Citations

Telomeres cap chromosome ends, protecting them from degradation, double‐strand breaks, and end‐to‐end fusions. Telomeres are maintained by telomerase, a reverse transcriptase encoded by TERT, and an RNA template encoded by TERC. Loci in the TERT and adjoining CLPTM1L region are associated with risk of multiple cancers. We therefore investigated associations between variants in 22 telomere structure and maintenance gene regions and colorectal, breast, prostate, ovarian, and lung cancer risk. We performed subset‐based meta‐analyses of 204,993 directly‐measured and imputed SNPs among 61,851 cancer cases and 74,457 controls of European descent. Independent associations for SNP minor alleles were identified using sequential conditional analysis (with gene‐level p value cutoffs ≤3.08 × 10−5). Of the thirteen independent SNPs observed to be associated with cancer risk, novel findings were observed for seven loci. Across the DCLRE1B region, rs974494 and rs12144215 were inversely associated with prostate and lung cancers, and colorectal, breast, and prostate cancers, respectively. Across the TERC region, rs75316749 was positively associated with colorectal, breast, ovarian, and lung cancers. Across the DCLRE1B region, rs974404 and rs12144215 were inversely associated with prostate and lung cancers, and colorectal, breast, and prostate cancers, respectively. Near POT1, rs116895242 was inversely associated with colorectal, ovarian, and lung cancers, and RTEL1 rs34978822 was inversely associated with prostate and lung cancers. The complex association patterns in telomere‐related genes across cancer types may provide insight into mechanisms through which telomere dysfunction in different tissues influences cancer risk.

Abstract Despite advances in prevention and early detection strategies, colorectal cancer (CRC) remains the second leading cause of cancer death in the United States. The majority of CRC arises from adenomatous polyps. However, only 5% of all people who receive a colonoscopy will have a lesion determined to be at high risk for malignant transformation. Adenomas are primarily classified based on histology, size, degree of dysplasia and limited genetic mutation information. Further characterization of what contributes to or underlies the malignant transformation of polyps is currently lacking, but is essential to further understand the transition from polyp to cancer and factors that lead only a small portion of polyps to progress onto cancer. Although telomere length is a recognized biomarker in multiple cancers, few studies have addressed the comprehensive role of telomeres in CRC progression. Telomere shortening is a fundamental feature of dividing cells related to the age of the cell and is implicated in carcinogenesis. Telomere length is not solely determined by attrition resulting from cell division, but also through telomere maintenance mechanisms (TMM), which are often exploited in malignant cells. In this study, we will measure the telomere profile (consisting of telomere length and TMM) in normal colon epithelium, polyps and the adjacent tumor trios and normal colon epithelium and cancer-free polyp pairs. Our aim is to determine which telomere features distinguish cancer adjacent polyps from cancer free polyps. This is an innovative approach for identifying the definable telomere-related properties that distinguish polyps with malignant potential that remain benign from those that transform to cancer and progress to invasive lesions. Further characterization of the molecular profile of polyps undergoing the transition to cancer that differentiate cancer free polyps has the potential to be utilized in designing optimal treatment options for patients with polyps that carry the risk of CRC. Citation Format: Brooke R. Druliner, Ruth Johnson, Xiaoyang Ruan, Russell Vanderboom, Donna Felmlee Devine, Jill Washechek Aletto, Lisa A. Boardman. Time lapse to cancer: Defining the transition from polyp to colorectal cancer based on telomere metrics. [abstract]. In: Proceedings of the AACR Special Conference on Computational and Systems Biology of Cancer; Feb 8-11 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 2):Abstract nr B2-04.

Abstract Introduction: Activation of telomerase and telomere lengthening in patient peripheral blood leukocytes (PBL) is associated with longer survival in cancer patients. There are few studies to date that have measured telomerase activity in clinical PBL samples, and it is unclear how initial collection and processing of PBL samples impact the measurement of telomerase activity. In this study, we assessed if telomerase activity measurement varies based on the method of blood collection, processing and storage. Methods: We first collected whole blood drawn in EDTA tubes from two healthy patients that were then processed three different ways. The first whole blood sample was spun down and the buffy coat was pipetted from the top of the red blood cells, and then placed directly into the -80°C freezer. The next two whole blood samples were isolated using an ACCUSPIN™ tube, which utilizes density gradient separation to recover the PBL without contamination of red blood cells. The resulting PBL from these two samples were suspended in freezing media containing RPMI and DMSO, with one sample also containing penicillin streptomycin glutamine, and then both samples were stored in liquid nitrogen following a slow freeze. We then measured enzymatic telomerase activity in each of the three PBL samples from the two different patients. Results: We detected a comparable level of telomerase activity in all three PBL samples. These findings indicate that telomerase activity can be measured in buffy coat samples from patients independent of how the samples were processed and stored. Conclusions: These findings are important because many biorepositories may not have collected or processed PBL samples by identical processes, therefore not allowing direct comparison across samples. Additionally, our finding that telomerase activity levels are comparable between PBL samples containing red blood cells and those processed using an ACCUSPIN™ tube is valuable because it eliminates the necessity for the extra cost of the ACCUSPIN™ tube, freezing media, antibiotics and use of liquid nitrogen storage tanks. Future studies will focus on the determining the relationship of telomerase activity between the PBL, normal colon and cancer tissue in colorectal cancer patients. Citation Format: Ruth A. Johnson, Brooke R. Druliner, Jill Washechek Alleto, Donna Felmlee Devine, Xiaoyang Ruan, Russell Vanderboom, Lisa A. Boardman. How collection, processing and storage of PBL samples impact the measurement of telomerase activity. [abstract]. In: Proceedings of the AACR Special Conference on Computational and Systems Biology of Cancer; Feb 8-11 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 2):Abstract nr B1-46.

Adolescent and young adult (AYA) patients with cancer have not attained the same improvements in overall survival as either younger children or older adults. One possible reason for this disparity may be that the AYA cancers exhibit unique biologic characteristics, resulting in differences in clinical and treatment resistance behaviors. This report from the biologic component of the jointly sponsored National Cancer Institute and LiveStrong Foundation workshop entitled “Next Steps in Adolescent and Young Adult Oncology” summarizes the current status of biologic and translational research progress for 5 AYA cancers; colorectal cancer breast cancer, acute lymphoblastic leukemia, melanoma, and sarcoma. Conclusions from this meeting included the need for basic biologic, genomic, and model development for AYA cancers as well as translational research studies to elucidate any fundamental differences between pediatric, AYA, and adult cancers. The biologic questions for future research are whether there are mutational or signaling pathway differences (for example, between adult and AYA colorectal cancer) that can be clinically exploited to develop novel therapies for treating AYA cancers and to develop companion diagnostics. Cancer 2016;122:1017–1028. © 2016 American Cancer Society

Abstract Purpose: Methylated DNA markers can discriminate upper from lower GI neoplasms (Gastroenterology 2013;144(5):S84). It is unknown if organ-specific molecular prediction of GI tumor site can be achieved. Experimental design: An unbiased search for neoplasm markers at each major GI organ site was conducted by reduced representation bisulfite sequencing. Top marker candidates were then blindly validated by methylation specific PCR on independent tissue DNA samples from normal mucosa [17 esophagus (E), 13 stomach (S), 33 pancreas (P), 35 bile duct/liver (BD), 21 colorectal (CR)] and neoplasia [42 E, 43 S, 36 P, 48 BD, 97 CR]. Recursive partitioning (rPart) trees modeled all stepwise combinations of single markers to classify controls vs neoplasms and then neoplasms by site (CR, gastroesophageal or pancreaticobiliary). A 2nd model was designed from tissue data for blinded clinical piloting on 42 independent plasma DNA samples (14 normal and 28 cancers (14 P, 14 CR). Results: From the top 100 markers, 95 were validated and used for rPart modeling. A 3- marker panel (chr12.133484978-5047, BMP3, chr11.123301058-255) was selected for the universal detection of GI neoplasms, as it classified neoplasms and control tissues with 95% accuracy. Two markers (chr7.25896389-501, QKI) then assigned lower vs upper GI neoplasms with 94% accuracy. Finally, 3 markers (PDGFD, ELOVL2, PCBP3) called pancreaticobiliary vs gastroesophageal neoplasms with 94% accuracy. All 8 markers applied in a single model (Table) accurately predicted control vs tumor by site [88% (p <0.0001)]. In plasma, a 2-marker panel (BMP3, QKI) was 78% accurate in assigning patients to cancer or control groups (p<0.0009) and 83% accurate in assigning cancer origin to CR or P sites (p<0.0001). Predicted by ModelTissue SourceControlsColorectal neoplasiaPancreatico-biliary cancerGastro-esophageal neoplasiaControls1169102Colorectal neoplasia18617Pancreatico-biliary cancer10654Gastro-esophageal neoplasia12872 Conclusion: Panels of methylated DNA markers were identified for both universal detection of GI neoplasia and accurate prediction of tumor site. Pilot testing of these markers in plasma supports potential clinical feasibility. Citation Format: John B. Kisiel, William R. Taylor, Tracy C. Yab, Hassan M. Ghoz, Patrick H. Foote, Mary E. Devens, Douglas W. Mahoney, Thomas C. Smyrk, Lisa A. Boardman, Gloria M. Petersen, Navtej S. Buttar, Lewis R. Roberts, Graham P. Lidgard, David A. Ahlquist. Accurate site prediction of gastrointestinal cancer by novel methylated DNA markers: Discovery & validation. [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 4252. doi:10.1158/1538-7445.AM2015-4252

Abstract Telomeres are complex structures that cap chromosome ends, protecting them from degradation, double strand breaks and end-to-end fusions. Telomeres are maintained by the enzyme telomerase, which is made up of a reverse transcriptase encoded by TERT, and an RNA template encoded by TERC. The telomere structure itself is composed of proteins encoded by ACD, ACYP2, BICD1, DKC1, DCLRE1B, MPHOSPH6, NAF1, NOLA1, NOLA2, NOLA3, OBFC1, PIK3C3, POT1, RTEL1, TEP1, TERF1, TERF2, TERF21P, TINF2, TNKS/PINX1, TPP1 and ZNF208. Several single nucleotide polymorphisms (SNPs) in the TERT and adjoining CLPTM1L gene region are associated with multiple cancer types, and some are associated with both increased and decreased risks across different cancer types. We systematically characterized the patterns of association between variants in these 25 telomere structure and maintenance genes and risk across five cancer types in the Genetic Association and Mechanisms in Oncology (GAME-ON) consortium. We performed a subset-based meta-analysis (ASSET) of 209,367 directly measured and imputed SNPs, one megabase up- and downstream of these genes, across genome-wide association studies of colorectal (5,100 cases, 4,831 controls), lung (12,160 cases, 16,838 controls), breast (15,748 cases, 18,084 controls), ovarian (4,369 cases, 9,123 controls) and prostate (14,160 cases, 12,724 controls) cancers. Correlations (r2) between SNPs were examined in Haploview using the 1000 Genomes Project CEU population. A total of 87 TERT, 123 TERC and 26 DCLRE1B SNPs were associated with cancer risk at gene-level Bonferroni-corrected p-values of 4.2-7.8×10−6. Patterns of association were similar for prostate and colorectal cancers in DCLRE1B. Much stronger associations were observed in TERT and TERC, with 63 and 24 SNPs reaching genome-wide significance (p<5.0×10−8), respectively. Of these, 9 TERT and 9 TERC SNPs were correlated at r2<0.75. The most strongly associated SNPs in TERT (rs37004, p = 2.6×10−11) and TERC (rs76925190, p = 1.5×10−15) are in regions containing documented risk loci for lung and prostate cancers, respectively. TERT rs37004 (p = 1.2×10−13) and 6 of the other 9 SNPs in TERT were associated only with lung cancer risk. For the other 2 SNPs, there was a suggestion that patterns of risk were opposite for lung and prostate cancers. While TERC rs76925190 was strongly associated with prostate cancer risk (p = 5.4 x10−17), it was also suggestively associated with colorectal cancer risk (p<0.005). This pattern was similar for another 2 of the 9 SNPs, while for the rest, associations were limited to prostate cancer. The complex patterns of association in telomere structure and maintenance genes observed across cancer types may provide insight about the mechanisms through which telomere dysfunction in different tissues influences cancer risk. Citation Format: Sara Karami, Younghun Han, Fredrick R. Schumacher, Zsofia Kote-Jarai, Sara Lindstrom, John S. Witte, Iona Cheng, Shenying Fang, Jiali Han, Peter Kraft, Fengju Song, Rayjean J. Hung, James McKay, Stephen J. Chanock, Mala Pande, Angela Risch, Hongbing Shen, Christopher A. Haiman, Lisa Boardman, Cornelia M. Ulrich, Graham Casey, Ulrike Peters, Nilanjan Chatterjee, Brandon Pierce, Wei Zheng, Christopher I. Amos, Jennifer A. Doherty. Risk loci in telomere structure and maintenance genes across five cancer types: GAME-ON Consortium. [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 4609. doi:10.1158/1538-7445.AM2015-4609

ABSTRACT The ability to assemble genomes from metagenomic sequencing avoids the need for culture and any associated culture biases. We assembled 24 essentially complete draft genomes from metagenomic pair-end and size-selected mate pair sequencing from 4 stool samples, 2 from subjects diagnosed with colorectal cancer and 2 from healthy controls.

Abstract Purpose: Discriminant markers for pancreatic cancer detection are needed. We sought to identify and validate methylated DNA markers for pancreatic cancer using next-generation sequencing unbiased by known targets. Experimental Design: At a referral center, we conducted four sequential case–control studies: discovery, technical validation, biologic validation, and clinical piloting. Candidate markers were identified using variance-inflated logistic regression on reduced-representation bisulfite DNA sequencing results from matched pancreatic cancers, benign pancreas, and normal colon tissues. Markers were validated technically on replicate discovery study DNA and biologically on independent, matched, blinded tissues by methylation-specific PCR. Clinical testing of six methylation candidates and mutant KRAS was performed on secretin-stimulated pancreatic juice samples from 61 patients with pancreatic cancer, 22 with chronic pancreatitis, and 19 with normal pancreas on endoscopic ultrasound. Areas under receiver-operating characteristics curves (AUC) for markers were calculated. Results: Sequencing identified >500 differentially hyper-methylated regions. On independent tissues, AUC on 19 selected markers ranged between 0.73 and 0.97. Pancreatic juice AUC values for CD1D, KCNK12, CLEC11A, NDRG4, IKZF1, PKRCB, and KRAS were 0.92*, 0.88, 0.85, 0.85, 0.84, 0.83, and 0.75, respectively, for pancreatic cancer compared with normal pancreas and 0.92*, 0.73, 0.76, 0.85*, 0.73, 0.77, and 0.62 for pancreatic cancer compared with chronic pancreatitis (*, P = 0.001 vs. KRAS). Conclusions: We identified and validated novel DNA methylation markers strongly associated with pancreatic cancer. On pilot testing in pancreatic juice, best markers (especially CD1D) highly discriminated pancreatic cases from controls. Clin Cancer Res; 21(19); 4473–81. ©2015 AACR.

Abstract PURPOSE: Cell free circulating microvesicles (in particular, exosomes) contain proteins and nuclei acids that may serve as biomarkers for disease diagnosis and prognosis. The goal of this study was to identify exosomal microRNAs that may predict clinical outcome of patients with castrate resistant prostate cancer (CRPC). EXPERIMENTAL DESIGN: In discovery stage, we performed RNA sequencing using plasma exosomal RNAs derived from 36 patients with CRPC. We mapped the sequence reads to known miRbase (Release 19, 2043 entries) and normalized the miRNA abundance by sequence counts per million mappable reads. We applied Cox regression analysis to identify the miRNAs that were associated with overall survival of CRPC. In validation stage, we first examined an exosomal small RNA sequencing dataset consisting of 192 individuals with various health conditions. We used Normfinder and Bestkeeper to select most stably expressed miRNAs as candidates for normalization references. We then applied real-time qRT-PCR assays to test selected candidate reference miRNAs and survival-related miRNAs in additional 100 CRPC patients. From validated miRNAs, we constructed multivariate models to predict overall survival for this group of patients. RESULTS RNA sequencing generated over 6 million mappable reads per patient in the initial discovery cohort. Among these reads, ∼45% were mapped to known miRNAs for a total of 483 known and 275 novel miRNAs with normalized read counts ≥ 5. The median follow-up time for the 36 CRPC patients was 35.6 months during which 10 patients had died. Cox regression analysis identified four microRNAs (miR-1290, -1246, -375 and a predicted miRNA at chromosome 12) that were associated with overall survival (FDR<0.05). Of six selected candidate miRNAs, miR-30a/e-5p performed the best in their stability as endogenous references for real-time qRT-PCR. When normalized by these internal controls, we confirmed two of the three known miRs (miR-1290 and -375) showing significant association with overall survival in the validation cohort (p<0.001) with a median follow up time of 12.5 (range: 1-41) months at which time 28 patients had died. The median overall survival for patients with high (N=21) and low (N=40) expression in both miR-1290 and -375 was 7.2 (1-26.7) months and 15.3 (1-41) months, respectively (p<0.05). The miR-1290/-375-based prediction model consistently showed better predictive performance with AUC=72% than clinical variable-based model with AUC=64%. CONCLUSIONS: Plasma exosomal miRNAs provide an easily accessible resource for biomarker development in prognosis of advanced prostate cancer. Exosomal miR-1290 and miR-375 are associated with overall survival in CRPC patients. miR-30a/e-5p, especially the geometric mean of miR-30a/e-5p, are qualified endogenous references for real-time qPCR. Further confirmation of these findings is needed for prognostic and predictive biomarker in development of CRPC stage. Citation Format: Xiaoyi Huang, Tiezheng Yuan, Meihua Liang, Meijun Du, Shu Xia, Rachel Louise Dittmar, Zhifu Sun, Yan Lu, Stephen N. Thibodeau, Lisa Boardman, Manish Kohli, Liang Wang. Exosomal miR-1290 and miR-375 as prognostic markers in metastatic castrate resistant prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 574. doi:10.1158/1538-7445.AM2014-574