Bruce M. Brenner

Genomic instability measurement in the diagnosis of thyroid neoplasms

Clinically palpable thyroid nodules are present in approximately 10% of the population, although only 5% to 7% of these nodules harbor malignancy. Fine‐needle aspiration has become one of the central tools in the diagnostic armamentarium of the surgeon/endocrinologist. There is, however, up to a 30% indeterminate diagnostic rate associated with this technique, resulting in unnecessary surgical interventions for patients harboring benign disease. A second issue of clinical importance is the unreliability of predicting outcomes based either on histologic findings alone or in combination with clinical staging. To address these diagnostic and clinical shortcomings, we have used measurement of genomic instability as a diagnostic and prognostic indicator for thyroid neoplasms.

Increased Frequency of Serrated Aberrant Crypt Foci Among Smokers

OBJECTIVES:The American College of Gastroenterology has published guidelines recently that suggest that smokers with a history of >20 pack years may need screening for colorectal cancer (CRC) at an earlier age than non-smokers. Aberrant crypt foci (ACF) may represent important precursors for colorectal neoplasms and potential surrogate biomarkers. Clarifying the role of ACF in relation to known CRC risk factors such as smoking may have important implications for screening as well as our understanding of tobacco use and colorectal carcinogenesis. Our goal was to examine whether smoking at least 20 pack years was associated with an increased frequency of ACF.METHODS:We gathered detailed smoking history, personal and family history of CRC, and other epidemiologic data (age, gender, height, weight, ethnicity, and medication use) from 125 patients undergoing routine screening or surveillance colonoscopy. We used a magnifying colonoscope (Olympus Close Focus Colonoscope XCF-Q160ALE, Olympus Corporation, Tokyo, Japan) and examined the distal 20 cm section of colon after staining with 0.5% methylene blue. ACF were counted and characterized histologically. Hyperplastic ACF were further characterized as either serrated or non-serrated.RESULTS:Smoking at least 20 pack years was associated with an increased likelihood (adjusted odds ratio (OR)=3.45; 95% confidence interval (CI)=1.93–6.18) of having more than the median number of ACF (≥15) compared with non-smokers. Similarly, patients with a personal history of advanced neoplasia were more likely (adjusted OR=3.42; 95% CI=1.01–11.67) to have a greater than median number of ACF compared with patients without this diagnosis. Smokers were more likely than non-smokers to have serrated ACF (P=0.002).CONCLUSIONS:Smoking at least 20 pack years seems to be associated with increased number of ACF in the rectum and distal sigmoid, especially those with serrated histology. Our data support ACG guidelines for earlier screening for CRC among smokers and add to our understanding of how colorectal carcinogenesis is related to tobacco use.

Vitamin D resistance and colon cancer prevention

Observational studies have been largely consistent in showing an inverse association between vitamin D and an individuals risk of developing colorectal cancer. Vitamin D protection is further supported by a range of preclinical colon cancer models, including carcinogen, genetic and dietary models. A large number of mechanistic studies in both humans and rodents point to vitamin D preventing cancer by regulating cell proliferation. Counterbalancing this mostly positive data are the results of human intervention studies in which supplemental vitamin D was found to be ineffective for reducing colon cancer risk. One explanation for these discrepancies is the timing of vitamin D intervention. It is possible that colon lesions may progress to a stage where they become unresponsive to vitamin D. Such a somatic loss in vitamin D responsiveness bears the hallmarks of an epigenetic change. Here, we review data supporting the chemopreventive effectiveness of vitamin D and discuss how gene silencing and other molecular changes somatically acquired during colon cancer development may limit the protection that may otherwise be afforded by vitamin D via dietary intervention. Finally, we discuss how understanding the mechanisms by which vitamin D protection is lost might be used to devise strategies to enhance its chemopreventive actions.

High-throughput SNP/CGH approaches for the analysis of genomic instability in colorectal cancer.

Colorectal cancer remains a major cancer killer in the US and worldwide. A better understanding of colorectal carcinogenesis may lead to an improved ability to diagnose, treat, and even prevent this disease. Since a genetic model for colorectal cancer was first proposed significant strides have been made in defining the genomic events that occur during colorectal carcinogenesis. Numerous tumor suppressor genes and oncogenes are clearly involved in this process, which is associated with genomic instability and a mutator phenotype. Newer, high-throughput techniques have accelerated our ability to identify and characterize critical genomic events that contribute to this process. The most commonly used of these techniques include array-comparative genomic hybridization and single nucleotide polymorphism arrays. These technologies allow the entire genome to be mapped at high resolution using tens or even hundreds of thousands of markers in a single experiment. These and other high-throughput genomic techniques are also beginning to be applied to patient care in predicting prognosis and response to treatment in patients with colorectal cancer. The development of Next-generation sequencing technologies that can quickly sequence an entire tumor genome will certainly lead to even more advances in our understanding of colorectal carcinogenesis and our ability to diagnose, prevent and treat this disease.

Regulation of VDR Expression in Apc-Mutant Mice, Human Colon Cancers and Adenomas

One variable that may affect the ability of vitamin D to reduce colon cancer risk is the expression of its high-affinity receptor, VDR. Here, we show that vitamin D does not reduce tumor formation in ApcΔ14/+ mice and that VDR expression is lost in the majority of the colon tumor cells. The extent of VDR loss corresponded inversely to the level of β-catenin nuclear localization and could be observed in early lesions composed of just a few crypts. Analysis of reported VDR regulators showed that the repressing class I histone deacetylases (HDAC) were significantly elevated in the tumors (up to 4-fold), whereas the VDR-activating retinoid X receptors (RXR) were downregulated (∼50%). Expression of the Slug repressor was also increased, but was found primarily in stromal cells. Analysis of epigenetically active compounds on colon cell lines and intestinal organoids showed that HDAC inhibitors were particularly adept at stimulating VDR expression. Treatment of tumor-bearing ApcΔ14/+ mice with the HDAC inhibitor panobinostat increased VDR expression in the tumors and normal mucosa. The RXR agonist bexarotene failed to activate VDR expression, indicating that RXR ligands were not limiting. Analysis of human microarray data indicated that VDR mRNA is frequently downregulated in colon adenomas, which correlated positively with RXRA expression and inversely with HDAC 2 and 8 expression. Human adenomas showed variable VDR protein expression levels, both between and within individual lesions. Determining the mechanisms of VDR regulation in colon neoplasms may significantly enhance our ability to use vitamin D as a cancer prevention agent. Cancer Prev Res; 8(5); 387–99. ©2015 AACR.

Proximal Aberrant Crypt Foci Associate with Synchronous Neoplasia and Are Primed for Neoplastic Progression

Aberrant crypt foci (ACF) are the earliest morphologically identifiable lesion found within the human colon. Despite their relatively high frequency in the distal colon, few studies have examined the molecular characteristics of ACF within the proximal colon. In the following study, clinical participants (n = 184) were screened for ACF using high-definition chromoendoscopy with contrast dye-spray. Following pathologic confirmation, ACF biopsies were subjected to laser capture microdissection (LCM), and epithelial cells were evaluated for somatic mutations with a customized colorectal cancer mutation panel using DNA-mass spectrometry. Samples were further characterized for microsatellite instability (MSI). Logistic models were used to associate proximal ACF with synchronous (detected during the same procedure) neoplasia. Thirty-nine percent of participants had at least one histologically confirmed proximal ACF. Individuals with a proximal ACF were significantly more likely to present with a synchronous neoplasm (P = 0.001), and specifically, a proximal, tubular, or tubulovillous adenoma (multivariable OR = 2.69; 95% confidence interval, 1.12–6.47; P = 0.027). Proximal ACF were more likely to be dysplastic (52%) compared with distal ACF (13%; P < 0.0001). Somatic mutations to APC, BRAF, KRAS, NRAS, and ERBB2 were detected in 37% of proximal ACF. Hyperplastic ACF were more often MSI-high, but there were no differences in MSI status observed by colonic location. In summary, ACF are identified in the proximal colons of approximately 40% of individuals undergoing chromoendoscopy and more often in patients with synchronous proximal adenomas. Implications: This study provides the most complete set of data, to date, that ACF represent the earliest step in the adenoma–carcinoma sequence but remain below the detection limit of conventional endoscopy. Visual Overview: http//mcr.accrjournals.org/content/molcanres/16/3/486/F1.large.jpg. Mol Cancer Res; 16(3); 486–95. ©2017 AACR. Visual Overview

Genomic Instability Measured by Inter-(Simple Sequence Repeat) PCR and High-Resolution Microsatellite Instability are Prognostic of Colorectal Carcinoma Survival After Surgical Resection

BackgroundDuring the multiyear progression to colorectal cancer, numerous genomic alterations arise in events ranging from single base mutations to gains or losses of entire chromosomes. A single genetic change might not stand out as an independent predictor of outcome. The goal of this study was to determine if more comprehensive measurements of genomic instability provide clinically relevant prognostic information.MethodsOur study included 65 sporadic colorectal cancer patients diagnosed from 1987 to 1991 with last follow-up ascertained in 2006. We estimated an overall tally of alterations using the genome-wide sampling technique of inter-(simple sequence repeat [SSR]) polymerase chain reaction (PCR), and evaluated its relationship with all-cause survival. We also extended and sensitized the Bethesda criteria for microsatellite instability (MSI), by analyzing 348 microsatellite markers instead of the normal five. We expanded the MSI categories into four levels: MSI stable (MSS), very low-level MSI, moderately low-level MSI, and classical high-level MSI.ResultsTumors with genomic instability above the median value of 2.6% as measured by inter-SSR PCR, were associated with far greater risk of death compared to tumors with lower levels of genomic instability. Adverse outcome was most pronounced for patients presenting with stage 3 disease. A gradient of increased survival was observed across increasing MSI levels but did not reach statistical significance.ConclusionOur findings suggest genomic instabilities quantified by inter-SSR PCR and increased precision in MSI values may be clinically useful tools for estimating prognosis in colorectal cancer.

Gallbladder cancer gene hypermethylation: genetics or environment?

The article by House et al.1 documents the progression of tumor suppressor gene hypermethylation leading to gallbladder cancer. Aberrant methylation was present in 7% of normal gallbladders, 28% of gallbladders with chronic cholecystitis secondary to cholelithiasis, and 72% with cancer. Hypermethylation occurred with equal frequency in cancers from the United States (Johns Hopkins Medical Institutions) and from Chile (Pontificia Universidad Catolica de Chile). The most commonly methylated tumor suppressor genes were p16 (56%; 50% United States, 60% Chile), p73 (28%; 13% United States, 40% Chile, P .04), and APC (26%; 42% United States, 14% Chile, P .03). As with most good studies, this article asks more questions than it answers. Perhaps the most interesting question raised by this analysis is whether the differences observed between the tumors from the United States and Chile are genetic or environmental. Surprisingly, the manuscript by House et al.1 provides no information about the race of the patients. The patients with tumors from the United States and Chile were the same age (median, 60 years) and gender (female, 70%), and were reasonably well matched by tumor stage. Presumably, most of the patients in the Johns Hopkins group were white, but some may have been black, Asian, or Hispanic. Similarly, the Chilean patients may have been Amerindian, Hispanic, or Maori. A subanalysis comparing gene methylation between whites and Amerindians would have been interesting. The relatively small numbers of patients in this type of subanalysis, however, may have prevented the authors from drawing any meaningful conclusions. The inclusion of patients with normal gallbladders and with chronic cholecystitis secondary to cholelithiasis in the study by House et al.1 was important. Although most of these “control’’ patients, as those with cancer, were female; they were significantly younger, and they were all from Chile. One group that could have been chosen as “normal’’ controls from the United States were elderly patients with colorectal liver metastases requiring cholecystectomy at the time of liver resection. Similarly, inclusion of elderly patients having cholecystectomy for chronic cholecystitis and cholelithiasis from the United States would have enhanced the analysis. The statistically significant difference in APC and p73 gene methylation observed by House et al.1 between tumors from the United States and Chile suggests that genetic differences may have led to the unique tumor biology. Recent studies of several common polygenic disorders, however, suggest that approximately one third of these diseases are genetic, whereas two thirds are environmental.2 This relative contribution of genetics and environment probably also applies to many cancers. Thus, another interesting question raised by House et al.1 is what environmental factors may have differed between their patients from the United States and those from Chile. Environmental candidates include diet, chemicals, radionuclides, and the specifics of gallbladder inflammation. Multiple dietary factors can play a role in the pathogenesis of biliary as well as other gastrointestinal malignancies. Nitrosamines have been implicated in inhabitants of northeast Thailand with biliary malignancies and in animal models, but probably are not major carcinogens in the United States or Chile.3 Capsaicin has been suggested as a potential dietary carcinogen in Chilean gallbladder cancer patients, whereas the high fat Western diet common in the United States may be an important factor in both gallstone formation and in the development of a number of malignancies. Several chemicals, Received July 23, 2003; accepted August 22, 2003. From the Department of Surgery, Medical College of Wisconsin, Milwaukee, Wisconsin. Address correspondence to: Henry A. Pitt, MD, Department of Surgery, Medical College of Wisconsin, 9200 West Wisconsin Ave., Milwaukee, WI 53226; Fax: 414-259-9225; E-mail: hapitt@mcw.edu.