Vanderboom, Russell J.

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.

Abstract Introduction: Peripheral blood leukocyte (PBL) telomere length is associated with colon cancer (CC) risk, but the relationship of PBL telomere length to survival in CC patients receiving chemotherapy is unknown. Methods: We measured relative telomere length (RTL) by qPCR of PBL DNA from 2,748 cases (53% male, 85% white, median age 58 yrs) in the North American Phase III Intergroup Trial N0147 that compared disease-free survival (DFS) among completely resected Stage III CC treated with mFOLFOX6 +/- cetuximab. Tumor markers were assessed, and 12% were BRAF mutant; 35% Kras mutant, and 12% deficient mismatch repair (dMMR). Linear and non-linear prognostic associations of PBL RTL to the primary endpoint of DFS were assessed by Cox models. Optimal categories of RTL were developed using cumulative martingale residual plots, designated from shortest to longest RTL (Table 1). Results: Never smokers had longer PBL RTL (p = 0.0006) than past or current smokers. Those with diabetes mellitus (DM) had longer PBL RTL than those without DM (p = 0.0165). PBL RTL was not associated with any tumor markers (MMR status; p = 0.7174; BRAF mutant vs wild-type (wt); p =0.4039; Kras mutant vs wt; p= 0.1149). PBL RTL was significantly associated with DFS in a non-linear form (p = 0.0389). Cases in the longer RTL category had more favorable DFS than those with shorter or longest RTL, (Table), which remained after adjusting for age, sex , T/N stage, grade, tumor markers, and treatment (p = 0.0230). We observed no significant interaction (p = 0.1490) between RTL categories and treatment. Conclusion: We observed a non-linear association between RTL and DFS in stage III CC, suggesting that an optimal range telomere length can influence tumor behavior in CC patients. Future studies of telomere maintenance mechanisms may help to explain the association of RTL with improved DFS. RTL is associated with smoking and DM in CC cases but further studies are needed to determine if either tobacco use or DM affect survival from CC by modulating PBL telomere length. Citation Format: Nivedita Basu, Ruth A. Johnson, Qian Shi, Garth D. Nelson, Julie M. Cunningham, Richard M. Goldberg, Frank A. Sinicrope, Daniel J. Sargent, Steven R. Alberts, Gloria M. Petersen, Stephen N. Thibodeau, Halcyon G. Skinner, Kristin Litzelman, Songwon Seo, Russell J. Vanderboom, Ronald E. Gangnon, Corinne D. Engelman, David N. Rider, Lisa A. Boardman. Telomeres and survival in stage III colon cancers: Findings from NCCTG N0147 (Alliance) study. [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 5102. doi:10.1158/1538-7445.AM2014-5102

Abstract Background: Both shorter and longer telomeres in peripheral blood leukocyte (PBL) DNA have been associated with cancer risk. However, associations remain inconsistent across studies of the same cancer type. This study compares DNA preparation methods to determine telomere length from patients with colorectal cancer. Methods: We examined PBL relative telomere length (RTL) measured by quantitative PCR (qPCR) in 1,033 patients with colorectal cancer and 2,952 healthy controls. DNA was extracted with phenol/chloroform, PureGene, or QIAamp. Results: We observed differences in RTL depending on DNA extraction method (P < 0.001). Phenol/chloroform-extracted DNA had a mean RTL (T/S ratio) of 0.78 (range 0.01–6.54) compared with PureGene-extracted DNA (mean RTL of 0.75; range 0.00–12.33). DNA extracted by QIAamp yielded a mean RTL of 0.38 (range 0.02–3.69). We subsequently compared RTL measured by qPCR from an independent set of 20 colorectal cancer cases and 24 normal controls in PBL DNA extracted by each of the three extraction methods. The range of RTL measured by qPCR from QIAamp-extracted DNA (0.17–0.58) was less than from either PureGene or phenol/chloroform (ranges, 0.04–2.67 and 0.32–2.81, respectively). Conclusions: RTL measured by qPCR from QIAamp-extracted DNA was less than from either PureGene or phenol/chloroform (P < 0.001). Impact: Differences in DNA extraction method may contribute to the discrepancies between studies seeking to find an association between the risk of cancer or other diseases and RTL. Cancer Epidemiol Biomarkers Prev; 22(11); 2047–54. ©2013 AACR.