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Adenomatous polyposis coli

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Retired Sailor overcomes bleak diagnosis, maintains positive attitude


Retired Sailor overcomes bleak diagnosis, maintains positive attitude

TRIPLER ARMY MEDICAL CENTER, Hawaii, USA – Daniel Shockley, a retired Sailor living on Oahu, meets with Lt. Col. Ronald Gagliano, chief, Colon and Rectal Surgery and director, Surgical Research, TAMC, to discuss recovery and post-operative…

Starting at age 50, it is encouraged that everyone get regular colonoscopies (in the USA), an examination that use a fiber-optic camera to view your lower gastrointestinal tract, to screen for colon cancer.In September 2011 when Daniel Shockley, a retired Sailor living on Oahu, went for his annual physical exam at Spark M. Matsunaga Veterans Affairs Medical Center, he thought besides a little weight loss, he had a clean bill of health.Since Shockley had just turned 50, he was referred to a Hawaii Pacific Health clinic in downtown Honolulu for his first colonoscopy.

Due to his hectic work schedule, Shockley rescheduled the screening a couple times and it wasn’t until May 8, 2012, when he got the colonoscopy.

“They usually schedule colonoscopies for 1-hour blocks of time, but they found so much wrong during mine that he had to spend a lot of time documenting and taking pictures,” Shockley explained. “What they found was approximately 100 polyps embedded throughout my colon, rectum and anus. And at the traverse colon, the junction between the large and small intestine, they found a large tumor that was creating an 80 percent blockage.”

Shockley was referred to Tripler Army Medical Center’s general surgery clinic, and the week following the screening, he met with Susan Donlon, a certified genetic counselor at Tripler.

Donlon performed DNA tests on Shockley and within three weeks the tests had come back confirming that Shockley has a gene mutation known as Adenomatous Polyposis Coli, which increases a person’s risk of developing colorectal cancer. As a result of the mutation, Shockley was diagnosed with Attenuated Familial Adenomatous Polyposis, a condition in which numerous polyps form mainly in the large intestine.

“I knew surgery was inevitable and I was willing to accept the worst case scenario the whole time,” Shockley said.

On July 13, Shockley underwent a total proctocolectomy with ileostomy surgery, which removed portions of his large intestine to include the entire colon, rectum and anus.

Shockley spent about two weeks in Tripler’s general inpatient surgery ward recovering before he was able to go home. It was nine weeks before he was able to go back to work.

Lt. Col. Ronald Gagliano, chief, Colon and Rectal Surgery and director, Surgical Research, TAMC, performed Shockley’s surgery and has followed up with him to ensure he is not only well-informed, but also well-educated.

“He knew nothing of his disease and its many facets before we met and our team (at Tripler) began his personal education in order to promote effective counseling regarding his diagnostic and therapeutic options,” Gagliano explained. “Finally we educated him regarding his genetic situation so that he could choose (how to best) inform his family. By giving him great care, we essentially treat an entire family cohort.”

“(Dr. Gagliano and his team) have passion for what they do, and my care was phenomenal,” Shockley expressed. “I cannot say enough good things about my stay and the care they provided.”

Gagliano is very pleased with Shockley’s recovery thus far and attributes it to his attitude.

“I tend not to think about things I can’t control,” Shockley explained. “Medical issues are not something I can control, but what I can control is my attitude and after 51 years on God’s green earth my positive attitude has gotten me this far and I am not going to change it.”

Because of Shockley’s surgeries, he now has an ostomy pouching system, a prosthetic medical device that provides a means for the collection of waste. Nina Lum, certified wound, ostomy and continence nurse, TAMC, who helped care for Shockley throughout his recovery, echoed Gagliano’s remarks.

“Shockley’s resilience in the face of challenges including his tremendous enthusiasm for life, regardless of setbacks, certainly played a huge role in his recovery,” Lum said. “He has always maintained a positive outlook, been fully engaged in his care from the beginning, reached out to the ostomy community not only for support, but also to offer support and advise based on his personal experience.

“He is selfless in trying to reach out to others,” Lum added.

Shockley has embraced his diagnosis and challenged it from the start. He acts as a patient advocate and an ambassador for colon cancer awareness.

“(I want to) share my story with others on behalf of those patients that have gone before me and who were unable to share their story,” Shockley explained. “My catchphrase is ‘AFAP-Seize the disease!'”

Shockley wants to spread the information about his diagnosis and experience so he can inspire others to get the screening and be aware of the condition. Additionally, there is not a lot of information about AFAP available, so he hopes that talking about his diagnosis will help the medical community.

“By maintaining a positive attitude, the opportunity for a success story is much higher,” Shockley said. “This in turn allows me a better chance of overcoming adversities I am faced with during my lifetime.”

Read More;

Polyposis Syndromes – Information for Patients

 

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Squaring Genetic vs Clinical Findings in Familial Polyposis


Journal CoverPrevalence and Phenotypes of APC and MUTYH Mutations in Patients With Multiple Colorectal Adenomas

Grover S, Kastrinos F, Steyerberg EW, et al

JAMA. 2012;308:485-492

Summary

Familial adenomatous polyposis (FAP) is caused by mutations in the APC gene and 2 different, or biallelic mutations, in the MUTYH gene. However, not all patients with colorectal polyposis are found to carry mutations on these genes. In addition, it is unclear how the extent of polyp burden or the age at development of the first adenoma corresponds to the likelihood of finding mutations in either of these 2 genes.

In an effort to better characterize the mutation frequency in patients with multiple colorectal adenomas, this study tested for APC and MUTYH mutations in 8676 individuals over 8 years. Each person’s cancer history, adenoma count, and family history of cancer or colorectal adenomas was reported by clinicians ordering the genetic testing.

The study found that patients with classic polyposis were very likely to carry an APC mutation: 80% of those with ≥ 1000 colorectal adenomas and 56% of those with 100-999 adenomas carried an APC mutation. APC mutations were prevalent even in individuals with fewer than 100 adenomas, with mutations seen in 10% of those with 20-99 adenomas and in 5% of those with 10-19 adenomas.

With regard to MUTYH mutations, the frequency was low in individuals with≥ 1000 adenomas (2%) but was fairly consistent in those with 10 colonic adenomas, those who present with multiple adenomas at an unusually young age, or those who have a family history consistent with FAP. The findings of the current study support testing in these individuals and demonstrate that the greater the number of polyps, the greater the likelihood of identifying a mutation.

However, multiple factors can complicate the value of genetic testing in clinical practice. The clinical phenotype of biallelic MUTYH mutations is quite varied; reports show that some mutation carriers can have hundreds of polyps, whereas others with colon cancer have no reported polyps.[2] Also, overlap among the clinical phenotypes of Lynch syndrome, MUTYH-associated disease, and attenuated FAP or other polyposis conditions may require clinical expertise for appropriate diagnosis and management. Finally, some controversy remains with regard to risk (if any) for colon cancer in persons with only 1 MUTYH mutation, and management in these patients is uncertain.[3]

At the same time, not all individuals manifesting colonic polyposis harbor a mutation in APC or MUTYH, and management is not straightforward in patients with polyposis but no identified mutation. Clearly, there are cases of unknown etiology, and there are probably as-yet unidentified genes that may predispose to adenomatosis. But changing technologies and testing standards can also affect interpretation of genetic test results. For example, polyposis testing was once only pursued in persons with > 20 polyps, whereas guidelines now recommend that testing be done in all patients who have ≥ 10 adenomas,[1] so historically “negative” tests may need to be revisited in the future.

Similarly, individuals tested before the availability of APC deletion/duplication analysis and MUTYH testing must be reassessed. Indeed, in the past few months, new and more efficient molecular testing modalities, so-called next-generation sequencing, have allowed the commercial launch of several cost-efficient gene panels that can test multiple genes at once for polyposis and nonpolyposis mutations. This may prove particularly helpful in evaluating patients with low polyp counts.

Current recommendations note that individuals with multiple adenomas or a family history of colon cancer be referred for genetic counseling. However, a lack of family history does not exclude the possibility of FAP, because an individual can harbor a de novo mutation; genetic testing for a hereditary cancer syndrome can thus be pursued on the basis of age, polyp count, and family history. In the absence of an identified mutation, family history as well as clinical presentation can be used to determine whether the individual may be at increased risk for other syndromes, and an empiric screening and prevention protocol can be established.

via Squaring Genetic vs Clinical Findings in Familial Polyposis.

Much Of The Population Genetic Risk Of Colorectal Cancer Is Likely To Be Mediated Through Susceptibility To Adenomas


Gastroenterology (journal)

Gastroenterology (journal) (Photo credit: Wikipedia)

Several single nucleotide polymorphisms (SNPs) have been associated with colorectal cancer (CRC) susceptibility. Most CRCs arise from adenomas, and SNPs might therefore affect predisposition to CRC by increasing adenoma risk. We found that 8 of 18 known CRC-associated SNPs (rs10936599, rs6983267, rs10795668, rs3802842, rs4444235, rs1957636, rs4939827, and rs961253) were over-represented in CRC-free patients with adenomas, compared with controls. Ten other CRC-associated SNPs (rs6691170, rs6687758, rs16892766, rs7136702, rs11169552, rs4779584, rs9929218, rs10411210, rs4813802, and rs4925386) were not significantly associated with adenoma risk. Genetic susceptibility to CRC in the general population is likely to be mediated in part by predisposition to adenomas.

ScienceDirect.com – Gastroenterology – Much Of The Population Genetic Risk Of Colorectal Cancer Is Likely To Be Mediated Through Susceptibility To Adenomas. Carvajal-Carmona et al In Press September 2012

The Elucidation of Familial Colorectal Cancer Type X


The Elucidation of Familial Colorectal Cancer Type X

Gene Discovery in Familial Cancer Syndromes by Exome Sequencing

Ku et al Modern Pathology 2012;25(8):1055-1068l Mod Pathol. 2012;25(8):1055-1068

Abstract

Recent advances in genotyping and sequencing technologies have provided powerful tools with which to explore the genetic basis of both Mendelian (monogenic) and sporadic (polygenic) diseases. Several hundred genome-wide association studies have so far been performed to explore the genetics of various polygenic or complex diseases including those cancers with a genetic predisposition. Exome sequencing has also proven very successful in elucidating the etiology of a range of hitherto poorly understood Mendelian disorders caused by high-penetrance mutations. Despite such progress, the genetic etiology of several familial cancers, such as familial colorectal cancer type X, has remained elusive. Familial colorectal cancer type X and Lynch syndrome are similar in terms of their fulfilling certain clinical criteria, but the former group is not characterized by germline mutations in DNA mismatch-repair genes. On the other hand, the genetics of sporadic colorectal cancer have been investigated by genome-wide association studies, leading to the identification of multiple new susceptibility loci. In addition, there is increasing evidence to suggest that familial and sporadic cancers exhibit similarities in terms of their genetic etiologies. In this review, we have summarized our current knowledge of familial colorectal cancer type X, discussed current approaches to probing its genetic etiology through the application of new sequencing technologies and the recruitment of the results of colorectal cancer genome-wide association studies, and explore the challenges that remain to be overcome given the uncertainty of the current genetic model (ie, monogenic vs polygenic) of familial colorectal cancer type X.

Introduction

Recent developments in high-throughput sequence capture methods and next-generation sequencing technologies have made exome sequencing a viable approach to the identification of pathological mutations, both from a technical standpoint and in terms of being cost-effective.[1–4] The advent of exome sequencing has already contributed significantly toward the identification of new causal mutations (and genes) for a number of previously unresolved Mendelian disorders such as Kabuki syndrome, Miller syndrome, Sensenbrenner syndrome, and Fowler syndrome to name just a few. Further, exome sequencing has proven to be an effective tool to interrogate the genetic basis of Mendelian disorders in samples derived from both families and unrelated individuals.[5–8] Since the inception of the idea of using exome sequencing as both a discovery[9] and a diagnostic tool[10] for Mendelian disorders, this field has advanced very considerably.[11] Accompanied and aided by other technical advances such as the development of computational and statistical approaches to interrogate the myriad variants identified by exome sequencing,[12, 13] including algorithms to detect copy number variants using exome sequencing data,[14] and the idea (and practical demonstration) of using single-nucleotide polymorphism genotypes extracted from exome sequencing data to perform accurate genetic linkage mapping to reduce the ‘search space’ for genetic variants,[15] exome sequencing has emerged as a mature analytical approach.

Although major progress has been made in understanding the genetic basis of Mendelian disorders over the past 3 years using exome sequencing, so far only limited studies have interrogated familial forms of cancer, ie, familial pancreatic cancer[16] and hereditary pheochromocytoma (a rare neural crest cell tumor).[17] By harnessing the latest technological advances, Jones et al [16] identified a germline truncating mutation in PALB2 through exome sequencing a single patient with familial pancreatic cancer. That this patient might have a familial form of pancreatic cancer was suggested by the fact that his sister had also developed the disease. In similar manner, mutations in MAX, the MYC-associated factor X gene, were also identified through sequencing the exomes of three unrelated individuals with hereditary pheochromocytoma.[17]

Since 2005, >100 genome-wide association studies have been performed to interrogate the genetic basis of various sporadic or polygenic forms of cancer (such as colorectal, prostate, breast, and lung) for which numerous statistically robust and novel single-nucleotide polymorphisms or genetic loci have been identified.[18, 19] In addition to their polygenic nature, these cancers are multifactorial, involving a complex interaction of multiple genetic and environmental factors. By contrast, little progress has so far been achieved in the context of ‘familial’ cancers (ie, cancers displaying a very evident family history with clustering of multiple affected family members). More specifically, familial forms of cancer typically occur in more individuals in a given family than would be expected by chance alone. Familial cancers are often characterized by their occurrence at a comparatively early age, thereby indicating the potential presence of a gene mutation that increases the risk of cancer. However, familial clustering of cases may also be a sign of a shared environment or lifestyle, or alternatively chance alone. By contrast, sporadic cancers lack any obvious family history of the disease.

The slow progress of research into familial cancer has been illustrated, for example, in hereditary diffuse gastric cancer. CDH1 was the first causal gene identified for this cancer in 1998,[20] and it remains the only known gene underlying hereditary diffuse gastric cancer. However, germline mutations in this gene account for only a proportion of hereditary diffuse gastric cancer cases,[21] suggesting that an as-yet-to-be identified gene(s) is likely to be responsible for the remaining cases unexplained by CDH1. Similarly, BRCA1 and BRCA2 are the only high-penetrance genes for familial breast cancer, although numerous novel single-nucleotide polymorphisms and genetic loci conferring low-to-moderate risk or effect size (odds ratio <1.5) have been identified by genome-wide association studies of polygenic breast cancer.[22, 23] Some of these common alleles have been reported to modify risk in BRCA1 and BRCA2 mutations carriers.[24] However, so far the results from genome-wide association studies have limited value for individual risk prediction,[25] as compared with the high-penetrance inherited mutations in causal genes for familial breast cancer which can prompt drastic clinical intervention such as mastectomy. An analysis to evaluate the potential for individualized disease risk stratification based on common single-nucleotide polymorphisms identified by genome-wide association studies in breast cancer came to the conclusion that the clinical utility of single, common, low-penetrance genes for breast cancer risk prediction is currently quite limited.[26]

In the context of familial colorectal cancer, the genetic causes of familial adenomatous polyposis and Lynch syndrome have been well documented; in most instances, they are accounted for by germline mutations in the APC gene and DNA mismatch-repair genes (ie, MSH2, MLH1, MSH6, and PMS2), respectively. For example, ~90% of familial adenomatous polyposis cases are caused by germline mutations in the APC gene. The majority of these mutations introduce a premature stop codon resulting in a truncated protein. Similarly, the MSH2 and MLH1 genes harbor >90% of the germline mutations found in Lynch syndrome patients.[27, 28] By contrast, the genetic etiology of familial colorectal cancer type X remains largely unknown.[29] It is widely anticipated that new insights generated from studies on familial colorectal cancer type X will lead to the molecular characterization of a novel form of familial colorectal cancer which will necessitate the reclassification of subsets of families with a strong history of colorectal cancer.

How to Interrogate the Genetics of Familial Colorectal Cancer Type X?

The nature of the disease determines the study design required to unravel the causal mutations or risk-predisposing variants for familial colorectal cancer type X. However, there is little evidence to show whether familial colorectal cancer type X is a monogenic or polygenic disease or whether it is somewhere in between. The evidence suggesting that familial colorectal cancer type X is a monogenic disease comes mainly from the fulfillment of Amsterdam Criteria. The Amsterdam Criteria state that at least three relatives must have colorectal cancer. However, the familial aggregation, with multiple affected family members in one family, could also be due to shared non-genetic factors, which would not therefore necessarily be compatible with the monogenic model. Such environmental factors would be expected to interact with multiple genetic risk factors causing colorectal cancer, a multifactorial disease model proposed for polygenic disease. This therefore raises the question as to whether the Amsterdam Criteria are sufficient to support a monogenic basis for familial colorectal cancer type X. Furthermore, some of the clinical features of familial colorectal cancer type X implied that it could have a polygenic basis. This uncertainty in the nature of the disease for familial colorectal cancer type X presents substantial challenges in terms of deciding upon an optimal approach to interrogate its genetic basis.

The targeted sequencing of causal genes, already applied in the context of other familial cancers (such as CDH1 (hereditary diffuse gastric cancer), BRCA1 and BRCA2 (familial breast cancer), and the genes underlying hereditary pheochromocytoma), appears to be a worthwhile approach to identify deleterious germline mutations for familial colorectal cancer type X. The rationale is that germline mutations in these genes could underlie different familial cancers, as for example in the case of the PALB2 germline mutations that have been found in both familial pancreatic and breast cancers.[16, 63] Another notable example is provided by the germline mutations in the BRCA2 gene that not only increase the risk of breast and ovarian cancer, but also pancreatic cancer.[101] This targeted sequencing approach has been greatly aided by high-throughput enrichment methods and next-generation sequencing technologies to selectively enrich for regions of interest. Hundreds of genes can be sequenced efficiently, leveraging these technological advances compared with traditional PCR-based Sanger sequencing. The efficiency of this approach has been exemplified in a targeted sequencing study of germline mutations in 21 tumor suppressor genes for 360 women with inherited ovarian, peritoneal, or fallopian tube carcinoma.[102] This study harnessed the power of the Sure-Select enrichment system and the Illumina sequencing platform to sequence these genes; 24% of the patients were found to carry germline loss-of-function mutations in 12 genes, six of which had not previously been implicated in inherited ovarian carcinoma. Although this targeted approach has limited discovery value, as these genes had already been implicated in causing familial cancers, it could still have some novelty value by identifying germline mutations in known genes for cancers, which have not yet been linked to these genes.

This targeted approach can be expanded to include the entire set of exons in all genes in the human genome. Exome sequencing on its own or coupled with linkage analysis has already unravelled multiple new causal mutations and genes for Mendelian disorders.[7, 8] Furthermore, these discoveries were made by exome sequencing fewer than 10 patient samples in most of the studies reported. As such, it is also widely anticipated that exome sequencing will represent a powerful tool to reveal the genetic causes of familial colorectal cancer type X by identifying rare and deleterious or high-penetrance mutations within gene coding regions. However, the appropriate selection of cases will have a key role in determining the success or otherwise of exome sequencing in this context. In addition to fulfilling the Amsterdam Criteria, and excluding germline mutations in mismatch-repair genes, selecting cases with a very early onset of disease, severe clinico-pathological manifestations or the ‘extreme’ familial colorectal cancer type X phenotypes are expected to enrich for the ‘monogenic’ component and hence enhance our chances of identifying high-penetrance mutations. Recurrent mutations (similar mutations in different samples) or genes harboring several different deleterious mutations (which include single-nucleotide variants and small indels) across multiple samples can then be prioritized for further studies using a larger sample of cases.

On the other hand, if we assume that familial colorectal cancer type X has a polygenic component, then genome-wide association studies would represent the ideal approach to identify common single-nucleotide polymorphisms associated with this disease. Further, whole-genome genotyping arrays would also allow copy number variants to be investigated to a certain extent for their associations with familial colorectal cancer type X within a single genome-wide association study. High-density genotyping arrays have been used to identify copy number variants in a cohort of 41 colorectal cancer patients who were below 40 years of age at diagnosis and/or who exhibited an overt family history.[103] Multiple copy number variants, encompassing genes such as CDH18, GREM1, and BCR, were identified in six patients as well as two deletions encompassing two microRNA genes, hsa-mir-491/KIAA1797 and hsa-mir-646/AK309218. Interestingly, these copy number variants had not previously been reported in relation to colorectal cancer predisposition, nor had they been encountered in large control cohorts. This illustrates the potential power of copy number variant investigation to identify novel causal or susceptibility genes or genetic loci for both familial and sporadic colorectal cancers. Through another interesting observation, multiple genomic aberrations including copy number gains and losses in different chromosomes have also been detected in 30 mismatch repair-proficient familial colorectal cancers. In particular, the frequency of 20q gain is remarkably increased when compared with sporadic colorectal cancer, suggesting that the 20q gain is involved in the genetic etiology of these mismatch repair-proficient familial colorectal cancers.[104] The finding that most of these genomic aberrations were also observed in sporadic colorectal cancer further suggests that familial and sporadic colorectal cancers could share genetic predisposition to a certain extent.

It is however noteworthy that genome-wide association studies represent an indirect association study design, based on linkage disequilibrium, to detect the disease-causing variants, as compared with direct sequencing. To achieve the required statistical power and significance threshold to detect common single-nucleotide polymorphisms conferring small effect sizes (odds ratio <1.5), several thousands of cases and controls are required for the initial genome-wide genotyping and subsequent replication studies.[105] Although the cost of genotyping arrays is steadily becoming much cheaper, a hefty investment is still required to analyze thousands of samples. In addition to this cost, collecting the adequate sample size of patients to embark on a genome-wide association study is a considerable challenge if this is to be achieved without an international consortium (because of the rarity of familial colorectal cancer type X as compared with sporadic colorectal cancer cases). The polygenic basis of familial colorectal cancer type X is still a speculative issue. Bearing in mind this uncertainty, an alternative is to leverage the results from genome-wide association studies of colorectal cancer by genotyping the robust single-nucleotide polymorphism associations in a familial colorectal cancer type X cohort. This approach might be more feasible in terms of cost-effectiveness and sample size (without the need of a stringent significance threshold to account for several hundred thousand single-nucleotide polymorphisms). The penalty of multiple testing imposed in genome-wide association studies should increase the attractiveness of this approach in the context of testing single-nucleotide polymorphisms identified by genome-wide association studies for familial colorectal cancer type X. One may speculate that if familial colorectal cancer type X has a polygenic component, some of these polymorphisms should also be associated with familial colorectal cancer type X, which would then warrant a comprehensive genome-wide association study for familial colorectal cancer type X in the future. This speculation appears reasonable because common shared single-nucleotide polymorphisms or genetic loci have been found in several different cancers. There have been several examples of the practical utility of genome-wide association study results in the context of familial cancers. These studies have provided evidence to suggest that low-penetrance variants may explain the increased cancer risk in familial colorectal cancer[106–108] and in familial testicular germ cell tumors.[100]

Finally, the genes or genetic loci implicated in colorectal cancer by genome-wide association studies can be captured and sequenced. This targeted sequencing approach is very cost-effective as up to 96 samples can be multiplexed through barcoding for massively parallel sequencing. This targeted sequencing approach will interrogate both rare variants and common single-nucleotide polymorphisms in the loci identified by genome-wide association studies. The promise of this approach in unravelling rare variants in loci implicated by genome-wide association studies has already been demonstrated.[51,53–55] For example, deep resequencing of such loci has identified independent rare variants associated with inflammatory bowel disease.[55]

Perspectives and Conclusions

The genetic and clinical differences between Lynch syndrome and familial colorectal cancer type X have been well documented. However, the genetic etiologies of familial colorectal cancer type X remain to be determined. There is also a paucity of evidence to indicate one way or the other whether familial colorectal cancer type X is a monogenic or a polygenic disease. On the other hand, the genetics of sporadic/polygenic colorectal cancer have been comprehensively investigated by >10 genome-wide association studies over the past few years. One striking observation is the sharing of common single-nucleotide polymorphisms or genetic loci across different cancers. It is therefore reasonable to speculate that if familial colorectal cancer type X has a polygenic basis, some of the single-nucleotide polymorphisms identified by genome-wide association studies as conferring risk of colorectal cancer might be expected to show associations with familial colorectal cancer type X as well. Given the expense and logistic challenges involved in collecting a large number of familial colorectal cancer type X cases to embark on a genome-wide association study, together with the uncertainty of the disease model, we believe that the genotyping of genome-wide association study-identified single-nucleotide polymorphisms in familial colorectal cancer type X would be a more feasible first approach to explore the genetic etiology of this disease. However, given the low incidence of familial colorectal cancer type X (ie, only ~2–3% of colorectal cancer families meet Amsterdam Criteria and about half of these are Lynch syndrome cases), collecting an adequate large sample size is difficult and challenging especially for studying the association of single-nucleotide polymorphisms with modest effect sizes. Thus, National or International Consortia involving many centers are likely to be needed to recruit large numbers of patients. Alternatively, the genes or loci identified by genome-wide association studies could be investigated using a targeted sequencing approach to unravel rare variants of larger effect size.

One of the limitations of genome-wide association studies is that they are based upon an indirect association study design, which is reliant on linkage disequilibrium to identify the disease functional variants. As a result, the surrogate markers (ie, the associated single-nucleotide polymorphisms) identified by genome-wide association studies generally lack functional significance. Furthermore, to enhance the statistical power, genome-wide association studies have tended to lump all colorectal cancers in the disease group, even although it is well recognized that colorectal cancers are inherently heterogeneous. These challenges have led to the notion and conceptualization of ‘molecular pathological investigation’, which is a relatively new field of epidemiology based upon the molecular classification of cancer. It is a multidisciplinary field involving the investigation of the interrelationship between exogenous and endogenous (eg, genetic) factors, tumoral molecular signatures, and tumor progression. Further, integrating genome-wide association studies with molecular pathological investigation allows examination of the relationship between susceptibility alleles identified by genome-wide association studies and specific molecular alterations/subtypes, which can help to elucidate the function of these alleles and provide insights into whether the detected susceptibility alleles are truly causal. Although there are challenges, molecular pathological epidemiology has unique strengths, and can provide insights into the pathogenic process.

In addition, exome sequencing of multiple ‘well-selected’ cases could be performed, assuming a monogenic basis in which high-penetrance mutations are predicted to underlie the genetic etiology of familial colorectal cancer type X. Exome sequencing of families with multiple affected individuals also represents a promising study design. This family-based design has the advantage that it allows for the genetically heterogeneous nature of familial colorectal cancer type X. Comparing unrelated individuals or probands from different families to identify ‘common/shared’ putative pathological variants or genes harboring putative pathological variants might not be a successful strategy for genetically heterogeneous diseases. However, it still depends on the degree of genetic heterogeneity (ie, allelic heterogeneity versus locus heterogeneity) characterizing the disease and this remains unknown. Although the family design is robust with respect to genetic heterogeneity (comparing affected and unaffected members in a family), one must recognize that it could also be problematic because the penetrance of disease mutations for familial colorectal cancer type X is likely to be lower than that for Lynch syndrome.

Moving forward, it is arguable that whole-genome sequencing should probably be considered instead of exome sequencing, as the cost differential between the two approaches (given a small patient sample size) would not be substantial, and because the former approach will generate genetic data for the entire genome rather than just 1–2% as for exome sequencing. However, one should select the study design that best fits the hypothesis where rare deleterious mutations in coding regions underlie the genetic etiology of a Mendelian disorder or familial cancer. So far, all the discoveries made by whole-genome sequencing could also have been achieved using exome sequencing for Mendelian disorders.  Furthermore, the genetic variants in most of the non-coding regions revealed by whole-genome sequencing remain ‘uninterpretable’ biologically. In taking a practical (rather than theoretical) point of view, whole-genome sequencing still presents a very substantial technical challenge as well as a challenge in terms of analyzing and interpreting the sequence data generated.

The disease models underpinning multiple familial cancers such as familial nasopharyngeal carcinoma,familial testicular germ cell tumor, familial chronic lymphocytic leukemia,and familial colorectal cancer (familial colorectal cancer type X) remain contentious as the high-penetrance mutations are yet to be identified. By contrast, multiple low-penetrance variants that confer an effect size of odds ratio <1.5 have been revealed through genome-wide association studies for the sporadic cases of these cancers; interestingly, some of these single-nucleotide polymorphisms have also been found to be associated with the familial cases (nasopharyngeal carcinoma,[115] testicular germ cell tumor,[116, 117] chronic lymphocytic leukemia, and colorectal cancer). In the context of familial colorectal cancer type X, we believe that the disease model and its genetic basis are likely to become more apparent when the approaches that we have outlined and discussed are applied in practice. This should facilitate the iterative interrogation of the genetics of familial colorectal cancer type X and other familial cancers of similar nature before embarking on either a comprehensive genome-wide association studies or whole-genome sequencing approach.

MUTYH-associated polyposis (MAP)


MUTYH-associated polyposis (MAP)

Clinical characteristics

MUTYH-associated polyposis (MAP), caused by biallelic mutations in MUTYH (formerly known as MYH), is characterized by a greatly increased lifetime risk of colorectal cancer (43% to almost 100% in the absence of timely surveillance). Although typically associated with ten to a few hundred colonic adenomatous polyps that are evident at a mean age of about 50 years, colonic cancer develops in some individuals with biallelic MUTYH mutations in the absence of polyposis (Wang et al. 2004). Duodenal adenomas are found in 17%-25% of individuals with MAP; the lifetime risk of duodenal cancer is about 4%. Also noted are a modestly increased risk for rather late-onset malignancies of the ovary, bladder, and skin, and some evidence for an increased risk for breast and endometrial cancer. More recently, thyroid abnormalities (multinodular goiter, single nodules, and papillary thyroid cancer) have been reported in some studies. Some affected individuals develop sebaceous gland tumors.

Biallelic mutations in MUTYH have been found to account for approximately 10% of polyposis patients, but <1% of all colorectal cancer (Halford et al. 2003; Wang, Baudhuin et al. 2004).  The largest population study to date indicates that approximately 0.2% of all colorectal cancer is caused by biallelic mutations in MYH (Webb et al. 2006).  It was demonstrated in the same study that monoallelic MUTYH mutations are not associated with an increased risk of colorectal cancer.  The MAP phenotype is similar AFAP, with extra-colonic manifestations consisting of duodenal polyps but not intra-abdominal desmoids although occasionally patients may have up to several thousand polyps.  Among Caucasians approximately 80% of mutations in MUTYH causing MAP are Y165C or G382D (Sieber, Lipton et al. 2003).  https://familyhistorybowelcancer.files.wordpress.com/2012/08/myh.png

The E466X mutation is a common founder mutation among Pakistani populations.  The family tree above shows 2 Pakistani brothers who were affected with colorectal cancer and polyps in their 30s due to this founder mutation.  Y90X is a founder mutation in Indian populations (Sieber, Lipton et al. 2003).

Around 2530% of polyposis cases with more than 20 polyps and without evidence of a dominant inheritance pattern, in whom genetic analysis has not identified an APC mutation, are due to bi-allelic mutations in the base excision repair (BER) gene, MUTYH. Polyps can be exclusively adenomatous or mixed adenomatous/hyperplastic. Since the mode of inheritance is autosomal recessive, lack of vertical transmission of the polyposis phenotype in the family should raise the possibility of MUTYH-associated polyposis  (MAP). Siblings are at 25% risk of carrying bi-allelic deleterious mutations. Children of a bi-allelic carrier are at high risk if the other parent also carries at least one mutant allele. Large, systematic studies of MUTYH mutation frequency in colorectal cancer cases and controls suggest penetrance in bi-allelic carriers is very high, and probably >90%.

The heterozygote carrier frequency in the UK is around 2% and around 1:10 000 homozygous or compound heterozygotes for two MUTYH mutations. The proportion of polyposis syndromes due to MUTYH in clinical practice is less clear because studies have so far focused on selected research case series of multiple polyps that have been screened negative for APC mutations. In one study 4% of multiple polyp cases (3100) and 8% of APC mutation negative polyposis cases carry MUTYH mutations.

Molecular Pathogenesis

Damaged DNA is repaired by several mechanisms, one of which involves a family of enzymes involved in base-excision repair (BER). The MUTYH gene (also known as MYH) encodes a DNA glycosylase involved in the repair of the oxidative lesion 8-oxoguanine, a by-product of cellular metabolism and oxidative damage of DNA.

(8-oxoG, left), in syn conformation, forming a...

(8-oxoG, left), in syn conformation, forming a with (dATP, right). Created using ACD/ChemSketch 10.0 and . (Photo credit: Wikipedia)

(8-oxoG, left), in syn conformation, forming a with (dATP, right). Created using ACD/ChemSketch 10.0 and . (Photo credit: Wikipedia)

The products of three BER repair genes, OGG1, MTH1 and MYH work together to prevent 8-oxo-G induced mutagenesis.  Mutations in MUTYH cause an autosomal recessive colorectal cancer and polyposis syndrome MYH-associated polyposis (MAP; OMIM 608456) (Al-Tassan et al. 2002).  Somatic mutations in the APC gene in polyps from individuals affected with MAP are almost invariably G to T transversions (Sieber et al. 2003), and it was by understanding the underlying DNA repair mechanism of this mutation, base-excision repair, that MUTYH was identified as a candidate-predisposition

English: Schematic of base excision repair

English: Schematic of base excision repair (Photo credit: Wikipedia)

Schematic of base excision repair (Photo credit: Wikipedia)

gene. G to T transversion mutations were also identified in KRAS in codon 12 (Lipton et al. 2003).  The adenoma to carcinoma pathway in MAP does not involve BRAF V600E, SMAD4 or TGFBIIR mutations, or microsatellite instability, and the cancers are near-diploid (Lipton, Halford et al. 2003).  Thus, tumours with germline MUTYH mutations tend to follow a distinct pathway.

Colorectal surveillance & screening

Treatment of manifestations: Suspicious polyps identified on colonoscopy should be removed until polypectomy alone cannot manage the large size and density of the polyps, at which point either subtotal colectomy or proctocolectomy is performed. Duodenal polyps showing dysplasia or villous changes should be excised during endoscopy. Abnormal findings on thyroid ultrasound examination should be evaluated by a thyroid specialist to determine what combination of monitoring, surgery, and/or fine needle aspiration (FNA) is appropriate. Surveillance: Individuals with biallelic MUTYH germline mutations: Evaluation of relatives at risk: Offer molecular genetic testing for the familial mutations to all siblings of an individual with genetically confirmed MAP in order to reduce morbidity and mortality through early diagnosis and treatment.

Counselling: MAP is inherited in an autosomal recessive manner. At conception, each sib of an affected individual has a 25% chance of being affected, a 50% chance of being a carrier with a small increased risk of CRC, and a 25% chance of being unaffected and not a carrier. Carrier testing for at-risk family members and prenatal diagnosis for pregnancies at increased risk are possible if the disease-causing mutations in the family have been identified.

UK Recommendations: Large bowel surveillance colonoscopy every 23 years is recommended from age 25 years for patients who are biallelic MUTYH carriers (or homozygous carriers of other BER gene defects). Colonoscopy is the preferred modality because of the likelihood of polyps requiring polypectomy.

Experience is limited because the role of MUTYH and other BER genes has only relatively recently been demonstrated. Hence, available evidence comes from pooled descriptive experience and opinion. However, there is a substantial colorectal cancer risk for those who are bi-allelic carriers.  Although indirect evidence suggests colonoscopic surveillance and polypectomy may be effective in colorectal cancer control, this has yet to be definitively determined. Indeed, we are not aware in the literature to date of any control subjects with bi-allelic MUTYH mutations who have reached the age of 55 years without developing colorectal cancer or polyposis . Hence, the risk may be sufficiently high to merit at least considering prophylactic colectomy and ileorectal anastomosis or even proctocolectomy and ileo-anal pouch if dense rectal polyposis is a feature. The patient should be counselled about the limited evidence available to guide decisions on either surveillance or pre-emptive surgical strategies

Familial adenomatous polyposis (FAP)


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Familial adenomatous polyposis (FAP)

Multiple polyp patients are a clinically heterogeneous group. Classical familial adenomatous polyposis (FAP; OMIM 175100) is caused by a germline mutation of the APC gene (at the locus 5q22-21) which activates the Wnt pathway (Bodmer et al. 1987; Groden et al. 1991; Clevers 2006). APC is also somatically mutated in approximately 70% of sporadic colorectal cancer.  However these cases are not caused by inherited mutations in the gene.

Polyposis (carpeting the rectum 20 years following a previous ileorectal anastamosis)

FAP is characterised by over a hundred colonic adenomas, and a high penetrance of colorectal cancer with an average age of cancer presentation of 39 years. There are also extra-colonic manifestations including intra-abdominal desmoids, duodenal adenomas and congenital hypertrophy of the retinal pigment epithelium(CHRPE).

In classical FAP, the risk of developing colorectal cancer exceeds 90% by age 70 years without prophylactic surgery.  The risk of gastroduodenal cancer is about 7%.  Around 25% of all cases are due to new mutations in the APC gene and so there is no previous family history. Nonetheless, children of individuals with a new mutation are at 50% risk of inheriting the condition.

The population prevalence of FAP is estimated at 1:14 000.  Owing to highly effective surgical prophylaxis, FAP accounts for only 0.07% of incident colorectal cancers in modern practice.  As registries and genetic services improve detection of at-risk family members, the proportion of colorectal cancer cases due to FAP should reduce, limited only by the proportion due to new mutations, which account for 25% of cases.

In attenuated FAP (AFAP) there is a later age of onset of colorectal cancer with a lower penetrance. The polyps number 10-100 in affected individuals. This arbitrary distinction is based on clinical characteristics, merely representing different ends of the same phenotypic spectrum of FAP. Germline mutations in APC account for up to 15% of patients with 5–100 adenomas and can be categorised AFAP.

English: CHRPE - congenital hypertrophy of the...

English: CHRPE – congenital hypertrophy of the retinal pigment epithelium (Photo credit: Wikipedia)

English: CHRPE – congenital hypertrophy of the retinal pigment epithelium (Photo credit: Wikipedia)

Somatic mutations in the APC gene

With regard to APC mutations, the most important functional domains of the APC gene appear to be the first serine alanine methionine proline (SAMP) (axin binding) repeat at codon 1580(Smits et al. 1999) and the first, second and third 20-amino acid repeats (20AARs) involved in ß-catenin binding and degradation. The great majority of pathogenic APC mutations truncate the protein before the first SAMP repeat and leave a stable, truncated protein that encodes 0-3 20AARs.

The ‘just-right’ model. The figure shows the multiple domains of the APC protein and the correlation between the position of the germline mutation and that of the somatic mutation. (a) Germline mutations between the first and the second 20AAR are associated with LOH. (b) Germline mutations before the first 20AAR are associated with somatic mutations between the second and third 20AAR. (c) Germline mutations after the second 20AAR are associated with somatic mutations before the first 20AAR(Segditsas and Tomlinson 2006).

APC is a classic tumour suppressor gene, requiring two hits for inactivation (Knudson 1971). In colorectal tumours from FAP patients, the germline wild-type allele either undergoes loss of heterozygosity (LOH) or acquires a protein-truncating mutation. Most somatic mutations occur in a restricted region of the gene, the mutation cluster region (MCR) (Miyoshi, Nagase et al. 1992). The reason for the MCR and relatively low frequency of LOH at APC was discovered from studies of FAP (Lamlum et al. 1999). It was found that LOH is strongly associated with germline mutations between the first and second 20AAR (codons 1285-1379). Germline mutations before codon 1280 are associated with somatic mutations between the second and third 20AAR (codons 1400 and 1495); and germline mutations after codon 1400 are associated with somatic mutations before codon 1280 (Lamlum, Ilyas et al. 1999; Albuquerque et al. 2002; Crabtree et al. 2003), Most tumours end up with APC alleles that encode a total of two 20AARs(Figure 1‑4). Similar associations exist for sporadic colorectal cancers. This association has been proposed to cause an optimal level of Wnt signalling/ß-catenin activation (Lamlum, Ilyas et al. 1999; Albuquerque, Breukel et al. 2002). Whatever the case, it is clear that selective constraints act on colorectal tumours such that some combinations of APC mutations provide a superior growth advantage for the tumour cell. This is known as the ‘just right’ hypothesis.

Germline APC mutation and phenotype

There is a genotype-phenotype relationship determined by the precise location of the APC mutation. AFAP is associated with germline mutations in three regions of APC: 5’ (codon 1580); and the alternatively spliced region of exon 9 (Knudsen et al. 2003). Mutations close to codon 1300 are the most commonly found and are associated with a severe phenotype, typically producing over 2000 polyps and earlier-onset colorectal cancer (Nugent et al. 1994; Debinski et al. 1996). De novo mutations of APC occur in approximately 20% of FAP. In a small study de novo mutations of APC were found to be more commonly of paternal origin (Aretz 2004).

Somatic mutations in the Wnt signalling pathway in genes other than APC

Figure 1. Wnt doesn't bind to the receptor. Ax...

Figure 1. Wnt doesn’t bind to the receptor. Axin, GSK and APC form a “destruction complex,” and β-Cat is destroyed. Compare to Figure 2. See the article main text for details. (Photo credit: Wikipedia)

Figure 1. Wnt doesn’t bind to the receptor. Axin, GSK and APC form a “destruction complex,” and β-Cat is destroyed. Compare to Figure 2. See the article main text for details. (Photo credit: Wikipedia)

The Wnt signalling pathway is activated in approximately 75% of colorectal cancer, and is one of the key signalling pathways in cancer, regulating cell growth, motility and differentiation. APC binds to the ß-catenin protein which functions in cell adhesion andas a downstream transcriptional activator in the Wnt signallingpathway (Wong and Pignatelli 2002). Somatic mutations in ß-CATENIN usually delete the whole of exon 3 or target individual serine or threonine residues encoded by this exon (Ilyas et al. 1997; Morin et al.). These residues are phosphorylated by the degradation complex that contains APC, and hence their mutation causes ß-catenin to escape from proteosomal degradation. These mutations are particularly associated with HNPCC tumours (but not sporadic MSI tumours) (Johnson et al. 2005). However, less than 5% of all sporadic colorectal cancer has mutation in ß-CATENIN. In addition somatic mutations have been reported in AXIN1 (Webster et al. 2000) and AXIN2 (Suraweera et al. 2006), the importance of which is uncertain.
Screening and Managment of FAP

Establishment of FAP registries

Families with FAP should be referred to the regional clinical genetics service or other specialist service that can facilitate risk assessment, genetic testing and screening of family members. Some regional services have specific FAP registers that facilitate regular follow-up. FAP registries have been shown to improve outcomes by systematic and structured delivery of management, monitoring interventions and surveillance, as well as serving as a focus for audit.

Large bowel surveillance for FAP family members Annual flexible sigmoidoscopy and alternating colonoscopy should be offered to mutation carriers from diagnosis until polyp load indicates a need for surgery.198 In a small minority of families where no mutation can be identified and genetic linkage analysis is not possible, family members at 50% risk should have annual surveillance from age 13e15 until age 30 years, and every 35 years thereafter until age 60. Surveillance might also be offered as a temporary measure for people with documented APC gene mutations and a significant polyp load but who wish to defer prophylactic surgery for personal reasons. Such individuals should be offered 6-monthly flexible sigmoidoscopy and annual colonoscopy. As in Lynch syndrome, chromoendoscopy or narrow band endoscopy may have a place in surveillance for attenuated FAP, but the utility of these techniques merits further appraisal and must not replace conventional endoscopic approaches. Surgery can be deferred if careful follow-up is instigated and the patient is fully aware of the risks of cancer. This is especially the case for attenuated FAP but can also be useful in the management of classical FAP for individuals who have a low polyp burden in terms of size, multiplicity and degree of dysplasia. The cancer risk increases substantially after 25 years, and so surgery should be undertaken before then unless polyps are sparse and there is no high-grade dysplasia. If colectomy and ileorectal anastomosis are performed, the rectum must be kept under review annually for life because the risk of cancer in the retained rectum is 1229%.The anorectal cuff after restorative proctocolectomy should also be kept under annual review for life.

Prophylactic colorectal surgery

Patients with typical FAP should be advised to undergo prophylactic surgery between the ages of 16 and 25 years, but the exact timing of surgery should be guided by polyp numbers, size and dysplasia and fully informed patient choice influenced by educational and child-bearing issues. Surgical options include proctocolectomy and ileoanal pouch or a colectomy with ileorectal anastomosis.

People with proven FAP require prophylactic surgery to remove the majority of at-risk large bowel epithelium. Colectomy and ileorectal anastomosis is associated with a 1229% risk of cancer in the retained rectum, whereas restorative proctocolectomy is associated with a very low risk of cancer in the pouch or in the retained mucosa at anorectum. Ileoanal pouch construction may be associated with impaired fertility.  It is clear that case identification and prophylactic surgery have markedly improved survival in FAP.

Upper gastrointestinal surveillance in FAP

Because of the substantial risk of upper gastrointestinal malignancy in FAP, surveillance of this tract is recommended. While gastroduodenal polyposis is well recognised in FAP and surveillance practice is established practice in the overall management, there is limited evidence on which to gauge the potential benefit of surveillance. However, the approach seems reasonable, and 3-yearly upper gastrointestinal endoscopy is recommended from age 30 years with the aim of detecting early curable cancers. Patients with large numbers of duodenal polyps should undergo annual surveillance.

Gastroduodenal and periampullary malignancies account for a small, but appreciable, number of deaths in patients with FAP. Duodenal polyposis occurs in approximately 90% of FAP patients and the overall lifetime risk of periampullary cancer is 35%.Advancing age and mutation location within the APC gene appear to have an effect on duodenal carcinoma risk.  Almost all FAP patients have some abnormality on inspection and biopsy of the duodenum by age 40.  The degree of duodenal polyposis can be assessed using an endoscopic/histological scoring system (Spigelman classification143), which can be helpful in predicting the risk of duodenal cancer. The worst stage (IV) has a 10-year risk of 36% and stage 0 negligible risk.207 Hence, it seems reasonable to offer 3-yearly upper gastrointestinal surveillance from age 30 years and more frequently if there is extensive polyposis. However, it should be noted that the effectiveness of this intervention in reducing mortality is unknown, especially since duodenal polypectomy is unsatisfactory208 and prophylactic duodenectomy is a major undertaking with substantial attendant morbidity and mortality.

 

Twenty-five years since landmark bowel cancer discovery – Cancer Research UK – Science Update blog


 

Twenty-five years since landmark bowel cancer discovery – Cancer Research UK – Science Update blog.

 

Twenty-five years since landmark bowel cancer discovery

 

Professor Sir Walter Bodmer

 

Professor Sir Walter Bodmer who helped locate the APC gene 25 years ago.

 

There’s a lot more to do before we can say we’ve beaten cancer, but every now and then, it’s good to sit back and reflect on how far we’ve already come.

 

Back in June, when the country was celebrating the Diamond Jubilee, we took time to think about how much cancer research has changed since the Queen came to the throne.

 

And this month, we’re proud to look back at one of our key achievements, which has played a big role in the lives of the one in twenty patients who’s bowel cancer is inherited.

 

It’s 25 years this month since we discovered the location in the DNA of our cells of a gene called APC. Thanks to this discovery, members of families in which many cases of bowel cancer occur – often at a young age – can now be offered genetic tests to look for inherited faults in this gene, and potentially life-saving screening if they carry them.

 

We now know that inherited faults in APC – which causes a disease called familial adenomatous polyposis (FAP) – account for about one in every hundred cases of bowel cancer.

 

When it’s working normally, the APC gene helps protect us from cancer by preventing cells from multiplying out of control.

 

But when it’s faulty, it greatly increases the risk of bowel cancer. And as well as its role in inherited disease, it’s also involved in non-inherited (or ‘sporadic’) bowel cancers. Overall, about eight out of ten cases of bowel cancer are fuelled by faults in the APC gene – making it a crucial part of the pathway that leads to bowel cancer.

 

And it was one of our former Chief Executive Officers, Professor Walter Bodmer, who made the breakthrough in 1987.  You can find out all about how Professor Bodmer tracked down the gene on this blog post.

 

Since Professor Bodmer’s discovery in the late 80s, death rates from bowel cancer in the UK have dropped by a third, thanks to improvements in prevention and screening, and better treatments.

 

But the story doesn’t stop there – and won’t stop until we beat the disease.

 

Ongoing story

 

Scientists have continued to study APC for the past two decades. They’re now getting a much clearer understanding of how APC works and what happens when it goes wrong.

 

Much of this groundbreaking work has been carried out by Cancer Research UK-funded scientists. For example, our scientists in Dundee discovered that APC helps bowel cells to know how to divide in an organised manner. And a team at the Cancer Research UK Beatson Research Institute in Glasgow discovered that blocking the activity of the APC gene in healthy bowel cancer stem cells causes tumours to grow.

 

Iron levels are the key

 

Earlier this month, there was yet more exciting news from our scientists in Glasgow. They found that levels of iron play a crucial role in controlling the development of bowel cancer in mice who carry a faulty APC gene. High levels of iron on their own do not cause bowel cancer, but they come into play if someone also has a faulty APC gene.

 

Although we’d known for a long time that people with a faulty APC gene have an increased risk of bowel cancer, it was not clear how this caused the disease. This latest research could help to answer that question. As study author Professor Owen Sampson says: “We’ve made a huge step in understanding how bowel cancer develops.”

 

Plans are afoot to investigate if treatments that cut the levels of iron in the bowel can help reduce the chances of bowel cancer developing in people who are at a greater risk of the disease.

 

Evolving science

 

In 25 years’ time, we’ll undoubtedly be looking back on this year’s breakthroughs and where they’ve taken us. For example, our landmark study that has completely reclassified breast cancer into ten new subtypes – and the in-depth research that has transformed the way we look at how cancers evolve.

 

How will we have built on these key discoveries? And how will things have changed for cancer patients as a result? As yet, we don’t have the answers, we’ll have to wait and see how the science unfolds. But it’s a tantalising prospect.

 

Cancer Research UK

Cancer Research UK (Photo credit: Wikipedia)

 

  • Polyposis (familyhistorybowelcancer.wordpress.com)

 

Rare variants as low-penetrance risk alleles for colorectal cancer


Rare variants as low-penetrance alleles

 

Rare variants will not be detectable by population association studies based on the use of linked polymorphic markers, even with very large case/control cohort studies.  This is because of low allelic frequency and individually small contributions to the overall inherited susceptibility of a disease.  These variants are less common than those studied in association studies (i.e. minor allele frequency (MAF) <0.05) but not as rare as obvious mutations (MAF >0.01), although such mutations may also be identified.  Finding rare variants requires nomination of candidate genes likely to have a role in disease aetiology, which are then directly screened for sequence variants which may affect protein function.  This is known as the ‘common-disease/rare-variant’ hypothesis (Pritchard 2001).

Allele frequency and effect sizes for genetic variants associated with colorectal cancer. Hindorff L A et al. Carcinogenesis 2011;32:945-954

So far there have been few rare variants identified in colorectal cancer, partially because candidate genes are not easily identified, and because there have only been a few studies performed.   In one such study variants in APC I1307K and E1317Q, in AXIN1, CTNNB1, and the mismatch repair genes hMLH1 and hMSH2 were more common in 124 multiple adenoma cases than in controls (Fearnhead et al. 2004).   Studies of other candidate genes have produced results of low or no significance however (Dallosso et al. 2008; Zogopoulos et al. 2008).

Labelling APC I1307K a rare variant may not be accurate, as the frequency of the polymorphism in the Ashkenazi population where it is present is 6%, thus potentially suitable for large association studies.  This distinction underlines the arbitrary nature of how such polymorphisms are labelled as rare or common variants.

Although the population attributable risk (PAR) of rare variants may be relatively high, the relative influence of these common variants is low, with reported odds ratios below 2 and peaking at approximately 1.2 (Easton and Eeles 2008).  Most rare variants have odds ratios a little higher than 2 but not above 5, with a mean of 3.7 in observations thus far (Bodmer and Bonilla 2008).  Their individual contributions are small, and they do not give rise to familial concentrations of cases.  As techniques improve to interrogate genetic sequence in an inexpensive, high-throughput and efficient manner this method of identifying variants is likely to generate a higher yield of significant results in the near future.

A candidate gene approach demonstrated rare novel low penetrance breast cancer predisposition loci in three genes, PALB2, BRIP1, and RAD51C.  (Seal et al 2006; Rahman et al 2007; Meindl et al 2010).   This discovery was assisted by the identification of breast cancer cases in Fanconi Anaemia pedigrees.  In general however, it is not a simple task to prioritize candidates for rare variant studies.  In the short term, it is likely that discovery efforts will be focused largely on sequencing candidate genes. Nevertheless, it is becoming feasible to sequence entire genomes to discover variants, due to decreased costs and increased efficiency of such methods.  In a proof of principle study, complete exomic sequencing of a patient with familial pancreatic cancer identified a germline truncating mutation in PALB2 which appeared responsible for this individual’s predisposition to the disease (Jones et al 2009), although mutations in this gene are thought to be rare events in familial pancreatic cancer (Tischkowitz et al 2010).

The above mentioned rare variant loci for breast cancer in PALB2, BRIP1, and RAD51C were present in 10, 8 and 2 cases and 0, 1 and 0 controls respectively.  Due to lack of power rare variants are difficult to validate by frequency alone in an association-type study. If we assume that a single variant or a set of related variants (for example, in the same gene) occurs at a general population frequency of 0.01–0.001, as many as 1000 unselected cases or controls will be required to detect with probability of about 0.7 more than one variant in a discovery screen (Bodmer & Tomlinson 2010).

Nevertheless, in principle the more common a variant is in the population the less its biological impact, thus allowing it to be passed on through generations without affecting reproductive ability.  Rare variants are likely to reveal more about the pathophysiology of the disease process than common variants, as they are likely to have functional significance, as opposed to common variants which are probably in linkage disequilibrium with the causative mutations.

However it is more problematic to design useful studies of rare variants, as random variation identified cannot be readily assumed to be of functional significance, for example over 1500 variants of uncertain significance (VUSs) have been identified in BRCA1 using a sequencing based approach in breast cancer cases.  The difficulty with rare variant discovery, particularly with whole exomic sequence analysis, will be to sort out the candidate functional variation from an almost overwhelming background of functionally irrelevant variation.  The choice of targets will, in general, require some a priori assessment of functional effects.  In silico biometric approaches have been developed with increasing predictive ability, although in vitro demonstration of effects are generally preferable in order to determine functional effects, for example simple effects on expression or protein truncation.

Studying a cohort of affected cases and subsequently examining a control set for variants identified can cause ascertainment bias.  Thus it would be preferable to search for them in affected individuals and controls with equal rigour, and to use a statistical framework to determine whether variants are truly more common in the affected.  These studies are likely to require extremely large and/or enriched data sets in order to identify and verify significant rare variants.  Nevertheless it is becoming increasingly cost and time effective to perform even whole genome sequencing to determine genetic predisposition to both common and rare disease.

 

Copyright, Dr Kevin Monahan

Adenoma to carcinoma sequence – Colorectal cancer development


 

Colorectal Cancer Development

Pathway from normal colorectal epithelium to cancer

 

Colorectal cancer develops via an adenoma to carcinoma sequence with the accumulation of a number of genetic and epigenetic mutations (Figure 1‑3) (Morson 1968; Fearon and Vogelstein 1990).  The mutations accumulated vary in hereditary cancer depending on the initiating mutation.  In their normal state, tumour suppressor genes inhibit cell proliferation.  Growth inhibition is lost when both alleles are inactivated by mutation and/or epigenetic changes, such as promoter methylation which stifles expression of the gene.  Tumour suppressor genes broadly conform to Knudson’s classic two-hit hypothesis, where inactivation of both alleles is required for tumour suppressor genes to lose their normal function (Knudson 1971).  In contrast, proto-oncogenes act by promoting cell proliferation.  Mutation of these genes leads to abnormal oncogenic over-expression or increased activity of the protein.

The adenoma-to-carcinoma sequence for colorectal cancer is probably most commonly initiated by bi-allelic mutation of the APC tumour suppressor gene.  APC mutations have been found in microadenomas (Otori et al. 1998), the earliest lesion on the pathway (also called aberrant crypt foci (Roncucci et al. 1991)), and in ~60-80% of early sporadic adenomas and carcinomas (Cottrell et al. 1992; Miyoshi et al. 1992; Nakamura et al. 1992).  APC is a key member of the canonical Wnt signalling pathway, and the key mechanism by which mutation of this gene contributes to carcinogenesis is by activation of this pathway.  However, further accumulated mutations in additional genes are required for progression of the early lesions to cancer.

The adenoma-to-carcinoma sequence: A: Aberrant crypt foci are seen using chromoendoscopy with x200 magnification (centre and 2 o’clock) surrounded by normal crypts. An early initiating mutation occurs here, usually a tumour suppressor gene such as APC. B: Adenomatous Polyp: Mutations in proto-oncogenes such as KRAS and BRAF lead to adenomatous polyp formation. C: Other genetic and epigenetic alterations such as promoter hypo-/hyper-methylation cause progression. There are hyperchromatic nuclei with prominent nucleoli indicate highly dysplastic crypts on the left side of this image. D; Adenocarcinoma, with invasion through the muscularis layer, and mucin producing glands with abnormal polarity. This is often associated with genomic copy number variation of regions such as 18q and 17p (P53).

Activating mutations of the oncogenes KRAS (Kirsten rat sarcoma viral oncogene homolog) and BRAF (v-raf murine sarcoma viral oncogene homolog B1), both members of the MAPK (mitogen activated protein kinase) signalling pathway, are found in the transition from early to an intermediate lesion in approximately 50% and 10% of cases respectively (Bos et al. 1987; Rajagopalan et al. 2002; Yuen et al. 2002).  Mutations of codons 12 and 13 in exon 2 of KRAS tend to occur in 30-60% of colorectal carcinomas (Kressner et al. 1998). The KRASgene product,a 21 kDa protein located at the inner plasma membrane, is involvedin the transduction of mitogenic signals.  The Ras protein isactivated transiently as a response to extracellular signalssuch as growth factors, cytokines and hormones that stimulate cell surface receptors (Campbell et al. 1998), and mutations in KRAS constitutively activate the Ras protein.

BRAF V600E substitution mutation

The substitution mutation of BRAF V600E is present in 4-12% of unselected colorectal tumours, and it is associated with sporadic MSI tumours but not HNPCC tumours (Vandrovcova et al. 2006).  MSI tumours outside the context of HNPCC are usually caused by methylation of the MLH1 promoter.  Indeed, there is a hypothesis that some tumours might develop through a separate hyperplastic polyp-serrated adenoma pathway (Spring et al. 2006).

Epigenetic changes such as promoter hypo-/hyper-methylation can cause disregulation of expression of many genes important in colorectal cancer (Hitchins et al. 2005; Hitchins et al. 2006).  Further progression to late type adenoma is associated with loss of 18q in 50% of large adenomas and 75% of carcinomas (Vogelstein et al. 1988; Fearon et al. 1990).  This causes loss of SMAD2 and SMAD4, members of the TGF-ß signalling pathway.  Point mutations of these genes have also been identified in colorectal cancer (Eppert et al. 1996; Hahn et al. 1996; Thiagalingam et al. 1996).  The adenoma to carcinoma transition appears to be associated with loss of 17p (Fearon et al. 1987; Rodrigues et al. 1990; Akiyama et al. 1998).  The 17p locus contains the P53 gene (Baker et al. 1990), the so-called gatekeeper of the cell which has important roles in the regulation of the cell cycle and apoptosis.  Loss of heterozygosity of 17p correlates with missense and truncating mutations in P53 (Baker et al. 1989; Baker, Preisinger et al. 1990).  Tumour invasion and metastasis are associated with loss of 8p (Hughes et al. 2006), and loss of E-cadherin function, a component of adherens-junctions (Hao et al. 1997; Christofori and Semb 1999).

The progression from adenoma to invasive carcinoma probably takes 10-40 years (Ilyas et al. 1999).  However, not all lesions will undergo malignant transformation, the reason for which is unclear.  It may be that the necessary mutations do not accumulate because of death, or because of environmental influences such as diet.

Genetic instability and colorectal cancer

 

Colorectal cancer may be subdivided genetically by the types of mutations which accumulate genome-wide during carcinogenesis.  It had been observed nearly a century ago that most cancers were aneuploid, and it has been noted that the degree of aneuploidy in colorectal cancer correlates with the severity of the neoplastic behaviour (Heim and Mitelman 1989).  A series of deletions, duplications, and rearrangements occur.  Allelic losses appear to be important in the progression from premalignant to malignant neoplasia in the colorectum.  This process is called chromosomal instability (CIN) and accounts for approximately 75% of colorectal cancers.  Ten to 15% are not CIN but do have smaller mutational events which are caused by loss of DNA mismatch repair (Fishel et al. 1993) and are referred to as MSI tumours.  The CpG island mutator phenotype (CIMP) is associated with methylation of promoter regions, CpG rich regions, which causes silencing of genes.  Tumours are often both MIN and CIMP, as methylation of mismatch repair gene promoters usually occurs in sporadic MSI tumours (Kane et al. 1997).

The role of genomic instability in causing and promoting tumour growth remains controversial (Lengauer et al. 1998; Tomlinson and Bodmer 1999).  Some argue that instability is necessary for tumourigenesis (Loeb 1991), while others take the viewthat Darwinian selection is the driving force. It is becoming clear that many cancers harbour multiple mutations, the great majority of which probably have no significant effect on tumour growth.  It may well be that some tumours with an inherited DNA repair defect accumulate more mutations than others.

 

Hereditary Colorectal Cancer Syndromes


Hereditary colorectal cancer syndromes

Germline mutations which predispose to multiple polyps

Familial adenomatous polyposis (FAP)

Multiple polyp patients are a clinically heterogeneous group.  Classical familial adenomatous polyposis (FAP; OMIM 175100) is caused by mutation of the APC gene which activates the Wnt pathway (Bodmer et al. 1987; Groden et al. 1991; Clevers 2006).  This gene is somatically mutated in approximately 70% of sporadic colorectal cancer.

Polyposis (carpeting a rectum after a previous ileocolonic anastamosis)

FAP is characterised by over a hundred colonic adenomas, and a high penetrance of colorectal cancer with an average age of cancer presentation of 39 years.  There are also extra-colonic manifestations including intra-abdominal desmoids, duodenal adenomas and congenital hypertrophy of the retinal pigment epithelium (CHRPE).  In attenuated FAP (AFAP) there is a later age of onset of colorectal cancer with a lower penetrance.  The polyps number 10-100 in affected individuals.  This arbitrary distinction is based on clinical characteristics, merely representing different ends of the same phenotypic spectrum of FAP.  Germline mutations in APC account for up to 15% of patients with 5–100 adenomas and can be partitioned out as AFAP.

English: CHRPE - congenital hypertrophy of the...

English: CHRPE – congenital hypertrophy of the retinal pigment epithelium (Photo credit: Wikipedia)

Somatic mutations in the APC gene

From the perspective of APC mutations, the most important functional domains of the APC gene appear to be the first serine alanine methionine proline (SAMP) (axin binding) repeat at codon 1580(Smits et al. 1999) and the first, second and third 20-amino acid repeats (20AARs) involved in ß-catenin binding and degradation. The great majority of pathogenic APC mutations truncate the protein before the first SAMP repeat and leave a stable, truncated protein that encodes 0-3 20AARs.

The ‘just-right’ model. The figure shows the multiple domains of the APC protein and the correlation between the position of the germline mutation and that of the somatic mutation. (a) Germline mutations between the first and the second 20AAR are associated with LOH. (b) Germline mutations before the first 20AAR are associated with somatic mutations between the second and third 20AAR. (c) Germline mutations after the second 20AAR are associated with somatic mutations before the first 20AAR(Segditsas and Tomlinson 2006).

APC is a classic tumour suppressor gene, requiring two hits for inactivation (Knudson 1971).  In colorectal tumours from FAP patients, the germline wild-type allele either undergoes loss of heterozygosity (LOH) or acquires a protein-truncating mutation.  Most somatic mutations occur in a restricted region of the gene, the mutation cluster region (MCR) (Miyoshi, Nagase et al. 1992). The reason for the MCR and relatively low frequency of LOH at APC was discovered from studies of FAP (Lamlum et al. 1999).  It was found that LOH is strongly associated with germline mutations between the first and second 20AAR (codons 1285-1379). Germline mutations before codon 1280 are associated with somatic mutations between the second and third 20AAR (codons 1400 and 1495); and germline mutations after codon 1400 are associated with somatic mutations before codon 1280 (Lamlum, Ilyas et al. 1999; Albuquerque et al. 2002; Crabtree et al. 2003), Most tumours end up with APC alleles that encode a total of two 20AARs(Figure 1‑4).  Similar associations exist for sporadic colorectal cancers. This association has been proposed to cause an optimal level of Wnt signalling/ß-catenin activation (Lamlum, Ilyas et al. 1999; Albuquerque, Breukel et al. 2002).  Whatever the case, it is clear that selective constraints act on colorectal tumours such that some combinations of APC mutations provide a superior growth advantage for the tumour cell.  This is known as the ‘just right’ hypothesis.

Germline APC mutation and phenotype

There is evidence of a genotype-phenotype relationship with regard to APC mutations.  AFAP is associated with germline mutations in three regions of APC: 5’ (codon 1580); and the alternatively spliced region of exon 9 (Knudsen et al. 2003).  Mutations close to codon 1300 are the most commonly found and are associated with a severe phenotype, typically producing over 2000 polyps and earlier-onset colorectal cancer (Nugent et al. 1994; Debinski et al. 1996).  De novo mutations of APC occur in approximately 20% of FAP.  In a small study de novo mutations of APC were found to be more commonly of paternal origin (Aretz 2004).

Somatic mutations in the Wnt signalling pathway in genes other than APC

Figure 1. Wnt doesn't bind to the receptor. Ax...

Figure 1. Wnt doesn’t bind to the receptor. Axin, GSK and APC form a “destruction complex,” and β-Cat is destroyed. Compare to Figure 2. See the article main text for details. (Photo credit: Wikipedia)

The Wnt signalling pathway is activated in approximately 75% of colorectal cancer, and is one of the key signalling pathways in cancer, regulating cell growth, motility and differentiation.  APC binds to the ß-catenin protein which functions in cell adhesion andas a downstream transcriptional activator in the Wnt signallingpathway (Wong and Pignatelli 2002).  Somatic mutations in ß-CATENIN usually delete the whole of exon 3 or target individual serine or threonine residues encoded by this exon (Ilyas et al. 1997; Morin et al.).  These residues are phosphorylated by the degradation complex that contains APC, and hence their mutation causes ß-catenin to escape from proteosomal degradation.  These mutations are particularlyassociated with HNPCC tumours (but not sporadic MSI tumours) (Johnson et al. 2005).  However, less than 5% of all sporadic colorectal cancer has mutation in ß-CATENIN.  In addition somatic mutations have been reported in AXIN1 (Webster et al. 2000) and AXIN2 (Suraweera et al. 2006), the importance of which is uncertain.

MYH-associated polyposis (MAP)

Damaged DNA is repaired by several mechanisms, one of which involves a family of enzymes involved in base-excision repair (BER). The MYH gene encodes a DNA glycosylaseinvolved in the repair of the oxidative lesion 8-oxoguanine, a by-product of cellular metabolism and oxidative damage of DNA.

(8-oxoG, left), in syn conformation, forming a...

(8-oxoG, left), in syn conformation, forming a with (dATP, right). Created using ACD/ChemSketch 10.0 and . (Photo credit: Wikipedia)

The products of three BER repair genes, OGG1, MTH1 and MYH work together to prevent 8-oxo-G induced mutagenesis.  Mutations in MYH cause an autosomal recessive colorectal cancer and polyposis syndrome MYH-associated polyposis (MAP; OMIM 608456) (Al-Tassan et al. 2002).  Somatic mutations in the APC gene in polyps from individuals affected with MAP are almost invariably G to T transversions (Sieber et al. 2003), and it was by understanding the underlying DNA repair mechanism of this mutation, base-excision repair, that MYHwas identified as a candidate-predisposition

English: Schematic of base excision repair

English: Schematic of base excision repair (Photo credit: Wikipedia)

gene. G to T transversion mutations were also identified in KRAS in codon 12 (Lipton et al. 2003).  The adenoma to carcinoma pathway in MAP does not involve BRAF V600E, SMAD4 or TGFBIIR mutations, or microsatellite instability, and the cancers are near-diploid (Lipton, Halford et al. 2003).  Thus, tumours with germline MYH mutations tend to follow a distinct pathway.

The term MYH-associated polyposis (MAP) may be misleading as up to 20% of biallelic MYH mutation carriers are diagnosed with colorectal cancer without polyposis (Wang et al. 2004).  Biallelic mutations in MYH have been found to account for approximately 10% of polyposis patients, but <1% of all colorectal cancer (Halford et al. 2003; Wang, Baudhuin et al. 2004).  The largest population study to date indicates that approximately 0.2% of all colorectal cancer is caused by biallelic mutations in MYH (Webb et al. 2006).  It was demonstrated in the same study that monoallelic MYH mutations are not associated with an increased risk of colorectal cancer.  The MAP phenotype typically falls in to the AFAP group, with extra-colonic manifestations consisting of duodenal polyps but not intra-abdominal desmoids.  Among Caucasians approximately 80% of mutations in MYH causing MAP are Y165C or G382D (Sieber, Lipton et al. 2003).  The E466X mutation is a common founder mutation among Pakistani populations, and the most common mutation in the St Mark’s Hospital MAP population (unpublished data).  Y90X is a founder mutation in Indian populations (Sieber, Lipton et al. 2003).

Hereditary mixed polyposis syndrome (HMPS)

Hereditary mixed polyposis syndrome (HMPS OMIM 601228) is a mixed colorectal tumour syndrome which has been linked to the CRAC1 locus on 15q13-14 (Thomas et al. 1996; Jaeger et al. 2003).  It is a rare condition found in a few families of Ashkenazi descent, with an autosomal dominant inheritance, mixed juvenile, adenomatous and hyperplastic polyps, as well as colorectal cancer (Whitelaw et al. 1997).  The best screening protocol for polyps in HMPS is not clear as the condtion is rare.  In addition genome-wide association revealed common low-penetrance predisposition alleles at the CRAC1 locus which are linked to sporadic colorectal cancer risk (Jaeger et al. 2008).  The gene which causes HMPS was recently identified as a 40kb duplication upstream of the gene GREM1 at the CRAC1 locus (Jaeger et al 2012) which causes disruption of the BMP pathway, a pathway also disrupted in Juvenile Polyposis Syndrome.

The hyperplastic polyp and serrated adenoma pathway

The first series of mixed hyperplastic-adenomatous polyps were described in 1990 (Longacre and Fenoglio-Preiser 1990), and have been an increasingly recognised phenomenon.   Most hyperplastic polyps have no malignant potential, although some recent studies have indicated that some have malignant potential, especially those with serrated architecture (sessile serrated adenomas – SSAs), large hyperplastic polyps, mixed polyps and polyps on the right side of the colon (Torlakovic et al. 2003).

Intermediate magnification micrograph of a SSA.

Intermediate magnification micrograph of a SSA. There are sawtooth serrations at the bases of the crypts which helps differentiate this from hyperplastic polyps (Photo credit: Wikipedia)

Some evidence suggests that some but not all of these tumours develop along a ‘serrated pathway’ separate from the classical adenoma-carcinoma sequence (Sawyer et al. 2002; Spring, Zhao et al. 2006). This serrated pathway involves one group who accumulate BRAF V600E mutations and another separate pathway which involves KRAS mutations(Carvajal-Carmona et al. 2007).  In addition the tumours often have methylation of the MLH1 promoter with subsequent microsatellite instability and CIMP phenotype(Jass 2005).

An inherited hyperplastic polyposis syndrome (HPS) has also been increasingly recognised (Cohen et al. 1981; Sumner et al. 1981). In HPS, multiple serrated polyps develop in the colorectum, and approximately 50% of cases present with at least one CRC (Ferrandez et al. 2004; Young and Jass 2006).  In the WHO criteria, Burt and Jass defined HPS as at least five HPs proximal to the sigmoid colon, two of which are > 1 cm diameter, or more than 30 HPs at any site in the large bowel (Burt 2000). Rashid et al, however, used a different classification system, in which HPS was defined as any person with more than 20 HPs, and separate classes were used for patients with large (>1 cm diameter) or multiple (5-10) HPs (Rashid et al. 2000).  These differing classification systems reflect a syndrome which may be both genetically and phenotypically heterogeneous, but one which is becoming increasingly recognised.

This is a mixed histology polyp from an affected individual in a large hyperplastic polyposis family. It contains both villous, serrated portions, the latter contains a zone of high grade dysplasia with BRAF mutations, the villous section was BRAF wild type.

HPS (sometimes known as the ‘serrated pathway syndrome’ (SPS)) may, in fact, be a heterogeneous group of conditions leading to sporadic and inherited cases of colorectal neoplasia.  There are two alternative clinical criteria for the diagnosis of HPS families (Burt 2000; Rashid, Houlihan et al. 2000).  This syndrome is usually associated with somatic mutations in either BRAF or KRAS, but not both together (Carvajal-Carmona, Howarth et al. 2007), providing further evidence of molecular as well as phenotypic heterogeneity.  BRAF mutations are associated with low-grade microsatellite instability due to methylation in CpG islands (CIMP)(Young, Jenkins et al. 2007).  This may result in loss of expression of DNA repair genes MLH1 and MGMT (O(6)-methylguanine-DNA methyltransferase) in dysplastic mixed polyps from HPS patients, possibly as a result of promoter methylation (Oh et al. 2005).

Linkage analysis in a large family affected with hyperplastic polyposis syndrome deomstrated a maximum LOD score of 2.71 on the short arm of chromosome 8 (8p.21; Monahan et al 2007).

The Galway Family: A large Irish family affected with Hyperplastic Polyposis Syndrome

Other causes of multiple colorectal polyp predisposition

Germline mutations in exon 7 of the AXIN2 gene have recently been very rarely associated with a predisposition to colorectal polyposis and tooth agenesis ((Lammi et al. 2004) OMIM 608615).   Somatic mutations have been found in AXIN2 previously, but germline mutations have not been found in other studies (Lejeune et al. 2006).

Other mutated genes which cause polyps such as SMAD4, PTEN and BMPR1A lead to multiple polyp syndromes with clinically recognisable differences from the above conditions, such as Juvenile Polyposis (OMIM 174900) and Peutz-Jeghers syndrome (OMIM 175200).  The BMPR1A gene product, mutated in Juvenile Polyposis, is a receptor for bone-morphogenetic proteins (BMPs) which are members of TGF-β superfamily and part of the BMP pathway which regulates colonocyte growth and proliferation (Howe et al. 2001).  Germline mutations in PTEN can cause a number of polyposis and multi-systemic syndromes including Cowden syndrome (CS) and Bannayan-Riley-Ruvalcaba syndrome (BRRS), and the umbrella term ‘PTEN-mutation spectrum’.  We recommend the Cleveland Calculator which can help determine the likelihood of a germline mutation in PTEN for any of these conditions and thus the need for genetic testing;

Cleveland Calculator: http://www.lerner.ccf.org/gmi/ccscore/index.php

Unknown genetic predispositions account for over 50% of all patients who develop 10-100 colorectal adenomas during their lifetime, and for about 20% of those with more than 100 polyps(Lamlum et al. 2000) (Spirio et al. 1993).  To develop as many as 10-100 colorectal adenomas is a priori indicative of an inherited predisposition and many of these patients have a family history of multiple polyps. It is overwhelmingly likely, therefore, that the remaining multiple polyp patients have an inherited disease of an unknown genetic origin. Molecular characterisation of tumours from these patients remains deficient.

Predisposition to colorectal cancer in patients without multiple polyps

Lynch Syndrome/Hereditary non-polyposis colorectal cancer (HNPCC) and related syndromes

Lynch Syndrome (also known as Hereditary non-polyposis colorectal cancer(HNPCC; OMIM 120435)) accounts for approximately 2.2-4% of all colorectal cancer (Hampel et al. 2005).  Lynch Syndrome is a familial cancer syndrome which accounts for approximately 2-3% of all colorectal cancer in the UK.  It has formerly been known as Hereditary Non-Polyposis Colorectal Cancer Syndrome (HNPCC), however the phenotype is more complex with multiple extracolonic tumours, for example, so this term has now been largely abandoned.

An Irish family tree with Lynch Syndrome caused by an inherited mutation in MSH2.  Members of this family are affected predominantly with colorectal cancer (CRC), but also small bowel cancer (SBCa), Gastric, Pancreatic, Uterine and other cancers, as well as conditions not linked to Lynch Syndrome such as Crohn’s disease.

LS is an autosomally dominant inherited condition commonly caused by germline mutation in one of four DNA mismatch repair genes, MLH1, MSH2, MSH6 and PMS2.  A minority of these families may be identified because they have multiple affected members diagnosed at an early age.   The Amsterdam Criteria I and II (Vasen et al. 1993; Vasen et al. 1999)(see below) identify patients for colonoscopic and other screening.  Approximately 40-80% of patients meet these criteria, with 50% of the remainder meeting the modified criteria which include extracolonic cancers.  The revised Bethesda criteria (Umar et al. 2004) are used to identify patients for molecular screening of HNPCC, i.e. microsatellite instability ± immunohistochemistry studies.  Approximately 80% of patients are identified using the Bethesda criteria, although the specificity is low.

Immunohistochemistry and microsatellite instability analysis for Lynch Syndrome

Amsterdam I Criteria

  • ≥3 1st degree relatives with colorectal cancer (CRC)
  • ≥2 generations affected
  • One family member below age 50 years of age
  • Exclude familial adenomatous polyposis

Amsterdam II Criteria

  • As for Amsterdam I except that CRC may be substituted by cancer of endometrium, small bowel, or pelviureter.

Most families with LS, however, do not fulfil the Amsterdam criteria. The Revised Bethesda criteria are another set of diagnostic criteria designed to increase the diagnostic yield of testing for LS [7]. For example, all individuals diagnosed under the age of 50 years should be tested for the molecular features of LS in their tumours.  If molecular testing is diagnostic of LS, it can subsequently determine which families should undergo colonoscopic and other investigations, and to screen other high risk family members. The Revised Bethesda guidelines are designed to streamline the clinical diagnostic pathways used to identify mutation carriers in patients with colorectal cancer who might or might not fulfil the Amsterdam criteria, thus increasing diagnostic yield screening for LS.

The identification of such families with Lynch syndrome involves an extensive diagnostic work up comprising of various screening tools combined with genetic and immunohistochemical tests.  Initially the tumour from an affected individual may be tested for features suggestive of this condition by either immunohistochemistry of the mismatch repair proteins and/or DNA microsatellite instability (a hallmark of faulty DNA mismatch repair).  If either of these tests are abnormal, then germline testing may be performed to identify a putative heritable mutation in one of the causative genes.

Patient selection using Amsterdam and revised Bethesda criteria have been applied to clinical pathways in the United Kingdom through the use of national guidelines.  Given the implication of family history and known mortality benefit, the early recognition of Lynch syndrome is highly desirable. There have been concerns over the sensitivity, specificity, and predictive value of already existing guidelines. About 22% of affected individuals do not fulfil either Amsterdam or the Revised Bethesda criteria. As Barnetson et al argues, there might be multiple reasons for this such as small family size, unknown or inadequately taken family history, adoption, and patients without available tumour data [9]. A number of alternative screening models have been developed, such as MMRpredict, PREMM 1,2,6, MMRPro, and MsPath whilst searching for a careening tool that is simple, accurate, and clinically useful for predicting the likelihood of Lynch Syndrome.

Bethesda (revised) Criteria (Umar et al 2003)

  • 1 CRC below age 50 yr
  • Multiple CRC or HNPCC-related cancers
  • CRC with MSI-related histology under 60 years of age
  • CRC or HNPCC-related cancer in ≥1 1st degree relative, < 50 years of age
  • CRC or HNPCC-related cancer in at least two 1st or 2nd degree relatives, any age

MSI-type Histology: Using the revised Bethesda Criteria patients aged 50-60 years should have tumour testing

There is a slight preponderance of right-sided tumours (70% proximal to the splenic flexure) in Lynch Syndrome.  It is a highly penetrant condition which also features extracolonic cancers such as endometrial and gastric cancer.  The adenoma to carcinoma sequence is rapid with interval cancers occurring in 5% of patients despite two-yearly colonoscopic surveillance (Jarvinen, Aarnio et al. 2000).  The tumours are characteristically associated with a local lymphocytic infiltrate and a good prognosis when surgically resected (Jass 2000; Takemoto et al. 2004).

Screening tumours for Lynch Syndrome – is it cost effective?

There are clinical and economic trade-offs when implementing screening protocol on a large scale. As nondirected germline mutation testing for Lynch syndrome is prohibitively expensive at £1000 per gene, MSI and IHC are the screening tests of choice. In view of high costs of testing of all colorectal cancers for MSI or loss or MMR protein, an approach described by Heather Hampel of The Ohio State University, the Revised Bethesda Guidelines were felt to be an appropriate tool to select patients for genetic testing. However, the question remains open: is the “reflex” molecular tumour testing justified clinically and economically? Kastrinos et al, have looked into the popularity of the universal testing across several centres in US. Unsurprisingly, a pessimistic picture emerged showing the low uptake of the concept. The benefits of the universal testing are counterbalanced by practical problems such as an informed consent controversy, practicalities of dealing with the complexity of test results and the resultant implications. The fact that the cost effectiveness of this approach has not been yet validated plays heavily against such approach.

In US, Ramsey et al have carried out a study looking at cost-effectiveness of different strategies for identifying of persons with Lynch syndrome. The average cost per carrier detected using Bethesda guidelines was $15,787, and expanding this strategy to include costs and benefits for first degree relatives greatly improves the cost effectiveness of the program. Expanding the program to first degree relatives leads to savings from intensive screening to exceed the cost of testing.

In Europe, Pinol et al, has carried out a similar study evaluating cost-minimization analysis of identification strategies for MSH2/MLH1-associated Lynch syndrome. Authors concluded that clinical selection of patients using the Revised Bethesda Guidelines followed by either MSI analysis (€11,989 per detected mutation) or IHC (€10,644 per detected mutation) has proved to be more cost effective than performing any of these tests directly (€32,140 and €37,956 per detected mutation, respectively).

Further research has been carried out by Dinh et al in 2010 looking at the cost effectiveness of MMR gene mutations screening, and reached the conclusion that it is comparable to that of already established cancer screening protocols such as colorectal, cervical, and breast cancer screening. Authors argue that primary screening of individuals for MMR gene mutations, starting with the risk assessment between the ages of 25 and 35, followed by genetic testing of those whose risk exceeds 5%, is a strategy that could improve health outcomes in a cost effective manner relative to current practise with the average cost-effectiveness ratio of $26,000 per QALY.

These results echo several European studies, such as that carried out by Pinol V et al, 2005 in Spain, where authors suggest that MSI and IHC testing are equivalent strategies in terms of cost effectiveness when it comes to screening selected patients for MMR mutations

Other non-polyposis predisposition to colorectal neoplasia

About 15% of sporadic colorectal cancers are also microsatellite unstable and feature loss of protein staining on immunohistochemistry but are not caused by germline mutations in mismatch repair genes.  Often they are acquired sporadic type cancers caused by methylation of MLH1.  These associated with a particular genetic pathway which differs from HNPCC by the presence of BRAF V600E mutations, the absence of β-CATENIN exon 3 mutations and a methylator genotype (Young et al. 2005) (Oliveira et al. 2005).  Recently kindreds demonstrating some inheritance of MLH1 promoter methylation have been identified (Suter et al. 2004; Hitchins, Williams et al. 2005), although the evidence for this inherited epimutation is limited to a few case studies and may be related in imprinting (Chong et al. 2007; Hitchins and Ward 2007).

In addition there are a number of families which fulfil Amsterdam criteria but do not demonstrate microsatellite instability (Dove-Edwin, de Jong et al. 2006).  These families are termed by one group familial colorectal cancer type X (Lindor et al. 2005), and have a lower incidence of colorectal cancer occurring at a later age.  The genetic aetiology is not known for these families.

Approximately 93% of colorectal cancer occurs after the age of 50 years, and thus those young patients who develop cancer are likely to have an inherited or other risk factor such as chronic colitis.  The genetic risk is partially made up by inherited mutations which cause HNPCC.  However, there are likely to be a number of other lower penetrance genes which cause cancer predisposition, many of which may have a recessive form of inheritance and few polyps, and therefore a less clearly identifiable phenotype.

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