Early Event In Tumorigenesis Research Articles

Abstract LB-355: Whole-exome sequencing of intracranial germ cell tumors reveals frequent mutations in KIT and RAS pathways and histone demethylases.

Abstract Intracranial germ cell tumors (IGCTs) are a group of rare pediatric brain tumors affecting mainly male adolescents and young adults. The incidence of IGCTs varies significantly according to geographic locations with the highest incidence in Japan and other East Asian countries. Taken together, these characteristics suggest predisposing germline factors. IGCTs can be divided into two main groups, pure germinoma and nongerminomatous germ cell tumor (NGGCT). The former in general responds well to chemoradiotherapy whereas the latter has a rather poor prognosis. Currently the only known gene associated with this disease is KIT, which is mutated in ~25% of IGCTs. Due to the scarcity of tissues available for research, the molecular biology of IGCTs remains largely unknown. Thus, deeper understanding of the pathogenesis of these unusual tumors should lead to new insights into the underlying cancer biology and the development of novel therapeutics for this rare and deadly disease. Using whole-exome sequencing and SNP array, we have analyzed 28 IGCT tumors (12 pure germinomas, 12 NGGCTs and 4 mixed GCTs) collected from Japan (21), Hong Kong (4) and United States (3). Somatic mutations of KIT were detected in 29% of cases, primarily in germinomas. In an additional 38% of the cases, deleterious somatic mutations were identified in important downstream mediators of KIT signal transduction including KRAS, NRAS, MTOR and their respective regulators, NF1 and PTEN. Mutations in KIT were mutually exclusive with mutations in RAS. Furthermore, 21% of patients showed inactivation of tumor suppressor genes (TSGs) including BCORL1, PTEN, NF1 and SMAD3. Inactivation of TSGs coexisted in tumors with activation of KIT/RAS oncogenes, suggesting these mutations act cooperatively in tumorigenesis. Copy-number analysis revealed almost exclusively arm-level gains and losses. Over 90% of the germinomas exhibited chromosomal imbalance and uniparental disomy of multiple chromosomes, suggesting aberrant meiotic division may possibly play a role in the pathogenesis of this disease. Patterns of chromosome imbalance within a given tumor revealed subclonal structure in 58% of the analyzed cases. Finally, we found in this cohort a significant enrichment (39%) of novel or rare germline variants in histone demethylases, compared with the control cohorts, suggesting epigenetic deregulation may be an important early event in tumorigenesis. One histone demethylase harbored a risk allele present at significantly higher frequency in our male cases compared to a Japanese control population, a finding that could explain both the population prevalence and sex differences in this disease. This study has important implications for our understanding of the pathogenesis of IGCTs, and suggests potential targets for the development of therapeutics focused on inhibitors of KIT/RAS activation. Citation Format: Linghua Wang, Shigeru Yamaguchi, Keita Terashima, Matthew D. Burstein, Jiayi M. Sun, Tomonari Suzuki, Ryo Nishikawa, Hideo Nakamura, Atsushi Natsume, Shunsuke Terasaka, Ho-Keung Ng, Robert C. Dauser, William E. Whitehead, Adesina M. Adekunle, Donna M. Muzny, Richard A. Gibbs, Ching C. Lau, David A. Wheeler. Whole-exome sequencing of intracranial germ cell tumors reveals frequent mutations in KIT and RAS pathways and histone demethylases. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-355. doi:10.1158/1538-7445.AM2013-LB-355

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Distinct ERG rearrangement prevalence in prostate cancer: higher frequency in young age and in low PSA prostate cancer

Background:The TMPRSS2-ERG gene fusion resulting in ERG overexpression has been found in around 50% of prostate cancers (PCa) and is a very early event in tumorigenesis. Most studies have reported on selected surgical cohorts with inconsistent results. We hypothesized that ERG gene rearrangements impact tumor development and investigated the frequency of ERG overexpression in the context of clinicopathological tumor characteristics.Methods:ERG overexpression (ERG+ or ERG-) was determined by immunohistochemistry (IHC) in 1039 radical prostatectomy (RP) tumors and association with PSA, D'Amico risk score, histopathology, biochemical recurrence, body mass index and age of PCa cases was analyzed.Results:ERG+ was associated with younger age at diagnosis (P<0.0001), lower serum PSA (P=0.002) and lower prostate volume (PV) (P=0.001). It was most frequent in the youngest age quartile (⩽55 years, 63.9% ERG+) and decreased constantly with increasing age to 40.8% in the oldest age quartile (⩾67 years, P<0.0001). In the PSA range <4 ng ml−1 the frequency of ERG positivity was 60.2% compared with 47.5 and 49.1% in the PSA ranges 4–10 and ⩾10 ng ml−1, respectively. In the first age quartile, ERG+ patients had lower median serum PSA and fPSA% and smaller PV. In the highest age quartile tumor volume (TV) was increased. Similar differences were observed in the low PSA range. Multivariate analysis identified the first age quartile as a predictor for ERG status (odds ratios (OR) 2.05, P=0.007). No association was found with the D'Amico progression risk score and with biochemical tumor recurrence.Conclusions:ERG+ tumors manifest clinically at lower PSA levels and their prevalence is age dependent. This suggests acceleration of tumor development by ERG overexpression that results in earlier tumor detection in young patients. Long-term results are warranted to determine the impact of ERG overexpression on disease outcome.

Differential mucin phenotypes and their significance in a variation of colorectal carcinoma

To investigate mucin expression profiles in colorectal carcinoma (CRC) histological subtypes with regard to clinicopathologic variables and prognosis. Mucin (MUC)2 and MUC5AC expressions were assessed by immunohistochemistry for a total of 250 CRC cases that underwent surgical resection. CRCs included 63 well-to-moderately differentiated adenocarcinomas (WMDAs), 91 poorly differentiated adenocarcinomas (PDAs), 81 mucinous adenocarcinoma (MUAs), and 15 signet-ring cell carcinomas (SRCCs). MUC2 and MUC5AC were scored as positive when ≥ 25% and ≥ 1% of cancer cells were stained positive, respectively. The human mutL homolog 1 and human mutS homolog 2 expressions were assessed by immunohistochemistry in PDAs to investigate mismatch-repair (MMR) status. Tumors that did not express either of these two were considered MMR-deficient. Results were analyzed for associations with clinicopathologic variables and the prognosis in individual histological CRC subtypes. MUC2-positive and MUC5AC-positive WMDA percentages were 49.2% and 30.2%, respectively. In contrast, MUC2-positive and MUC5AC-positive PDA percentages were 9.5% and 51.6%, respectively. MUC2 levels tended to decrease and MUC5AC levels tended to increase from WMDA to PDA. In 21 tumors comprising both adenoma and adenocarcinoma components in a single tumor (4 WMDAs, 7 PDAs, and 10 MUAs), MUC2 was significantly downregulated in PDA and MUC5AC was downregulated in PDA and MUA in the adenoma-carcinoma sequence. These results suggested that MUC2 levels might be associated with malignant potential and that MUC5AC expression was an early event in tumorigenesis. Despite worse prognoses than WMDA, high MUC2 expression levels were maintained in MUA (95.1%) and SRCC (71.5%), which suggested a pathogenesis for these subtypes distinct from that of WMDA. No significant associations were found between MUC2 expression and any clinicopathologic variables in any histological subtype. MUC5AC expression in PDA was closely associated with right-sided location (P = 0.017), absence of nodal metastasis (P = 0.010), low tumor node metastasis stage (P = 0.010), and MMR deficiency (P = 0.003). MUC2 expression in WMDA was a marginal prognostic factor for recurrence/metastasis-free survival (RFS) by univariate Cox analysis (P = 0.077) but not by multivariate Cox analysis (P = 0.161). MUC5AC expression in PDA was a significant prognostic factor for RFS by univariate Cox analysis (P = 0.007) but not by multivariate Cox analysis (P = 0.104). Kaplan-Meier curves and log-rank tests revealed that MUC2 expression was marginally associated with a better WMDA prognosis [P = 0.064 for RFS and P = 0.172 for overall survival (OS)] but not for PDA. In contrast, MUC5AC expression was significantly and marginally associated with a better PDA prognosis in terms of RFS and OS, respectively (P = 0.004 for RFS and P = 0.100 for OS), but not for WMDA and MUA. Mucin core protein expression profiles and clinical significance differ according to histological CRC subtypes. This may reflect different pathogeneses for these tumors.

CD36 Repression Activates a Multicellular Stromal Program Shared by High Mammographic Density and Tumor Tissues

Although high mammographic density is considered one of the strongest risk factors for invasive breast cancer, the genes involved in modulating this clinical feature are unknown. Tissues of high mammographic density share key histologic features with stromal components within malignant lesions of tumor tissues, specifically low adipocyte and high extracellular matrix (ECM) content. We show that CD36, a transmembrane receptor that coordinately modulates multiple protumorigenic phenotypes, including adipocyte differentiation, angiogenesis, cell-ECM interactions, and immune signaling, is greatly repressed in multiple cell types of disease-free stroma associated with high mammographic density and tumor stroma. Using both in vitro and in vivo assays, we show that CD36 repression is necessary and sufficient to recapitulate the above-mentioned phenotypes observed in high mammographic density and tumor tissues. Consistent with a functional role for this coordinated program in tumorigenesis, we observe that clinical outcomes are strongly associated with CD36 expression. CD36 simultaneously controls adipocyte content and matrix accumulation and is coordinately repressed in multiple cell types within tumor and high mammographic density stroma, suggesting that activation of this stromal program is an early event in tumorigenesis. Levels of CD36 and extent of mammographic density are both modifiable factors that provide potential for intervention.

EGFRvIII gene rearrangement is an early event in glioblastoma tumorigenesis and expression defines a hierarchy modulated by epigenetic mechanisms

Amplification and rearrangements of the epidermal growth factor receptor (EGFR) gene are frequently found in glioblastoma multiforme (GBM). The most common variant is EGFR variant III (EGFRvIII). Research suggests that EGFRvIII could be a marker for a cancer stem cell or tumor-initiating population. If amplification and rearrangement are early events in tumorigenesis, this implies that they should be preserved throughout the tumor. However, in primary GBM, EGFRvIII expression is focal and sporadic. Unexpectedly, we found EGFR amplification and rearrangement throughout the tumor, including regions with no EGFRvIII expression, suggesting that mechanisms exist to modulate EGFRvIII expression even in the presence of high gene amplification. To study this phenomenon, we characterized three GBM cell lines with endogenous EGFRvIII. EGFRvIII expression was heterogeneous, with both positive and negative populations maintaining the genetic alterations, akin to primary tumors. Furthermore, EGFRvIII defined a hierarchy where EGFRvIII-positive cells gave rise to additional positive and negative cells. Only cells that had recently lost EGFRvIII expression could re-express EGFRvIII, providing an important buffer for maintaining EGFRvIII-positive cell numbers. Epigenetic mechanisms had a role in maintaining heterogeneous EGFRvIII expression. Demethylation induced a 20-60% increase in the percentage of EGFRvIII-positive cells, indicating that some cells could re-express EGFRvIII. Surprisingly, inhibition of histone deacetylation resulted in a 50-80% reduction in EGFRvIII expression. Collectively, this data demonstrates that EGFR amplification and rearrangement are early events in tumorigenesis and EGFRvIII follows a model of hierarchical expression. Furthermore, EGFRvIII expression is restricted by epigenetic mechanisms, suggesting that drugs that modulate the epigenome might be used successfully in glioblastoma tumors.

Transcriptional Regulation of the Protocadherin β Cluster during Her-2 Protein-induced Mammary Tumorigenesis Results from Altered N-Glycan Branching

Changes in the levels of N-acetylglucosaminyltransferase V (GnT-V) can alter the function of several types of cell surface receptors and adhesion molecules by causing altered N-linked glycan branching. Using a her-2 mammary tumor mouse model, her-2 receptor signaling was down-regulated by GnT-V knock-out, resulting in a significant delay in the onset of her-2-induced mammary tumors. To identify the genes that contributed to this GnT-V regulation of early events in tumorigenesis, microarray analysis was performed using her-2 induced mammary tumors from wild-type and GnT-V-null mice. We found that 142 genes were aberrantly expressed (>2.0-fold) with 64 genes up-regulated and 78 genes down-regulated after deletion of GnT-V. Among differentially expressed genes, the expression of a subgroup of the cadherin superfamily, the protocadherin β (Pcdhβ) cluster, was up-regulated in GnT-V-null tumors. Altered expression of the Pcdhβ cluster in GnT-V-null tumors was not due to changes in promoter methylation; instead, impaired her-2-mediated signaling pathways were implicated at least in part resulting from reduced microRNA-21 expression. Overexpression of Pcdhβ genes inhibited tumor cell growth, decreased the proportion of tumor-initiating cells, and decreased tumor formation in vivo, demonstrating that expression of the Pcdhβ gene cluster can serve as an inhibitor of the transformed phenotype. Our results suggest the up-regulation of the Pcdhβ gene cluster as a mechanism for reduced her-2-mediated tumorigenesis resulting from GnT-V deletion.

ESE3/EHF Controls Epithelial Cell Differentiation and Its Loss Leads to Prostate Tumors with Mesenchymal and Stem-like Features

Cancer stem cells (CSC) play a significant role in tumor progression, disease recurrence, and treatment failure. Here, we show that the endogenously expressed ETS transcription factor ESE3/EHF controls prostate epithelial cell differentiation and stem-like potential. We found that loss of ESE3/EHF induced epithelial-to-mesenchymal transition (EMT), stem-like features, and tumor-initiating and metastatic properties in prostate epithelial cells, and reexpression of ESE3/EHF inhibited the stem-like properties and tumorigenic potential of prostate cancer cells. Mechanistically, ESE3/EHF repressed the expression of key EMT and CSC genes, including TWIST1, ZEB2, BMI1, and POU5F1. Analysis of human tissue microarrays showed that reduced ESE3/EHF expression is an early event in tumorigenesis, frequently occurring independently of other ETS gene alterations. Additional analyses linked loss of ESE3/EHF expression to a distinct group of prostate tumors with distinctive molecular and biologic characteristics, including increased expression of EMT and CSC genes. Low ESE3/EHF expression was also associated with increased biochemical recurrence of prostate cancer and reduced overall survival after prostatectomy. Collectively, our findings define a key role for ESE3/EHF in the development of a subset of prostate tumors and highlight the clinical importance of identifying molecularly defined tumor subgroups.

Abstract 10: Oncogenic variant EGFRvIII defines a hierarchy in glioblastoma and expression is restricted by epigenetic mechanisms despite the presence of gene amplification

Abstract Glioblastoma multiforme (GBM) is the most common and deadly primary brain tumor. Amplification and rearrangements of the epidermal growth factor receptor (EGFR) gene are frequently found in GBM. The most common variant is EGFRvIII, an exon 2-7 deletion mutant resulting from amplification and rearrangement of the EGFR locus. We have hypothesized that EGFRvIII could mark a cancer stem cell or tumor initiating cell population. If amplification and rearrangement are early events in tumorigenesis, this implies that they should be preserved throughout the tumor. However, in primary GBM EGFRvIII expression was focal and sporadic. To understand the relationship between EGFRvIII expression and the underlying genomic alterations, we utilized manual dissection to separate EGFRvIII positive and negative cells and a quantitative PCR assay that detects independently both EGFR amplification and rearrangement. Unexpectedly, we found EGFR amplification and rearrangement throughout the tumor, including regions with no EGFRvIII expression. This supports our hypothesis and further suggests that mechanisms exist to modulate EGFRvIII expression even in the presence of high gene amplification. To study this phenomenon, we characterized three GBM cell lines with endogenous EGFRvIII expression and corresponding EGFR amplification and rearrangement, confirmed by Western blot, RT-PCR and FISH. By flow cytometry analysis EGFRvIII expression was heterogeneous, with 9-50% EGFRvIII positive cells in each cell line. Both positive and negative populations maintained the genetic alterations, recapitulating what we observed in primary GBM. Importantly, EGFRvIII defined a hierarchy where EGFRvIII-positive cells gave rise to additional positive and negative cells. However, only cells that had recently lost EGFRvIII expression could re-express EGFRvIII. Epigenetic mechanisms played a role in modulating EGFRvIII expression to maintain a heterogeneous population. Demethylation induced a 20-60% increase in the percentage of EGFRvIII-positive cells, indicating that some cells could re-express EGFRvIII. Surprisingly, inhibition of histone deacetylation resulted in a 50-80% reduction in EGFRvIII expression. Collectively, this data demonstrates that EGFRvIII does follow a stem cell model for hierarchical expression and sheds light on the existence of a transient population that is able to re-express EGFRvIII, an important buffer for maintaining EGFRvIII-positive cell numbers. Furthermore, we provide the first evidence that EGFRvIII expression can be silenced by epigenetic mechanisms, suggesting that drugs which modulate the epigenome might be used successfully in glioblastoma tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 10. doi:1538-7445.AM2012-10

Abstract LB-254: PRMT5 is upregulated in malignant and metastatic melanoma, and regulates expression of the MITF transcription factor

Abstract Protein arginine methyltransferase 5 (PRMT5) is an enzyme which catalyzes the covalent attachment of methyl groups to arginine residues of various proteins. PRMT5 binds and/or methylates both nuclear (e.g. histones) and cytoplasmic (e.g. p53, CRAF) proteins to regulate cellular functions including gene expression, cell cycle, and apoptosis among others. PRMT5 has been characterized in leukemia, lymphoma, glioma, and breast cancer; however little is known regarding its role in malignant melanoma. We hypothesized that PRMT5 plays a unique role in regulating melanoma cell biology. PRMT5 expression was measured by IHC analysis of formalin-fixed samples from patients with melanoma (n=133 primary; n=66 metastases), benign nevi (n=24), and normal adjacent epidermis (n=21). PRMT5 expression was significantly elevated in melanoma samples compared to normal adjacent epidermis (p&amp;lt;0.001, Fisher's exact test). The nuclear and cytoplasmic localization of PRMT5 was heterogeneous within individual melanoma specimens. PRMT5 was significantly higher in the nucleus, but not the cytoplasm, of malignant tissue compared to metastatic tissue (p=0.0004 vs p=0.6, Wilcoxon rank sum). PRMT5 protein was also significantly elevated in benign melanocytic nevi (compound, junctional, and intradermal) compared to normal adjacent epidermis (p&amp;lt;0.0001), suggesting that elevated PRMT5 may represent an early event in tumorigenesis. Analysis of human melanoma cell lines revealed that PRMT5 expression was predominantly cytoplasmic and its expression did not differ based on B-Raf or N-Ras mutational status. Co-immunoprecipitation experiments revealed PRMT5 associated with its enzymatic co-factor MEP-50, but not cyclin D1 or STAT3, in both bulk cultured or synchronized cells, as has been reported in other cell types. Depletion of PRMT5 with siRNA led to decreased transcript and protein level of MITF-M (microphthalmia-associated transcription factor) in human melanoma cell lines with V600E-mutated B-Raf (A375, Hs294T, 1106 MEL, FO1), or wild-type B-Raf (CHL-1, Wm1366). Furthermore, depletion of PRMT5 led to a significant growth inhibition in 1106 MEL, Wm1366, and FO1 cells at 48 and 72 hours post-transfection. These data uncover important differences between the biology of PRMT5 in melanoma cells as compared to other tumor types, and are the first to demonstrate that PRMT5 is a regulator of MITF expression, a key transcription factor regulating melanoma cell biology and cell proliferation. The upregulation of PRMT5 in human melanoma specimens and cell lines further suggests that PRMT5 inhibition deserves exploration as a novel therapeutic strategy for melanoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-254. doi:1538-7445.AM2012-LB-254

Copy Number Aberrations in Benign Serous Ovarian Tumors: A Case for Reclassification?

Serous ovarian carcinomas are the predominant epithelial ovarian cancer subtype and it has been widely believed that some or all of these may arise from precursors derived from the ovarian surface epithelium or fimbriae, although direct molecular evidence for this is limited. This study aimed to conduct copy number (CN) analysis using a series of benign and borderline serous ovarian tumors to identify underlying genomic changes that may be indicative of early events in tumorigenesis. High resolution CN analysis was conducted on DNA from the epithelial and fibroblast components of a cohort of benign (N = 39) and borderline (N = 24) serous tumors using the Affymetrix OncoScan assay and SNP6.0 arrays. CN aberrations were detected in the epithelium of only 2.9% (1 of 35) of serous cystadenomas and cystadenofibromas. In contrast, CN aberrations were detected in the epithelium of 67% (16 of 24) of the serous borderline tumors (SBT). Unexpectedly, CN aberrations were detected in the fibroblasts of 33% (13 of 39) of the benign serous tumors and in 15% (3 of 20) of the SBTs. Of the 16 cases with CN aberrations in the fibroblasts, 12 of these carried a gain of chromosome 12. Chromosome 12 trisomy has been previously identified in pure fibromas, supporting the concept that a significant proportion of benign serous tumors are in fact primary fibromas with an associated cystic mass. This is the first high resolution genomic analysis of benign serous ovarian tumors and has shown not only that the majority of benign serous tumors have no genetic evidence of epithelial neoplasia but that a significant proportion may be more accurately classified as primary fibromas.

Toward an Integrative Analysis of the Tumor Microenvironment in Ovarian Epithelial Carcinoma

Ovarian epithelial carcinomas are heterogeneous malignancies exhibiting great diversity in histological phenotypes as well as genetic and epigenetic aberrations. A general early event in tumorigenesis is regional dissemination into the peritoneal cavity. Initial spread to the peritoneum is made possible by cooperative signaling between a wide array of molecules constituting the tissue microenvironment in the coelomic epithelium. Changes in the activity of key microenvironmental components not constitutively expressed in normal tissue, including several disclosed adhesion molecules, growth factors, proteases, and G-protein coupled receptors (GPCRs), coordinate the transition. Remodeling of the extracellular matrix (ECM) and subsequent cell surface interactions enable transformation by promoting chromosomal instability (CIN) and stimulating several common signal transduction cascades to prepare the tissue for harboring and facilitating growth, angiogenesis and metastasis of the developing tumor.

Another road leads to HIF-1 activation: implications for prostate cancer progression

Another road leads to HIF-1 activation: implications for prostate cancer progression

Circulating Angiogenic Cytokines Are Elevated in Patients with Smoldering Myeloma; Implications Into Disease Biology

Abstract 5059Angiogenesis is a critical step in the evolution of carcinogenesis in solid tumors and hematologic malignancies and is considered to be an early event in tumorigenesis. Multiple myeloma (MM) is a hematologic malignancy in which a preceding monoclonal gammopathy (MGUS) is considered a precursor. Asymptomatic/smoldering MM (SMM) is associated with a substantial risk of progression to MM and according to current recommendations these patients should be followed without therapy. Thus evolution from MGUS to SMM and to symptomatic myeloma is now considered as the model describing the natural history of the disease. In order to study the role and evolution of circulating angiogenesis related cytokines, we studied their levels in patients with MGUS, asymptomatic MM and symptomatic MM, before the initiation of first line therapy. We also studied possible associations of these cytokines with features of the disease in patients with SMM that could help identify possible markers of early evolution.We measured serum levels of vascular endothelial growth factor (VEGF), angiogenin, angiopoietin (angp)-1 and -2, using standard ELISA methodology (R&D Systems, Minneapolis, MN, USA). The definition of MGUS, SMM and symptomatic MM was based on the published IMWG criteria. All above cytokines were also measured in 21 individuals with MGUS, in 174 newly diagnosed, untreated MM patients (31 with SMM) and in 44 age- and gender-matched healthy controls.We focused our analysis on patients with SMM. The median age was 63.5 years (range 40–83 years) and 55% were males. The median bone marrow infiltration in trephine biopsy was 20% (range: 12%-75%). Sixty-one per cent had IgG, 29% had IgA isotype while 3% had light chain only myeloma and 6% had biclonal myeloma.The median (range) serum levels for VEGF were 406.5 pg/ml (186.3–797.6 pg/ml), for angp-1 were 31064 pg/ml (18220–50856 pg/ml), for angp-2 were 1434 pg/ml (486.1–4004.5 pg/ml), for angp-1 to angp-2 ratio were 20.8 (6.5–78.1), for angiogenin were 262732.6 pg/ml (138670–1003040 pg/ml) and for bFGF were 12.082 pg/ml (non-detected to 123.37 pg/ml). There were no significant correlations of the levels of angiogenesis related cytokines with serum beta-2 microglobulin levels, the levels of the monoclonal protein, IgA versus IgG isotype, serum LDH levels or age. Patients with extensive bone marrow infiltration (≥60%) had significantly higher levels of ang-2 (p=0.017) and significantly lower angp-1/angp-2 ratio (p=0.004) compared to all others. Compared to healthy controls, patients with SMM had higher levels of angp-1 (p=0.05) and angp-2 (p=0.03) but their respective ratio was not significantly different (p=0.272). Serum levels of VEGF were significantly higher in SMM patients than in controls (mean 429 pg/ml vs. 196 pg/ml, p<0.001). Similarly serum levels of angiogenin were significantly higher in SMM (mean 304028 pg/ml vs 190245 pg/ml, p<0.001). When patients with SMM compared to MGUS patients, there were no significant differences for any of the studied angiogenesis related cytokines. Compared to patients with symptomatic MM, patients with SMM had higher levels of angp-1 (p<0.001) and lower level of angp-2 (p<0.001) resulting in a significantly lower angp-1/angp-2 ratio, indicating a switch to increased vessel-formation activity, while the levels of VEGF were similar.The above results indicate that early in the evolution of the disease (MGUS to SMM to Symptomatic MM) there is an angiogenic switch which is manifested by an increase in the levels of angiogenic cytokines that promote neovasculogenesis (such as VEGF, angp-2 and angiogenin) and a gradual suppression of cytokines that balance their effects (such as angp-1). A possible prognostic significance of the circulating levels of angiogenic cytokines in patients with SMM that could help identify patients at higher risk for progression to symptomatic MM needs further study. Disclosures:No relevant conflicts of interest to declare.

FOXL2 mutation and large-scale genomic imbalances in adult granulosa cell tumors of the ovary

Adult granulosa cell tumors (AGCTs) are a rare class of ovarian tumors with recurrent cytogenetic abnormalities including trisomy 12, trisomy 14, monosomy 16/deletion 16q, and monosomy 22. Over 90% contain a missense point mutation (C134W) in the FOXL2 gene at 3q22.3. The relationship between FOXL2 mutation and cytogenetic abnormalities is unclear, although both are presumably early events in tumorigenesis. In addition, FOXL2 C134W mutant allele imbalance has been noted in a minority of AGCTs, but the mechanism for allelic imbalance has not yet been described. We used a microarray platform designed for formalin-fixed, paraffin-embedded (FFPE) tissue specimens, the Affymetrix OncoScan FFPE Express 330K Molecular Inversion Probe (MIP) array, to explore the correlation between genomic imbalances detected by microarray and FOXL2 mutation status detected by pyrosequencing in a series of 21 archived AGCTs. Tumors were characterized by histopathologic features, stage, and alpha-inhibin expression by immunohistochemistry. All tumors were positive for inhibin, and 18/21 tumors contained a FOXL2 mutation. The most common genomic imbalances were a gain of 14q, a loss of 16q, and a loss of 22q. Three tumors showed evidence of FOXL2 mutant allele imbalance by pyrosequencing; microarray revealed a 32.5 Mb deletion encompassing FOXL2 in 1 case and a 70.9 Mb stretch of homozygosity encompassing FOXL2 in the other case. The third case, with a FOXL2 mutant allele imbalance, showed a diminished mutant allele population (32%) despite high estimated tumor content (>90%), suggesting tumor heterogeneity for the mutation. This study provides the first correlation of FOXL2 mutation status and genomic imbalances in AGCTs, and it further elucidates the mechanisms for mutant allele imbalance in cancer.

Heparan sulfate D-glucosaminyl 3-O-sulfotransferase-3B1, a novel epithelial-mesenchymal transition inducer in pancreatic cancer

Epithelial-mesenchymal transition (EMT) is a critical early event in tumorigenesis. The contribution of heparan sulfate (HS) to EMT has not been fully elucidated. HS D-glucosaminyl 3-O-sulfotransferase-3B1 (3-OST-3B1) participates in the final step of HS fine structure biosynthesis, whose involvement in cancer has yet to be determined. This study demonstrated that following treatment with trichostatin-A, a histone deacetylase inhibitor, 3-OST-3B1 gene expression was activated in the pancreatic cancer cell line, PANC-1. By chromatin immunoprecipitation analysis, permissive histone modifications including an increase in histone H3 lysine 9 monoactylation (H3 ac K9) but a decrease in methylated histone H3 (H3 me K9) were observed accompanying transcriptional activation of 3-OST-3B1. Functional, results revealed that increased 3-OST-3B1 levels were involved in the promotion of EMT processes. In vitro studies demonstrated that overexpression of 3-OST-3B1 in both pancreatic cancer cells and vascular endothelial cells could trigger an EMT-like phenotype as evidenced by the up-regulation of Snail at the mRNA and protein level, and its nuclear translocation. And 3-OST-3B1 appeared to be sufficient for the development of a more mesenchymal phenotype in vivo. Together, the results from this study unveiled a distinct function for 3-OST-3B1 as an EMT inducer in cancer and provided a link between histone modification and EMT modulation.

DNA hypermethylation in lung cancer is targeted at differentiation-associated genes

Aberrant DNA hypermethylation of tumor suppressor genes is thought to be an early event in tumorigenesis. Many studies have reported the methylation status of individual genes with known involvement in cancer, but an unbiased assessment of the biological function of the collective of hypermethylated genes has not been conducted so far. Based on the observation that a variety of human cancers recapitulate developmental gene expression patterns (that is activate genes normally expressed in early development and suppress late developmental genes), we hypothesized that the silencing of differentiation-associated genes in cancer could be attributed in part to DNA hypermethylation. To this end, we investigated the developmental expression patterns of genes with hypermethylated CpG islands in primary human lung carcinomas and lung cancer cell lines. We found that DNA hypermethylation primarily affects genes that are expressed in late stages of murine lung development. Gene ontology characterization of these genes shows that they are almost exclusively involved in morphogenetic differentiation processes. Our results indicate that DNA hypermethylation in cancer functions as a selective silencing mechanism of genes that are required for the maintenance of a differentiated state. The process of cellular de-differentiation that is evident on both the microscopic and transcriptional level in cancer might at least partly be mediated by these epigenetic events. Our observations provide a mechanistic explanation for induction of differentiation upon treatment with DNA methyltransferase inhibitors.

Expression of BDNF, TrkB, and p53 in early-stage squamous cell carcinoma of the uterine cervix

TrkB and BDNF play a critical role in cancer invasion and metastasis. We investigated the potential role of these proteins, together with p53, in squamous cell carcinoma (SCC) of the uterine cervix by focusing on the clinicopathological correlation and outcome. We performed RT-PCR analysis of TrkB and BDNF mRNA expression in cervical cancer cell lines, as well as tumour and normal tissue of the cervix. We analysed TrkB, BDNF, and p53 protein expression using immunohistochemistry in 80 patients with cervical SCC. The expression of BDNF and TrkB mRNA was higher in human cervical cancer cell lines and SCC tissues than in normal tissues. Positive TrkB expression was significantly correlated with low FIGO stage (p = 0.032), low T stage (p = 0.027), less than 1/2 stromal invasion of the cervix (p = 0.018), 4 cm or smaller for the greatest dimension of the tumour (p = 0.015) and absent lymphatic invasion (p = 0.032). Positive BDNF expression was significantly related to low FIGO stage (p = 0.001), less than 1/2 stromal invasion of the cervix (p = 0.040), absent lymphatic invasion (p = 0.026), and absent lymph node metastasis (p = 0.003). In contrast, p53 expression was significantly related to advanced T stage (p = 0.001), greater than 4 cm for the greatest dimension of the tumour (p = 0.002), more than 1/2 stromal invasion of the cervix (p = 0.005), and poor survival (p = 0.0218). TrkB and BDNF expression might play a significant role in the early events in tumorigenesis of cervical SCC. However, p53 is involved late in the process of tumour progression and therefore is predictive of a poor prognosis in SCC.

MicroRNAs in body fluids—the mix of hormones and biomarkers

Since the discovery of microRNAs (miRNAs), the study of these small noncoding RNAs has steadily increased and more than 10,000 papers have already been published. The great interest in miRNAs reflects their central role in gene-expression regulation and the implication of miRNA-specific aberrant expression in the pathogenesis of cancer, cardiac, immune-related and other diseases. Another avenue of current research is the study of circulating miRNAs in serum, plasma, and other body fluids--miRNAs may act not only within cells, but also at other sites within the body. The presence of miRNAs in body fluids may represent a gold mine of noninvasive biomarkers in cancer. Since deregulated miRNA expression is an early event in tumorigenesis, measuring circulating miRNA levels may also be useful for early cancer detection, which can contribute greatly to the success of treatment. In this Review, we discuss the role of fluid-expressed miRNAs as reliable cancer biomarkers and treatment-response predictors as well as potential new patient selection criteria for clinical trials. In addition, we explore the concept that miRNAs could function as hormones.

Abstract 4002: Epigenetic control of a checkpoint for miRNA tolerance

Abstract The interferon (IFN) pathway controls the cellular response to viral infection and double-stranded RNA. We have found that this pathway provides a novel checkpoint for enhanced expression of miRNAs. The IFN pathway is abrogated in fibroblasts from Li-Fraumeni Syndrome (LFS) patients during spontaneous cellular immortalization, a necessary step in carcinogenesis. Interferon stimulated genes (ISGs) were down-regulated by epigenetic silencing during immortalization and some of these same ISGs were activated by replicative senescence. Bisulfite sequencing of the promoter regions of IFN-regulatory transcription factors (IRF5 and IRF7) revealed that IRF7 but not IRF5 was epigenetically silenced by methylation of CpG islands in immortal LFS cells. The re-activation of ISGs by those transcription factors is sufficient to inhibit cell proliferation and induce a senescence-related phenotype in immortal LFS cells. Similar epigenetic silencing of IRF7 also results in disruption of IFN pathway in ∼30% lung cancer cells and primary tissues. Examination of innate immunity status in normal bronchial epithelial cell line Beas2B, its ras transformed derivative BVK cells and tumor-generating clones reveals increasing loss of functional IFN pathway as cells become more cancerous. Furthermore, microarray profiling of differentially expressed microRNAs (miRNAs) in LFS cell lines revealed most miRNAs were up-regulated in IFN-pathway-defective MDAH087-10 fibroblasts compared to MDAH087-N cells with relatively normal IFN signaling. Overexpression of Dicer, a critical enzyme in miRNA biogenesis, promoted cell growth and colony formation in MDAH087-10 cells. However, double-stranded miRNA produced by Dicer enhanced the expression of ISGs in MDAH087-N cells resulting in significant cell death and reduced cell growth. In addition, manipulation of the IFN pathway in immortal LFS fibroblasts through transcription factor IRF7 reversed their response to Dicer overexpression due to altered activation of the IFN pathway. Dicer-overexpressing MDAH087-N cells contained lower levels of miRNA than vector control, and conversely much higher miRNA expression was detected in Dicer transfected MDAH087-10 cells. The higher miRNA tolerance in immortal LFS fibroblasts with a disrupted IFN pathway indicates for the first time that the IFN pathway as mediated through the transcription factor IRF7 must be disrupted to permit miRNA up-regulation to occur in early carcinogenesis. This IFN pathway appears to provide a checkpoint for miRNA level tolerance and its abrogation by epigenetic silencing leads to cellular immortalization and early events in tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4002. doi:10.1158/1538-7445.AM2011-4002