteins can be perturbed by HER2Mab and HER3 plays a key role in sensing the perturbation.
Abstract: Aberrant expression and activation of EGFR and ERBB2 (HER2) have been successfully targeted for cancer therapeutics. Recent evidence from both basic and clinical studies suggests that ERBB3 (HER3) serves as a key activator of downstream signaling through dimerization with other ERBB proteins and plays a critical role in the widespread clinical resistance to EGFR and HER2 targeting cancer therapies. As a result, HER3 is actively pursued as an antibody therapeutic target for cancer. Ligand binding is thought to be a prerequisite for dimerization of HER3 with other ERBB proteins, which results in phosphorylation of its c-terminal tyrosine residues and activation of downstream AKT and MAPK signaling pathways. In this study, we report that an anti-HER2 monoclonal antibody (HER2Mab), which blocks HER2 dimerization with HER3, induces HER3 dimerization with EGFR in both low and high HER2 expressing cancer cells. Treatment of the low HER2 expressing MCF7 cancer cells with HER2Mab promoted cell proliferation and migration in the absence of HER3 ligand stimulation. Follow-up studies revealed that HER2Mab-induced HER3 signaling via EGFR/HER3 dimerization and activation of downstream AKT signaling pathways. These results suggest that equilibrium of dimerization among the ERBB proteins can be perturbed by HER2Mab and HER3 plays a key role in sensing the perturbation.
TL;DR: It is confirmed that CXCL‐8 expression plays a critical role in regulating multiple cellular phenotypes associated with melanoma growth and metastasis, and is accompanied by increased microvessel density in vivo, as compared with vector control‐transfected cells.
Abstract: CXCL-8, a chemokine secreted by melanoma and stromal cells, serves as a growth and angiogenic factor for melanoma progression. This study evaluated how modulation of CXCL-8 levels in melanoma cell lines with different tumorigenic and metastatic potentials affected multiple tumor phenotypes. A375P cells (CXCL-8 low expressor) were stably transfected with a CXCL-8 mammalian expression vector to overexpress CXCL-8, whereas A375SM cells (CXCL-8 high expressor) were transfected with a CXCL-8 antisense expression vector to suppress CXCL-8 expression. Subsequent cell proliferation, migration, invasion, and soft-agar colony formation were analyzed, and in vivo tumor growth and metastasis were evaluated using mouse xenograft models. Our data demonstrate that overexpression of CXCL-8 significantly enhanced primary tumor growth and lung metastasis, accompanied by increased microvessel density in vivo, as compared with vector control-transfected cells. We also observed increased clonogenic ability, growth, and invasive potential of CXCL-8 overexpressing cells in vitro. Knockdown of CXCL-8 using an antisense vector resulted in increased cell death and reduced tumor growth relative to control. Taken together, these data confirm that CXCL-8 expression plays a critical role in regulating multiple cellular phenotypes associated with melanoma growth and metastasis.
TL;DR: This study has successfully introduced FISH for diagnosing KIF5B/RET positive lung adenocarcinoma and facilitates the molecular evaluation for RET fusions and could be applicable in clinical practice to detect lung cancer that may be responsive to RET inhibitors.
Abstract: RET encodes the tyrosine kinase receptor of growth factors belonging to the glial-derived neurotrophic factor family. Recently, RET gene rearrangements with N-terminal of KIF5B gene were identified in lung adenocarcinomas from large-scale sequencing. We investigated RET mRNA expression by real-time reverse-transcriptase polymerase chain reaction (RT-PCR) assay using LightCycler, and KIF5B/RET gene rearrangements using newly established fluorescence in situ hybridization (FISH) analysis in surgically treated nonsmall cell lung cancer (NSCLC) cases. RET protein expression was also investigated by immunohistochemistry (IHC). This study included 157 surgically removed NSCLC cases for mRNA level analyses. The RET/β actin mRNA levels were not significantly different between lung cancer (6.359 ± 15.268) and adjacent normal lung tissues (8.205 ± 28.931, P = 0.6332). Tumor/normal (T/N) ratio of RET/β actin mRNA levels was not different within gender, stage, smoking status, and pathological subtypes. T/N ratio of RET/β actin mRNA levels was significantly higher in KIF5B/RET rearrangement samples (161.763 ± 123.488) than in wild-type samples (5.9013 ± 17.148, P = 0.044). Although RET IHC positivity was not perfectly correlated with KIF5B/RET arrangement, we have detected the KIF5B/RET rearrangements using FISH analysis. Thus, we have successfully introduced FISH for diagnosing KIF5B/RET positive lung adenocarcinoma. This method facilitates the molecular evaluation for RET fusions and could be applicable in clinical practice to detect lung cancer that may be responsive to RET inhibitors.
TL;DR: The results identify the IL‐1β /CEBPβ/MMP pathway as a putative target in the design of anti‐metastatic therapies for the treatment of advanced RCC.
Abstract: Effective treatment of metastatic renal cell carcinoma (RCC) remains a major medical concern, as these tumors are refractory to standard therapies and prognosis is poor. Although molecularly targeted therapies have shown some promise in the treatment of this disease, advanced RCC tumors often develop resistance to these drugs. Dissecting the molecular mechanisms underlying the progression to advanced disease is necessary to design alternative and improved treatment strategies. Tumor-associated macrophages (TAMs) found in aggressive RCC tumors produce a variety of inflammatory cytokines, including interleukin-1β (IL-1β). Moreover, the presence of TAMs and high serum levels of IL-1β in RCC patients correlate with advanced disease. We hypothesized that IL-1β in the tumor microenvironment promotes the development of aggressive RCC tumors by directing affecting tumor epithelial cells. To address this, we investigated the role of IL-1β in mediating RCC tumor cell invasion as a measure of tumor progression. We report that IL-1β induced tumor cell invasion of RCC cells through a process that was dependent on the activity of matrix metalloproteinases (MMPs) and was independent of migration rate. Specifically, IL-1β induced the expression of MMP-1, MMP-3, MMP-10, and MT1-MMP in a mechanism dependent on IL-1β activation of the transcription factor CCAAT enhancer binding protein β (CEBPβ). Consistent with its role in MMP gene expression, CEBPβ knockdown significantly reduced invasion, but not migration, of RCC tumor cells. These results identify the IL-1β /CEBPβ/MMP pathway as a putative target in the design of anti-metastatic therapies for the treatment of advanced RCC.
TL;DR: The presence of LVSI is an independent predictive indicator of nodal metastasis and is associated with worse clinical outcome of patients with epithelial ovarian cancer.
Abstract: While the prognostic significance of lymphovascular space invasion (LVSI) is well established in endometrial and cervical cancer, its role in ovarian cancer is not fully understood. First, a training cohort was conducted to explore whether the presence and quantity of LVSI within the ovarian tumor correlated with nodal metastasis and survival (n = 127). Next, the results of the training cohort were applied to a different study population (validation cohort, n = 93). In both cohorts, histopathology slides of epithelial ovarian cancer cases that underwent primary cytoreductive surgery including pelvic and/or aortic lymphadenectomy were examined. In a post hoc analysis, the significance of LVSI was evaluated in apparent stage I cases (n = 53). In the training cohort, the majority of patients had advanced-stage disease (82.7%). LVSI was observed in 79.5% of cases, and nodal metastasis was the strongest variable associated with the presence of LVSI (odds ratio [OR]: 7.99, 95% confidence interval [CI]: 1.98-32.1, P = 0.003) in multivariate analysis. The presence of LVSI correlated with a worsened progression-free survival on multivariate analysis (hazard ratio [HR]: 2.06, 95% CI: 1.01-4.24, P = 0.048). The significance of the presence of LVSI was reproduced in the validation cohort (majority, early stage 61.3%). In apparent stage I cases, the presence of LVSI was associated with a high negative predictive value for nodal metastasis (100%, likelihood ratio, P = 0.034) and with worsened progression-free survival (HR: 5.16, 95% CI: 1.00-26.6, P = 0.028). The presence of LVSI is an independent predictive indicator of nodal metastasis and is associated with worse clinical outcome of patients with epithelial ovarian cancer.
TL;DR: In conclusion, there was no detectable excess risk of lung cancer due to welding fumes among moderate to heavy smokers; but among light smokers the authors found an excess risk related to both types of welding fumes.
Abstract: We investigated relationships between occupational exposure to gas and arc welding fumes and the risk of lung cancer among workers exposed to these agents throughout the spectrum of industries. Two population-based case-control studies were conducted in Montreal. Study I (1979-1986) included 857 cases and 1066 controls, and Study II (1996-2001) comprised 736 cases and 894 controls. Detailed job histories were obtained by interview and evaluated by an expert team of chemist-hygienists to estimate degree of exposure to approximately 300 substances for each job. Gas and arc welding fumes were among the agents evaluated. We estimated odds ratios (ORs) and 95% confidence intervals (CIs) of lung cancer using logistic regression, adjusting for smoking history and other covariates. The two studies provided similar results, so a pooled analysis was conducted. Among all subjects, no significant association was found between lung cancer and gas welding fumes (OR = 1.1; 95% CI = 0.9-1.4) or arc welding fumes (OR = 1.0; 95% CI = 0.8-1.2). However, when restricting attention to light smokers, there was an increased risk of lung cancer in relation to gas welding fumes (OR = 2.9; 95% CI = 1.7-4.8) and arc welding fumes (OR = 2.3; 95% CI = 1.3-3.8), with even higher OR estimates among workers with the highest cumulative exposures. In conclusion, there was no detectable excess risk of lung cancer due to welding fumes among moderate to heavy smokers; but among light smokers we found an excess risk related to both types of welding fumes.
TL;DR: It is demonstrated that Id4 is down‐regulated in prostate cancer due to promoter hypermethylation, which strongly support the role of Id4 as a tumor suppressor.
Abstract: The inhibitor of DNA-binding (Id) proteins, Id1–4 are negative regulators of basic helix-loop-helix (bHLH) transcription factors. As key regulators of cell cycle and differentiation, expression of Id proteins are increasingly observed in many cancers and associated with aggressiveness of the disease. Of all the four Id proteins, the expression of Id1, Id2, and to a lesser extent, Id3 in prostate cancer and the underlying molecular mechanism is relatively well known. On the contrary, our previous results demonstrated that Id4 acts as a potential tumor suppressor in prostate cancer. In the present study, we extend these observations and demonstrate that Id4 is down-regulated in prostate cancer due to promoter hypermethylation. We used prostate cancer tissue microarrays to investigate Id4 expression. Methylation specific PCR on bisulfite treated DNA was used to determine methylation status of Id4 promoter in laser capture micro-dissected normal, stroma and prostate cancer regions. High Id4 expression was observed in the normal prostate epithelial cells. In prostate cancer, a stage-dependent decrease in Id4 expression was observed with majority of high grade cancers showing no Id4 expression. Furthermore, Id4 expression progressively decreased in prostate cancer cell line LNCaP and with no expression in androgen-insensitive LNCaP-C81 cell line. Conversely, Id4 promoter hypermethylation increased in LNCaP-C81 cells suggesting epigenetic silencing. In prostate cancer samples, loss of Id4 expression was also associated with promoter hypermethylation. Our results demonstrate loss of Id4 expression in prostate cancer due to promoter hypermethylation. The data strongly support the role of Id4 as a tumor suppressor.
TL;DR: The observed associations of diabetes and perineural invasion as well as reduced frequency of pain as early symptom of pancreatic cancer support the hypothesis that diabetes may contribute to pancreatic progression via the mechanism of nerve damage.
Abstract: Diabetes and perineural invasion are frequently observed in pancreatic cancer. In this study, we tested possible relations between diabetes and perineural invasion in patients with resected pancreatic cancer. We conducted a retrospective study in 544 cases of resected pancreatic adenocarcinoma seen at the University of Texas MD Anderson Cancer Center during 1996-2011. Information on tumor characteristics, diabetes history, and survival time was collected by personal interview and medical record review. Patients with diabetes before or at the time of the pancreatic cancer diagnosis were considered diabetes only. Pearson χ(2) test was used to compare categorical variables in diabetic and nondiabetic groups. Kaplan-Meier plot, log-rank test, and Cox proportional regression models were applied in survival analysis. The prevalence of diabetes and perineural invasion was 26.5% and 86.9%, respectively, in this study population. Patients with diabetes had a significantly higher prevalence of perineural invasion (92.4%) than those without diabetes (85%) (P = 0.025, χ(2) test). Diabetes was not associated with other pathological characteristics of the tumor, such as tumor size, lymphovascular invasion, tumor grade, lymph node metastasis, and resection margin status. Diabetic patients had a significantly lower frequency of abdominal pain (P = 0.01), but a slightly higher frequency of weight loss (P = 0.078) as early symptoms of their cancer. Both diabetes and perineural invasion were related to worse survival and increased risk of death after adjusting for tumor grade and margin and node status (P = 0.036 and 0.019, respectively). The observed associations of diabetes and perineural invasion as well as reduced frequency of pain as early symptom of pancreatic cancer support the hypothesis that diabetes may contribute to pancreatic progression via the mechanism of nerve damage.
TL;DR: Women who used TAM/AIs had a large reduction in risk of subsequent breast cancer compared with nonusers and women with high adherence who used AIs exclusively and had positive ER or PR receptor status had the greatest risk reduction.
Abstract: Tamoxifen (TAM) has been prescribed for decades and aromatase inhibitors (AIs) have been used since the early 2000s in preventing subsequent breast cancer. However, outside of clinical trials, the effectiveness of AIs is not established. We examined the long-term risk of subsequent breast cancer among survivors treated with TAM and AIs in a large health plan. The study included 22,850 survivors, diagnosed with initial breast cancer (stages 0-IV) from 1996 to 2006, and followed 13 years maximum. We compared the risk of subsequent breast cancer in those who used TAM and/or AIs versus nonusers (the reference group). Hazard ratios (HR) adjusted for patient, tumor, treatment, and health-care characteristics were estimated using Cox models with time-dependent drug use status. Women who used TAM/AIs had a large reduction in risk of subsequent breast cancer compared with nonusers. While confidence intervals (CI) for all hormone treatment groups overlapped, women with high adherence (medication possession ratio ≥80%) who used AIs exclusively and had positive ER or PR receptor status had the greatest risk reduction (HR = 0.34, 95% CI: 0.28-0.41), followed by those who switched from TAM to AIs (HR = 0.39, 95% CI: 0.30-0.49), and those who used TAM exclusively (HR = 0.42, 95% CI: 0.36-0.47). Women with high adherence had the greatest risk reduction in subsequent breast cancer, but the results were not substantially different from women who took the drugs less regularly. Compared with nonusers, the reduction in subsequent breast cancer risk ranged from 58% to 66% across the hormone treatment groups and degree of adherence.
TL;DR: It is shown that RFP was expressed in 62% of ovarian cancer patients and its positivity significantly correlated with drug resistance, and depletion of RFP by RNA interference in ovarian cancer cell lines, SKOV3 and HEY, significantly increased carboplatin‐ or paclitaxel‐induced apoptosis and resulted in reduced anticancer drug resistance.
Abstract: Resistance to platinum- and taxane-based chemotherapy is a major cause of treatment failure in ovarian cancer. Thus, it is necessary to develop a predictive marker and molecular target for overcoming drug resistance in ovarian cancer treatment. In a previous report, using an in vitro model, we found that the RET finger protein (RFP) (also known as tripartite motif-containing protein 27, TRIM27) confers cancer cell resistance to anticancer drugs. However, the significance of RFP expression in cancer patients remains elusive. In this study, we showed that RFP was expressed in 62% of ovarian cancer patients and its positivity significantly correlated with drug resistance. Consistent with clinical data, depletion of RFP by RNA interference (RNAi) in ovarian cancer cell lines, SKOV3 and HEY, significantly increased carboplatin- or paclitaxel-induced apoptosis and resulted in reduced anticancer drug resistance. In a nude mouse tumor xenograft model, inoculated RFP-knockdown ovarian cancer cells exhibited lower carboplatin resistance than control cells. These findings suggest that RFP could be a predictive marker for chemoresistance in ovarian cancer patients and also a candidate for a molecular-targeted agent.
TL;DR: A decrease in SUVmax was associated with improved PFS and OS, and results further support the incorporation of 18‐FDG PET/CT scans in advanced BCC management.
Abstract: The use of 18-fluorodeoxyglucose (FDG) positron emission tomography with computed tomography (PET/CT) in subjects with advanced basal cell carcinoma (BCC) has not been fully explored due to the rarity of disease presentation. This study evaluated PET/CTs from subjects with advanced BCC participating in a phase I dose-escalation clinical trial of vismodegib. Fourteen subjects with BCC were imaged with 18-FDG PET/CT for lesion identification and response categorizing (European Organisation for Research and Treatment for Cancer [EORTC] and PET response criteria in solid tumors [PERCIST] 1.0). Several parameters including metabolic activity of target lesions, site of disease presentation and spread, treatment response, and prognostic significance of metabolic activity following therapy were evaluated. All subjects exhibited at least one hypermetabolic lesion. Most subjects had only four organ systems involved at study enrollment: skin-muscle (93%), lung (57%), lymph nodes (29%), and bone (21%). SUVmax measured across all lesions decreased (median 33%, SD ± 45%) following therapy with metabolic activity normalizing or disappearing in 42% of lesions. No significant difference was observed between EORTC and PERCIST 1.0. Subjects that demonstrated at least a 33% reduction in SUVmax from baseline had a significantly longer progression-free survival (PFS) (median 17 months, 95% confidence interval [CI] ±4 months vs. 9 months, 95% CI ±5 months, P = 0.038) and overall survival (OS) (median 24 months, 95% CI ±4 months vs. 17 months, 95% CI ±13 months, P = 0.019). BCC lesions are hypermetabolic on 18-FDG PET/CT. A decrease in SUVmax was associated with improved PFS and OS. These results further support the incorporation of 18-FDG PET/CT scans in advanced BCC management.
TL;DR: The blocked Notch activity in MCF‐7 cells was blocked by expressing a dominant‐negative MAML‐GFP (dnMAML) construct, which inhibits signaling through all Notch receptors, and quantified the effect on tumor‐initiating activity.
Abstract: Studies have suggested the potential importance of Notch signaling to the cancer stem cell population in some tumors, but it is not known whether all cells in the cancer stem cell fraction require Notch activity. To address this issue, we blocked Notch activity in MCF-7 cells by expressing a dominant-negative MAML-GFP (dnMAML) construct, which inhibits signaling through all Notch receptors, and quantified the effect on tumor-initiating activity. Inhibition of Notch signaling reduced primary tumor sphere formation and side population. Functional quantification of tumor-initiating cell numbers in vivo showed a significant decrease, but not a complete abrogation, of these cells in dnMAML-expressing cells. Interestingly, when assessed in secondary assays in vitro or in vivo, there was no difference in tumor-initiating activity between the dnMAML-expressing cells and control cells. The fact that a subpopulation of dnMAML-expressing cells was capable of forming primary and secondary tumors indicates that there are Notch-independent tumor-initiating cells in the breast cancer cell line MCF-7. Our findings thus provide direct evidence for a heterogeneous cancer stem cell pool, which will require combination therapies against multiple oncogenic pathways to eliminate the tumor-initiating cell population.
TL;DR: The studies reported here support the role of CIP2A in bladder cancer progression and its usefulness for the surveillance of recurrence or progression of human bladder cancer.
Abstract: Bladder cancer is one of the most common cancers in the United States. Numerous markers have been evaluated for suitability of bladder cancer detection and surveillance. However, few of them are acceptable as a routine tool. Therefore, there exists a continuing need for an assay that detects the presence of bladder cancer in humans. It would be advantageous to develop an assay with a protein that is associated with the development of bladder cancer. We have identified the cancerous inhibitor of PP2A (CIP2A) protein as a novel bladder cancer biomarker. In this study, Western blot analysis was used to assess the expression level of CIP2A protein in bladder cancer cell lines and bladder cancer patient tissues (n = 43). Our studies indicated CIP2A protein was abundantly expressed in bladder cancer cell lines but not in nontumor epithelial cell lines. Furthermore, CIP2A was specifically expressed in transitional cell carcinoma (TCC) of the bladder tumor tissues but not in adjacent nontumor bladder tissue. Our data showed that CIP2A protein detection in high-grade TCC tissues had a sensitivity of 65%, which is 3.4-fold higher than that seen in low-grade TCC tissues (19%). The level of CIP2A protein expression increased with the stage of disease (12%, 27%, 67%, and 100% for pTa, pT1, pT2, and pT3 tumor, respectively). In conclusion, our studies suggest that CIP2A protein is specifically expressed in human bladder tumors. CIP2A is preferentially expressed in high-grade and high-stage TCC tumors, which are high-risk and invasive tumors. Our studies reported here support the role of CIP2A in bladder cancer progression and its usefulness for the surveillance of recurrence or progression of human bladder cancer.
TL;DR: It is shown that preserved overexpression of Wnt5a protein is associated with increased time to biochemical recurrence in localized low‐grade prostate cancer patients after radical prostatectomy, emphasizing that Wnt 5a can be used as a predictive biomarker, and favoring the view of Wtsn5a as a future therapeutic target in prostate cancer Patients with tumor cells displaying low expression of WNT5a.
Abstract: Wnt5a, a member of non-canonical wingless-related MMTV integration site family is a secreted glycoprotein that plays important roles in development and disease. Recent studies have shown that Wnt5a protein levels are up-regulated in prostate cancer, but contrasting reports exist on the role of Wnt5a to predict outcome after radical prostatectomy in patients with localized prostate cancer. Our group has recently shown that preserved high protein expression of Wnt5a in prostate cancer is associated with longer relapse-free time after radical prostatectomy. The present tissue microarray study emphasizes the role of Wnt5a protein expression in a different, well-defined, and independent cohort consisting of 312 prostate cancer patients. Kaplan–Meier curves plotted between Wnt5a expression and time to biochemical recurrence revealed that in low-grade prostate cancer, patients with preserved high-Wnt5a protein levels in their tumor cells have a lower risk of recurrence after radical prostatectomy compared to patients with low-Wnt5a protein expression. When Wnt5a protein expression was added to a Cox regression multivariate analysis, both Wnt5a protein expression and surgical margin status independently predict biochemical free survival. Herein we confirm Wnt5a positivity as a prognostic factor and show that preserved overexpression of Wnt5a protein is associated with increased time to biochemical recurrence in localized low-grade prostate cancer patients after radical prostatectomy. Our results emphasize that Wnt5a can be used as a predictive biomarker, and favoring the view of Wnt5a as a future therapeutic target in prostate cancer patients with tumor cells displaying low expression of Wnt5a.
TL;DR: It is demonstrated in human breast cancer cell lines that expressions of TRF1, TIN2, and POT1 are upregulated by dexamethasone, suggesting activation of the glucocorticoid receptor, whereas TERT, TRF2, TRf1,TIN2 and Pot1 areUpregulated by tumor necrosis factor‐α (TNF‐α), suggestingactivation of the nuclear factor kappa B transcription factor.
Abstract: Telomere dysregulation occurs in both the in situ and invasive stages of many carcinomas, including breast. Knockout experiments have identified several telomere-associated proteins required for proper telomere function and maintenance, including telomere repeat-binding factor 1 and 2 (TRF1 and TRF2), protection of telomeres (POT1), and TRF1-interacting nuclear factor 2 (TIN2). Using telomere content assays and quantitative reverse transcription-polymerase chain reaction (RT-PCR), we examined the relationship between telomere length and the mRNA levels of telomere-associated proteins in breast tumors. The levels of TRF2, TRF1, TIN2, and POT1 mRNA, but not telomerase reverse transcriptase (TERT) RNA, are inversely correlated with telomere content in breast tumors. Significant associations were identified between the mRNA levels of TRF1, TIN2, and POT1; however, there were no significant associations with the mRNA levels of TRF2 or TERT. These associations suggest that a complex transcriptional program coordinately regulates the expression of these mRNAs. We examined the promoter regions of the telomere-associated proteins to identify transcription factors consistent with the observed patterns of presumed coordinate expression. We demonstrated in human breast cancer cell lines that expressions of TRF1, TIN2, and POT1 are upregulated by dexamethasone, suggesting activation of the glucocorticoid receptor, whereas TERT, TRF2, TRF1, TIN2, and POT1 are upregulated by tumor necrosis factor-α (TNF-α), suggesting activation of the nuclear factor kappa B transcription factor. These findings link telomere content in breast tumors to the coordinate expression of several telomere-associated proteins previously shown to be negative regulators of telomere length in cell lines. The results further suggest a possible link between the expressions of the telomere-associated proteins and mediators of stress and inflammation.Telomere content assays and quantitative RT-PCR demonstrate that the levels of TRF2, TRF1, TIN2, and POT1 mRNA, but not telomerase reverse transcriptase (TERT) RNA, are inversely correlated with telomere content in breast tumors. Within human breast cancer cell lines, expressions of TRF1, TIN2, and POT1 are upregulated by dexamethasone, suggesting activation of the glucocorticoid receptor, whereas TERT, TRF2, TRF1, TIN2, and POT1 are upregulated by TNF-α, suggesting activation of the NFκB transcription factor. These findings link telomere content in breast tumors to the expression of several telomere-associated proteins previously shown to be negative regulators of telomere length in cell lines and suggest a link between the expressions of the telomere-associated proteins and mediators of stress and inflammation.
TL;DR: It is concluded that nested PCR detection of HPV followed by short target DNA sequencing can be used for screening and genotyping to formulate a paradigm in clinical management of HPV‐related disorders in a rapidly developing economy.
Abstract: The incidence of cervical cancer is expected to rise sharply in China. A reliable routine human papillomavirus (HPV) detection and genotyping test to be supplemented by the limited Papanicolaou cytology facilities is urgently needed to help identify the patients with cervical precancer for preventive interventions. To this end, we evaluated a nested polymerase chain reaction (PCR) protocol for detection of HPV L1 gene DNA in cervicovaginal cells. The PCR amplicons were genotyped by direct DNA sequencing. In parallel, split samples were subjected to a Digene HC2 HPV test which has been widely used for “cervical cancer risk” screen. Of the 1826 specimens, 1655 contained sufficient materials for analysis and 657 were truly negative. PCR/DNA sequencing showed 674 infected by a single high-risk HPV, 188 by a single low-risk HPV, and 136 by multiple HPV genotypes with up to five HPV genotypes in one specimen. In comparison, the HC2 test classified 713 specimens as infected by high-risk HPV, and 942 as negative for HPV infections. The high-risk HC2 test correctly detected 388 (57.6%) of the 674 high-risk HPV isolates in clinical specimens, mislabeled 88 (46.8%) of the 188 low-risk HPV isolates as high-risk genotypes, and classified 180 (27.4%) of the 657 “true-negative” samples as being infected by high-risk HPV. It was found to cross-react with 20 low-risk HPV genotypes. We conclude that nested PCR detection of HPV followed by short target DNA sequencing can be used for screening and genotyping to formulate a paradigm in clinical management of HPV-related disorders in a rapidly developing economy.
TL;DR: Some issues faced during the experience in conducting an epidemiologic case–control study of inflammatory breast cancer in North Africa are illustrated to summarize important issues for conducting future Epidemiologic studies in these or similar low‐ or middle‐income countries.
Abstract: This article illustrates some issues we faced during our experience in conducting an epidemiologic case–control study of inflammatory breast cancer in North Africa. We expect that some of the questions we had to ask in order to address these issues might be helpful to others in setting up epidemiologic studies in developing regions. We describe our experience from different angles including the use of multiple sites to achieve adequate sample size, standardizing diagnosis of disease, identifying cancer cases at the time of diagnosis, control selection procedures, logistics of study implementation, questionnaire development and interviewing, biologic specimens, and procedures for protection of human subjects. We have developed a brief checklist to summarize important issues for conducting future epidemiologic studies in these or similar low- or middle-income countries.
TL;DR: The results reveal that EPCR is expressed not only by a wide range of human malignant hematological cells but also the detection of plasma sEPCR levels provides a powerful insight into thrombotic risk assessment in cancer patients, especially when it surpasses 200 ng/mL.
Abstract: Elevated plasma level of soluble endothelial protein C receptor (sEPCR) may be an indicator of thrombotic risk. The present study aims to correlate leukemia-associated hypercoagulability to high level plasma sEPCR and proposes its measurement in routine clinical practice. EPCR expressions in leukemic cell lines were determined by flow cytometry, immunocytochemistry, and reverse transcription polymerase chain reaction (RT-PCR). EPCR gene sequence of a candidate cell line HL-60 was also determined. Plasma samples (n = 76) and bone marrow aspirates (n = 72) from 148 patients with hematologic malignancies and 101 healthy volunteers were analyzed by enzyme-linked immunosorbent assay (ELISA) via a retrospective study for sEPCR and D-dimer. All leukemic cell lines were found to express EPCR. Also, HL-60 EPCR gene sequence showed extensive similarities with the endothelial reference gene. All single nucleotide polymorphisms (SNPs) originally described and some new SNPs were revealed in the promoter and intronic regions. Among these patients 67% had plasma sEPCR level higher than the controls (100 ± 28 ng/mL), wherein 16.3% patients had experienced a previous thrombotic event. These patients were divided into: group-1 (n = 45) with amount of plasmatic sEPCR below 100 ng/mL, group-2 (n = 45) where the concentration of sEPCR was between 100 and 200, and group-3 (n = 20) higher than 200 ng/mL. The numbers of thrombotic incidence recorded in each group were four, six, and eight, respectively. These results reveal that EPCR is expressed not only by a wide range of human malignant hematological cells but also the detection of plasma sEPCR levels provides a powerful insight into thrombotic risk assessment in cancer patients, especially when it surpasses 200 ng/mL.
TL;DR: Data suggest that ER expression does not always correlate with ER transcriptional activity, and determination of ERE activity as an ER functional marker will be helpful for analysis of a variety of diverse breast cancer cases and the subsequent course of treatment.
Abstract: To predict the efficacy of hormonal therapy at the individual-level, immunohistochemical methods are used to analyze expression of classical molecular biomarkers such as estrogen receptor (ER), progesterone receptor (PgR), and HER2. However, the current diagnostic standard is not perfect for the individualization of diverse cases. Therefore, establishment of more accurate diagnostics is required. Previously, we established a novel method that enables analysis of ER transcriptional activation potential in clinical specimens using an adenovirus estrogen response element–green fluorescence protein (ERE-GFP) assay system. Using this assay, we assessed the ERE transcriptional activity of 62 primary breast cancer samples. In 40% of samples, we observed that ER protein expression was not consistent with ERE activity. Comparison of ERE activity with clinicopathological information revealed that ERE activity was significantly correlated with the ER target gene, PgR, rather than ER in terms of both protein and mRNA expression. Moreover, subgrouping of Luminal A-type breast cancer samples according to ERE activity revealed that ERα mRNA expression correlated with ER target gene mRNA expression in the high-, but not the low-, ERE-activity group. On the other hand, the low-ERE-activity group showed significantly higher mRNA expression of the malignancy biomarker Ki67 in association with disease recurrence in 5% of patients. Thus, these data suggest that ER expression does not always correlate with ER transcriptional activity. Therefore, in addition to ER protein expression, determination of ERE activity as an ER functional marker will be helpful for analysis of a variety of diverse breast cancer cases and the subsequent course of treatment.
TL;DR: It is demonstrated that testing pancreatic tumor samples for a biomarker‐driven, multicenter study in metastatic pancreas cancer is feasible and enriching for a subset of second/third line pancreatic cancer patients treated with a Src inhibitor based on a biomarkers would improve 6‐month survival.
Abstract: Src tyrosine kinases are overexpressed in pancreatic cancers, and the oral Src inhibitor saracatinib has shown antitumor activity in preclinical models of pancreas cancer. We performed a CTEP-sponsored Phase II clinical trial of saracatinib in previously treated pancreas cancer patients, with a primary endpoint of 6-month survival. A Simon MinMax two-stage phase II design was used. Saracatinib (175 mg/day) was administered orally continuously in 28-day cycles. In the unselected portion of the study, 18 patients were evaluable. Only two (11%) patients survived for at least 6 months, and three 6-month survivors were required to move to second stage of study as originally designed. The study was amended as a biomarker-driven trial (leucine rich repeat containing protein 19 [LRRC19] > insulin-like growth factor-binding protein 2 [IGFBP2] “top scoring pairs” polymerase chain reaction [PCR] assay, and PIK3CA mutant) based on preclinical data in a human pancreas tumor explant model. In the biomarker study, archival tumor tissue or fresh tumor biopsies were tested. Biomarker-positive patients were eligible for the study. Only one patient was PIK3CA mutant in a 3′ untranslated region (UTR) portion of the gene. This patient was enrolled in the study and failed to meet the 6-month survival endpoint. As the frequency of biomarker-positive patients was very low (<3%), the study was closed. Although we were unable to conclude whether enriching for a subset of second/third line pancreatic cancer patients treated with a Src inhibitor based on a biomarker would improve 6-month survival, we demonstrate that testing pancreatic tumor samples for a biomarker-driven, multicenter study in metastatic pancreas cancer is feasible.