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DNA mismatch repair

This tag is associated with 11 posts

Aspirin for Hereditary Colorectal Cancer


The study shows that regularly taking the medicine cuts the risk of bowel cancer by more than 60% in those with a particular genetic predisposition to get the disease – as well as reducing the risk of other hereditary cancers.

Scientists who led the study said people with several family members with cancers other than breast, blood and prostate might be advised to start taking aspirin daily from the age of 45.

They said those without a family history of the disease might also consider doing so, but that they should make a personal assessment of the risks and benefits and get medical advice. Anyone thinking of taking the drug regularly should consult their doctor first.

Doctors already prescribe low, daily doses of aspirin to people at increased risk of heart attacks and strokes, and evidence has been growing of anti-cancer properties for 20 years. However, this is the first long-term, randomised controlled trial to show such an effect.

The trial involved people with Lynch syndrome, a genetic abnormality that predisposes carriers to develop bowel cancer and other solid organ cancers including endometrial, ovarian, stomach, kidney, oesophageal, brain and skin tumours.

The condition affects at least one in 1,000 people. Carriers are around 10 times as likely to develop cancer and often do so at a young age.

Professor John Burn of Newcastle University, who led the study, estimated that if all 30,000 or so people with Lynch syndrome in the UK were to start taking two aspirin tablets a day then some 10,000 cancers would be prevented over the next 30 years, saving about a thousand lives. The downside of the treatment is that around an extra thousand people would develop stomach ulcers as a side-effect.

“People with a genetic susceptibility are a model system,” said Burn, whose work is published online in the Lancet on Friday. “They are more sensitive to the environmental triggers to cancer.

“If we can do something to change cancer progression in people at high genetic risk, then that’s telling us what we might all benefit. But we are not making a recommendation for the general population. Everyone can take this evidence and make their own choice.

“In between you have the people who have a family history [of cancer]. Those individuals may well decide to put themselves on aspirin and that would be a reasonable conclusion from the data currently available.”

Between 1999 and 2005, about half of a group of 861 Lynch syndrome carriers were given two aspirins (600mg) a day, while the rest took placebos.

By 2010 those who had taken aspirin for at least two years were 63% less likely to have developed bowel cancer.

Looking at all forms of the disease, almost 30% of those in the placebo group developed a Lynch syndrome-related cancer, compared with 15% for those given aspirin.

The most common side effects associated with taking aspirin are gastrointestinal ulcers and stomach bleeding. There is also an very small increased risk of haemorrhagic stroke, in which a blood vessel in the brain bursts.

There was no difference in the proportions of the study groups suffering such side-effects.

Burn added that he takes low-dose aspirin tablets as a preventative measure. “That was a balanced judgment based on weighing risks and benefits. I know I might get an ulcer or a cerebral bleed but I’d rather not have a heart attack, stroke or cancer. That’s my choice.”

Aspirin is a synthetic version of the active component of willow bark, salicylic acid, which has been used as a medicine for its anti-inflammatory properties for hundreds of years. Salicylates also trigger programmed cell death to help diseased plants contain the spread of infection.

“It’s not a huge stretch to think that if salicylate induces programmed cell death in plants to kill infected cells, maybe it’s doing similar things in the animal kingdom to enhance the death of aberrant cells causing cancer,” said Prof Burn.

“This adds to the growing body of evidence showing the importance of aspirin, and aspirin-like drugs, in the fight against cancer and emphasises how critical it is to carry out long-term international research,” said Prof Chris Paraskeva, a bowel cancer expert at the University of Bristol.

The CAPP team have launched a website to recruit 3,000 people with Lynch syndrome worldwide to take part in a five-year trial to determine the best dose of aspirin to take.

 

A trial of methotrexate for cancer that has spread in people with a faulty MSH2 gene (MESH)


A trial of methotrexate for cancer that has spread in people with a faulty MSH2 gene (MESH)

This trial is looking at the chemotherapy drug methotrexate for people with a MSH2 gene fault who have cancer that started in the bowel, stomach, womb (endometrium), bladder, or lining of the urinary system (urothelium) and has spread.

Every cell contains DNA. This is the genetic information which controls how cells behave. In cancer cells, the DNA is changed or damaged. Cancers can have different types of changes in the DNA. One of these is when a gene called MSH2 is not working properly.

Doctors often use chemotherapy to treat cancer. But sometimes the cancer comes back after treatment and spreads elsewhere in the body.

Methotrexate is a chemotherapy drug that is used to treat some types of cancer. We know from research that methotrexate kills cancer cells when the MSH2 gene is not working properly. Researchers want to find out if it will help people with a faulty MSH2 gene who have cancer that has spread.

The aims of this trial are to

  • See how much methotrexate helps people in this situation
  • Learn more about the side effects

Recruitment

Start 08/04/2009

End 08/04/2014

Phase

Phase 2

Who can enter

You can enter this trial if you

  • Have cancer that started in your bowel, stomach, womb, bladder or urothelium and has grown into surrounding tissue, or has spread elsewhere in the body
  • Have cancer that did not respond to, or has come back after, treatment with standard chemotherapy, or you cannot have standard treatment for some reason
  • Have a MSH2 gene fault (the doctors will do tests to confirm this)
  • Have satisfactory blood test results
  • Are well enough to take part in the trial (performance status 0, 1 or 2)
  • Are at least 18 years old
  • Are willing to use reliable contraception during the trial and for 6 months afterwards if there is any chance you or your partner could become pregnant

You cannot enter this trial if you

  • Have already had treatment with methotrexate unless it was for a non cancerous condition and you finished treatment at least 5 years before you were diagnosed with cancer
  • Have had any other cancer in the last 10 years apart from non melanoma skin cancer or carcinoma of the cervix and the trial doctor thinks this could affect you taking part in this trial (If you have Lynch syndrome, you may be able to take part if you have had other cancers – the doctors will advise you on this)
  • Have had radiotherapy to a single area of cancer (a lesion) that the researchers will be measuring in this trial, unless the lesion has got bigger since you had radiotherapy
  • Have another medical condition that cannot be controlled with medication
  • Are pregnant or breastfeeding

Trial design

This phase 2 trial will recruit 56 people. Everybody taking part will have methotrexate.

You have methotrexate as an injection into a vein. The treatment only takes a few minutes. You have another injection a week later and then 2 weeks without any treatment. Each 3 week period is called a cycle of treatment. You have up to 6 cycles of treatment. But if the treatment is helping you, your doctor may talk to you about having it for longer.

During the trial, the researchers will take samples of blood, urine and a hair follicle (such as from an eyebrow). And they will get a sample of the tissue taken when you had surgery to remove your cancer or when you had a biopsy.

The researchers will use the samples to try to find substances they can measure in the body to help them tell how well the treatment is working. They call these substances biomarkers. And they will use the blood samples to look at your genes. This is to learn more about how genetic changes can lead to cancer and whether certain changes affect how people respond to treatment.

The trial team may also ask your permission to take an extra biopsy during treatment. This is to learn more about what effect the treatment has on the genetic make up of your cancer. If you don’t want to have this extra biopsy, you don’t have to. You can still take part in the trial.

All samples will be stored safely and may be used in the future, but only for research purposes.

You will be asked to fill out a questionnaire before you start treatment, just before the 2nd and 4th cycle of chemotherapy, and every 3 months for a year after you finish treatment. The questionnaire will ask about any side effects you have had and how you have been feeling. This is called a quality of life study.

The trial team will also ask you to fill out a short questionnaire which asks about other members of your family who have had cancer.

People taking part in this trial may also be asked to join extra studies looking at PET scans and MRI scans. Doctors want to find out if these scans can provide more information about bowel cancer with a faulty MSH2 gene.

You may be able to take part in one or both of these studies. Whether or not you are asked to take part will depend on where you are having your treatment and also where in your body the cancer is.

Hospital visits

You will see the doctors and have some tests before you start treatment. The tests include

  • Physical examination
  • Blood tests
  • CT scan
  • Chest X-ray
  • Heart trace (ECG)

You go to hospital twice in each 3 week cycle of treatment. You have regular blood tests. And after 9 weeks of treatment you have a CT scan to check that your cancer has not got any bigger. If the scan shows the cancer has grown, you will stop having the trial treatment and the doctors will discuss other treatment options with you. If the cancer has stayed the same size or got smaller, you will have the next 3 cycles of treatment and then another CT scan.

After you finish treatment you will see the trial doctors and have a CT scan every 3 months for up to 1 year.

If you do take part in the MRI or PET scan study (or both), you will have extra scans

  • Before you start treatment
  • After 2 weeks of treatment
  • When you finish treatment

Having an MRI scan takes about 15 to 30 minutes. If you have PET scans, you have an injection of a small amount of a radioactive drug first. Then you have to wait an hour before having the scan. The scan itself can take up to an hour.

Side effects

The side effects of methotrexate include

There is more information about the side effects of methotrexate on CancerHelp UK.

Location of trial

Top of Form

  • London
  • Sutton

For more information

Please note: we cannot help you to join a specific trial. Unless we state otherwise in this trial summary, you need to print this page and take it to your own doctor to discuss.

Find out how to join a trial or contact our cancer information nurses for other questions about cancer by phone (0808 800 4040), by email, or at

The Information Nurses

Cancer Research UK

Angel Building

407 St John Street

London
EC1V 4AD

Chief Investigator

Professor David Cunningham

Supported by: Institute of Cancer Research (ICR), The Royal Marsden NHS Foundation Trust

JAMA: Identification of Lynch Syndrome Among Patients With Colorectal Cancer


Identification of individuals at increased risk of hereditary cancer allows for the possibility of screening and early cancer detection, possibly resulting in decreased disease-specific mortality, and is the justification for germline genetic testing for specific cancer risk alleles. However, factors of prevalence and age-specific penetrance, effectiveness and invasiveness of screening procedures, and efficacy of early detection influence the potential benefit of such an approach.

For one of the most common hereditary cancer syndromes, Lynch syndrome (LS), also known as hereditary nonpolyposis colorectal cancer (HNPCC), various sets of clinical criteria, combined with pathologic phenotypic characteristics of tumor tissues in probands, have been used to identify individuals at risk in whom it is important to consider germline genetic testing for deleterious mutations in 1 of 4 DNA mismatch repair genes (MLH1, MSH2, MSH6, or PMS2). Lynch syndrome was originally defined by the “Amsterdam” clinical criteria as a history of at least 3 family members with histologically confirmed colorectal cancer (CRC) involving 2 generations with at least 1 person diagnosed before age 50 years.1 Although this approach is fairly specific in identifying families with highly penetrant LS, it is also overly restrictive and does not consider the possibility of later-onset variants of the disease, the implications of extracolonic tumors, or the limitations imposed by small family size.

Many families with known LS do not meet the original Amsterdam criteria, and this approach misses many families because of poor sensitivity. Therefore, it has been suggested, given the availability of an immunohistochemistry-based screening test for mismatch repair that closely mimics phenotypic microsatellite instability, that testing all colorectal (and perhaps endometrial) cancers for loss of proteins associated with mismatch DNA repair deficiency, or all cancers below some age cutoff, may overcome the limitations of selective criteria. However, the effectiveness of such a “universal” approach to screening for LS has not been tested in a population-based manner.

In this issue of JAMA, Moreira and colleagues2 address this question by performing a pooled-data analysis of a large set of population-based patient cohorts from around the world to determine the sensitivity and efficiency of several different strategies for LS screening, including the “universal” approach. Using more than 10 000 CRC samples, the authors found overall that universal tumor testing for mismatch repair deficiency was superior in sensitivity to the Bethesda guidelines, which incorporate personal and family history information, and which have been reported to be more accurate than the Amsterdam criteria,3 or an approach using an age cut-off of 70 years old, but that a hybrid of testing all tumors in individuals 70 years or younger and in older patients who meet Bethesda guidelines provides a reasonable compromise that may result in substantial cost savings.

The study by Moreira et al2 confirms that the prevalence of LS is high enough among patients with CRC, 3.1% in the whole series, that screening should be considered. However, in the EPICOLON cohort, consisting of patients newly diagnosed with CRC in 20 community hospitals in Spain,3 the prevalence was only 0.9% compared with 2.9% to 3.5% in the other 3 cohorts analyzed. The current study does not address possible explanations for this difference. Some patients in the study by Moreira et al were not drawn from a population-based registry but were excluded from analyses on the performance characteristics of screening strategies. Because most germline tests were driven by abnormal tumor testing results and not all patients underwent “gold-standard” (germline) testing, the high sensitivity estimates for screening strategies are probably somewhat optimistic. In addition, the PMS2 gene was not tested in many patients, so some persons with PMS2 mutations were probably missed. In this study, microsatellite instability testing added little to immunohistochemistry, but not all previous studies have found similar results. Selective BRAF mutation testing or promoter methylation testing to identify sporadic CRC were not performed, but the focus of the study was on the sensitivity of screening strategies, not specificity.

These results highlight the limitations of various clinical criteria to identify persons with LS, particularly those with mutations in the MSH6 or PMS2 genes. The study results should remind clinicians that simply asking about a family history of CRC in a first-degree relative will miss the majority of patients with LS: only 43% of patients with LS had such a family history—approximately 50% of those with MLH1 and MSH2 gene mutations and less than 20% of those with MSH6 and PMS2 gene mutations. Furthermore, although the mean age at CRC diagnosis was 48 years in LS patients, only 45% were diagnosed with CRC at 50 years or younger.

The potential for individualized preventive medicine provides the rationale for screening for LS. In deciding whether to establish widespread screening for LS in selected subgroups, the same considerations that govern screening for CRC in the general population could be applied. The target condition must be common enough to justify screening. A long asymptomatic period must allow for effective interventions. The potential benefits must outweigh the risks. The aggregate costs of screening and its consequences must be acceptable.

Moreira et al2 confirm that a variety of strategies can identify a significant number of persons with LS among patients with CRC. The benefits of intensive CRC and adenoma screening and prophylactic hysterectomy-oophorectomy in LS are well established.4 – 7 This raises several questions. What is the balance between the potential benefits and harms of screening? Are the economic costs acceptable? Which screening strategies are preferred?

Several studies have addressed the potential psychological harms of testing for LS. These studies include select populations and are observational. Patients considering genetic testing can develop short-term increases in anxiety, distress, and fear of cancer or death, especially among mutation carriers.8 – 10 Generally, a person’s psychological state returns to baseline after several months. Longer follow-up has demonstrated no major adverse psychological consequences for either mutation carriers or noncarriers after 1 and 3 years.8 – 9 ,11 However, some groups, such as younger men affected by cancer, may be at higher risk for adverse psychological effects.12 Patients may express fear concerning discrimination in employment or health insurance. In the United States, patients may be counseled that such discrimination is illegal under the Genetic Information Nondiscrimination Act.13

In the absence of controlled studies evaluating the long-term consequences of different screening strategies for LS, computerized decision analytic modeling can be used to explore critical questions. Several modeling studies with long-term time horizons have suggested that screening for LS among persons with CRC is likely to be cost-effective.14 – 17 The same may apply to women with endometrial cancer.18 One study that incorporated the potential short-term adverse effects LS testing can have on quality of life19 found that the long-term gains in life expectancy are likely to outweigh any short-term decreases in quality of life, at acceptable costs.20

Key issues raised by the results of Moreira et al2 were explored in a modeling study.17 Moreira et al found a relatively small incremental yield of universal tumor screening vs a highly sensitive selection strategy based on clinical criteria. The modeling study suggested that tumor testing strategies are likely to be costly compared with clinical criteria strategies when both are implemented optimally.17 However, in the model, when the clinical criteria strategies failed to be implemented in as few as 15% of patients, tumor testing strategies became cost-effective. In clinical practice, routine testing of tumors in pathology laboratories may be more feasible than ensuring widespread application of clinical criteria.

Moreira et al2 address the important issue of age at CRC diagnosis as a factor to inform screening strategies. The modeling study17 estimated that immunohistochemistry-based tumor testing in all persons vs only in those 70 years and younger could be considered cost-effective depending on society’s willingness to pay for preventive services. Using the actual age distribution at CRC diagnosis in the study of Moreira et al, instead of the model’s original assumptions, would result in enhanced cost-effectiveness for the “universal” approach.

Lynch syndrome affects families, not only individuals. Patients often identify the potential benefits to their family, especially their children, as a motivating force driving acceptance of genetic testing.21 However, the published uptake rates for genetic testing among relatives at risk have varied from 34% to 52%.22 – 23 The number of relatives unaffected by cancer but at risk for LS who undergo genetic testing is a key determinant of the cost-effectiveness of any screening strategy.16 – 17 Future public health efforts must address this critical factor.

A recent survey of US hospitals reported that routine tumor testing with immunohistochemistry, microsatellite instability, or both is currently performed at 71% of National Cancer Institute comprehensive cancer centers, 36% of American College of Surgeons–accredited community hospital comprehensive cancer programs, but only 15% of community hospital cancer programs.24 Routine tumor testing with immunohistochemistry or microsatellite instability does not require written consent. The authors suggested that this approach to testing may reflect an emerging standard of care. Routine tumor testing programs require mechanisms to track results, contact patients in ways patients will accept, and facilitate consultation with genetics professionals. Those dealing with results must understand complicating factors, including variants of uncertain significance, and must handle difficult cases, such as patients with classic family pedigrees in whom no mutation is found by current methods.

In the not too distant future, advances in genomic sequencing will challenge current genetic testing approaches. Commercial panels of “cancer genes” are already emerging. With the anticipated further reductions in the costs of DNA sequencing, up-front germline testing at the time of CRC diagnosis could become the most cost-effective strategy to screen for LS.17 Such testing would require written informed consent. Population-based genomic profiling could revolutionize the approach to identifying persons with LS.

The majority of patients with CRC do not have LS. But in the haystack of patients with CRC, those with LS are more like large knitting needles than tiny sewing needles—and a systematic search can find them. The investments of effort and resources required for this search can be rewarded by reductions in cancer incidence and mortality that are possible among patients and their unsuspecting relatives.

Abstract

Context Lynch syndrome is the most common form of hereditary colorectal cancer (CRC) and is caused by germline mutations in DNA mismatch repair (MMR) genes. Identification of gene carriers currently relies on germline analysis in patients with MMR-deficient tumors, but criteria to select individuals in whom tumor MMR testing should be performed are unclear.

Objective To establish a highly sensitive and efficient strategy for the identification of MMR gene mutation carriers among CRC probands.

Design, Setting, and Patients Pooled-data analysis of 4 large cohorts of newly diagnosed CRC probands recruited between 1994 and 2010 (n = 10 206) from the Colon Cancer Family Registry, the EPICOLON project, the Ohio State University, and the University of Helsinki examining personal, tumor-related, and family characteristics, as well as microsatellite instability, tumor MMR immunostaining, and germline MMR mutational status data.

Main Outcome Measures Performance characteristics of selected strategies (Bethesda guidelines, Jerusalem recommendations, and those derived from a bivariate/multivariate analysis of variables associated with Lynch syndrome) were compared with tumor MMR testing of all CRC patients (universal screening).

Results Of 10 206 informative, unrelated CRC probands, 312 (3.1%) were MMR gene mutation carriers. In the population-based cohorts (n = 3671 probands), the universal screening approach (sensitivity, 100%; 95% CI, 99.3%-100%; specificity, 93.0%; 95% CI, 92.0%-93.7%; diagnostic yield, 2.2%; 95% CI, 1.7%-2.7%) was superior to the use of Bethesda guidelines (sensitivity, 87.8%; 95% CI, 78.9%-93.2%; specificity, 97.5%; 95% CI, 96.9%-98.0%; diagnostic yield, 2.0%; 95% CI, 1.5%-2.4%; P < .001), Jerusalem recommendations (sensitivity, 85.4%; 95% CI, 77.1%-93.6%; specificity, 96.7%; 95% CI, 96.0%-97.2%; diagnostic yield, 1.9%; 95% CI, 1.4%-2.3%; P < .001), and a selective strategy based on tumor MMR testing of cases with CRC diagnosed at age 70 years or younger and in older patients fulfilling the Bethesda guidelines (sensitivity, 95.1%; 95% CI, 89.8%-99.0%; specificity, 95.5%; 95% CI, 94.7%-96.1%; diagnostic yield, 2.1%; 95% CI, 1.6%-2.6%; P < .001). This selective strategy missed 4.9% of Lynch syndrome cases but resulted in 34.8% fewer cases requiring tumor MMR testing and 28.6% fewer cases undergoing germline mutational analysis than the universal approach.

Conclusion Universal tumor MMR testing among CRC probands had a greater sensitivity for the identification of Lynch syndrome compared with multiple alternative strategies, although the increase in the diagnostic yield was modest.

via JAMA Network | JAMA: The Journal of the American Medical Association | Identification of Lynch Syndrome Among Patients With Colorectal CancerLynch Syndrome and Colorectal Cancer.

Preoperative Testing For Lynch Syndrome Yields ‘Huge’ Benefit


Preoperative Testing For Lynch Syndrome Yields ‘Huge’ Benefit

Vancouver, British Columbia—Mayo Clinic researchers are calling for all young patients with colorectal cancer (CRC) to undergo preoperative testing for Lynch syndrome as the results can significantly alter surgical management.

“The benefit of this testing to the patient and their family is huge,” said Rajesh Pendlimari, MBBS, a research fellow at Mayo Clinic in Rochester, Minn., and a study investigator.

“If they have Lynch syndrome and will, therefore, be more prone to getting cancer, they can get screened more regularly. The knowledge gleaned can change the course of surgical treatment.”

At the 2011 annual meeting of the American Society of Colon and Rectal Surgeons, the Mayo team presented two studies examining the benefit of pre- and postoperative microsatellite instability (MSI) testing for Lynch syndrome.

In the first study, 210 of 258 newly diagnosed patients younger than age 50 years who underwent colorectal surgery at Mayo Clinic had MSI testing between 2003 and 2008. Of these, 82 underwent testing postoperatively, according to the hospital’s protocol requiring pathologists to complete MSI testing on operative specimens for all young patients who did not have the tests done prior to surgery. Overall, 13% of patients were found to have high levels of MSI and 33% of these would have been missed without the testing protocol.

The second, complementary paper retrospectively compared the surgical management of 210 patients who were tested pre- and postoperatively for MSI (n=103, n=107, respectively). (The number of patients in the postoperative group differs in the two studies: It is listed as 82 in the first study and 107 in the second because 25 patients underwent preoperative testing on the day of surgery; their results were not available to surgeons before operating.)

Results showed that the MSI test results significantly influenced surgical recommendations for total colectomy. Of patients with positive preoperative MSI tests (MSI-H), 94% underwent total colectomy, compared with 8% of patients whose status was not known until after surgery (P<0.0001). Moreover, there appears to be an increased rate of hysterectomy among women with MSI-H. Eight of 10 MSI-H women had a hysterectomy. There was only one female patient who was tested postoperatively and she did not have a hysterectomy.

“Probably the most significant result of this research is that it has stimulated our multidisciplinary team of geneticists, pathologists, gastroenterologists and surgeons to develop new clinical pathways that will direct patients at risk to providers experienced with management of Lynch syndrome,” said Eric Dozois, MD, professor of surgery at Mayo Clinic and lead researcher on the project.

Other gastroenterologists and surgeons applauded the paper, saying that preoperative testing for Lynch syndrome is easy to do and can dramatically affect surgical treatment.

“Virtually everybody [who] gets an operation for colorectal cancer has a colonoscopy or biopsy of the tumor before they go on to surgery. In my opinion, that is a golden opportunity, since you are taking a biopsy anyway, to get the cascade of evaluation going for the possible presence of HNPCC [hereditary nonpolyposis colorectal cancer],” said Patrick Lynch, MD, professor of medicine in the Department of Gastroenterology, Hepatology and Nutrition at the University of Texas MD Anderson Cancer Center in Houston.

He added that endoscopists should not skip the opportunity for testing during colonoscopic biopsy.

“It doesn’t take that much material, it’s easy to do and you’re doing it at the front end of a window of opportunity that exists between that time and when the patient goes to surgery. The surgeon and the patient can use that information to decide if they want to expand the surgery from a simple segmental resection to a subtotal colectomy.”

Preoperative testing could improve treatment for younger patients, a group that is showing increased incidence of CRC, said Michael Stamos, MD, professor and chair of surgery at the University of California, Irvine, where surgeons and gastroenterologists routinely do immunohistochemistry (IHC) staining testing prior to surgery.

The Mayo Clinic team has developed a new clinical pathway (Figure) for testing and treatment of patients at high risk for CRC. The protocol requires all high-risk patients to undergo either MSI or IHC to test for Lynch syndrome prior to surgery.

It’s still unclear which test is best to start with. Although MSI is the gold standard test for the DNA mismatch repair, it does have some disadvantages such as a slow turnaround time and it requires an advanced and experienced lab as well as more testing than IHC. Individuals with positive results still need to undergo IHC. On the other hand, IHC requires an experienced pathologist and detects abnormalities of only four major genes.

The best test depends on the local expertise, said Dr. Stamos.

Of every 35 patients with CRC, one has Lynch syndrome, the most common hereditary cause of colorectal and endometrial cancers.

The last guideline on screening was the 2004 Revised Bethesda Guideline, which calls for MSI screening for anyone with young-onset (<50 years old) CRC, synchronous or metachronous CRC or HNPCC-associated cancer at any age, CRC in a patient under age 60 years with tumor-infiltrating T lymphocytes, mucinous/signet ring differentiation or Crohn’s-like lymphocytic reaction, or for patients of any age with a first-degree relative with an HNPCC-related tumor before age 50, or two first- or second-degree relatives with HNPCC tumors at any age.

“Since those guidelines, we’ve discovered a new gene,” said Dr. Pendlimari. “Our understanding of this disease is changing considerably.”

Risks of Primary Extracolonic Cancers Following Colorectal Cancer in Lynch Syndrome


JNCI J Natl Cancer Inst From Win et al Journal of the National Cancer Institute September 2012

Background Lynch syndrome is a highly penetrant cancer predisposition syndrome caused by germline mutations in DNA mismatch repair (MMR) genes. We estimated the risks of primary cancers other than colorectal cancer following a diagnosis of colorectal cancer in mutation carriers.

Methods We obtained data from the Colon Cancer Family Registry for 764 carriers of an MMR gene mutation (316 MLH1, 357 MSH2, 49 MSH6, and 42 PMS2), who had a previous diagnosis of colorectal cancer. The Kaplan–Meier method was used to estimate their cumulative risk of cancers 10 and 20 years after colorectal cancer. We estimated the age-, sex-, country- and calendar period–specific standardized incidence ratios (SIRs) of cancers following colorectal cancer, compared with the general population.

Results Following colorectal cancer, carriers of MMR gene mutations had the following 10-year risk of cancers in other organs: kidney, renal pelvis, ureter, and bladder (2%, 95% confidence interval [CI] = 1% to 3%); small intestine, stomach, and hepatobiliary tract (1%, 95% CI = 0.2% to 2%); prostate (3%, 95% CI = 1% to 5%); endometrium (12%, 95% CI = 8% to 17%); breast (2%, 95% CI = 1% to 4%); and ovary (1%, 95% CI = 0% to 2%). They were at elevated risk compared with the general population: cancers of the kidney, renal pelvis, and ureter (SIR = 12.54, 95% CI = 7.97 to 17.94), urinary bladder (SIR = 7.22, 95% CI = 4.08 to 10.99), small intestine (SIR = 72.68, 95% CI = 39.95 to 111.29), stomach (SIR = 5.65, 95% CI = 2.32 to 9.69), and hepatobiliary tract (SIR = 5.94, 95% CI = 1.81 to 10.94) for both sexes; cancer of the prostate (SIR = 2.05, 95% CI = 1.23 to 3.01), endometrium (SIR = 40.23, 95% CI = 27.91 to 56.06), breast (SIR = 1.76, 95% CI = 1.07 to 2.59), and ovary (SIR = 4.19, 95% CI = 1.28 to 7.97).

Conclusion Carriers of MMR gene mutations who have already had a colorectal cancer are at increased risk of a greater range of cancers than the recognized spectrum of Lynch syndrome cancers, including breast and prostate cancers.

Risks of Primary Extracolonic Cancers Following Colorectal Cancer in Lynch Syndrome

Table 2.

Cumulative risks (percent) and corresponding 95% confidence intervals (CIs) of primary extracolonic cancers during the 10 and 20 years following diagnosis of colorectal cancer for carriers of mismatch repair gene mutations

Cancer site 10 years 20 years
Risk, % (95% CI) Risk,% (95% CI)
Both sexes
    Kidney etc.*
1.90
(0.87 to 3.17) 5.15 (2.86 to 7.68)
    Urinary bladder 1.61 (0.65 to 2.75) 3.15 (1.37 to 5.20)
    Small intestine 0.92 (0.28 to 1.73) 4.00 (1.92 to 6.41)
    Stomach 0.66 (0.13 to 1.40) 1.15 (0.19 to 2.48)
    Hepatobiliary tract 0.83 (0.16 to 1.69) 1.42 (0.42 to 2.73)
Men
    Prostate 2.74 (0.86 to 4.77) 5.90 (2.69 to 9.76)
Women
    Endometrium 12.12 (7.66 to 17.11) 23.99 (16.79 to 32.84)
    Breast 1.94 (0.58 to 3.83) 11.38 (0.63 to 16.69)
    Ovary 0.94 (0.00 to 2.11) 2.08 (0.50 to 4.14)
  • * Kidney etc. included kidney, renal pelvis, ureter and other and unspecified urinary organs.

  • † Hepatobiliary tract included liver and intrahepatic bile duct, gall bladder, and other and unspecified parts of biliary tract.

    Interpretation

    Patients who have had colorectal cancer and who are carriers of the DNA mismatch repair gene mutations that cause Lynch syndrome “have an increased risk of a greater range of cancers than the recognized spectrum of Lynch syndrome cancers, including breast and prostate cancers,” according to a study in the Journal of the National Cancer Institute.

    Previous studies had shown that mutation carriers “are at a substantially increased risk of cancers of the colon, rectum, endometrium, stomach, ovary, ureter, renal pelvis, brain, small bowel, hepatobiliary tract, and pancreas,” the authors noted. A major inherited cancer syndrome, Lynch syndrome is also known as hereditary nonpolyposis colorectal cancer (HNPCC).

    The study was based on data for 764 patients from the Colon Cancer Family Registry, evenly divided between men and women, who were carriers of the mismatch repair gene mutation and previously diagnosed with colorectal cancer. Most of the carriers (52%) were recruited in Australia and New Zealand, with 33% from the United States and 15% from Canada. The average age at diagnosis of colorectal cancer was 44 years.

    Compared with the general population, following colorectal cancer, carriers of mismatch repair gene mutations had a 70-fold increased risk for cancer of the small intestine, a 13-fold increased risk for cancer of the kidney, renal pelvis, and ureter or urethra, a 7-fold increased risk for cancer of the bladder, a 6-fold increased risk for hepatobiliary tract cancer, and a nearly 6-fold increased risk for gastric cancer. Men had a 2-fold increased risk of prostate cancer. The most common primary cancer following colorectal cancer for women with Lynch syndrome was endometrial cancer, with a 40-fold increased risk compared to the general population. There were 20 breast cancers and 6 ovarian cancers in the study population.

    “These new data provide further determination of cancer risks, potentially informing and justifying ongoing studies to create the evidence for effective screening methodologies and intervals in [mismatch repair] gene mutation carriers,” the researchers concluded. “Larger studies are needed to refine risk estimates separately for specific [mismatch repair] gene mutations to best inform policies on clinical risk management.” ■

 

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.

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.

 

Lynch Syndrome and other non-polyposis inherited cancer syndromes


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

Lynch Syndrome (OMIM 120435)) is a familial cancer syndrome which accounts for approximately 2.2-4% of all colorectal cancer in the USA (Hampel et al. 2005) and 2-3% of the total in the UK.  It was also 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, involved with the mismatch repair (MMR) pathway. This pathway functions to identify and remove single nucleotide mismatches or insertions and deletion loops. Mutations in four of the MMR genes can cause Lynch syndrome [Peltomaki 2003]. The functions of the mismatch repair genes can be disrupted by missense mutations, truncating mutations, splice site mutations, large deletions, or genomic rearrangements. In addition, germline deletion within EPCAM, which is not an MMR gene, can disrupt the MMR pathway. EPCAM deletions result in inactivating the adjacent MMR gene MSH2, even though MSH2 itself has not been mutated.

Identification of LS Families

A minority of Lynch Syndrome 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 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

Large bowel surveillance for Lynch syndrome family members and gene carriers

Total colonic surveillance (at least biennial) should commence at age 25 years. Surveillance colonoscopy every 18 months may be appropriate because of the occurrence of interval cancers in some series.  Surveillance should continue to age 7075 years or until co-morbidity makes it clinically inappropriate. If a causative mutation is identified in a relative and the consultand is a non-carrier, surveillance should cease and measures to counter general population risk should be applied.

The effectiveness of colonoscopic surveillance for people with MMR gene mutations and Lynch family members has been examined in retrospective casecontrol comparisons. Screened individuals were compared to control subjects who declined, or did not receive, regular colonoscopy with respect to outcomes of cancer incidence, tumour stage and mortality, or mortality alone. Surveillance appears to provide an average of 7 years of extra life for Lynch syndrome family members.  Thus, available evidence supports regular colonoscopic surveillance as a means of early colorectal cancer detection, leading to mortality reduction as well as reduction in cancer incidence.

Surveillance should consist of total colonoscopy, since the risk of polyps and cancer is high and a substantial proportion of patients have neoplasia restricted to the proximal colon.  Colonoscopy is preferable to flexible sigmoidoscopy combined with barium enema. Because the cancer risk is high, it is not appropriate to accept an incomplete colonoscopy until the next surveillance interval. Incomplete colonoscopy should be followed soon after, or even the same day, by completion CT colonography in centres skilled in providing this technique to a high quality, but repeated radiation exposure should be avoided wherever possible. Repeat full colonoscopy or barium enema remain as options. Chromoendoscopy and narrow wavelength visible light (narrow band) endoscopy may have a place in the detection of small or flat lesions, but there is very limited experience and evidence is restricted to descriptive studies of their use in Lynch syndrome surveillance. Hence, the utility of such techniques requires further assessment and is neither recommended nor discouraged in high risk surveillance, but should not replace conventional endoscopic approaches. Evidence for commencing surveillance at 25 years of age is based on observational data that indicate that the risk increases substantially from age 25 in groups defined by family history and in groups defined by presence of a mutation.

Colorectal resection has a place as prophylaxis and for established cancer in Lynch syndrome family members and/or MMR gene carriers.

Patients who have developed a colorectal malignancy and who come from a Lynch syndrome family, or carry a mutation in an MMR gene, should be counselled and offered a surgical procedure that includes both a cancer control element and prophylaxis to counter future cancer risk. At present there is no evidence to guide decision-making on primary prophylactic surgery for patients who do not yet have cancer.

People with MMR gene mutations or those from Amsterdam positive Lynch syndrome families who have cancer will require surgery unless treatment is palliative. Case series evidence shows that the risk of metachronous colorectal cancer is high following segmental resection (16%), but substantially lower after colectomy and ileorectal anastomosis (3%).  Hence, incorporating a prophylactic element to the cancer resection is appropriate. For patients with proximal tumours, colectomy and ileorectal anastomosis is most relevant, but the retained rectum must be screened because cancer risk in the retained rectum is 3% every 3 years for the first 12 years.

Upper gastrointestinal surveillance for Lynch syndrome family members and/or MMR gene carriers

In families manifesting gastric cancer as part of the phenotype, biennial upper gastrointestinal endoscopy should be considered. The evidence is limited and a pragmatic recommendation is to screen from age 50 since the incidence is very low until that age. Surveillance should continue to age 75 or until the causative mutation in that family has been excluded. This recommendation is based on observations that some Lynch syndrome families have a particular propensity for gastric cancer.  There is as yet no evidence that this reduces mortality.

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 LS 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.

UK BSG/ACPGBI Guidelines for Moderate Risk Family History/Colorectal Cancer Risk Groups


 

BSG/ACPGBI GUIDANCE ON LARGE BOWEL SURVEILLANCE FOR INDIVIDUALS WITH A FAMILY HISTORY INDICATING A MODERATE RISK (2010)

http://www.bsg.org.uk/clinical-guidelines/endoscopy/guidelines-for-colorectal-cancer-screening-and-surveillance-in-moderate-and-high-risk-groups-update-from-2002.html

Executive summary

Dedicated Clinics: Referrals on the basis of family history are best coordinated through centres with a specialist interest, such as regional genetics services or medical/surgical gastroenterology centres. Such centralisation enables audit of family history ascertainment, assigned level of risk, collection of outcome data and research.

Screening Procedure: Total colonoscopy is the preferred mode of surveillance for the moderate risk categories defined here, owing to the propensity for proximal colonic lesions and the opportunity for snare polypectomy. Incomplete colonoscopy should initiate an alternative imaging modality on the same day, such as double-contrast barium enema or CT colonography. A repeat colonoscopy soon after an incomplete examination is acceptable, but success must be assured. However, radiation exposure should be minimised and regular radiological surveillance is not recommended.

High moderate risk group inclusion criteria comprise familial aggregations where affected relatives are first-degree relatives of each other (first-degree kinship) with at least one being a first-degree relative of the consultand. If both parents are affected, these count as being within first-degree kinship:

– Three affected relatives any age in a first-degree kinship (eg, a parent and a blood-related aunt/uncle and/or grandparent), at least one of whom is a first-degree relative of the consultand, or two siblings/one parent or two siblings/one offspring combinations, or both parents and one sibling. However, there should be no affected relative <50 years old, as otherwise the family would fulfil high risk criteria.

– Two affected relatives aged <60 years in a first-degree kinship or mean age of two affected relatives <60 years. At least one relative must be a first-degree relative of the consultand and so this category includes a parent and grandparent, >2 siblings, >2 children or child+sibling. The risk is sufficiently increased to merit low-intensity surveillance comprising 5-yearly colonoscopy between age 50 and age 75 years. Polyps should be snared; adenoma surveillance applies thereafter if a benign neoplasm is confirmed.

Low-moderate risk group. Inclusion criteria are:

– One affected first-degree relative under 50 years old or

– Two affected first-degree relatives, aged 60 or older.

In both high-moderate and low-moderate categories, pathology tumour material from an affected relative may be available to test for Lynch syndrome gene involvement.

Excluding such instances, there is a modest excess risk meriting a single colonoscopy at age 55 (if older at presentation then instigate forthwith), in the lowmoderate group to identify polyp formers. Polyps should be snared; adenoma surveillance applies thereafter if a benign neoplasm is confirmed. If colonoscopy is clear, reassure and discharge with recommendations relevant to population risk (uptake of faecal occult blood test screening in the UK).

Early-onset colorectal cancer (<50 years). The elevation of risk in relatives of an early-onset case is modest. However, the heightened anxiety and emotive nature of cancer in this age group merit special mention because this frequently initiates requests for surveillance. Such cases are covered by the above risk categorisation, but algorithms can also be used to predict whether the affected relative is a carrier of a mutation in a Lynch syndrome gene. These approaches identify affected individuals where tumour immunohistochemistry and/or microsatellite instability analysis could lead to identification of a DNA mismatch repair gene mutation. Bethesda criteria are not discriminatory within this group because all patients fulfil these criteria due to age alone.

Low Risk Group: People with only one affected relative and who do not fulfil any of the above criteria, and do not fulfil high risk criteria, should be reassured and encouraged to avail themselves of population-based screening measures. The low level residual risk over that of the general population should be explained.

Outcome of screening in Moderate Risk Groups (Dove-Edwin et al BMJ 2005)

Advanced neoplasia and age at initial colonoscopy.

Colonoscopic surveillance is effective in preventing colorectal cancer in individuals from families with hereditary non-polyposis colorectal cancer (group 4) and in individuals with a family history of colorectal cancer that does not meet the Amsterdam criteria. However, colonoscopic surveillance in the families at moderate risk seems not indicated until age 45 (or even 50), and this is true even for the relatives of young patients. Furthermore, surveillance intervals of more than five years may be appropriate in individuals with a moderate risk family history (groups 1-3) in whom no advanced pathology is found.

Colonoscopic polypectomy has been shown to decrease the incidence of colorectal cancer in a large cohort study as well as in clinical practice and to decrease both the incidence and mortality of colorectal cancer in individuals with a family history of hereditary non-polyposis colorectal cancer. It is also considered by some to be a safe tool for population screening. Clear guidelines exist for colorectal surveillance in hereditary non-polyposis colorectal cancer families, but guidelines and practices for individuals at moderate risk on the basis of their family history are heterogeneou.

Concerns exist about colonoscopic surveillance in individuals with a moderate risk family history, as some will not be at increased risk. Dunlop et al calculated that if surveillance were offered to individuals aged 30-70 who have two direct relatives affected or one under age 45 then 235 000 individuals would be eligible in the United Kingdom. Even if the age of initiating surveillance is raised, the potential burden on resources is immense. Colonoscopy is associated with a small risk of serious complications, and this may substantially outweigh any benefits in people at low risk.

In this study, only one incident cancer was detected on surveillance in an individual with a moderate risk family history during 9281 person years of follow-up. In families at moderate risk, advanced neoplasia is very rare below the age of 45 and, if not seen initially, it remains uncommon (under age 65) if follow-up colonoscopy is carried out within six years. These findings are important because individuals with a moderate risk family history who are under age 65 with no advanced neoplasia can be considered to be at low risk and extended surveillance intervals may be sufficient. Individuals with a moderate risk family history in whom advanced neoplasia is seen on initial colonoscopy should continue with colonoscopy every three years. The low yield of advanced neoplasia under the age of 45 is true also of those with a first degree relative affected under age 45. Only 4% of 139 individuals in group 1—families with one case of colorectal cancer diagnosed under age 45, and no other cases—screened under age 45 (mean age 33) had an adenoma of any description. Despite the increased risk of colorectal cancer in this group individuals’ absolute risk therefore remains small and the benefit of screening seems minimal below the age of 45.

 

Colorectal Cancer Aetiology


 

Epidemiology

 

Colorectal cancer is diagnosed in approximately 35,000 people in the United Kingdom each year, the third most commonly diagnosed cancer after breast and lung cancer (NICR 2005; ONS 2005, ISD_Online, 2005 #540).  With 16,148 deaths in 2004 it was the second most common-cause of cancer death.  This is also true in other Caucasian populations (Jemal et al. 2007).

Age-standardised death rates from Colon and re...

Age-standardised death rates from Colon and rectum cancers by country (per 100,000 inhabitants). (Photo credit: Wikipedia)

Colorectal cancer, however,is not equally common throughout the world (Boyle and Langman 2000).  In the ‘westernised’countries colorectal cancerrepresents ~13.4% of all incident cases compared to ~7.8% elsewhere.

The occurrence of colorectal cancer is strongly related to age, with 93% occurring after the age of 50 years (ONS 2005).  Before this age there are equal numbers of males and females diagnosed, although after males predominate, with an eventual overall male to female ratio of 1.2:1.  Using data for England and Wales the overall lifetime risk of colorectal cancer is 1 in 18 for men and 1 in 20 for women (CRUK. 2006).  Sixty two percent of cancers are situated in the colon and the remainder in the rectum (ONS 2005).  Seventy percent are left-sided and the remainder right-sided (Toms 2004).

English: Gross appearance of an opened colecto...

Gross appearance of an opened colectomy specimen containing an invasive colorectal carcinoma and two adenomatous polyps. (Photo credit: Wikipedia)

More than 90% of colorectal cancers are adenocarcinomas arising from benign precursor adenomas.  A small proportion may arise from other types of polyps including hyperplastic and serrated polyps (Young et al. 2007).  The remainder of colorectal malignant tumours include carcinoids and lymphomas.  The National Polyp Study showed that endoscopic removal of adenomas prevents colorectal cancer (Winawer et al. 1993).  Endoscopic surveillance and polyp removal is a vital tool making colorectal cancer also one of the most preventable cancers as polyps removed at an early stage can prevent the cancer ever occurring.   Of course the endoscopic procedure performed must be technically perfect, performed at the correct stage in cancer development and in appropriate groups, e.g. for age, risk etc.  The resources are not available to perform colonoscopy on whole populations.  Identifying individuals at high risk due to genetic or environmental factors will help us undertake these preventative measures with the highest levels of efficacy and efficiency.

The concordance rates of cancer in monozygous and dizygous twins suggest that about one-third of the variation in cancer risk might typically be ascribed to genetic factors (Lichtenstein et al. 2000).  Highly penetrant (Mendelian) genetic syndromes account for 5–10% (Cannon-Albright et al. 1988) (Houlston et al. 1992) (Johns and Houlston 2001).  These include Familial Adenomatous Polyposis (FAP), due to germline mutations in the tumour suppressor gene APC, and Hereditary Non-Polyposis Colorectal cancer (HNPCC), due to germline mutations DNA mismatch repair enzymes MLH1, MSH2, PMS2 and MSH6. HNPCC accounts for 1-4% of colorectal cancer and is characterised by cancers with DNA microsatellite instability (MSI).  There is good evidence that endoscopic surveillance of patients with high-risk genetic predisposition also prevents cancer (Jarvinen et al. 2000; Dove-Edwin et al. 2005; Dove-Edwin et al. 2006).

Proportion of colorectal cancer due to environmental factors is about 65%, with 5-10% caused by cancer syndromes and the remainder due to low penetrance heritability

Environmental factors such as physical activity, obesity, diet and other exogenous factors also contribute to the aetiology of colorectal cancer (Linseisen et al. 2002; Bingham et al. 2003; Ferrari et al. 2007).    Diet and lifestyle has been found to modify risk across the cancer spectrum e.g., smoking and lung cancer (Doll and Hill 1950).  However, there are difficulties studying the effects of ‘five pieces of fruit a day’ in population studies due to analytical challenges with accurate food monitoring, variation in the fruit types and other confounding variables such as smoking, BMI, etc, and results of studies of environmental factors often vary widely.  Large international studies such as the European Prospective Investigation into Cancer and Nutrition (EPIC) are attempting to unravel the evidence of sometimes contradictory epidemiological studies (Gonzalez 2006).  Genetic predisposition alleles with a low penetrance may be more susceptible to environmental influences which modify an individual’s risk of colorectal neoplasia. There have been very few studies that clearly address the mechanisms of mutational induction by the environment in colorectal cancer.  Although the contribution of individual environmental factors is uncertain, they play an important role in the multifactorial aetiology of colorectal cancer.

 

The normal large bowel

 

The large bowel is embryologically derived from midgut and hindgut which in have their junction at the splenic flexure.  The vascular supply and lymphatic drainage of the large bowel is similarly divided, and this junction is sometimes taken to separate the ‘right’ and ‘left’ colorectum.  In adults, the large bowel lumen is continuous with nine anatomical regions: The caecum, ascending colon, hepatic flexure, transverse colon, splenic flexure, descending colon, sigmoid colon and the rectum.  The mucosa lies over a basement membrane, underneath is the muscularis mucosa, lamina propria and muscularis propria (Figure 1‑1).

 

 

 

The Wnt signalling pathway and colonic crypt homeostasis

 

The Wnt, and to a lesser extent the TGF-ß, BMP, Notch, and Par polarity pathways are the major players in homeostatic control of the adult epithelium (Sancho et al. 2004).  The surface of the colon is covered by an epithelium composed of four differentiated cell types (enterocytes, enteroendocrine,goblet and Paneth cells) that invaginates at regular intervalsto form crypts.  The bottom of the crypts is occupied by a few stem cells that give rise to actively dividing precursorcells that populate the bottom two-thirds of the crypts. The epithelial cell precursors migrate upward in an ordered fashion, which isalso controlled by Wnt factors, and they stop proliferating when they reach the top third of the crypt, possibly because they are too far from the Wnt source. Meanwhile, they continue their migration and colonise the surface of the colon.After about a week, epithelial cells undergo apoptosis and areshed in the lumen of the gut.  The Paneth cells constitute anexception, as they migrate downward and occupy the very bottomof the crypt (van Es et al. 2005).

Thus, the epithelium of the colon is under perpetual renewal.  Wnt growth factors activate a cascade of intracellular events, which is known as the canonical Wnt pathway that ultimately leads to the expression of a genetic program controlling the co-ordinated expansion, fate and sorting of the epithelial cell population (Figure 1‑2).

The Wnt signalling pathway (McDonald SAC et al. (2006) Mechanisms of Disease: from stem cells to colorectal cancer)

The canonical Wnt signalling cascade controls cell behaviour by activating the transcriptional properties of DNA-binding proteins of the T-cell factor(TCF)/lymphoid enhancer factor-1 family (Clevers 2006). Wnt induces stabilisation of cytosolic ß-catenin, which is transported to the nucleus and associates with TCFs, leading to the expression of specific target genes (McDonald et al. 2006). In the absence of Wnt signalling, ß-catenin is targeted for degradation in proteosomes, and TCFs block expression of Wnt target genes.  Thus activation of the pathway may be demonstrated by nuclear localisation of ß-catenin.  In colorectal cancer mutations in components of the Wnt pathway mimic the effect of a permanent Wnt stimulation, causing epithelial cells to proliferate inappropriately.

Wnt factors can also trigger the activation of at least twoother, ß-catenin-independent, non-canonical pathways. They are referredto as non-canonical signalling and their implication in colorectal cancer, if any, is not yet elucidated.  The list of genes targeted by the Wnt pathway is very long (http://www.stanford.edu/%7ernusse/pathways/targets.html), indicating its wide range of roles in cellular homeostasis.

The transforming growth factor-ß pathway

The transforming growth factor-ß (TGF-ß) superfamily comprises a large number of structurally related polypeptide growth factors and each of these is capable of regulating a complex array of cellular processes (Hata et al. 1998).  In normal epithelial cells including intestinal epithelium, TGF-ß has a major inhibitory role and a substantial body of evidence suggests a role as tumour suppressor.  Neoplastic transformation results in loss of normal growth inhibitory responses to TGF-ß (Kurokowa et al. 1987).  Inhibition of intestinal cell proliferation by TGF-ß treatment results in cell cycle arrest from mid-to-late G1 (Ko et al. 1998).   To date, some of the most important contributors to the growth response seen on TGF-ß exposure appear to involve cell cycle regulatory genes, including C-MYC, CDC25A, and the cyclin-dependent kinase inhibitors (CDKIs) p15INK4B, p21WAF1/CIP1, and p27KIP1 (Ko et al. 1994; Ko et al. 1995).  Other targets of this pathway include the SMAD (Small mothers against decapentaplegic) and BMP genes (bone morphogenetic proteins), important mediators of signal transduction.  In vivo, there is increased expression of both TGF-ß1 and the TGF-ß type II receptor in intestinal epithelial cells as they migrate from the proliferative compartment toward the lumen in both the small intestine and the colon (Winesett et al. 1996).  This pattern of expression is inversely correlated with the mitotic activity in the gut epithelium.  Reduced expression of the TGF-ß type I receptor was been strongly linked to colorectal cancer predisposition (Valle et al. 2008).  However a larger study published subsequently did not demonstrate allele-specific expression or an association polymorphisms within this gene with colorectal neoplasia (Carvajal-Carmona et al 2010).

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 KRAS gene product,a 21 kDa protein located at the inner plasma membrane, is involved in the transduction of mitogenic signals.  The Ras protein is activated transiently as a response to extracellular signals such 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 instabilityis 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 ofwhich 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.

 

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