Xiaoyang Ruan

Colorectal Cancer with Residual Polyp of Origin: A Model of Malignant Transformation

The majority of colorectal cancers (CRCs) arise from adenomatous polyps. In this study, we sought to present the underrecognized CRC with the residual polyp of origin (CRC RPO +) as an entity to be utilized as a model to study colorectal carcinogenesis. We identified all subjects with biopsy-proven CRC RPO + that were evaluated over 10 years at Mayo Clinic, Rochester, MN, and compared their clinical and pathologic characteristics to CRC without remnant polyps (CRC RPO −). Overall survival and disease-free survival overlap with an equivalent hazard ratio between CRC RPO + and RPO − cases when age, stage, and grade are adjusted. The somatic genomic profile obtained by whole genome sequencing and the gene expression profiles by RNA-seq for CRC RPO + tumors were compared with that of age -and gender-matched CRC RPO − evaluated by The Cancer Genome Atlas. CRC RPO + cases were more commonly found with lower-grade, earlier-stage disease than CRC RPO −. However, within the same disease stage and grade, their clinical course is very similar to that of CRC RPO −. The mutation frequencies of commonly mutated genes in CRC are similar between CRC RPO + and RPO − cases. Likewise, gene expression patterns are indistinguishable between the RPO + and RPO − cases. We have confirmed that CRC RPO + is clinically and biologically similar to CRC RPO − and may be utilized as a model of the adenoma to carcinoma transition.

Time Lapse to Colorectal Cancer: Telomere Dynamics Define the Malignant Potential of Polyps

Objective:Whereas few adenomas become cancer, most colorectal cancers arise from adenomas. Telomere length is a recognized biomarker in multiple cancers, and telomere maintenance mechanisms (TMM) are exploited by malignant cells. We sought to determine whether telomere length and TMM distinguish cancer-associated adenomas from those that are cancer-free.Methods:Tissues were identified as cancer-adjacent polyp (CAP)—residual adenoma contiguous with cancer—and cancer-free polyp (CFP)—adenomas without malignancy. Telomere length, TMM, and expression were measured in 102 tissues including peripheral blood leukocytes (PBLs), normal colon epithelium, adenoma, and cancer (in CAP cases) from 31 patients. Telomere length was measured in a separate cohort of 342 PBL from CAP and CFP patients.Results:The mean differences in telomere length between normal and adenoma were greater in CAP than in CFP cases, P=0.001; telomere length in PBL was 91.7 bp greater in CAP than in CFP, P=0.007. Each 100 bp telomere increase was associated with a 1.14 (1.04–1.26) increased odds of being a CAP, P=0.0063. The polyp tissue from CAP patients had shorter telomeres and higher Telomerase reverse transcriptase (hTERT) expression compared with polyps from CFP patients, P=0.05. There was a greater degree of alternative lengthening of telomere (ALT) level difference in CFP polyps than in CAP polyps. The polyp telomere lengths of aggressive CAPs were significantly different from the polyps of non-aggressive CAPs, P=0.01.Conclusions:Adenomas that progress to cancer exhibit distinct telomere length and TMM profiles. We report for the first time that PBL telomeres differ in patients with polyps that become malignant, and therefore may have clinical value in adenoma risk assessment and management.

Genome-Wide Analysis of Loss of Heterozygosity in Breast Infiltrating Ductal Carcinoma Distant Normal Tissue Highlights Arm Specific Enrichment and Expansion across Tumor Stages

Studies have shown concurrent loss of heterozygosity (LOH) in breast infiltrating ductal carcinoma (IDC) and adjacent or distant normal tissue. However, the overall extent of LOH in normal tissue and their significance to tumorigenesis remain unknown, as existing studies are largely based on selected microsatellite markers. Here we present the first autosome-wide study of LOH in IDC and distant normal tissue using informative loci deduced from SNP array-based and sequencing-based techniques. We show a consistently high LOH concurrence rate in IDC (mean = 24%) and distant normal tissue (m = 54%), suggesting for most patients (31/33) histologically normal tissue contains genomic instability that can be a potential marker of increased IDC risk. Concurrent LOH is more frequent in fragile site related genes like WWOX (9/31), NTRK2 (10/31), and FHIT (7/31) than traditional genetic markers like BRCA1 (0/23), BRCA2 (2/29) and TP53 (1/13). Analysis at arm level shows distant normal tissue has low level but non-random enrichment of LOH (topped by 8p and 16q) significantly correlated with matched IDC (Pearson r = 0.66, p = 3.5E-6) (topped by 8p, 11q, 13q, 16q, 17p, and 17q). The arm-specific LOH enrichment was independently observed in tumor samples from 548 IDC patients when stratified by tumor size based T stages. Fine LOH structure from sequencing data indicates LOH in low order tissues non-randomly overlap (∼67%) with LOH that usually has longer tract length (the length of genomic region affected by LOH) in high order tissues. The consistent observations from multiple datasets suggest progressive LOH in the development of IDC potentially through arm-specific pile up effect with discernible signature in normal tissue. Our finding also suggests that LOH detected in IDC by comparing to paired adjacent or distant normal tissue are more likely underestimated.

Comprehensive nucleosome mapping of the human genome in cancer progression.

Altered chromatin structure is a hallmark of cancer, and inappropriate regulation of chromatin structure may represent the origin of transformation. Important studies have mapped human nucleosome distributions genome wide, but the role of chromatin structure in cancer progression has not been addressed. We developed a MNase-Transcription Start Site Sequence Capture method (mTSS-seq) to map the nucleosome distribution at human transcription start sites genome-wide in primary human lung and colon adenocarcinoma tissue. Here, we confirm that nucleosome redistribution is an early, widespread event in lung (LAC) and colon (CRC) adenocarcinoma. These altered nucleosome architectures are consistent between LAC and CRC patient samples indicating that they may serve as important early adenocarcinoma markers. We demonstrate that the nucleosome alterations are driven by the underlying DNA sequence and potentiate transcription factor binding. We conclude that DNA-directed nucleosome redistributions are widespread early in cancer progression. We have proposed an entirely new hierarchical model for chromatin-mediated genome regulation.

Peripheral Neutrophil to Lymphocyte Ratio Improves Prognostication in Colon Cancer

Micro‐Abstract Distinction of patients at a higher mortality risk beyond stage‐only prognostication is an active area of cancer research. Preoperative neutrophil to lymphocyte ratio (NLR) correlates with survival outcomes in colorectal cancer. In this study, the predictive value of NLR in 2536 patients with respectable tumors was compared with the American Joint Committee on Cancer and Memorial Sloan Kettering Cancer Center models. NLR improved the prognostication of these staging systems. Background: We studied the role of peripheral neutrophil to lymphocyte ratio (NLR) on survival outcomes in colon and rectal cancer to determine if its inclusion improved prognostication within existing staging systems. Patients and Methods: Disease‐free (DFS) and overall survival (OS) hazard ratios (HRs) of pretreatment NLR were calculated for 2536 patients with stage I to III colon or rectal cancer and adjusted for age, positive/total number of nodes, T stage, and grade. The association of NLR with clinicopathologic features and survival was evaluated and compared with the American Joint Committee on cancer (AJCC) TNM staging and Memorial Sloan Kettering Cancer Center (MSKCC) models. Results: High NLR was significantly associated with worse DFS (HR, 1.36; 95% confidence interval [CI], 1.08‐1.70; P = .009) and OS (HR, 1.65; 95% CI, 1.29‐2.10; P < .0005) in all stages for patients with colon, but not rectal, cancer. High NLR was significantly associated with site‐specific worse prognosis, which was stronger in the left versus right colon; an inverse relationship with grade was found. The impact of high NLR on DFS and OS occurred early, with the majority of deaths within 2 years following surgery. Adjusted HRs for 5‐year and 2‐year outcomes in colon cancer per each additional 2‐unit increase in NLR were 1.15 (95% CI, 1.08‐1.23) and 1.20 (95% CI, 1.10‐1.30), respectively. The addition of NLR enhanced the prognostic utility of TNM (TNM alone vs. TNM + NLR: concordance index, 0.60 vs. 0.68), and MSKCC (MSKCC alone vs. MSKCC + NLR: concordance index, 0.71 vs. 0.73) models for colon cancer patients. Conclusion: NLR is an independent prognostic variable for nonmetastatic colon cancer that enhances existing clinical staging systems.

Early genetic aberrations in patients with sporadic colorectal cancer

Chromosome instability (CIN) is widely observed in both sporadic and hereditary colorectal cancer (CRC). Defects in APC and WNT signaling are primarily associated with CIN in hereditary CRC, but the genetic causes for CIN in sporadic CRC remain elusive. Using high‐density SNP array and exome data from The Cancer Genome Atlas (TCGA), we characterized loss of heterozygosity (LOH) and copy number variation (CNV) in the peripheral blood, normal colon, and corresponding tumor tissue in 15 CRC patients with proficient mismatch repair (MMR) and 24 CRC patients with deficient MMR. We found a high frequency of 18q LOH in tumors and arm‐specific enrichment of genetic aberrations on 18q in the normal colon (primarily copy neutral LOH) and blood (primarily copy gain). These aberrations were specific to the sporadic, pMMR CRC. Though in tumor samples genetic aberrations were observed for genes commonly mutated in hereditary CRC (eg, APC, CTNNB1, SMAD4, BRAF), none of them showed LOH or CNV in the normal colon or blood. DCC located on 18q21.1 topped the list of genes with genetic aberrations in the tumor. In an independent cohort of 13 patients subjected to Whole Genome Sequencing (WGS), we found LOH and CNV on 18q in adenomatous polyp and tumor tissues. Our data suggests that patients with sporadic CRC may have genetic aberrations preferentially enriched on 18q in their blood, normal colon epithelium, and non‐malignant polyp lesions that may prove useful as a clinical marker for sporadic CRC detection and risk assessment.

Abstract B1-46: How collection, processing and storage of PBL samples impact the measurement of telomerase activity

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 B2-04: Time lapse to cancer: Defining the transition from polyp to colorectal cancer based on telomere metrics

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.