Deborah W. Neklason

Hereditary and Familial Colon Cancer

Between 2% to 5% of all colon cancers arise in the setting of well-defined inherited syndromes, including Lynch syndrome, familial adenomatous polyposis, MUTYH-associated polyposis, and certain hamartomatous polyposis conditions. Each is associated with a high risk of colon cancer. In addition to the syndromes, up to one-third of colon cancers exhibit increased familial risk, likely related to inheritance. A number of less penetrant, but possibly more frequent susceptibility genes have been identified for this level of inheritance. Clarification of predisposing genes allows for accurate risk assessment and more precise screening approaches. This review examines the colon cancer syndromes, their genetics and management, and also the common familial colon cancers with current genetic advances and screening guidelines.

Role of Multidrug Resistance P-glycoproteins in Cholesterol Esterification

Cholesterol esterification, catalyzed by acyl-CoA:cholesterol acyltransferase (ACAT), plays a central role in cellular cholesterol homeostasis and in physiologic processes that lead to coronary heart disease. Although ACAT resides in the endoplasmic reticulum (ER), the cholesterol substrate for esterification originates in the plasma membrane and must be transported to the ER for esterification. Progesterone inhibits esterification, possibly by blocking the transport of cholesterol to the ER. Recent studies suggest that progesterone acts by inhibiting the activity of one or more of the multidrug-resistant (MDR) P-glycoproteins. In the current manuscript, we demonstrate that progesterones ability to inhibit esterification is not mediated through the progesterone receptor. We evaluate a series of steroid hormones and find a strong correlation between a steroid hormones hydrophobicity and its ability to inhibit both cholesterol esterification and MDR-catalyzed drug efflux. We also find that cholesterol esterification is inhibited by nonsteroidal MDR inhibitors, and that this inhibition specifically affects the esterification of cholesterol derived from the plasma membrane. MDR inhibitors also inhibit cholesterol esterification in a wide range of cultured human cell lines. These observations suggest that MDR activity normally functions in a general process of intracellular cholesterol transport.

Maximum-likelihood estimation of recent shared ancestry (ERSA)

Accurate estimation of recent shared ancestry is important for genetics, evolution, medicine, conservation biology, and forensics. Established methods estimate kinship accurately for first-degree through third-degree relatives. We demonstrate that chromosomal segments shared by two individuals due to identity by descent (IBD) provide much additional information about shared ancestry. We developed a maximum-likelihood method for the estimation of recent shared ancestry (ERSA) from the number and lengths of IBD segments derived from high-density SNP or whole-genome sequence data. We used ERSA to estimate relationships from SNP genotypes in 169 individuals from three large, well-defined human pedigrees. ERSA is accurate to within one degree of relationship for 97% of first-degree through fifth-degree relatives and 80% of sixth-degree and seventh-degree relatives. We demonstrate that ERSAs statistical power approaches the maximum theoretical limit imposed by the fact that distant relatives frequently share no DNA through a common ancestor. ERSA greatly expands the range of relationships that can be estimated from genetic data and is implemented in a freely available software package.

Effect of Sulindac and Erlotinib vs Placebo on Duodenal Neoplasia in Familial Adenomatous Polyposis: A Randomized Clinical Trial

IMPORTANCE Patients with familial adenomatous polyposis (FAP) are at markedly increased risk for duodenal polyps and cancer. Surgical and endoscopic management of duodenal neoplasia is difficult and chemoprevention has not been successful. OBJECTIVE To evaluate the effect of a combination of sulindac and erlotinib on duodenal adenoma regression in patients with FAP. DESIGN, SETTING, AND PARTICIPANTS Double-blind, randomized, placebo-controlled trial, enrolling 92 participants with FAP, conducted from July 2010 through June 2014 at Huntsman Cancer Institute in Salt Lake City, Utah. INTERVENTIONS Participants with FAP were randomized to sulindac (150 mg) twice daily and erlotinib (75 mg) daily (n = 46) vs placebo (n = 46) for 6 months. MAIN OUTCOMES AND MEASURES The total number and diameter of polyps in the proximal duodenum were mapped at baseline and 6 months. The primary outcome was change in total polyp burden at 6 months. Polyp burden was calculated as the sum of the diameters of polyps. The secondary outcomes were change in total duodenal polyp count, change in duodenal polyp burden or count stratified by genotype and initial polyp burden, and percentage of change from baseline in duodenal polyp burden. RESULTS Ninety-two participants (mean age, 41 years [range, 24-55]; women, 56 [61%]) were randomized when the trial was stopped by the external data and safety monitoring board because the second preplanned interim analysis met the prespecified stopping rule for superiority. Grade 1 and 2 adverse events were more common in the sulindac-erlotinib group, with an acne-like rash observed in 87% of participants receiving treatment and 20% of participants receiving placebo (P < .001). Only 2 participants experienced grade 3 adverse events. [table: see text]. CONCLUSIONS AND RELEVANCE Among participants with FAP, the use of sulindac and erlotinib compared with placebo resulted in a lower duodenal polyp burden after 6 months. Adverse events may limit the use of these medications at the doses used in this study. Further research is necessary to evaluate these preliminary findings in a larger study population with longer follow-up to determine whether the observed effects will result in improved clinical outcomes. TRIAL REGISTRATION clinicaltrials.gov Identifier: NCT 01187901.

Common familial colorectal cancer linked to chromosome 7q31: a genome-wide analysis.

Present investigations suggest that approximately 30% of colorectal cancer cases arise on the basis of inherited factors. We hypothesize that the majority of inherited factors are moderately penetrant genes, common in the population. We use an affected sibling pair approach to identify genetic regions that are coinherited by siblings with colorectal cancer. Individuals from families with at least two siblings diagnosed with colorectal adenocarcinoma or high-grade dysplasia were enrolled. Known familial colorectal cancer syndromes were excluded. A genome-wide scan on 151 DNA samples from 70 kindreds was completed using deCODE 1100 short tandem repeat marker set at an average 4-cM density. Fine mapping on a total of 184 DNAs from 83 kindreds was done in regions suggesting linkage. Linkage analysis was accomplished with Merlin analysis package. Nonparametric linkage analysis revealed three genetic regions with logarithm of the odds (LOD) scores >or=2.0: Ch. 3q29, LOD 2.61 (P = 0.0003); Ch. 4q31.3, LOD 2.13 (P = 0.0009); and Ch. 7q31.31, LOD 3.08 (P = 0.00008). Affected siblings with increased sharing at the 7q31 locus have a 3.8-year (+/- 3.5) earlier age of colorectal cancer onset although this is not statistically significant (P = 0.11). No significant linkage was found near genes causing known syndromes or regions previously reported (8q24, 9q22, and 11q23). The chromosome 3q21-q24 region reported to be linked in colorectal cancer relative pairs is supported by our study, albeit a minor peak (LOD 0.9; P = 0.02). No known familial cancer genes reside in the 7q31 locus, and thus the identified region may contain a novel susceptibility gene responsible for common familial colorectal cancer.

Intron 4 Mutation in APC Gene Results in Splice Defect and Attenuated FAP Phenotype

The adenomatous polyposis coli (APC) protein is a tumor suppressor frequently involved in the development of inherited and sporadic colon cancers. Somatic mutations of the APC gene are found in 80% of all colon cancers. Inherited mutations result in familial adenomatous polyposis (FAP) as well as an attenuated form of this syndrome. FAP is characterized by the early age onset of hundreds to thousands of colonic adenomatous polyps and a virtual certainty of colon cancer unless the colon is removed. The attenuated form of FAP (AFAP) is characterized by fewer adenomas, later onset of adenomas and cancer, and a decreased lifetime cancer risk. We report a 37-year-old man with a history of more than 50 colonic adenomatous polyps, located predominately in the right colon. An insertion of a single thymidine between the second and third base pairs of intron 4 of the APC gene was identified (c.531+2_531+3insT). Monoallelic hybrid cells harboring a single copy of human chromosome 5 were generated from patient lymphoblasts. Sequencing of the APC cDNA product from these cells revealed a single RNA transcript with aberrant splicing in the mutant mRNA whereby exon 4 is deleted. The translational reading frame is shifted after codon 140 and a translational stop is generated predicting a truncated protein of 147 amino acids, thus indicating that the intronic mutation is disease causing. The lack of a secondary transcript from the mutant allele suggests that incomplete exon skipping is not the molecular mechanism behind the attenuated phenotype.

Activating mutation in MET oncogene in familial colorectal cancer

BackgroundIn developed countries, the lifetime risk of developing colorectal cancer (CRC) is 5%, and it is the second leading cause of death from cancer. The presence of family history is a well established risk factor with 25-35% of CRCs attributable to inherited and/or familial factors. The highly penetrant inherited colon cancer syndromes account for approximately 5%, leaving greater than 20% without clear genetic definition. Familial colorectal cancer has been linked to chromosome 7q31 by multiple affected relative pair studies. The MET proto-oncogene which resides in this chromosomal region is considered a candidate for genetic susceptibility.MethodsMET exons were amplified by PCR from germline DNA of 148 affected sibling pairs with colorectal cancer. Amplicons with altered sequence were detected with high-resolution melt-curve analysis using a LightScanner (Idaho Technologies). Samples demonstrating alternative melt curves were sequenced. A TaqMan assay for the specific c.2975C > T change was used to confirm this mutation in a cohort of 299 colorectal cancer cases and to look for allelic amplification in tumors.ResultsHere we report a germline non-synonymous change in the MET proto-oncogene at amino acid position T992I (also reported as MET p.T1010I) in 5.2% of a cohort of sibling pairs affected with CRC. This genetic variant was then confirmed in a second cohort of individuals diagnosed with CRC and having a first degree relative with CRC at prevalence of 4.1%. This mutation has been reported in cancer cells of multiple origins, including 2.5% of colon cancers, and in <1% in the general population. The threonine at amino acid position 992 lies in the tyrosine kinase domain of MET and a change to isoleucine at this position has been shown to promote metastatic behavior in cell-based models. The average age of CRC diagnosis in patients in this study is 63 years in mutation carriers, which is 8 years earlier than the general population average for CRC.ConclusionsAlthough the MET p.T992I genetic mutation is commonly found in somatic colorectal cancer tissues, this is the first report also implicating this MET genetic mutation as a germline inherited risk factor for familial colorectal cancer. Future studies on the cancer risks associated with this mutation and the prevalence in different at-risk populations will be an important extension of this work to define the clinical significance.

RNA Sequencing of Sessile Serrated Colon Polyps Identifies Differentially Expressed Genes and Immunohistochemical Markers

Background Sessile serrated adenomas/polyps (SSA/Ps) may account for 20–30% of colon cancers. Although large SSA/Ps are generally recognized phenotypically, small (<1 cm) or dysplastic SSA/Ps are difficult to differentiate from hyperplastic or small adenomatous polyps by endoscopy and histopathology. Our aim was to define the comprehensive gene expression phenotype of SSA/Ps to better define this cancer precursor. Results RNA sequencing was performed on 5′ capped RNA from seven SSA/Ps collected from patients with the serrated polyposis syndrome (SPS) versus eight controls. Highly expressed genes were analyzed by qPCR in additional SSA/Ps, adenomas and controls. The cellular localization and level of gene products were examined by immunohistochemistry in syndromic and sporadic SSA/Ps, adenomatous and hyperplastic polyps and controls. We identified 1,294 differentially expressed annotated genes, with 106 increased ≥10-fold, in SSA/Ps compared to controls. Comparing these genes with an array dataset for adenomatous polyps identified 30 protein coding genes uniquely expressed ≥10-fold in SSA/Ps. Biological pathways altered in SSA/Ps included mucosal integrity, cell adhesion, and cell development. Marked increased expression of MUC17, the cell junction protein genes VSIG1 and GJB5, and the antiapoptotic gene REG4 were found in SSA/Ps, relative to controls and adenomas, were verified by qPCR analysis of additional SSA/Ps (n = 21) and adenomas (n = 10). Immunohistochemical staining of syndromic (n≥11) and sporadic SSA/Ps (n≥17), adenomatous (n≥13) and hyperplastic (n≥10) polyps plus controls (n≥16) identified unique expression patterns for VSIG1 and MUC17 in SSA/Ps. Conclusion A subset of genes and pathways are uniquely increased in SSA/Ps, compared to adenomatous polyps, thus supporting the concept that cancer develops by different pathways in these phenotypically distinct polyps with markedly different gene expression profiles. Immunostaining for a subset of these genes differentiates both syndromic and sporadic SSA/Ps from adenomatous and hyperplastic polyps.

Colonic Adenoma Risk in Familial Colorectal Cancer – a Study of Six Extended Kindreds

OBJECTIVES:Most colorectal cancers (CRCs) arise from adenomatous polyps, but the effects of CRC family history on adenoma risk are not well known. This issue is clinically relevant since several medical societies currently recommend earlier and more rigorous colorectal screening for individuals with a strong family history of CRC.METHODS:Colonoscopies were performed in 236 first-, second-, and third-degree relatives of 40 index CRC cases from six large kindreds selected from a large population database. The kindreds were selected for significantly greater risk of CRCs compared with the overall population. Known hereditary colon cancer syndromes were clinically and genetically excluded.RESULTS:Thirty-seven percent of relatives were found to have adenomas on colonoscopy. The average age of diagnosis for colon cancer was 63 yr and advanced adenomas 56 yr. Independent predictors of adenomatous polyps in the relatives were advancing age (P < 0.0001), male gender (P < 0.001), and greater degree of relation to CRC cases (P < 0.01). There was no significant predilection of colorectal tumors for the right or left colon. A higher degree of relationship to CRC cases was a significant predictor of having simple and advanced adenomas, but not hyperplastic polyps after adjustment for age and gender.CONCLUSIONS:These data support the current recommendations for colonoscopy starting before the age of 50 yr in individuals with a strong family history of CRC.

Disease-associated casein kinase I δ mutation may promote adenomatous polyps formation via a Wnt/β-catenin independent mechanism

The Wnt signaling pathway is critical for embryonic development and is dysregulated in multiple cancers. Two closely related isoforms of casein kinase I (CKIδ and ϵ) are positive regulators of this pathway. We speculated that mutations in the autoinhibitory domain of CKIδ/ϵ might upregulate CKIδ/ϵ activity and hence Wnt signaling and increase the risk of adenomatous polyps and colon cancer. Exons encoding the CKIϵ and CKIδ regulatory domains were sequenced from DNA obtained from individuals with adenomatous polyps and a family history of colon cancer unaffected by familial adenomatous polyposis or hereditary nonpolyposis colorectal cancer (HNPCC). A CKIδ missense mutation, changing a highly conserved residue, Arg324, to His (R324H), was found in an individual with large and multiple polyps diagnosed at a relatively young age. Two findings indicate that this mutation is biologically active. First, ectopic ventral expression of CKIδ(R324H) in Xenopus embryos results in secondary axis formation with an additional distinctive phenotype (altered morphological movements) similar to that seen with unregulated CKIϵ. Second, CKIδ(R324H) is more potent than wildtype CKIδ in transformation of RKO colon cancer cells. Although the R324H mutation does not significantly change CKIδ kinase activity in an in vitro kinase assay or Wnt/β‐catenin signal transduction as assessed by a β‐catenin reporter assay, it alters morphogenetic movements via a β‐catenin‐independent mechanism in early Xenopus development. This novel human CKIδ mutation may alter the physiological role and enhance the transforming ability of CKIδ through a Wnt/β‐catenin independent mechanism and thereby influence colonic adenoma development.