Dietary Supplement Use and Colorectal Adenoma Risk in Individuals with Lynch Syndrome: The GEOLynch Cohort Study

Background and Aims

Individuals with Lynch syndrome have a high lifetime risk of developing colorectal tumors. In this prospective cohort study of individuals with Lynch syndrome, we examined associations between use of dietary supplements and occurrence of colorectal adenomas.

Materials and Methods

Using data of 470 individuals with Lynch syndrome in a prospective cohort study, associations between dietary supplement use and colorectal adenoma risk were evaluated by calculating hazard ratios (HR) and 95% confidence intervals (CI) using cox regression models adjusted for age, sex, and number of colonoscopies during person time. Robust sandwich covariance estimation was used to account for dependency within families.

Results

Of the 470 mismatch repair gene mutation carriers, 122 (26.0%) developed a colorectal adenoma during an overall median person time of 39.1 months. 40% of the study population used a dietary supplement. Use of any dietary supplement was not statistically significantly associated with colorectal adenoma risk (HR = 1.18; 95%CI 0.80–1.73). Multivitamin supplement use (HR = 1.15; 95%CI 0.72–1.84), vitamin C supplement use (HR = 1.57; 95%CI 0.93–2.63), calcium supplement use (HR = 0.69; 95%CI 0.25–1.92), and supplements containing fish oil (HR = 1.60; 95%CI 0.79–3.23) were also not associated with occurrence of colorectal adenomas.

Conclusion

This prospective cohort study does not show inverse associations between dietary supplement use and occurrence of colorectal adenomas among individuals with Lynch syndrome. Further research is warranted to determine whether or not dietary supplement use is associated to colorectal adenoma and colorectal cancer risk in MMR gene mutation carriers.     

Distinct Gene Expression Signatures in Lynch Syndrome and Familial Colorectal Cancer Type X

Introduction

Heredity is estimated to cause at least 20% of colorectal cancer. The hereditary nonpolyposis colorectal cancer subset is divided into Lynch syndrome and familial colorectal cancer type X (FCCTX) based on presence of mismatch repair (MMR) gene defects.

Purpose

We addressed the gene expression signatures in colorectal cancer linked to Lynch syndrome and FCCTX with the aim to identify candidate genes and to map signaling pathways relevant in hereditary colorectal carcinogenesis.

Experimental design

The 18 k whole-genome c-DNA-mediated annealing, selection, extension, and ligation (WG-DASL) assay was applied to 123 colorectal cancers, including 39 Lynch syndrome tumors and 37 FCCTX tumors. Target genes were technically validated using real-time quantitative RT-PCR (qRT-PCR) and the expression signature was validated in independent datasets.

Results

Colorectal cancers linked to Lynch syndrome and FCCTX showed distinct gene expression profiles, which by significance analysis of microarrays (SAM) differed by 2188 genes. Functional pathways involved were related to G-protein coupled receptor signaling, oxidative phosphorylation, and cell cycle function and mitosis. qRT-PCR verified altered expression of the selected genes NDUFA9, AXIN2, MYC, DNA2 and H2AFZ. Application of the 2188-gene signature to independent datasets showed strong correlation to MMR status.

Conclusion

Distinct genetic profiles and deregulation of different canonical pathways apply to Lynch syndrome and FCCTX and key targets herein may be relevant to pursue for refined diagnostic and therapeutic strategies in hereditary colorectal cancer.     

Reduced Intestinal Tumorigenesis in APCmin Mice Lacking Melanin-Concentrating Hormone

Background

Melanin-concentrating hormone (MCH) is an evolutionary conserved hypothalamic neuropeptide that in mammals primarily regulates appetite and energy balance. We have recently identified a novel role for MCH in intestinal inflammation by demonstrating attenuated experimental colitis in MCH deficient mice or wild type mice treated with an anti-MCH antibody. Therefore, targeting MCH has been proposed for the treatment of inflammatory bowel disease. Given the link between chronic intestinal inflammation and colorectal cancer, in the present study we sought to investigate whether blocking MCH might have effects on intestinal tumorigenesis that are independent of inflammation.

Methodology

Tumor development was evaluated in MCH-deficient mice crossed to the APCmin mice which develop spontaneously intestinal adenomas. A different cohort of MCH−/− and MCH+/+ mice in the APCmin background was treated with dextran sodium sulphate (DSS) to induce inflammation-dependent colorectal tumors. In Caco2 human colorectal adenocarcinoma cells, the role of MCH on cell survival, proliferation and apoptosis was investigated.

Results

APCmin mice lacking MCH developed fewer, smaller and less dysplastic tumors in the intestine and colon which at the molecular level are characterized by attenuated activation of the wnt/beta-catenin signaling pathway and increased apoptotic indices. Form a mechanistic point of view, MCH increased the survival of colonic adenocarcinoma Caco2 cells via inhibiting apoptosis, consistent with the mouse studies.

Conclusion

In addition to modulating inflammation, MCH was found to promote intestinal tumorigenesis at least in part by inhibiting epithelial cell apoptosis. Thereby, blocking MCH as a therapeutic approach is expected to decrease the risk for colorectal cancer.     

High DNA methylation pattern intratumoral diversity implies weak selection in many human colorectal cancers

Background

It is possible to infer the past of populations by comparing genomes between individuals. In general, older populations have more genomic diversity than younger populations. The force of selection can also be inferred from population diversity. If selection is strong and frequently eliminates less fit variants, diversity will be limited because new, initially homogeneous populations constantly emerge.

Methodology and Results

Here we translate a population genetics approach to human somatic cancer cell populations by measuring genomic diversity within and between small colorectal cancer (CRC) glands. Control tissue culture and xenograft experiments demonstrate that the population diversity of certain passenger DNA methylation patterns is reduced after cloning but subsequently increases with time. When measured in CRC gland populations, passenger methylation diversity from different parts of nine CRCs was relatively high and uniform, consistent with older, stable lineages rather than mixtures of younger homogeneous populations arising from frequent cycles of selection. The diversity of six metastases was also high, suggesting dissemination early after transformation. Diversity was lower in DNA mismatch repair deficient CRC glands, possibly suggesting more selection and the elimination of less fit variants when mutation rates are elevated.

Conclusion/Significance

The many hitchhiking passenger variants observed in primary and metastatic CRC cell populations are consistent with relatively old populations, suggesting that clonal evolution leading to selective sweeps may be rare after transformation. Selection in human cancers appears to be a weaker than presumed force after transformation, consistent with the observed rarity of driver mutations in cancer genomes. Phenotypic plasticity rather than the stepwise acquisition of new driver mutations may better account for the many different phenotypes within human tumors.     

Comparative sequencing analysis reveals high genomic concordance between matched primary and metastatic colorectal cancer lesions

Background

Colorectal cancer is the second leading cause of cancer death in the United States, with over 50,000 deaths estimated in 2014. Molecular profiling for somatic mutations that predict absence of response to anti-EGFR therapy has become standard practice in the treatment of metastatic colorectal cancer; however, the quantity and type of tissue available for testing is frequently limited. Further, the degree to which the primary tumor is a faithful representation of metastatic disease has been questioned. As next-generation sequencing technology becomes more widely available for clinical use and additional molecularly targeted agents are considered as treatment options in colorectal cancer, it is important to characterize the extent of tumor heterogeneity between primary and metastatic tumors.

Results

We performed deep coverage, targeted next-generation sequencing of 230 key cancer-associated genes for 69 matched primary and metastatic tumors and normal tissue. Mutation profiles were 100% concordant for KRAS, NRAS, and BRAF, and were highly concordant for recurrent alterations in colorectal cancer. Additionally, whole genome sequencing of four patient trios did not reveal any additional site-specific targetable alterations.

Conclusions

Colorectal cancer primary tumors and metastases exhibit high genomic concordance. As current clinical practices in colorectal cancer revolve around KRAS, NRAS, and BRAF mutation status, diagnostic sequencing of either primary or metastatic tissue as available is acceptable for most patients. Additionally, consistency between targeted sequencing and whole genome sequencing results suggests that targeted sequencing may be a suitable strategy for clinical diagnostic applications.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0454-7) contains supplementary material, which is available to authorized users.     

Somatic Mutations,Allele Loss,and DNA Methylation of the Cub and Sushi Multiple Domains 1 (CSMD1) Gene Reveals Association with Early Age of Diagnosis in Colorectal Cancer Patients

Background

The Cub and Sushi Multiple Domains 1 (CSMD1) gene, located on the short arm of chromosome 8, codes for a type I transmembrane protein whose function is currently unknown. CSMD1 expression is frequently lost in many epithelial cancers. Our goal was to characterize the relationships between CSMD1 somatic mutations, allele imbalance, DNA methylation, and the clinical characteristics in colorectal cancer patients.

Methods

We sequenced the CSMD1 coding regions in 54 colorectal tumors using the 454FLX pyrosequencing platform to interrogate 72 amplicons covering the entire coding sequence. We used heterozygous SNP allele ratios at multiple CSMD1 loci to determine allelic balance and infer loss of heterozygosity. Finally, we performed methylation-specific PCR on 76 colorectal tumors to determine DNA methylation status for CSMD1 and known methylation targets ALX4, RUNX3, NEUROG1, and CDKN2A.

Results

Using 454FLX sequencing and confirming with Sanger sequencing, 16 CSMD1 somatic mutations were identified in 6 of the 54 colorectal tumors (11%). The nonsynonymous to synonymous mutation ratio of the 16 somatic mutations was 15∶1, a ratio significantly higher than the expected 2∶1 ratio (p = 0.014). This ratio indicates a presence of positive selection for mutations in the CSMD1 protein sequence. CSMD1 allelic imbalance was present in 19 of 37 informative cases (56%). Patients with allelic imbalance and CSMD1 mutations were significantly younger (average age, 41 years) than those without somatic mutations (average age, 68 years). The majority of tumors were methylated at one or more CpG loci within the CSMD1 coding sequence, and CSMD1 methylation significantly correlated with two known methylation targets ALX4 and RUNX3. C:G>T:A substitutions were significantly overrepresented (47%), suggesting extensive cytosine methylation predisposing to somatic mutations.

Conclusions

Deep amplicon sequencing and methylation-specific PCR reveal that CSMD1 alterations can correlate with earlier clinical presentation in colorectal tumors, thus further implicating CSMD1 as a tumor suppressor gene.     

BAY61-3606 affects the viability of colon cancer cells in a genotype-directed manner

Background

K-RAS mutation poses a particularly difficult problem for cancer therapy. Activating mutations in K-RAS are common in cancers of the lung, pancreas, and colon and are associated with poor response to therapy. As such, targeted therapies that abrogate K-RAS-induced oncogenicity would be of tremendous value.

Methods

We searched for small molecule kinase inhibitors that preferentially affect the growth of colorectal cancer cells expressing mutant K-RAS. The mechanism of action of one inhibitor was explored using chemical and genetic approaches.

Results

We identified BAY61-3606 as an inhibitor of proliferation in colorectal cancer cells expressing mutant forms of K-RAS, but not in isogenic cells expressing wild-type K-RAS. In addition to its anti-proliferative effects in mutant cells, BAY61-3606 exhibited a distinct biological property in wild-type cells in that it conferred sensitivity to inhibition of RAF. In this context, BAY61-3606 acted by inhibiting MAP4K2 (GCK), which normally activates NFκβ signaling in wild-type cells in response to inhibition of RAF. As a result of MAP4K2 inhibition, wild-type cells became sensitive to AZ-628, a RAF inhibitor, when also treated with BAY61-3606.

Conclusions

These studies indicate that BAY61-3606 exerts distinct biological activities in different genetic contexts.     

HotSpotter: efficient visualization of driver mutations

Background

Driver mutations are positively selected during the evolution of cancers. The relative frequency of a particular mutation within a gene is typically used as a criterion for identifying a driver mutation. However, driver mutations may occur with relative infrequency at a particular site, but cluster within a region of the gene. When analyzing across different cancers, particular mutation sites or mutations within a particular region of the gene may be of relatively low frequency in some cancers, but still provide selective growth advantage.

Results

This paper presents a method that allows rapid and easy visualization of mutation data sets and identification of potential gene mutation hotspot sites and/or regions. As an example, we identified hotspot regions in the NFE2L2 gene that are potentially functionally relevant in endometrial cancer, but would be missed using other analyses.

Conclusions

HotSpotter is a quick, easy-to-use visualization tool that delivers gene identities with associated mutation locations and frequencies overlaid upon a large cancer mutation reference set. This allows the user to identify potential driver mutations that are less frequent in a cancer or are localized in a hotspot region of relatively infrequent mutations.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1044) contains supplementary material, which is available to authorized users.     

Glycomic profiling of carcinoembryonic antigen isolated from human tumor tissue

Background

Carcinoembryonic antigen (CEA) is a protein commonly found in human serum, with elevated CEA levels being linked to the progression of a wide range of tumors. It is currently used as a biomarker for malign tumors such as lung cancer and colorectal cancer [Urol Oncol: Semin Orig Invest 352: 644–648, 2013 and Lung Cancer 80: 45-49, 2013]. However, due to its low specificity in clinical applications, CEA can be used for monitoring only, rather than tumor diagnosis. The function of many glycoproteins is critically dependent on their glycosylation pattern, which in turn has the potential to serve in tumor detection. However, little is known about the detailed glycan patterns of CEA.

Methods

To determine these patterns, we isolated and purified CEA proteins from human tumor tissues using immunoaffinity chromatography. The glycan patterns of CEA were then analyzed using a Matrix-Assisted Laser Desorption/Ionization-Time of Flight-Mass Spectrometry³ (MALDI-TOF-MS³) approach.

Results

We identified 61 glycoforms in tumor tissue, where CEA is upregulated. These glycosylation entities were identified as bi-antennary, tri-antennary and tetra-antennary structures carrying sialic acid and fucose residues, and include a multitude of glycans previously not reported for CEA.

Conclusion

Our findings should facilitate a more precise tumor prediction than currently possible, ultimately resulting in improved tumor diagnosis and treatment.

Electronic supplementary material

The online version of this article (doi:10.1186/s12014-015-9088-3) contains supplementary material, which is available to authorized users.     

Deep sequencing reveals clonal evolution patterns and mutation events associated with relapse in B-cell lymphomas

Background

Molecular mechanisms associated with frequent relapse of diffuse large B-cell lymphoma (DLBCL) are poorly defined. It is especially unclear how primary tumor clonal heterogeneity contributes to relapse. Here, we explore unique features of B-cell lymphomas - VDJ recombination and somatic hypermutation - to address this question.

Results

We performed high-throughput sequencing of rearranged VDJ junctions in 14 pairs of matched diagnosis-relapse tumors, among which 7 pairs were further characterized by exome sequencing. We identify two distinctive modes of clonal evolution of DLBCL relapse: an early-divergent mode in which clonally related diagnosis and relapse tumors diverged early and developed in parallel; and a late-divergent mode in which relapse tumors developed directly from diagnosis tumors with minor divergence. By examining mutation patterns in the context of phylogenetic information provided by VDJ junctions, we identified mutations in epigenetic modifiers such as KMT2D as potential early driving events in lymphomagenesis and immune escape alterations as relapse-associated events.

Conclusions

Altogether, our study for the first time provides important evidence that DLBCL relapse may result from multiple, distinct tumor evolutionary mechanisms, providing rationale for therapies for each mechanism. Moreover, this study highlights the urgent need to understand the driving roles of epigenetic modifier mutations in lymphomagenesis, and immune surveillance factor genetic lesions in relapse.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0432-0) contains supplementary material, which is available to authorized users.     

Comprehensive Re-Sequencing of Adrenal Aldosterone Producing Lesions Reveal Three Somatic Mutations near the KCNJ5 Potassium Channel Selectivity Filter

Background

Aldosterone producing lesions are a common cause of hypertension, but genetic alterations for tumorigenesis have been unclear. Recently, either of two recurrent somatic missense mutations (G151R or L168R) was found in the potassium channel KCNJ5 gene in aldosterone producing adenomas. These mutations alter the channel selectivity filter and result in Na⁺ conductance and cell depolarization, stimulating aldosterone production and cell proliferation. Because a similar mutation occurs in a Mendelian form of primary aldosteronism, these mutations appear to be sufficient for cell proliferation and aldosterone production. The prevalence and spectrum of KCNJ5 mutations in different entities of adrenocortical lesions remain to be defined.

Materials and Methods

The coding region and flanking intronic segments of KCNJ5 were subjected to Sanger DNA sequencing in 351 aldosterone producing lesions, from patients with primary aldosteronism and 130 other adrenocortical lesions. The specimens had been collected from 10 different worldwide referral centers.

Results

G151R or L168R somatic mutations were identified in 47% of aldosterone producing adenomas, each with similar frequency. A previously unreported somatic mutation near the selectivity filter, E145Q, was observed twice. Somatic G151R or L168R mutations were also found in 40% of aldosterone producing adenomas associated with marked hyperplasia, but not in specimens with merely unilateral hyperplasia. Mutations were absent in 130 non-aldosterone secreting lesions. KCNJ5 mutations were overrepresented in aldosterone producing adenomas from female compared to male patients (63 vs. 24%). Males with KCNJ5 mutations were significantly younger than those without (45 vs. 54, respectively; p<0.005) and their APAs with KCNJ5 mutations were larger than those without (27.1 mm vs. 17.1 mm; p<0.005).

Discussion

Either of two somatic KCNJ5 mutations are highly prevalent and specific for aldosterone producing lesions. These findings provide new insight into the pathogenesis of primary aldosteronism.     

Deep sequencing of multiple regions of glial tumors reveals spatial heterogeneity for mutations in clinically relevant genes

Background

The extent of intratumoral mutational heterogeneity remains unclear in gliomas, the most common primary brain tumors, especially with respect to point mutation. To address this, we applied single molecule molecular inversion probes targeting 33 cancer genes to assay both point mutations and gene amplifications within spatially distinct regions of 14 glial tumors.

Results

We find evidence of regional mutational heterogeneity in multiple tumors, including mutations in TP53 and RB1 in an anaplastic oligodendroglioma and amplifications in PDGFRA and KIT in two glioblastomas (GBMs). Immunohistochemistry confirms heterogeneity of TP53 mutation and PDGFRA amplification. In all, 3 out of 14 glial tumors surveyed have evidence for heterogeneity for clinically relevant mutations.

Conclusions

Our results underscore the need to sample multiple regions in GBM and other glial tumors when devising personalized treatments based on genomic information, and furthermore demonstrate the importance of measuring both point mutation and copy number alteration while investigating genetic heterogeneity within cancer samples.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0530-z) contains supplementary material, which is available to authorized users.     

A survey of tandem repeat instabilities and associated gene expression changes in 35 colorectal cancers

Background

Colorectal cancer is a major contributor to cancer morbidity and mortality. Tandem repeat instability and its effect on cancer phenotypes remain so far poorly studied on a genome-wide scale.

Results

Here we analyze the genomes of 35 colorectal tumors and their matched normal (healthy) tissues for two types of tandem repeat instability, de-novo repeat gain or loss and repeat copy number variation. Specifically, we study for the first time genome-wide repeat instability in the promoters and exons of 18,439 genes, and examine the association of repeat instability with genome-scale gene expression levels. We find that tumors with a microsatellite instable (MSI) phenotype are enriched in genes with repeat instability, and that tumor genomes have significantly more genes with repeat instability compared to healthy tissues. Genes in tumor genomes with repeat instability in their promoters are significantly less expressed and show slightly higher levels of methylation. Genes in well-studied cancer-associated signaling pathways also contain significantly more unstable repeats in tumor genomes. Genes with such unstable repeats in the tumor-suppressor p53 pathway have lower expression levels, whereas genes with repeat instability in the MAPK and Wnt signaling pathways are expressed at higher levels, consistent with the oncogenic role they play in cancer.

Conclusions

Our results suggest that repeat instability in gene promoters and associated differential gene expression may play an important role in colorectal tumors, which is a first step towards the development of more effective molecular diagnostic approaches centered on repeat instability.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1902-9) contains supplementary material, which is available to authorized users.     

Colorectal Tumors from APC*I1307K Carriers Principally Harbor Somatic APC Mutations outside the A8 Tract

Purpose

APC*I1307K (c.3920T>A) is an inherited variant associated with colorectal tumour risk found almost exclusively in those of Ashkenazi Jewish ancestry. A single nucleotide substitution creates an oligo-adenine tract (A8) that appears to be inherently prone to further mis-pairing and slippage. The reported multiple tumor phenotype of carriers is not easily reconciled with molecular and population genetics data. We postulated that some c.3920T>A carriers with multiple adenomas have other unidentified APC germ line or somatic mutations.

Methods

DNA from 82 colonic tumours and accompanying normal tissue collected from 29 carriers with multiple colorectal tumors was directly sequenced between codons 716 and 1604. We also assessed APC gene loss of heterozygosity.

Results

One patient (3.4%) was found to have an additional APC germ line mutation. Twenty-five of the tumours showed no significant somatic molecular change, 36 showed one change, 20 showed two, and one tumour showed more than 2 changes. Our data suggest a correlation between advancing histology and fewer beta-catenin binding sites remaining in the mutant proteins.

Conclusions

There were no other common germ line variants identified within the region of the APC gene examined, suggesting that any effect from this region on tumour production is attributable to the c.3920T>A allele. Our findings further suggest the only somatic genetic change clearly attributable to the c.3920T>A mutation is the c.3924_3925insA.     

Towards accurate characterization of clonal heterogeneity based on structural variation

Background

Recent advances in deep digital sequencing have unveiled an unprecedented degree of clonal heterogeneity within a single tumor DNA sample. Resolving such heterogeneity depends on accurate estimation of fractions of alleles that harbor somatic mutations. Unlike substitutions or small indels, structural variants such as deletions, duplications, inversions and translocations involve segments of DNAs and are potentially more accurate for allele fraction estimations. However, no systematic method exists that can support such analysis.

Results

In this paper, we present a novel maximum-likelihood method that estimates allele fractions of structural variants integratively from various forms of alignment signals. We develop a tool, BreakDown, to estimate the allele fractions of most structural variants including medium size (from 1 kilobase to 1 megabase) deletions and duplications, and balanced inversions and translocations.

Conclusions

Evaluation based on both simulated and real data indicates that our method systematically enables structural variants for clonal heterogeneity analysis and can greatly enhance the characterization of genomically instable tumors.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2105-15-299) contains supplementary material, which is available to authorized users.