Short Presentations in Emerging Concepts (SPEC) Emerging Concepts in Colorectal Cancer: Hereditary Non-Polyposis Cancer (Lynch Syndrome) Short Presentations in Emerging Concepts (SPEC)

Colorectal Cancer: Molecular Pathways Colon cancer is a relatively common malignancy. It is now recognized that colon cancer can arise through two distinct molecular pathways. The majority of colon cancers are the product of the chromosomal instability pathway. These tumors are characterized by frequent mutations in the ras, FAP, and p53 genes, and frequently display chromosomal aneuploidy. Approximately 20% of all colon cancers arise by another mechanism called the hypermutability pathway. This pathway does not result in gross chromosomal rearrangements and losses, but rather is accompanied by relatively small deletions and insertions in specific types of DNA sequences. The purpose of this presentation is to explain why it is important to recognize tumors which arise through the hypermutability pathway from those which do not. 3

Molecular pathways in colon cancer Both genetic pathways progress through similar histologic intermediaries. This progression through adenomatous polyps by way of increasing degrees of dysplasia in the chromosomal instability pathway is accompanied by mutations in specific genes. In the hyper mutability pathway a number of genes that are involved have been identified. What is remarkable is that these genes code for proteins that are involved in DNA repair. Mistakes that are introduced into the DNA during the course of replication, or secondary to exposure to radiation or certain mutagenic chemicals are recognized, and the mistakes are repaired. Tumor cells in which one or more of these DNA repair genes have been inactivated are not able to remove those mistakes from the DNA. The accumulation of mutations in genes which the affect differentiation and proliferation ultimately result in malignancy. Note that while it appears that this hyper mutability pathway also progresses through the similar histologic intermediates, we really don’t know whether the rate of progression is the same as in the chromosomal instability pathway.

What value is there in recognizing MSI-H colorectal tumors? Prognosis Response to chemotherapy Screen for Lynch Syndrome (HNPCC)

Prognostic significance of MSI-H in sporadic CRC This publication examined the survival of patients with MSI tumors relative to the survival of patients with and MSS tumors. HNPCC tumors are known to have a better survival than similar stage sporadic cancers. You can see that sporadic MSI tumors are associated with a better survival than microsatellite stable tumors (MSS). So it appears that sporadic MSI tumors not only look like the tumors that arise in HNPCC, but they behave similarly. From N Engl J Med, Gryfe, R et al, Tumor microsatellite instability and clinical outcome in young patients with colorectal cancer, 342, 69-77 Copyright © 2000 Massachusetts Medical Society. Reprinted with permission. from Massachusetts Medical Society. Gryfe,R et al, NEJM 2000; 342:69-77

Tumor Microsatellite-Instability Status as a Predictor of Benefit from Fluorouracil-Based Adjuvant Chemotherapy for Colon Cancer Ribic, C.R., et al. New Engl J Med 349:247-57 (2003) This graph from Ribic et al.,(New Engl J Med 349:247-57 (2003)) indicates that MSI-H tumors do not respond to 5FU therapy as well as MSS tumors. Panel B shows that patients with treated MSI colon cancers showed a poorer Overall Survival. Close inspection of the Ribic data, however, suggested that patients with MSI tumors treated with 5FU have a poorer survival than if they had not been treated at all. Consequently, many oncologists avoid the use of 5FU regimens for patients with MSI-H tumors. From N Engl J Med, Ribic, CM et al, Tumor Microsatellite-instability status as a predictor of benefit, 349, 247-257 Copyright © 2003 Massachusetts Medical Society. Reprinted with permission. from Massachusetts Medical Society.

Colorectal Cancer: Molecular Pathways In the chromosomal instability pathway, the archetype inherited predisposition syndrome is FAP – all the syndromes associated with polyposis. Approximately 1% of all CRC are of this type. Similarly, Lynch Syndrome represents an inherited predisposition to CRC that occurs through the hypermutability pathway. These constitute about 3% of all CRCs and 15 - 20% of tumors that are defective in DNA mis-match repair. Identifying patients whose tumors display MMR deficiency increase the likelihood of the patient having Lynch Syndrome. 8

Lynch Syndrome (HNPCC) HNPCC – Hereditary Non-Polyposis Colon Cancer Historically: Lynch Syndrome I – restricted to colon Lynch Syndrome II – colon and extracolonic sites Accounts for 3-4% of all colon cancers Accounts for 15-20% of MSI tumors Inherited predisposition to many different cancers, including colon cancer HNPCC accounts for about 3-4% of all colon cancers. It is important to recognize these patients because they are at increased risk of developing further colon cancers. Furthermore, the rate at which cancers develop in HNPCC seems to be faster than what we see in “conventional” polyps so screening needs to be more frequent. It should also be obvious that relatives of affected patients will also be at risk if they inherited the same defective mismatch repair gene. It is somewhat unfortunate that this syndrome’s name is associated with colon cancer because affected patients are also susceptible to numerous extra colonic malignancies.

Lynch Syndrome: Cardinal Features Autosomal dominant inheritance Gene penetrance for CRC of 85-90% Develop CRC at an early age - 45 yrs Most CRC (70%) proximal to splenic flexure Multiple CRC’s common - synchronous and metachronous Prognosis better than sporadic CRC Associated pathologic features Increased risk for other malignancies

Lynch Syndrome: Extracolonic Tumors Site Features Endometrium Second most common Stomach Older generations Small bowel Risk 25X in HNPCC Hepatobiliary tract 5% risk Ureter and pelvis 14-20% risk Skin Muir-Torre Syndrome Pancreas Trend for increase Brain GBM in some HNPCC (Turcot’s) Hematologic Case reports Soft tissue Larynx Case report This slide lists some of those tumors. Of particular importance are tumors of the endometrium, stomach and urinary tract.

Lynch Syndrome: Cumulative cancer risk in LS carriers by age 70 Site of tumor Finnish population (%) HNPCC families (%) Colon/rectum 1.6 82 Endometrium 1.3 60 Stomach 0.8 13 Ovary 12 Bladder, ureter, urethra 0.7 4.0 Brain 0.9 3.7 Kidney 3.3 Biliary tract, gallbladder 0.2 2.0 This report evaluated the incidence of various tumors in HNPCC families in Finland. Note the marked increase in risk due to HNPCC for cancers of the endometrium, stomach, and ovary. Aarnio M, et al, Int J Cancer 1999; 81:214-218.

Lynch Syndrome: Cumulative cancer risk by age 70 By age 70, the risk for endometrial cancer exceeds that of colon cancer: Site Incidence by age 70 in women Endometrium 60% Colon 54% For women, the risk of endometrial cancer by age 70 exceeded that of colon cancer. It is clear that patients with LS should be monitored not only for colon cancer, but for these extra colonic malignancies as well, with particular attention given to endometrial cancer. It has also been suggested, given the high likelihood of endometrial cancer in HNPCC carriers, that such patients consider prophylactic hysterectomy after the childbearing years should they be undergoing abdominal surgery for other purposes. Aarnio, M et al, Int J Cancer 1999; 81:214-18

Lynch Syndrome: Pathological features of colorectal cancer Poor differentiation Increased signet cells Medullary features Peritumoral lymphocyte infiltration Crohn’s like reaction Tumor infiltrating lymphocytes (TIL’s) These are features that a pathologist may note that should suggest the possibility of Lynch Syndrome, and should warrant further studies to evaluate the tumor for mismatch repair deficiency.

How to recognize Lynch Syndrome Amsterdam Criteria Clinical guidelines for when to suspect Lynch Syndrome Bethesda Guidelines Guidelines for when to do MSI testing Screen all new colon cancers? Clinical criteria for when to suspect HNPCC were put forth in the early 1990’s, in the Amsterdam Criteria, and later in the Bethesda Guidelines. The Amsterdam Criteria were revised in 1999 to acknowledge that any HNPCC associated cancer should be considered in addition to colon cancer. These are referred to as the Amsterdam Criteria II.

Lynch Syndrome - Amsterdam Criteria II (1999) At least three family members with a Lynch Syndrome-associated cancer, two of whom are first-degree relatives. At least two generations represented. At least 1 individual younger than 50 years at diagnosis. FAP should be excluded. Tumors should be verified by pathologic examination. It is important to note that the Amsterdam Criteria II include all LS associated cancers, not just colon cancer. These criteria were originally established for the purpose of formal studies of HNPCC families and were thought to too restrictive for clinical purposes. Consequently, an NCI consensus conference developed the following Bethesda Guidelines. Vasen et al, Gastroenterology 1999;116:1453-56

Revised Bethesda Guidelines for testing colorectal tumors for MSI - 2004 Tumors from individuals should be tested for MSI in the following situations: Colorectal cancer in a patient less than 50 years of age. Presence of synchronous, metachronous colorectal, or other HNPCC associated tumors, regardless of age. Colorectal cancer with the MSI-H histology diagnosed in a patient less than 60 yr. Colorectal cancer diagnosed in one or more first-degree relatives with an HNPCC-related tumor, with one of the cancers being diagnosed under age 50 yr. Colorectal cancer diagnosed in two or more first- or second-degree relatives with HNPCC-related tumors, regardless of age. Points to note: Guideline #2: multiple HNPCC tumors regardless of age. Hence, elderly patients with CRC and bladder cancer would qualify for MSI testing. Likewise, a patient with CRC and a history of endometrial cancer would also qualify. The point here is that suspicion for Lynch Syndrome is a multidisciplinary responsibility. Guideline #3: Histology is important. This responsibility falls on the pathologist. Umar, et al., 2004. Revised Bethesda Guidelines for Hereditary Nonpolyposis Colorectal Cancer (Lynch Syndrome) and Microsatellite Instability, J Natl Cancer Inst 96:261-268. Umar, et al., 2004, Testing guidelines for hereditary non-polyposis colorectal cancer, Nature Reviews 4: 153-158. Umar, et al., J Natl Cancer Inst 2004; 96:261-8

Lynch Syndrome: Mismatch repair gene mutations Frequency in HNPCC MSH2 ~39% MLH1 ~32% PMS1 Rare PMS2 ~14% GTBP/MSH6 Other ? A number of genes involved in mismatch repair have been identified. Individuals with LS will typically harbor mutations in one of these genes, with most occurring in MSH2 and MLH1. Patients who have MSI-H tumors and are suspected to have LS can be tested for germline mutations in MSH2 and MLH1. Identifying a specific mutation allows for the possibility of testing relatives for carrier status. While these two genes account for most of the mutations found in LS families, they do not account for all of them.

Immunohistochemistry for MMR Protein Expression MSH2 MLH1 MSH6 PMS2 Immunohistochemistry can also be performed for the major MMR proteins. The absence of specific protein expression suggests an underlying mutation in the corresponding gene. However, some genes are not mutated, but simply “turned off” due to hypermethylation. This mechanism accounts for those MSI-H cases not due to LS, i.e. “sporadic” MSI-H cases. The vast majority of these are due to MLH1 hypermethlation. Microsatellite analysis and MMR IHC have comparable sensitivities for identifying LS related MMR deficiency (90-95%). The specificities of the two methodologies are not identical, and each method will fail to identify a small number of patients. Some authors have advocated using both methods to be as thorough as possible is identifying potential LS patients. Loss of expression Due to mutation Lynch Syndrome Due to methylation Sporadic MSI CRC

Universal screening Recommendations from the EGAPP Working Group: genetic testing strategies in newly diagnosed individuals with colorectal cancer aimed at reducing morbidity and mortality from Lynch syndrome in relatives Evaluation of Genomic Applications in Practice and Prevention Working Group Some large centers have implemented “universal screening” of all newly identified CRC patients in order to capture the maximum number of potential LS patients. This EGAPP study found that there was moderate evidence to support that practice. Genetics in Medicine 11:35-41 (2009)

Significance of Lynch Syndrome The patient is at risk for other cancers and needs appropriate surveillance. The patient’s relatives will also be at increased risk if they carry the same mutation, and will need appropriate surveillance. Relatives can be tested to determine their risk, and level of surveillance. The value of universal screening comes not only from the benefit to the patient, but from benefits to family members affected by LS who also need more intense surveillance for CRC and other LS related malignancies.

Summary MSI-H tumors account for about 20% of all colon cancers. Lynch Syndrome tumors account for 15 - 20% of MSI-H colon cancers, and about 4% of all colon cancers. MSI-H colon cancers are biologically distinctive in their behavior. MSI testing should be performed if indicated by Bethesda Guidelines. MSI testing can be performed on fixed tissue. Patients with MSI-H tumors are candidates for genetic counseling and further genetic testing.

Selected Resources Lynch HT, et al. Hereditary nonpolyposis colorectal carcinoma and HNPCC-like families: problems in diagnosis,m surveillance, and management. Cancer .2004 ;100:53-64. EGAPP Working Group. Recommendations from the EGAPP Working Group: genetic testing strategies in newly diagnosed individuals with colorectal cancer aimed at reducing morbidity and mortality from Lynch syndrome in relatives. Genet Med. 2009 ;11(1):35-41. Vasen HF, Blanco I, Aktan-Collan K, et al. Revised guidelines for the clinical management of Lynch Syndrome (HNPCC): recommendations by a group of European experts. Gut. 2013;62(6):812-823.

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