Matched Tumor Samples Research Articles

Abstract A08: Analysis of MRGBP and PPIL1 genes in colon cancer

Abstract Colorectal cancer is one of the most common causes of cancer death throughout the world. Colorectal carcinogenesis is a multi-step process and in addition to genetic changes significant changes in DNA methylation and epigenetic events such as histone modifications can also lead to deregulation of gene expression. The histone proteins play a dynamic role in the chromatin structure and transcription. Aberrant protein acetylation, particularly on histones, has been related to cancer while abnormal expression of histone deacetlytransferases (HDACs) are observed in a broad range of cancer types. Therefore, HDACs have emerged as promising targets in cancer therapeutics. The MRG-binding protein (MRGBP / C20orf20) is a component of the NuA4 histone acetyltransferase (HAT) complex and is involved in the transcriptional activation of specific genes. The MRGBP gene is frequently upregulated in tumors and is thought to play a role in cancer development. On the other hand removal of introns from pre-mRNA is carried out by a large macromolecular spliceosome complex. PPIL1 (Peptidyl-prolylisomerase-like 1) is a component of the human spliceosome and plays a primary role in pre-mRNA splicing for catalyzing isomerization of the peptide bonds. Overexpression of the PPIL1 protein in cancer suggest a potential role of PPIL1 in the in the development and promotion of colon cancer. In the present study we analyzed MRGBP and PPIL1 gene expression levels in matched tumor and normal tissue samples from patients with colorectal cancer. Tissue samples were collected from 51 patients with colorectal cancer who underwent surgery for tumor resection. The normal tissue specimens used in the study were histologically confirmed to be free of cancer. The HPRT (hypoxanthine-Guanine phosphoribosyltransferase) gene was used as reference. The target and reference genes were amplified in the same multiplex PCR using the LightCycler 480 System. 8-9 nucleotide long UPL probes were labeled with fluorescein (FAM) and TAMRA at the 5′ and 3′ ends, respectively. Expression levels were determined using the Basic Relative Quantification analysis software and the results were evaluated by the Mann-Whitney U test. Although the median MRGBP expression levels in the tumor tissue were higher than normal tissue (1,72 vs.1,41) the difference was not significant. The median expression levels of PPIL1 in the tumors and normal tissue samples (1,97 vs 1,86) were also similar. Our results indicate that neither MRGBP nor PPIL1 gene expression levels are upregulated in colon cancer cells indicating that these are not directly associated with colorectal cancer. Citation Format: Ebru Akisik, Nejat Dalay, Sumer Yamaner, Dursun Bugra. Analysis of MRGBP and PPIL1 genes in colon cancer. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr A08.

Decreased Expression of Alpha-L-Fucosidase Gene FUCA1 in Human Colorectal Tumors

In previous studies we described a decreased alpha-l-fucosidase activity in colorectal tumors, appearing as a prognostic factor of tumoral recurrence. The aim of this work was to extend the knowledge about tissue alpha-l-fucosidase in colorectal cancer by quantifying the expression of its encoding gene FUCA1 in tumors and healthy mucosa. FUCA1 mRNA levels were measured by RT-qPCR in paired tumor and normal mucosa tissues from 31 patients. For the accuracy of the RT-qPCR results, five candidate reference genes were validated in those samples. In addition, activity and expression of alpha-l-fucosidase in selected matched tumor and healthy mucosa samples were analyzed. According to geNorm and NormFinder algorithms, RPLP0 and HPRT1 were the best reference genes in colorectal tissues. These genes were used for normalization of FUCA1 expression levels. A significant decrease of more than 60% in normalized FUCA1 expression was detected in tumors compared to normal mucosa (p = 0.002). Moreover, a gradual decrease in FUCA1 expression was observed with progression of disease from earlier to advanced stages. These findings were confirmed by Western blot analysis of alpha-l-fucosidase expression. Our results demonstrated diminished FUCA1 mRNA levels in tumors, suggesting that expression of tissue alpha-l-fucosidase could be regulated at transcriptional level in colorectal cancer.

HES1, a target of Notch signaling, is elevated in canine osteosarcoma, but reduced in the most aggressive tumors

BackgroundHairy and enhancer of split 1 (HES1), a basic helix-loop-helix transcriptional repressor, is a downstream target of Notch signaling. Notch signaling and HES1 expression have been linked to growth and survival in a variety of human cancer types and have been associated with increased metastasis and invasiveness in human osteosarcoma cell lines. Osteosarcoma (OSA) is an aggressive cancer demonstrating both high metastatic rate and chemotherapeutic resistance. The current study examined expression of Notch signaling mediators in primary canine OSA tumors and canine and human osteosarcoma cell lines to assess their role in OSA development and progression.ResultsReverse transcriptase - quantitative PCR (RT-qPCR) was utilized to quantify HES1, HEY1, NOTCH1 and NOTCH2 gene expression in matched tumor and normal metaphyseal bone samples taken from dogs treated for appendicular OSA at the Colorado State University Veterinary Teaching Hospital. Gene expression was also assessed in tumors from dogs with a disease free interval (DFI) of <100 days compared to those with a DFI > 300 days following treatment with surgical amputation followed by standard chemotherapy. Immunohistochemistry was performed to confirm expression of HES1. Data from RT-qPCR and immunohistochemical (IHC) experiments were analyzed using REST2009 software and survival analysis based on IHC expression employed the Kaplan-Meier method and log rank analysis. Unbiased clustered images were generated from gene array analysis data for Notch/HES1 associated genes.Gene array analysis of Notch/HES1 associated genes suggested alterations in the Notch signaling pathway may contribute to the development of canine OSA. HES1 mRNA expression was elevated in tumor samples relative to normal bone, but decreased in tumor samples from dogs with a DFI < 100 days relative to those with a DFI > 300 days. NOTCH2 and HEY1 mRNA expression was also elevated in tumors relative to normal bone, but was not differentially expressed between the DFI tumor groups. Survival analysis confirmed an association between decreased HES1 immunosignal and shorter DFI.ConclusionsOur findings suggest that activation of Notch signaling occurs and may contribute to the development of canine OSA. However, association of low HES1 expression and shorter DFI suggests that mechanisms that do not alter HES1 expression may drive the most aggressive tumors.

Abstract 2987: VHL dependent and independent regulation of HDAC expression and activity in renal cell carcinomas.

Abstract Background: Histone modifications include addition and/or deletion of acetyl and methyl groups to lysine, serine and arginine residues of the histone tails leading to transcriptional inactivation or activation. The enzymes governing these modifications are histone acetyl transferases (HATs), histone deacetylases (HDACs), histone methylases (HMTs) and histone demethylases (HDMTs). HDAC inhibitors exert their inhibitory effects on tumor cell population through cell cycle inhibition, apoptosis, decreased cell proliferation as well as their induction of tumor suppressor genes (for eg: p53). Clear cell renal cell carcinoma (ccRCC) represents the dominant major subtype of RCC malignancy with frequent mutations, deletions or methylation in the tumor suppressor gene Von-Hippel Lindau (VHL). Class I HDACs have been reported to be overexpressed in ccRCCs and VHL protein is present in the same complex containing class I HDACs. The present study was aimed at defining the role of VHL in the regulation of HDAC expression and activity. Methods: Approximately 15 ccRCC tumors and matched normal kidney samples from nephrectomies were collected. Western blot analysis of UMRC-2 (VHL−/− RCC cell line) and UMRC2 (wt-VHL RCC cell line) were carried out under conditions of normoxia, hypoxia and in the presence of proteasomal inhibitor MG-132. Tumor cells (1 x 107) were injected subcutaneously in SCID mice to establish tumor xenografts. Results: Our preliminary studies in matched clinical tumor samples show overexpression of specific Class I (2 fold increase; p&amp;lt;0.05) and II HDACs (5 fold increase for HDAC 4). Conversely, HDAC 6 expression (2 fold decrease, p&amp;lt;0.01) and activity was down regulated in tumor samples. To investigate the role of VHL in RCCs, UMRC2 and UMRC2-VHL were utilized for HDAC expression and activity. Western blot analysis indicated HDAC 1 down regulation (1.5 fold) and HDAC 6 overexpression (1.5 fold) in the UMRC2 cells as compared to the UMRC2-VHL cells. In contrast, HDAC 6 activity, measured by expression of its target protein acetylated α-tubulin, was lower in the UMRC2 cells. Under hypoxic conditions both HDAC 1 and HDAC 6 were induced in the UMRC2-VHL cells suggesting VHL dependent hypoxic regulation of these HDACs. MG-132 induced HDAC 1 and 6 in UMRC2-VHL; whereas proteasome inhibitor down regulated HDAC 1 and 6 in UMRC2 cells suggesting a VHL independent mechanism of HDAC regulation. In vivo studies of tumor xenografts generated from the cell lines demonstrated hypoxic induction of HDAC 1 (5 fold increase) and HDAC 6 in UMRC2 when compared to the UMRC2-VHL tumors. Conclusion: Our results suggest that specific HDACs are differentially regulated by VHL dependent (via hypoxia regulation) and independent mechanisms, providing a rationale for testing selective HDAC inhibitors in RCCs. Citation Format: Swathi Ramakrishnan, Paula Sotomayor, Leigh Ellis, Sreenivasulu Chintala, Roberto Pili. VHL dependent and independent regulation of HDAC expression and activity in renal cell carcinomas. [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 2987. doi:10.1158/1538-7445.AM2013-2987

High-Resolution Mutational Profiling Suggests the Genetic Validity of Glioblastoma Patient-Derived Pre-Clinical Models

Recent advances in the ability to efficiently characterize tumor genomes is enabling targeted drug development, which requires rigorous biomarker-based patient selection to increase effectiveness. Consequently, representative DNA biomarkers become equally important in pre-clinical studies. However, it is still unclear how well these markers are maintained between the primary tumor and the patient-derived tumor models. Here, we report the comprehensive identification of somatic coding mutations and copy number aberrations in four glioblastoma (GBM) primary tumors and their matched pre-clinical models: serum-free neurospheres, adherent cell cultures, and mouse xenografts. We developed innovative methods to improve the data quality and allow a strict comparison of matched tumor samples. Our analysis identifies known GBM mutations altering PTEN and TP53 genes, and new actionable mutations such as the loss of PIK3R1, and reveals clear patient-to-patient differences. In contrast, for each patient, we do not observe any significant remodeling of the mutational profile between primary to model tumors and the few discrepancies can be attributed to stochastic errors or differences in sample purity. Similarly, we observe ∼96% primary-to-model concordance in copy number calls in the high-cellularity samples. In contrast to previous reports based on gene expression profiles, we do not observe significant differences at the DNA level between in vitro compared to in vivo models. This study suggests, at a remarkable resolution, the genome-wide conservation of a patient’s tumor genetics in various pre-clinical models, and therefore supports their use for the development and testing of personalized targeted therapies.

Can Serum be Used for Analyzing the EGFR Mutation Status in Patients with Advanced Non-small Cell Lung Cancer?

Epidermal growth factor receptor (EGFR) mutations as prognostic or predictive marker in patients with non-small cell lung cancer (NSCLC) have been used widely. However, it may be difficult to get tumor tissue for analyzing the status of EGFR mutation status in large proportion of patients with advanced disease. We obtained pairs of tumor and serum samples from 57 patients with advanced NSCLC, between March 2006 and January 2009. EGFR mutation status from tumor samples was analyzed by genomic polymerase chain reaction and direct sequence and EGFR mutation status from serum samples was determined by the peptide nucleic acid locked nucleic acid polymerase chain reaction clamp. EGFR mutations were detected in the serum samples of 11 patients and in the tumor samples of 12 patients. EGFR mutation status in the serum and tumor samples was consistent in 50 of the 57 pairs (87.7%). There was a high correlation between the mutations detected in serum sample and the mutations detected in the matched tumor sample (correlation index 0.62; P<0.001). Twenty-two of 57 patients (38.5%) received EGFR-tyrosine kinase inhibitors as any line therapy. The response for EGFR-tyrosine kinase inhibitors was significantly associated with EGFR mutations in both tumor samples and serum samples (P<0.05). There was no significant differences in overall survival according to the status of EGFR mutations in both serum and tumor samples (P>0.05). Serum sample might be alternatively used in the difficult time of getting tumor tissue for analyzing the status of EGFR mutation status in patients with advanced NSCLC.

Global methylation profiling in serous ovarian cancer is indicative for distinct aberrant DNA methylation signatures associated with tumor aggressiveness and disease progression

ObjectiveTo characterize at high resolution the DNA methylation changes which occur in the genome of serous epithelial ovarian cancer (EOC) in association with tumor aggressiveness. MethodsMethylated DNA immunoprecipitation in combination with CpG island-tiling arrays was used to compare the methylation profiles of five borderline, five grade 1/stage III/IV, five grade 3/stage I and five grade 3/stage III/IV serous EOC tumors, to those of five normal human ovarian tissue samples. ResultsWe found widespread DNA hypermethylation that occurs even in low-malignant potential (borderline) tumors and which predominantly includes key developmental/homeobox genes. Contrary to DNA hypermethylation, significant DNA hypomethylation was observed only in grade 3 serous EOC tumors. The latter observation was further confirmed when comparing the DNA methylation profiles of primary cell cultures derived from matched tumor samples obtained prior to, and following chemotherapy treatment from two serous EOC patients with advanced disease. To our knowledge this is the first report that has shown the presence of massive DNA hypomethylation in advanced serous EOC, associated with tumor malignancy and disease progression. ConclusionsOur data raise the concern that demethylating drugs that are currently being used in advanced EOC disease (representing the majority of serous EOC cases) might have adverse effects due to activation of oncogenes and prometastatic genes. Understanding the relative roles of hypomethylation and hypermethylation in cancer could have clear implications on the therapeutic use of agents targeting the DNA methylation machinery.

Recurrent Mutations of MYD88 and TBL1XR1 in Primary Central Nervous System Lymphomas

Our objective was to identify the genetic changes involved in primary central nervous system lymphoma (PCNSL) oncogenesis and evaluate their clinical relevance. We investigated a series of 29 newly diagnosed, HIV-negative, PCNSL patients using high-resolution single-nucleotide polymorphism (SNP) arrays (n = 29) and whole-exome sequencing (n = 4) approaches. Recurrent homozygous deletions and somatic gene mutations found were validated by quantitative real-time PCR and Sanger sequencing, respectively. Molecular results were correlated with prognosis. All PCNSLs were diffuse large B-cell lymphomas, and the patients received chemotherapy without radiotherapy as initial treatment. The SNP analysis revealed recurrent large and focal chromosome imbalances that target candidate genes in PCNSL oncogenesis. The most frequent genomic abnormalities were (i) 6p21.32 loss (HLA locus), (ii) 6q loss, (iii) CDKN2A homozygous deletions, (iv) 12q12-q22, and (v) chromosome 7q21 and 7q31 gains. Homozygous deletions of PRMD1, TOX, and DOCK5 and the amplification of HDAC9 were also detected. Sequencing of matched tumor and blood DNA samples identified novel somatic mutations in MYD88 and TBL1XR1 in 38% and 14% of the cases, respectively. The correlation of genetic abnormalities with clinical outcomes using multivariate analysis showed that 6q22 loss (P = 0.006 and P = 0.01) and CDKN2A homozygous deletion (P = 0.02 and P = 0.01) were significantly associated with shorter progression-free survival and overall survival. Our study provides new insights into the molecular tumorigenesis of PCNSL and identifies novel genetic alterations in this disease, especially MYD88 and TBL1XR1 mutations activating the NF-κB signaling pathway, which may be promising targets for future therapeutic strategies.

Recurrent genetic alterations in primary central nervous system lymphoma of immunocompetent patients.

2023 Background: Little is known about the molecular pathogenesis of primary central nervous system lymphoma (PCNSL) in immunocompetent patients. Our objective was to identify the genetic changes involved in PCNSL oncogenesis and evaluate their clinical relevance. Methods: Twenty nine and four newly diagnosed, HIV-negative PCNSL patients were investigated using high-resolution single nucleotide polymorphism (SNPa) arrays (Infinium Illumina Human 610-Quad SNP array-Illumina; validated by real-time quantitative polymerase chain reaction) and whole-exome sequencing respectively. Molecular results were correlated with prognosis. Results: All PCNSLs were diffuse large B-cell lymphomas, and the patients received high-dose methotrexate-based polychemotherapy without radiotherapy as an initial treatment.SNPa analysis revealed recurrent large and focal chromosome imbalances that target candidate genes in PCNSL oncogenesis. The most frequent genomic changes were (i) 6p21.32 loss (79%), corresponding to the HLA locus; (ii) 6q loss (27-37%); (iii) CDKN2A homozygous deletions (45%); (iv) 12q12-q22 (27%); (v) chromosome 7q21 and 7q31 gains (20%). Sequencing of matched tumor and blood DNA samples identified novel somatic mutations in MYD88 (L265P hot spot mutation) and TBL1XR1 in 38% and 14% of the cases, respectively. The correlation of genetic abnormalities with clinical outcomes using multivariate analysis showed that 6q22 loss (p=0.006 and p=0.01), and CDKN2A homozygous deletion (p=0.02 and p=0.01) were significantly associated with shorter progression free survival and overall survival. Conclusions: Our study identified novel genetic alterations in PCNSL, such as MYD88 and TBL1XR1 somatic mutations, which would both contribute to the constitutive activation of the NFkB signaling pathway and represent potential promising targets for future therapeutic strategies.

Analysis of molecular mechanisms of response and resistance to vemurafenib (vem) in BRAFV600E melanoma.

8503^ Background: Vem induces frequent clinical responses (RR &gt;50%) and improved survival in patients (pts) with BRAF-mutated metastatic melanoma. Multiple mechanisms of escape from vem have been proposed. We performed a centralized analysis of pretreatment, cycle 1, day 15 (D15), and progression (DP) tumor samples collected during the phase II BRIM-2 trial (Sosman et al, NEJM 2012). Methods: Of 132 pts enrolled, archival tumor samples, biopsies taken at baseline (BL), D15, and DP were obtained from 84, 38, 26, and 22 pts, respectively, and analyzed by immunohistochemistry (IHC) for signaling molecules in the MAPK, PI3K/AKT, and cell cycle pathways. Genetic analyses of signaling genes were performed using Sequenom MASSarray and direct DNA sequencing. Results: High levels of ERK phosphorylation were seen at BL indicating constitutive MAPK signaling due to the BRAF mutation. In 19/22 paired samples, pERK levels were reduced following vem (BL vs D15). Mean absolute pERK reduction in pts with clinical response (RECIST criteria) to vem (n=14) was significantly greater than in non-responders (n=8; p=0.013). Baseline cytoplasmic PTEN H-score was higher in responders vs non-responders (H-score=88 vs 68; p=0.042). At progression, upregulation of pERK (H-score=181) was frequently, but not uniformly found vs D15 tumors (H-score=48.5). At progression, increases in Cyclin D1 and Ki67 were seen, without obvious changes in PTEN or pAKT. NRAS mutations occurred in 3/13 DP; 2 had paired BL samples without NRAS mutations. Only 1/82 pts had a concomitant NRAS and BRAF mutation at BL, and did not respond to vem. MAP2K1 (MEK1) codon 124 mutations occurred in 7/92 BL and 1/20 DP samples. 2 pts with BL MEK1 mutations had partial responses. Conclusions: MAPK signaling was effectively inhibited by vem early in treatment, and in the subset of patients with matched tumor samples the degree of pathway inhibition correlated with clinical response. MAPK signaling is upregulated in many lesions at progression. NRAS mutations appear to be a mechanism of acquired resistance in a few tumors (3/13), while the role of MEK1 mutations in resistance is less clear.

Abstract 4792: Biomarker discovery: Correlation of peripheral and tumoral markers in matched tumor and plasma samples

Abstract The heterogeneous nature of cancer necessitates the identification of markers to enable selection of appropriate patients for treatment and maximal clinical benefit. Archival tumor specimens are often analyzed for biomarkers, but may not reflect a patient's current state of disease. Predictive biomarkers from matrices amenable to longitudinal analyses and available through minimally invasive approaches could greatly improve patient outcomes by ensuring that the right patient receives the right drug. Peripheral markers are accessible and attractive, however, their relationship to tumoral markers, tumor biology as well as to primary or secondary clinical outcomes are not well understood. We examined the correlations among potential predictive biomarkers in these compartments by performing proteomic profiling of ∼300 analytes in 100 matched plasma and tumor tissue samples derived from patients with colorectal cancer (CRC), gastric, ovarian, and non-small cell lung cancer (NSCLC) of squamous cell carcinoma (SCC) or adenocarcinoma (ADC) histology. Using Luminex based assays, we identified markers which clearly distinguish each tumor type from another and may be useful for patient enrichment. VEGF has been implicated in processes essential to tumor growth and disease progression such as angiogenesis, vasculogenesis and hypoxia. Therefore, we examined the relationship between VEGF levels in plasma samples and tumor tissue within individual patients and explored the correlations among VEGF levels with those of other soluble proteins in the same matrices as well as with a marker of blood vessel formation in the tumors, microvessel density (MVD). We found the highest level of VEGF in the plasma of CRC patients but NSCLC patients displayed the highest level of VEGF in tumors. A poor correlation between tumoral or plasma VEGF levels was observed in all of the samples except for those from NSCLC SCC patients. MVD analysis of the tumors revealed that gastric and NSCLC ADC patients have a higher density of capillary vessels than other cancers. Surprisingly, no intra-patient correlation was detected between tumoral levels or plasma levels of VEGF and MVD. However, modest correlations between the MVD level in tumor tissues and select analytes in plasma have been observed for some tumor types. Further evaluation of these potential protein signatures in additional matched patient samples and animal disease models is warranted to generate clinically testable hypotheses for patient enrichment. The validity of these protein signatures as predictive markers for response to targeted therapies must be tested clinically. These novel and unexpected findings suggest that some peripheral markers may accurately represent the biology of the tumor and that matched sample analysis can provide an approach for biomarker discovery and hypothesis generation. 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 4792. doi:1538-7445.AM2012-4792

Transcriptional Profiling by Sequencing of Oropharyngeal Cancer

ObjectiveTo compare full transcriptome expression levels of matched tumor and normal samples from patients with oropharyngeal carcinoma stratified by known tumor etiologic factors. Patients and MethodsFull transcriptome sequencing was analyzed for 10 matched tumor and normal tissue samples from patients with previously untreated oropharyngeal carcinoma. Transcriptomes were analyzed using massively parallel messenger RNA sequencing and validated using the NanoString nCounter system. Global gene expression levels were compared in samples grouped by smoking status and human papillomavirus status. This study was completed between June 10, 2010, and June 30, 2011. ResultsGlobal gene expression analysis indicated tumor tissue from former smokers grouped more closely to the never smokers than the current smokers. Pathway analysis revealed alterations in the expression of genes involved in the p53 DNA damage-repair pathway, including CHEK2 and ATR, which display patterns of increased expression that is associated with human papillomavirus–negative current smokers rather than former or never smokers. ConclusionThese findings support the application of messenger RNA sequencing technology as an important clinical tool for more accurately stratifying patients based on individual tumor biology with the goal of improving our understanding of tumor prognosis and treatment response, ultimately leading to individualized patient care strategies.

Low-Level Expression of miR-375 Correlates with Poor Outcome and Metastasis While Altering the Invasive Properties of Head and Neck Squamous Cell Carcinomas

Small, noncoding microRNAs (miRNAs) have been shown to be abnormally expressed in every tumor type examined. We used comparisons of global miRNA expression profiles of head and neck squamous cell carcinoma (HNSCC) samples and adjacent normal tissue to rank those miRNAs that were most significantly altered in our patient population. Rank Consistency Score analysis revealed miR-375 to have the most significantly lowered miRNA levels in tumors relative to matched adjacent nonmalignant tissue from the same patient among 736 miRNAs that were evaluated. This result has been previously observed by other groups; however, we extend this finding with the unique observation that low miR-375 expression levels correlate significantly with cancer survival and distant metastasis. In a study of 123 primary HNSCC patients using multivariable Cox proportional hazard ratios (HR) and 95% confidence intervals (CI), both death from disease (HR: 12.8, 95% CI: 3 to 49) and incidence of distant metastasis (HR: 8.7, 95% CI: 2 to 31) correlated with lower expression levels of miR-375 regardless of the site or stage of the tumor. In addition, we found that oral cavity tumor cell lines (eg, UMSCC1 and UMSCC47) overexpressing miR-375 were significantly less invasive in vitro than their matched empty vector controls. We conclude that miR-375 represents a potential prognostic marker of poor outcome and metastasis in HNSCC and that it may function by suppressing the tumor's invasive properties.

OT3-01-03: Pre-Surgical Evaluation of the AKT Inhibitor MK-2206 in Patients with Operable Invasive Breast Cancer: New York Cancer Consortium Trial P8740.

Abstract Background: To serve as a bridge between pre-clinical and early clinical testing, pre-surgical studies are being designed to expose patients to a limited duration of an anti-cancer agent before launching a lengthy clinical trial program. In this model, modulation of predictive tissue and serum biomarkers are assessed. At Columbia University, we have completed 3 pre-surgical studies. The PI3K/Akt signaling pathway is an important signaling pathway in breast cancer (BC). The first allosteric Akt inhibitor to enter clinical development, MK-2206 is well-tolerated and has demonstrated anti-cancer properties in pre-clinical and early-phase clinical studies. The purpose of this study (NCI P8740) is to determine the effects of MK-2206 on women with newly diagnosed BC between breast biopsy and surgery. Trial Design: Patients (n=30) will receive 2 doses of weekly MK-2206 (200 mg): first dose at day −9 and second at day −2 from surgery. This drug administration consistency will decrease the potential for noise in detecting true biomarker response. This time period was selected based upon the pharmacokinetic profile of weekly MK-2206. The main eligibility criteria for this open-label pre-surgical trial include operable, clinical stage I (at least T1c) to IIIC invasive BC. Patients not considered for neoadjuvant chemotherapy are eligible. Specific Aims: Our hypothesis is that MK-2206 will decrease phospho-AktSer473 in tumor tissue after 2 weekly doses. To maintain sample quality and ensure that phospho-Akt evaluated on formalin-fixed paraffin-embedded (FFPE) tissue is comparable across samples (given the 20 minute half life of AKT), all samples will be processed rapidly by a standardized protocol. Secondary objectives include the following in FFPE: modulation of downstream PI3K/Akt pathway signaling [immunohistochemistry and reverse phase-protein microarray analysis (RPPA)]; change in tumor proliferation (Ki-67 staining); and exploration of whether PI3K/Akt signaling change depends upon tumor genetics (PIK3CA mutation or PTEN loss) or expression (Hormone Receptor/HER2 status). Pre- and post-MK-2206 blood will be collected for phospho-expression analysis in peripheral blood mononuclear cells (RPPA and western blotting). Statistical Methods: With the expectation that 20% of the matched pre- and post-treatment tissue samples will not be analyzable, we will enroll 30 patients to ensure that matched tumor samples from 24 patients are available for our primary analysis. A sample size of 24 patients will yield greater than 90% power to detect a difference in the mean score between pre-and-post treatment samples of 60 units, paired t-test (2-sided, 0.05 significance level). This trial was recently activated by the NCI and currently ready to accrue patients. Patients will be enrolled and treated at affiliated institutions in the New York Cancer Consortium. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr OT3-01-03.

Receptor for Activated Protein C Kinase 1 (RACK1) Is Overexpressed in Papillary Thyroid Carcinoma

The receptor for activated C kinase 1 (RACK1) has been shown to be overexpressed in several types of cancers such as breast, colon, melanomas, and lung. RACK1 is linked to Ras-Raf-mediated signal transduction and transformed foci formation of 3T3 cells in vitro, and since this pathway is central in papillary thyroid carcinoma (PTC) oncogenesis, we hypothesized that RACK1 could play a role in the development or maintenance of PTC. No report on RACK1 expression in thyroid tissue is available; the present study was therefore aimed at identifying possible correlation of RACK1 expression at the mRNA or protein level in normal thyroid tissue compared to PTC. We used TaqMan quantitative reverse transcriptase-polymerase chain reaction and immunohistochemistry to study the RACK1 gene and protein expression in matched tumor and nontumor samples from 59 PTC patients. The tumor samples were divided into two main categories, low-risk (group 1-3) and high-risk (group 4-6), in accordance with both histological classification and clinical appearance. RACK1 mRNA and protein levels were found highly overexpressed in tumor samples, whereas Ki-Ras mRNA was found to be relatively unchanged. B-Raf mRNA expression was low and detected only in tumor samples. Sequencing analysis detected no mutations in RACK1 or Ki-Ras, but 62.7% of the patients harbored the B-Raf single-nucleotide substitution T1799A (codon V600E). Phosphorylated extracellular signal-regulated kinase (pERK) immunohistochemistry analysis demonstrated activation of the mitogen-activated protein kinase (MAPK) pathway in tumor cells. Poorly differentiated and undifferentiated PTCs expressed significantly higher RACK1 mRNA levels than well-differentiated PTCs (p<0.017). Taken together, our findings point to an important role of RACK1 protein in PTC development and progression. Our data also emphasize the importance of assessing protein expression and not only mRNA levels.

Abstract B39: Results of a pilot study: Identification of ethnicspecific gene expression differences in normal breast tissue

Abstract Disparities in breast cancer stage of presentation and survival rates exist in patients of different ethnicities. These differences are undoubtedly a result of a combination of factors, including socio-economic, lifestyle, tumor characteristics and inherent factors, such as genetic composition. Our group remains focused on analyzing genetic/genomic contributions to these disparities, with the ultimate goal of increased biological understanding, leading ultimately to individualized, ethnic-specific diagnostic and therapeutic approaches. We have previously reported ethnicspecific expression patterns in matched tumor and adjacent samples from a cohort of triple negative breast cancer (BC) samples. Here we report results from a parallel study focusing on gene expression profiling in a multi-ethnic collection of normal breast tissues. Study samples were cut from FFPE (formalin fixed paraffin-embedded tissue) blocks saved from local reduction mammoplasty cases [5 Caucasians (CAU); 7 African-American (AA); and 4 Hispanics (HIS) women with no personal or family BC history]. These were sent to Almac Diagnostics for RNA isolation, cDNA preparation, and hybridization of cDNAs to a cancer focused gene expression array (Xcel) containing 110,961 probes, representing 19,905 unique known genes. Arrays were quantile normalized and log transformed to the median of all samples. The probes were filtered to remove variation within each ethnic group to ≤ 0.5 SD, while the variation between the three groups was maintained at ≥ 0.2 SD. Samples which had &amp;lt; 90 % similarity based on ‘Pearson correlation’ were removed from subsequent analyses. This removed three AA, 2 CAU and 1 HIS samples. Finally, the filtered subset of 68,145 probes common to the three ethnic groups was then compared on GeneSpring® analytical software. A mean centered Principal Component Analysis (PCA) was performed on the filtered subset of normalized data. In addition, a one way ANOVA between the three ethnic groups identified 1884 significantly differentially expressed probes representing 237 unknown and 1647 known genes (P ≤ 0.05; Benjamin-Hochberg multiple testing corrected). Post-hoc analysis of the 1884 significant probes identified 207 probes significantly differentially expressed between CAU and AA; 1863 probes were found to be differentially expressed between HIS and CAU, and 1873 probes were significantly differentially expressed between HIS and AA. These results suggests that the HIS group/samples have a unique gene signature in comparison to the other ethnic groups analyzed. When the differentially expressed gene list across ethnicities is reduced to those genes with 1.5 fold change, the number of statistically-significant differentially-expressed genes across ethnicities decreases dramatically (7 AA vs. Cau; approx 600 AA vs HIS; 600 CAU vs His), suggesting a much more select group of differentially-expressed genes in normal breast tissue across these ethnic groups. Since the overall sample size is small, we are continuing to validate these data on a larger set of samples. In addition, we are performing laser capture micro-disssection of these samples, to compare gene expression patterns in stroma vs. epithelial. We will present our latest study results, including pathway analysis of statistically-significant differentially-expressed genes across the three ethnic groups. Citation Information: Cancer Epidemiol Biomarkers Prev 2011;20(10 Suppl):B39.

An Epigenetic Marker Panel for Detection of Lung Cancer Using Cell-Free Serum DNA

We investigated the feasibility of detecting aberrant DNA methylation of some novel and known genes in the serum of lung cancer patients. To determine the analytic sensitivity, we examined the tumor and the matched serum DNA for aberrant methylation of 15 gene promoters from 10 patients with primary lung tumors by using quantitative methylation-specific PCR. We then tested this 15-gene set to identify the more useful DNA methylation changes in the serum of a limited number of lung cancer patients and controls. In an independent set, we tested the six most promising genes (APC, CDH1, MGMT, DCC, RASSF1A, and AIM1) for further elucidation of the diagnostic application of this panel of markers. Promoter hypermethylation of at least one of the genes studied was detected in all 10 lung primary tumors. In majority of cases, aberrant methylation in serum DNA was accompanied by methylation in the matched tumor samples. In the independent set, using a single gene that had 100% specificity (DCC), 35.5% (95% CI: 25-47) of the 76 lung cancer patients were correctly identified. For patients without methylated DCC, addition of a logistic regression score that was based on the five remaining genes improved sensitivity from 35.5% to 75% (95% CI: 64-84) but decreased the specificity from 100% to 73% (95% CI: 54-88). This approach needs to be evaluated in a larger test set to determine the role of this gene set in early detection and surveillance of lung cancer.

Influence of the metabolic syndrome on leptin and leptin receptor in breast cancer

Obesity and its associated metabolic syndrome (MetS) are recognized risk factors for breast cancer. The molecular basis for this association remains largely unknown. Adipokines, in particular leptin and adiponectin, are thought to form part of the mechanism linking obesity with cancer through their altered expression/production either systemically (endocrine pathway) or locally (paracrine/autocrine pathway). Using quantitative PCR, mRNA expression of adiponectin (AdipoQ) and leptin (Ob) in mammary adipose tissue (MAT), intratumoral leptin and associated ligand receptors (ObR, AdipoR1, and AdipoR2) was examined in 77 patients with complete anthropomorphic and serological data. Expression of Ob in MAT, and ObR in matched tumor tissue was significantly higher in patients with MetS compared to obese only or normal weight cancer patients (P < 0.005). There was no difference in intratumoral leptin adiponectin or its ligand receptors in the same groups. Individual features of MetS correlated with Ob and ObR expression, but not obesity markers (BMI, waist circumference). mRNA expression of leptin (Ob) and ObR, in adipose tissue and matched tumor samples, respectively, appear to be associated with obesity status in breast cancer. Increasing insulin resistance is a predominant feature of this higher Ob/ObR expression observed. These novel data indicate that the MetS may be an amenable risk factor for breast cancer.

Abstract PL02-02: The molecular pathology of acute leukemia

Abstract PL02-02: The molecular pathology of acute leukemia

Abstract 3741: Identification of differentially expressed genes in breast tumors from African American compared with Caucasian women

Abstract Background: Breast tumors from African American women (AAW) have less favorable pathological characteristics and higher mortality rates than Caucasian women. While socioeconomic status may influence prognosis, biological factors are also likely to contribute to tumor behavior. Methods: Patients treated within an equal-access military medical center were matched by age, grade and ER status. Tumors were subjected to laser microdissection and RNA hybridized to HG U133A 2.0 microarrays. Data was analyzed using Partek Genomics Suite using a cutoff of P&amp;lt;0.001, 1.5-fold change. Microarray results were validated by qRT-PCR. Results: Twenty-six pairs of matched tumor samples were identified. Clinico-pathological factors did not differ significantly for age at diagnosis, tumor size or stage, lymph node, hormone receptor, or HER2 status. Mortality was similar between the two groups. One putative and nineteen known genes were differentially expressed between populations with five genes expressed at higher and 15 genes at lower levels in tumors from AAW; hierarchical clustering classified 23/26 AAW and 26/26 CW correctly. Differential expression was validated (P&amp;lt;0.001) for PSPHL (higher in AAW) and LTF and PSD3 (lower in AAW). Conclusions: Despite matching of tumors by pathological characteristics and provision of medical care in an equal-access health care system, a molecular signature differentiating AAW from CW tumors was detected, supporting a model in which aggressive tumor phenotypes are driven by molecular differences within the tumors. These differentially expressed genes are involved in immune response, cell cycle and metastasis and thus may contribute to the poor outcome in AAW. 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 3741. doi:10.1158/1538-7445.AM2011-3741