Early Event In Tumorigenesis Research Articles

Abstract B02: DNA promoter methylation of HIST1H2BB as a potential early detection biomarker to reduce ovarian cancer disparities in the United States

Abstract Background: Ovarian cancer disproportionately affects African American women in the United States (US) at all stages of the disease, from presentation through treatment, and ultimately leads to increased mortality and decreased survival, compared to non-Hispanic White women. Till recently, epithelial ovarian cancers were thought to arise from the ovarian surface epithelial cells. However, recent studies suggest that the majority of high-grade serous carcinomas (HGSCs) arise from lesions in the fallopian tubes. DNA promoter hypermethylation of certain genes is often an early event in tumorigenesis, and it can be detected in blood and other bodily fluids, making it a feasible biomarker strategy for early detection of tumors. Purpose of study: Therefore, we sought to determine the methylation profile of HGSCs as compared to fallopian tube epithelium. Using the Infinium HumanMethylation 450 Array, that permits to quantitatively assess over 450,000 methylation sites, within and outside of CpG islands and more than 20,000 genes per sample, at single-nucleotide resolution, we have obtained the genome-wide methylation profile of HGSC and fallopian tubal epithelium. We conducted a pilot study by comparing the global methylation status in 12 HGSC tissue samples to 15 fallopian tube epithelium samples. In addition, we compared the methylation profile among long-term survivors (more than 5 years) and short-term survivors (2-3 years) of HGSCs. Results: Our results identified the HIST1H2BB promoter as the most differentially methylated region when comparing HGSC with normal fallopian tube samples. Furthermore, we also found that HIST1H2BB is differentially methylated in HGSC patients with long-term survival when compared to patients with short-term survival. Conclusions: Taken together, these results support the potential use of HIST1H2BB promoter methylation as a diagnostic and prognostic biomarker for ovarian cancer. Our findings could lead to the development of a biomarker for the diagnosis and/or prognosis of ovarian cancer. An early detection biomarker for ovarian cancer could contribute to the reduction and eventual elimination of ovarian cancer health disparities in the US. Citation Format: Blanca L. Valle, Elisabetta Kuhn, David Sidransky, Rafael Guerrero-Preston. DNA promoter methylation of HIST1H2BB as a potential early detection biomarker to reduce ovarian cancer disparities in the United States. [abstract]. In: Proceedings of the Seventh AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 9-12, 2014; San Antonio, TX. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2015;24(10 Suppl):Abstract nr B02.

Expression and DNA methylation status of the Rap2B gene in human bronchial epithelial cells treated by cigarette smoke condensate

Background: The relationship between lung cancer and smoking has been demonstrated. The Rap2B gene is usually overexpressed in lung cancers. This study was aimed to investigate the Rap2B gene expression and its promoter methylation in human bronchial epithelial cells (16HBE) treated by cigarette smoke condensate (CSC).Methods: 16HBE cells were treated with CSC (1/8 IC50). Soft ager assay, tumorigenicity test, chromosome aberrations analysis were used to identify the transformed cells. The expression level of mRNA and protein of Rap2B was detected using real time PCR and Western blotting, respectively. The genome DNA methylation level was detected using combined bisulfite restriction analysis (COBRA) and the methylation status of the target fragment in Rap2B gene promoter was determined by bisulfite sequencing PCR (BSP).Results: The 16HBE cells were successfully malignant transformed after the chronic exposure to CSC. The expression of Rap2B gradually increased in the process of malignant transformation. Meanwhile, global DNA was hypomethylated. However, no obvious change was observed in the methylation level of Rap2B gene promoter in transformed 16HBE cells.Conclusions: Rap2B gene may play an important role in the process of lung cancer and global DNA hypomethylation might be an early event in tumorigenesis.

Abstract AS15: DNA promoter hypermethylation of HIST1H2BB as a diagnostic and prognostic biomarker for ovarian cancer

Abstract Background: Until recently, epithelial ovarian cancers were thought to arise from the ovarian surface epithelial cells. However, recent studies suggest that the majority of high grade serous carcinomas (HGSCs) arise from lesions in the fallopian tubes. DNA promoter hypermethylation of certain genes is often an early event in tumorigenesis, and it can be detected in blood and other bodily fluids, making it a feasible biomarker strategy for early detection of tumors. Purpose of study: Therefore, we sought to determine the methylation profile of HGSCs as compared to fallopian tube epithelium. Using the Infinium HumanMethylation 450 Array, that permits to quantitatively assess over 450,000 methylation sites, within and outside of CpG islands and more than 20,000 genes per sample, at single-nucleotide resolution, we have obtained the genome-wide methylation profile of HGSC and fallopian tubal epithelium. We conducted a pilot study by comparing the global methylation in 12 HGSC tissue samples to 15 fallopian tube epithelium samples. In addition, we compared the methylation profile among long-term survivors (more than 5 years) and short-term survivors (2-3 years) of HGSCs. Results: Our results identified the HIST1H2BB promoter as the most differentially methylated region when comparing HGSC with normal fallopian tube samples. Furthermore, we also found that HIST1H2BB is differentially methylated in HGSC patients with long-term survival when compared to patients with short-term survival. Conclusions: Taken together, these results support the potential of HIST1H2BB promoter hypermethylation as a diagnostic and prognostic biomarker. Our findings could lead to the development of a biomarker for the diagnosis and/or prognosis of ovarian cancer and could contribute to the current understanding of the mechanisms of progression in ovarian cancer. Citation Format: Blanca L. Valle, Elisabetta Kuhn, David Sidransky, Rafael Guerrero-Preston. DNA promoter hypermethylation of HIST1H2BB as a diagnostic and prognostic biomarker for ovarian cancer [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr AS15.

Abstract 2960: DNA promoter hypermethylation of genes as potential diagnostic and prognostic biomarkers for ovarian cancer

Abstract Till recently, epithelial ovarian cancers were thought to arise from the ovarian surface epithelial cells. However, recent studies suggest that the majority of high grade serous carcinomas (HGSCs) arise from lesions in the fallopian tubes. DNA promoter hypermethylation of certain genes is often an early event in tumorigenesis, and it can be detected in blood and other bodily fluids, making it a feasible biomarker strategy for early detection of tumors. Therefore, we sought to determine the methylation profile of HGSCs as compared to fallopian tube epithelium, using the Infinium HumanMethylation 450 Array. We compared the methylation in 12 HGSC tissue samples to 15 fallopian tube epithelium samples. In addition, we compared the methylation profile among long-term survivors (more than 5 years) and short-term survivors (2-3 years) of HGSCs. Our results identified the promoter regions of HIST1H2BB, MAGI2, and EPSTI1 to be differentially methylated when comparing HGSC with normal fallopian tube samples. Furthermore, these genes were differentially methylated in HGSC patients with long-term survival when compared to patients with short-term survival. Quantitative methylation specific PCR analysis confirmed our results of differential methylation among short-term and long-term survivors, and show that these genes are also methylated in ovarian cancer cell lines. Taken together, we have identified genes that could have potential as diagnostic and prognostic biomarkers. Our findings could lead to the development of a biomarker panel for the diagnosis and/or prognosis of ovarian cancer. Citation Format: Blanca L. Valle, Elisabetta Kuhn, David Sidransky, Rafael Guerrero-Preston. DNA promoter hypermethylation of genes as potential diagnostic and prognostic biomarkers for ovarian cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2960. doi:10.1158/1538-7445.AM2015-2960

Abstract 611: Exploratory analyses suggest ovarian tumors with somatic or germline loss of function mutations in BRCA1 or BRCA2 are biologically similar and sensitive to PARP inhibition

Abstract The PARP inhibitor olaparib (LynparzaTM) elicits responses in ovarian cancer patients with inherited (germline) loss of function mutations in the BRCA1 or BRCA2 genes (gBRCAm). In the tumors of such patients the unmutated (wt) copy of the relevant BRCA gene is usually lost. Many patients are aware of their BRCA mutation status due to the wide availability of blood testing. However, in around a third of ovarian tumors with BRCA mutations the mutation has not been inherited but is tumor-specific (sBRCAm). Given the difference in origin of mutation, it is important to determine whether there are genotypic and phenotypic differences between sBRCAm and gBRCAm ovarian tumors. In a randomized, Phase II trial of 265 patients with platinum-sensitive relapsed serous ovarian cancer (NCT00753545; Study 19), maintenance treatment with olaparib (400 mg bid; capsules) led to a significant increase in progression-free survival (PFS) vs placebo in patients with a BRCA mutation (HR 0.18; 95% CI 0.10-0.31; P<0.00001). Diagnostic tumor samples from the study were sequenced by Foundation Medicine. After filtering for common polymorphisms, BRCA gene variants were classified using American College of Medical Genetics standards as mutant, wild type or variant of unknown significance (VUS). VUS were further classified on the basis of information from the Breast Information Core database. Mutation origin (g vs s) and zygosity were determined by tumor allele frequency analysis and/or by comparison to germline sequencing, and heterogeneity was determined using the Foundation Medicine SGZ algorithm, which compares allele frequency to model expectations, taking into account tumor purity, ploidy and local copy number. BRCA mutations were found in 53% of the 209 samples with data available. Known germline BRCA mutations were correctly classified as germline by the SGZ algorithm. Tumor BRCA mutations were identified in 40 patients without confirmation of a mutation in a blood sample. Twenty of these patients were classified as sBRCAm (12 BRCA1 and 8 BRCA2) by the algorithm. Only one case was called without loss of the wt BRCA allele. It has been hypothesized that TP53 mutation is an early event and was found to be consistent between tumors and precursor lesions. Allele frequencies/clonality for BRCA and TP53 mutations were comparable and there was no evidence of systematic subclonality in the sBRCAm cases. An exploratory analysis of PFS and overall survival (OS) was performed on the 20 patients (10 received treatment and 10 placebo). Consistent with results from the gBRCAm population, a trend in event rate in favor of treatment over placebo was observed (3/10 vs 8/10 events for PFS and 3/10 vs 7/10 for OS). Although from a small number of patients, data are consistent with sBRCA mutation as an early event in tumorigenesis and support phenotypic similarity of gBRCA and sBRCA mutated tumors. Citation Format: Brian Dougherty, Zhongwu Lai, Jonathan A. Ledermann, Jane D. Robertson, Tony W. Ho, Wenting Wu, Darren R. Hodgson, Maria C M Orr, Mark J. O'Connor, Matthew J. Hawryluk, Timothy A. Brennan, Roman Yelensky, James X. Sun, J Carl Barrett. Exploratory analyses suggest ovarian tumors with somatic or germline loss of function mutations in BRCA1 or BRCA2 are biologically similar and sensitive to PARP inhibition. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 611. doi:10.1158/1538-7445.AM2015-611

Decreased expression of 14‐3‐3 σ, an early event of malignant transformation of respiratory epithelium, also facilitates progression of squamous cell lung cancer

BackgroundIt has been shown that 14-3-3 σ serves as a tumor suppressor gene, and is downregulated in various tumor tissues. However, the role of 14-3-3 σ during the initiation and progression of lung squamous cell carcinoma (SqCC) is not well understood.MethodsThe expression status of 14-3-3 σ in archival tissue samples from 40 lung SqCC patients (36 with normal bronchia, 19 squamous metaplasia, and 17 dysplasia/carcinoma in situ, in their tissue samples) was examined by immunohistochemical analysis. The proliferation rate and tumor formation ability of the H520 cell transfected with 14-3-3 σ was tested with methyl thiazolyl tetrazolium assay and nude mice subcutaneous injection, respectively.ResultsIn the normal bronchial epithelia, 14-3-3 σ was highly expressed, whereas it was significantly decreased in precancerous and cancerous tissues. Compared with matched invasive cancer tissues, the expression level of 14-3-3 σ in squamous metaplasia was significantly higher (P = 0.049), while that in dysplasia/carcinoma in situ showed no significant changes (P = 0.135). Statistical analysis showed that the expression level of 14-3-3 σ in tumor tissue was associated with the differentiation grade of the tumor (P = 0.001) and the prognosis of the patient (P = 0.003). The overexpression of 14-3-3 σ significantly suppressed the proliferation of H520 cells in vitro and in vivo.ConclusionThe inactivation of 14-3-3 σ may be a very early event in tumorigenesis and could facilitate the initiation and progression of lung SqCC in a sustainable way.

New developments in the pathogenesis and therapeutic targeting of the IDH1 mutation in glioma.

In the last five years, IDH1 mutations in human malignancies have significantly shaped the diagnosis and management of cancer patients. Ongoing intense research efforts continue to alter our understanding of the role of the IDH1 mutation in tumor formation. Currently, evidence suggests the IDH1 mutation to be an early event in tumorigenesis with multiple downstream oncogenic consequences including maintenance of a hypermethylator phenotype, alterations in HIF signalling, and disruption of collagen maturation contributing to a cancer-promoting extracellular matrix. The most recent reports elucidating these mechanisms is described in this review with an emphasis on the pathogenesis of the IDH1 mutation in glioma. Conflicting findings from various studies are discussed, in order to highlight areas warranting further research. Finally, the latest progress in developing novel therapies against the IDH1 mutation is presented, including recent findings from ongoing phase 1 clinical trials and the exciting prospect of vaccine immunotherapy targeting the IDH1 mutant protein.

DNA Methylation Profiling of Sorted Cells from Myelofibrosis Patients reveals Aberrant Epigenetic Regulation of Immune Pathways and identifies Early MPN Driver Genes

Introduction:Primary myelofibrosis (PMF) belongs to the heterogeneous group of chronic myeloproliferative neoplasms (MPN) together with essential thrombocytosis (ET) and polycythemia vera (PV). It has been suggested that these neoplasms represent a biological continuum from early cancer stage (ET,PV) to advanced MF. Multiple studies report frequent mutations in epigenetic regulators. However, the association to epigenetic changes and the role of epigenetic aberrations in different cell populations is still unknown. We therefore performed DNA methylation profiling of sorted cells from MF patients to unravel pathways contributing to disease phenotype and gain insight into MF pathogenesis. As an aberrant DNA methylation pattern may be an early event in tumorigenesis and may be crucial for progression of the malignant clone towards the more aggressive forms of MPN, we further aimed to identify methylated candidate driver genes.Material and methods:Peripheral blood samples from 16 MF patients were together with BM (bone marrow) and peripheral blood from 3 healthy age matched controls sorted in CD34+ cells, granulocytes and mononuclear cells, and analysed for differential methylated regions using Illumina Infinium HumanMethylation 450K BeadChip. Candidate genes were validated by pyrosequencing in a second cohort of 30 MF patients. To identify potential driver genes the DNA methylation status of candidate genes were likewise analyzed in a larger cohort consisting of 60 ET and PV patients.Results:The number of differential methylated CpG sites between MF cells and the healthy counterparts differed extensively among the three cell populations analyzed. In MF CD34+ cells 1628 CpG sites were differential methylated compared to normal CD34+ cells, and 519 and 213 differential methylated CpG sites were observed in MF granulocytes and MF mononuclear cells, respectively (Δβ was set to 0.2 with an adjusted p-value < 0.05, T-test). Differentially methylated genes were mainly involved in cancer and embryogenic pathways in both the MF CD34+ and mononuclear cells, while mononuclear cells also showed aberrant methylation of genes involved in the inflammatory disease pathways. MF granulocytes showed significant aberrations in pathways involving immunological diseases, cell death and survival.Candidate genes have been identified and validation is ongoing. Interestingly, a gradual increase of the DNA methylation level of TRIM59 was observed from the healthy controls (31%) over ET (53%) to PV (64%) and MF (65%). ET patients could be distinguished from both healthy controls (P= 0.0004, Mann-Whitney test) and from the more progressed stages PV and MF (P=0.0132, Mann-Whitney test) based on the TRIM59 DNA methylation level. TRIM59 promoter methylation could, however, not discriminate between PV and MF (P=0.4721, Mann-Whitney test).Conclusion:Genome-wide DNA methylation profiling of sorted MF blood cells provided an exclusive insight into which pathways that are contributing to MF disease phenotype at a cell specific level. The MF CD34+ cells had the highest number of differential methylated CpG sites (n=1628) when comparing to granulocytes (n=519) and mononuclear cells (n=213) and should be cells of choice when exploring new treatment strategies. Interestingly, the mononuclear compartment show aberrant methylation of inflammatory genes supporting a role of aberrant immune regulation in the pathogenesis of MPN. Earlier studies have failed to identify aberrant methylation in early ET and PV, however, the preliminary data on the methylation of individual genes (TRIM59 promoter methylation) shows that it might be possible to identify early driver genes, and that it may be possible to select a panel of genes that can discriminate early MPN from the late MF stage. DisclosuresNo relevant conflicts of interest to declare.

Abstract B45: Combinatorial activation of PI3-kinase and Ras-Raf signaling in mouse models of advanced prostate cancer

Abstract Concomitant activation of the PI3K and MAP kinase pathways is a hallmark of aggressive prostate cancer. Here, we describe a series of new genetically engineered mouse models of prostate cancer in which PI3K-Akt-mTOR signaling is activated through inducible deletion of PTEN, while MAP kinase signaling is activated through inducible expression of oncogenic mutants of either BRAF or KRAS oncogenes [BRAFV600E (NPB mice) and KRASG12D (NPK mice), respectively]. Activation of PI3K-Akt-mTOR and MAPK signaling pathways in NPB mice results in the formation of lethal prostate tumors that are inherently resistant to castration. These tumors bypass cellular senescence and disseminate to lymph nodes, bone marrow, and lungs where they form overt metastases in approximately 30% of the cases. Molecularly, tumors in the NPB mice cooperate to up-regulate c-MYC oncogene and accordingly, therapeutic treatments with an mTOR inhibitor (rapamycin) and the MEK1/2 inhibitor (PD0325901) results in attenuation of c-MYC levels and reduction primary tumor and metastatic burden. These findings suggest a generalized therapeutic approach to target c-MYC activation in prostate cancer by combinatorial targeting of the PI3K-Akt-mTOR and ERK1/2 MAPK signaling pathways. On the other hand, activation of PI3K-Akt-mTOR and MAP kinase signaling pathways in the NPK mice, results in prostate tumors that are also inherently castration resistant and metastasize to distant organs with 100% penetrance. Our studies show that in the NPK mice, dissemination is an early event in tumorigenesis although overt metastatic growth only occurs in late stage tumors coinciding with the upregulation of the ETS family member ETV4 in the primary tumor. Functional studies have shown that silencing of Etv4 in a metastatic cell line derived from this mouse model (NPK mice) abrogates the metastatic phenotype but does not affect tumor growth. These findings indicate that ETV4 promotes metastasis in prostate tumors that have activation of PI3-kinase and Ras signaling, and therefore, ETV4 represents a potential target of therapeutic intervention for metastatic prostate cancer. Taken together, our analysis of the molecular mechanisms involved in the distinct metastatic potential of these two new mouse models provides us with clinically relevant tools to conduct preclinical studies. Citation Format: Jingqiang Wang, Alvaro Aytes, Cory Abate-Shen. Combinatorial activation of PI3-kinase and Ras-Raf signaling in mouse models of advanced prostate cancer. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr B45.

Increased centrosome amplification in aged stem cells of the Drosophila midgut

Age-related changes in long-lived tissue-resident stem cells may be tightly linked to aging and age-related diseases such as cancer. Centrosomes play key roles in cell proliferation, differentiation and migration. Supernumerary centrosomes are known to be an early event in tumorigenesis and senescence. However, the age-related changes of centrosome duplication in tissue-resident stem cells in vivo remain unknown. Here, using anti-γ-tubulin and anti-PH3, we analyzed mitotic intestinal stem cells with supernumerary centrosomes in the adult Drosophila midgut, which may be a versatile model system for stem cell biology. The results showed increased centrosome amplification in intestinal stem cells of aged and oxidatively stressed Drosophila midguts. Increased centrosome amplification was detected by overexpression of PVR, EGFR, and AKT in intestinal stem cells/enteroblasts, known to mimic age-related changes including hyperproliferation of intestinal stem cells and hyperplasia in the midgut. Our data show the first direct evidence for the age-related increase of centrosome amplification in intestinal stem cells and suggest that the Drosophila midgut is an excellent model for studying molecular mechanisms underlying centrosome amplification in aging adult stem cells in vivo.

Early inflammatory response in epithelial ovarian tumor cyst fluids.

Mortality rates for epithelial ovarian cancer (EOC) are high, mainly due to late-stage diagnosis. The identification of biomarkers for this cancer could contribute to earlier diagnosis and increased survival rates. Given that chronic inflammation plays a central role in cancer initiation and progression, we selected and tested 15 cancer-related cytokines and growth factors in 38 ovarian cyst fluid samples. We used ovarian cyst fluid since it is found in proximity to the pathology and mined it for inflammatory biomarkers suitable for early detection of EOC. Immunoprecipitation and high-throughput sample fractionation were obtained by using tandem antibody libraries bead and mass spectrometry. Two proteins, monocyte chemoattractant protein-1 (MCP-1/CCL2) and interleucin-8 (IL-8/CXCL8), were significantly (P < 0.0001) higher in the malignant (n = 16) versus benign (n = 22) tumor cysts. Validation of MCP-1, IL-8, and growth-regulated protein-α (GROα/CXCL1) was performed with ELISA in benign, borderline, and malignant cyst fluids (n = 256) and corresponding serum (n = 256). CA125 was measured in serum from all patients and used in the algorithms performed. MCP-1, IL-8, and GROα are proinflammatory cytokines and promoters of tumor growth. From 5- to 100-fold higher concentrations of MCP-1, IL-8 and GROα were detected in the cyst fluids compared to the serum. Significant (P < 0.001) cytokine response was already established in borderline cyst fluids and stage I EOC. In serum a significant (P < 0.01) increase of IL-8 and GROα was found, but not until stage I and stage III EOC, respectively. These findings confirm that early events in tumorigenesis can be analyzed and detected in the tumor environment and we conclude that ovarian cyst fluid is a promising source in the search for new biomarkers for early ovarian tumors.

Genetics of testicular germ cell tumors

Testicular cancer is by far the most common neoplasm among young males between the ages of 20 and 40years and with an increasing incidence rate worldwide. Congenital malformations of the male genitals, such as cryptorchidism or inguinal hernia are established risk factors. Men with a family history of testicular cancer are also associated with an increased risk of the disease. In the testes more than 90 % of tumors develop from germ cells (progenitor cells) and represent a histologically heterogeneous group. Germ cell tumors in extragonadal localizations are rare. Isochromosome i(12p), the typical marker chromosome in testicular germ cell tumors, occurs as an early event in tumorigenesis. Spermatocytic seminoma is a rare variant of germ cell tumors and according to the current classification is a distinct entity with different morphological, clinical and also cytogenetical features compared with other germ cell tumors.

MP23-07 REGIONAL GENETIC VARIABILITY DETECTED WITH RENAL TUMOR BIOPSIES: IMPLICATIONS IN BIOMARKER DEVELOPMENT

You have accessJournal of UrologyKidney Cancer: Basic Research I1 Apr 2014MP23-07 REGIONAL GENETIC VARIABILITY DETECTED WITH RENAL TUMOR BIOPSIES: IMPLICATIONS IN BIOMARKER DEVELOPMENT Alexander Sankin, Nina Mikkilineni, A. Ari Hakimi, Mikhail Silk, Yupu Liang, Roy Mano, Jeremy C. Durack, Jonathan Coleman, Paul Russo, and James Hsieh Alexander SankinAlexander Sankin More articles by this author , Nina MikkilineniNina Mikkilineni More articles by this author , A. Ari HakimiA. Ari Hakimi More articles by this author , Mikhail SilkMikhail Silk More articles by this author , Yupu LiangYupu Liang More articles by this author , Roy ManoRoy Mano More articles by this author , Jeremy C. DurackJeremy C. Durack More articles by this author , Jonathan ColemanJonathan Coleman More articles by this author , Paul RussoPaul Russo More articles by this author , and James HsiehJames Hsieh More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.875AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Intratumor heterogeneity can lead to an underestimation of the genetic landscape detected from single site biopsies in renal cell carcinoma (RCC). We sought to characterize the extent of clonal branching present in distinct regions of primary renal tumors. METHODS Ex vivo core needle biopsies were obtained from three to four geographically distinct regions of renal tumors that were resected via partial or radical nephrectomy at a single institution from 10/2012 to 6/2013. DNA was extracted and subjected to next-generation sequencing, focusing on the coding regions for nine genes with known associations to renal cell carcinoma (VHL, PBRM1, SETD2, BAP1, KDM5C, MTOR, PIK3CA, EGFR, PTEN). We then constructed phylogenetic trees for each tumor by inferring clonal evolution based on mutations present within each core. RESULTS We obtained 45 biopsy cores from 14 patients with clear cell histology RCC (median tumor size 4.5 cm, interquartile range 4.0 – 5.9 cm). A total of 9/14 tumors (64%) were ≥ grade 3 and 6/14 (43%) were ≥ stage 3. The mutation rates for each studied gene are represented in Figure 1. Four patients (29%) had no detectable mutations and three patients (21%) had a single mutation. Branching patterns of various complexities were observed in patients with two or more mutations (example: Figure 2). When VHL mutations were present, they were identical and occurred ubiquitously in all core samples, likely reflecting an early event in tumorigenesis. In contrast, when KDM5C mutations were present they were only detected in one or two cores, an example of regional mutational diversity. When SETD2, PBRM1, and BAP1 mutations were present, they were present in all cores 50%, 50%, and 25% of the time, respectively. CONCLUSIONS Ex vivo core needle biopsies reveal regional variations in gene mutations associated with clear cell RCC. Single site needle biopsy assessment of diagnostic or prognostic genetic mutations is unlikely to reflect the broader tumor landscape. This presents a challenge in the validation of biomarkers when utilizing a single core biopsy. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e244-e245 Peer Review Report Advertisement Copyright & Permissions© 2014MetricsAuthor Information Alexander Sankin More articles by this author Nina Mikkilineni More articles by this author A. Ari Hakimi More articles by this author Mikhail Silk More articles by this author Yupu Liang More articles by this author Roy Mano More articles by this author Jeremy C. Durack More articles by this author Jonathan Coleman More articles by this author Paul Russo More articles by this author James Hsieh More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

MP23-08 MICRORNA-29S INHIBITS CANCER CELL MIGRATION AND INVASION VIA TARGETING FOCAL ADHESION PATHWAYS IN RENAL CELL CARCINOMA

single site biopsies in renal cell carcinoma (RCC). We sought to characterize the extent of clonal branching present in distinct regions of primary renal tumors. METHODS: Ex vivo core needle biopsies were obtained from three to four geographically distinct regions of renal tumors that were resected via partial or radical nephrectomy at a single institution from 10/2012 to 6/2013. DNA was extracted and subjected to next-generation sequencing, focusing on the coding regions for nine genes with known associations to renal cell carcinoma (VHL, PBRM1, SETD2, BAP1, KDM5C, MTOR, PIK3CA, EGFR, PTEN). We then constructed phylogenetic trees for each tumor by inferring clonal evolution based on mutations present within each core. RESULTS: We obtained 45 biopsy cores from 14 patients with clear cell histology RCC (median tumor size 4.5 cm, interquartile range 4.0 e 5.9 cm). A total of 9/14 tumors (64%) were grade 3 and 6/14 (43%) were stage 3. The mutation rates for each studied gene are represented in Figure 1. Four patients (29%) had no detectable mutations and three patients (21%) had a single mutation. Branching patterns of various complexities were observed in patients with two or more mutations (example: Figure 2). When VHL mutations were present, they were identical and occurred ubiquitously in all core samples, likely reflecting an early event in tumorigenesis. In contrast, when KDM5C mutations were present they were only detected in one or two cores, an example of regional mutational diversity. When SETD2, PBRM1, and BAP1 mutations were present, they were present in all cores 50%, 50%, and 25% of the time, respectively. CONCLUSIONS: Ex vivo core needle biopsies reveal regional variations in gene mutations associated with clear cell RCC. Single site needle biopsy assessment of diagnostic or prognostic genetic mutations is unlikely to reflect the broader tumor landscape. This presents a challenge in the validation of biomarkers when utilizing a single core biopsy. Source of Funding: Sidney Kimmel Center for Prostate and Urologic Cancers

Comparative Time Course Profiles of Phthalate Stereoisomers in Mice

More efficient models are needed to assess potential carcinogenicity hazard of environmental chemicals based on early events in tumorigenesis. Here, we investigated time course profiles for key events in an established cancer mode of action. Using a case study approach, we evaluated two reference phthalates, di(2-ethylhexyl) phthalate (DEHP) and its stereoisomer di-n-octyl phthalate (DNOP), across the span of a two-year carcinogenicity bioassay. Male B6C3F1 mice received diets with no phthalate added (control), DEHP at 0.12, 0.60, or 1.20%, or DNOP at 0.10, 0.50, or 1.00% (n = 80-83/group) for up to 104 weeks with six interim evaluations starting at week 4. Mean phthalate doses were 139, 845, and 3147 mg/kg/day for DEHP and 113, 755, and 1281 mg/kg/day for DNOP groups, respectively. Incidence and number of hepatocellular tumors (adenoma and/or carcinoma) were greater at ≥ 60 weeks for all DEHP groups with time and dose trends, whereas DNOP had no significant effects. Key events supported a peroxisome proliferator-activated receptor alpha (PPARα) mode of action for DEHP, with secondary cytotoxicity at the high dose, whereas DNOP induced modest increases in PPARα activity without proliferative or cytotoxic effects. Threshold estimates for later tumorigenic effects were identified at week 4 for relative liver weight (+24%) and PPARα activity (+79%) relative to the control group. Benchmark doses (BMDs) for these measures at week 4 clearly distinguished DEHP and DNOP and showed strong concordance with values at later time points and tumorigenic BMDs. Other target sites included testis and kidney, which showed degenerative changes at higher doses of DEHP but not DNOP. Our results highlight marked differences in the chronic toxicity profiles of structurally similar phthalates and demonstrate quantitative relationships between early bioindicators and later tumor outcomes.

Hypermethylation of a New Distal Sodium/Iodide Symporter (NIS) Enhancer (NDE) Is Associated With ReducedNISExpression in Thyroid Tumors

In thyroid tumors, reduced radioiodine uptake is associated with worse patient outcome concomitantly with low sodium/iodide symporter (NIS) mRNA expression. Previous studies showed that CpG-island methylation in the NIS proximal promoter does not correlate with mRNA expression. The aim of the study was to identify new CpG-islands within the NIS 5'region and investigate the involvement of their methylation in NIS expression. DNA was obtained from 30 pairs of thyroid samples: tumor (T) and surrounding nontumor (NT) samples. All T samples had reduced NIS mRNA expression compared to NT samples. Methylation degree was quantified by bisulfite sequencing, and NIS expression by real-time PCR and Western blot. Reporter gene assays were performed to determine CpG-island functionality. Tumor cell cultures were treated with 5-Aza demethylating agent to determine NIS expression, methylation status, and (125)I uptake. We identified a new CpG-island2 with 14 CpG sites, located -2152/-1887 relative to ATG site. CpG-island2 was hypermethylated in T compared to NT samples, in both benign and malignant tumor groups. There was a significant inverse correlation between NIS mRNA expression and degree of CpG-island2 methylation in NT and T samples. This sequence increased the expression of a reporter gene; thus, it was considered a new enhancer. Cell culture treatments with 5-Aza reduced CpG-island2 methylation levels concomitantly with restoration of NIS mRNA and protein expression and (125)I uptake. We identified a new distal enhancer, NIS distal enhancer, that regulates gene expression through DNA methylation. This enhancer is hypermethylated in T compared to NT samples and is associated with decreased NIS expression in tumors. This epigenetic deregulation may be an early event in tumorigenesis.

MicroRNAs as regulators of tumor-associated stromal cells

The interplay between tumor and stromal cells represents an essential component of tumor progression and metastasis, and has become a focal point of current research efforts. Tumor-stromal interactions support early events in tumorigenesis, mediate the formation of pre-metastatic niches, and regulate the initiation and progression of distant metastases. Developing a better understanding of stromal regulation will be necessary for future therapeutic strategies, although progress has traditionally been hindered due to the complexity of the stromal microenvironment. Despite this, multiple studies have now examined the regulation of stromal microRNAs (miRNAs) during tumor progression, revealing the potential for miRNA-based therapeutic strategies to combat metastasis. We have recently described broad miRNA expression changes in osteoclasts, essential bone-resorbing stromal cells, during bone metastasis (Ell et al, Cancer Cell, 2013, 24;542-556). Osteoclastic activity drives lytic breast cancer bone metastasis, degrading bone and releasing bone-derived growth factors that enhance tumor growth. Thus, inhibiting osteoclast differentiation, through the blockage of osteoclast differentiation factors such as RANKL, or via direct inhibition with bisphosphonates, has proven to be a reliable method of limiting bone metastasis progression. Paramount within our study was the discovery that tumor-secreted factors could induce broad miRNA changes within osteoclasts, mimicking miRNA changes seen in physiological osteoclast differentiation. Mechanistically, osteoclast miRNA changes were induced through downstream signaling from soluble ICAM1 and other tumor-secreted factors. Importantly, specific ectopic expression of miR-141 or miR-219 was capable of inhibiting osteoclast differentiation and limiting bone metastasis in a mouse model of breast cancer metastasis. A similar study (Squadrito et al, Cell Reports, 2012, 1;141-154) discovered activity of miR-511-3p in tumor-associated macrophages (TAMs), well known mediators of tumor progression. The authors discovered that ectopic expression of miR-511-3p reduced the pro-tumoral activity of TAMs and inhibited the growth of Lewis lung carcinoma in mice. Interestingly, a comparable study has revealed a miRNA signature capable of recapitulating a cancer-associated fibroblast (CAF) phenotype in normal omental fibroblasts (Mitra et al, Cancer Discovery, 2012, 2;1100). Normal fibroblasts transfected with anti-miR-31, anti-miR-214, and miR-155 displayed enhanced migration, as well as elevated invasion and colony formation of co-cultured ovarian cancer cells, and increased tumor growth in vivo. Importantly, in an opposite set of experiments, elevating expression of miR-31 and miR-214 while inhibiting miR-155, abrogated the CAF phenotype. Together, these studies reveal the potential for utilizing miRNAs to target the stromal compartment and thereby limit tumor progression. An additional discovery within our study was the capability to utilize serum levels of miRNAs as biomarkers for bone metastasis progression in patients. It is currently difficult to predict which patients will develop bone metastasis, and standard biomarkers, such as N-terminal telopeptide, show only modest specificity as diagnostic markers. Interestingly, we found that two miRNAs, miR-16 and miR-378, were elevated in bone metastasis patients relative to healthy donors. It is not currently known exactly how miRNAs are secreted into the serum, although it has been hypothesized that miRNA trafficking could occur through the secretion of exosomes. While the miRNAs examined in our study did not show an overt effect on osteoclast differentiation or tumor cell proliferation, it is possible that secreted miRNAs can be laterally transferred between stromal and tumor cells, leading to enhanced metastatic potential. Indeed, TAM secreted miR-223 can enhance breast cancer invasiveness in vitro, and might indicate a novel mechanism of communication between macrophages and tumor cells (Yang et al, Molecular Cancer 2011, 10;117). These studies raise a number of interesting questions in regard to the therapeutic potential for miRNAs. Can miRNAs be useful as therapeutics, not only as direct inhibitors of metastatic cells, but also to disrupt tumor-stromal interactions? These preliminary studies show promising results, although rigorous follow-up studies will be necessary before advancing to clinical trials. Additionally, our preliminary results suggest a potential additive effect when miRNAs were combined with the bisphosphonate Zometa. It will be interesting to examine combinatorial treatments with miRNAs and current antiresorptive therapies, as well as the usefulness of miRNAs as inhibitors of other diseases associated with pathological osteoclast activity, including Paget's disease and osteoporosis. Beyond osteoclasts, can miRNAs be useful as inhibitors of other stromal cells, including TAMs, fibroblasts, and endothelial cells, and will these treatments be effective in limiting metastasis within their respective tumor models? Finally, a major concern within miRNA treatments involves off-target effects, and it will be essential to confirm specificity and low risk of side effects as development of these therapeutics progresses. In summary, these studies reveal the potential for utilizing miRNAs as therapeutic targets to limit the activity of tumor-associated stromal cells. In addition, secreted stromal miRNAs might prove useful as functional mediators of metastatic progression or clinical biomarkers. While a number of questions remain as to the utility, efficacy, and safety of miRNA-based therapeutic strategies, we hope that our efforts will facilitate the development of novel treatments for metastasis.

Abstract B117: Analysis of secondary mutations in BRCA1/2 genes as a mechanism of resistance to PM01183 in BRCA-mutation carriers.

Abstract Introduction: PM01183 is a modified analogue of trabectedine currently being evaluated in clinical trials, including germline BRCA mutation carriers. PM01183 binds to the DNA minor groove forming PM01183-DNA adducts that provoke DNA double strand (ds) breaks. The homologous recombination (HR) DNA repair pathway is the major mechanism involved in the repair of these DNA ds breaks, and BRCA mutated cells, which are HR deficient, show great sensitivity to these agents. In BRCA carriers, the somatic loss of the wild type copy of BRCA through loss-of-heterozygosity (LOH) is proposed to be an early event in tumorigenesis. Genomic characterization of somatic BRCA status can yield valuable information for therapeutic decisions and the development of new therapeutic strategies in BRCA carriers. Secondary mutations in BRCA1/2 genes restoring the gene and the HR function have been identified in up to 46% of cisplatin-resistant BRCA-associated ovarian tumors. Secondary mutations have also been identified in breast cancers with acquired resistance to olaparib. Based on the sensitivity of BRCA mutated cells to ds DNA-damaging, we hypothesized that BRCA genetic reversion could appear as one of the mechanism of resistance to PM01183. Methods: Patients with metastatic BRCA-associated breast cancer treated at our institution with PM01183 were enrolled in this study after signing the specifically designed, IRB-approved informed consent form. A blood sample was drawn prior to receiving treatment. Tumor biopsies were performed at baseline and at the time of progression to PM01183 from two patients exhibiting an initial clinical response. Whole exome sequencing (Illumina) was performed on DNA extracted from blood and from fresh-frozen paired tumor samples in order to analyze BRCA1/2 status, BRCA1/2 genetic reversion, and to detect novel genetic events acquired at disease progression. Findings: Sequencing of the BRCA genes in blood confirmed that one patient carried a germline mutation in BRCA1 (c5123C&amp;gt;A), with concomitant LOH found in both tumor samples. The second patient was confirmed a BRCA2-mutation carrier (9610 C&amp;gt;T), without LOH of BRCA2. Secondary mutations in BRCA1/2 genes were not identified in either tumor after progression to PM01183. Other de novo genetic events upon PM01183 progression are under review. Conclusions: Secondary functional-reverting mutations in BRCA1/2 genes have not been identified as a mechanism of resistance to PM01183 in any of the two patients analyzed. LOH of BRCA was not found in the metastatic disease of one patient, thus warranting further investigation of the mechanisms of tumorigenesis in BRCA tumors. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B117. Citation Format: Cristina Cruz, Ana Vivancos, Ginevra Caratu, Yasir Ibrahim, Violeta Serra, Judith Balmana. Analysis of secondary mutations in BRCA1/2 genes as a mechanism of resistance to PM01183 in BRCA-mutation carriers. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B117.

Sporadic colon cancer murine models demonstrate the value of autoantibody detection for preclinical cancer diagnosis

Although autoantibody detection has been proposed for diagnosis of colorectal cancer, little is known about their initial production and development correlation with cancer progression. Azoxymethane/dextran sodium sulfate (AOM/DSS)-treated mice developed colon adenocarcinoma in the distal colon similar to human sporadic colon cancer. We assessed this model together with AOM and DSS-only models for their applicability to early detection of cancer. All AOM/DSS-treated mice produced autoantibodies to tumor-associated antigens analogous to those observed in human colon cancer patients. Autoantibody response was related to tumor antigen overexpression. Cancer autoantibodies were detected 21 days after starting treatment, when no malignant histopathological features were detectable, and they increased according to tumor progression. When carcinogenesis was induced separately by AOM or DSS, only those mice that developed malignant lesions produced significant levels of autoantibodies. These findings demonstrate that autoantibody development is an early event in tumorigenesis and validates its use for preclinical colon cancer diagnosis.

Small gastrointestinal stromal tumor in the stomach: identification of precursor for clinical gastrointestinal stromal tumor using c-kit and α-smooth muscle actin expression

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the digestive tract. To find precursors for clinical GISTs of the stomach, small gastric stromal tumors of less than 3 cm were collected and examined immunohistochemically with analysis of the KIT mutation. Sixty-eight of 74 lesions were classified into 4 representative groups according to the expression of c-kit and α-smooth muscle actin (αSMA): group A, c-kit diffusely positive and αSMA negative (18 cases); group B, c-kit diffusely positive and αSMA focally positive (13); group C, c-kit focally positive and αSMA diffusely positive (27); and group D, c-kit negative and αSMA diffusely positive (10). Of the 4 groups, groups A and B of c-kit diffuse expression showed higher cellularity and labeling indices of p27(Kip1) and Ki-67 than did groups C and D of diffuse αSMA expression. Incidence of KIT exon 11 mutation in groups A and B was 86% (25/29), whereas that in groups C and D was 0% (0/20). Small gastric stromal tumors with c-kit diffuse expression were considered precursors for clinical GIST because they were significantly different from c-kit focally positive or negative tumors. The mutation of KIT is considered as an early event in tumorigenesis of GIST.