GENETICS of COLORECT AL NEOPLASIA Somatic Mutations Within an Individual Cell Give Rige to Colorectal Cancer Provides an Ideal Model to Study Tu- Transformation

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Terry C. Lairmore, MD, Jeffrey A. Norton, MD, St. Louis, Missouri

A mPle evidence has been discovered recently to sup- genetic analysis currently provides for accurate predictive port the fundamental concept that cancer is a ge- testing in patients at high risk for cancer development in a netic disease.Cancer resultsfrom the accumulation variety of familial cancer syndromes, allows prognostic of genetic damage within the nucleus of a cell that ulti- stratification by identifying specific genetic defects that are mately causestransformation to a neoplastic phenotype. In associatedwith increasedpotential for tumor aggressiveness patients with a familial cancer syndrome, a predisposition or metastases, and holds promise for future directed thera- to the development of cancer is conferred by the inheri- peutic interventions. tancefrom one parent of a mutatedgene in the germline. Altematively, in patients without a familial predisposition, GENETICS OF COLORECT AL NEOPLASIA somatic mutations within an individual cell give rige to Colorectal cancer provides an ideal model to study tu- transformation. A current model for neoplastic transfor- morigenesisbecause of the existence of distinct hereditary mation of a cell is the hypothesis that cancer results from syndromesassociated with a high risk for the development a stepwise accumulation of multiple genetic defects, result- of colon cancer and the stepwise transition from adenoma ing in the acquisition of increasingly more aggressiveand to carcinoma proposed for sporadic cancers. uncontrolled growth properties.l Genetic alterations in óne of three broad categories of Familial Adenomatous Polyposis (FAP) genes have now been connected to cancer development. Familial adenomatouspolyposis is a rafe autosomal dom- These include proto-oncogenes, tumor suppressorgenes, inant cancel syndrome in which affected individuals de- and most recently, genes that normally function in DNA velop 100 or more adenomatouspolyps of the colon by fue damage recognition and repair (Table). Mutation in one third or fourth decadeof life, and subsequentlycarry a very allele of a proto-oncogene, the normal cellular counterpart high risk of developing a colorectal malignancy. Tumors in of this gene, results in an oncogene with abnormal function patients with FAP probably account for less than 0.5% of or inappropriate expression. A mutation in one of these all colorectal cancers. However, this clinical phenotype is genes results in a gain-of-function, which is deleterious for important because the predisposition to neoplasia of the cellular growth regulation, and the mutated allele thus con- colonic mucosais transmitted by the inheritance of a germ- fers oncogenic potential in a dominant fashion. By contrast, line mutation in the adenomatouspolyposis coli (APC) tu- tumor suppressorgenes encode products that normallyexert mor suppressorgene located on the long arm of chromo- negative control or a brake on cellular growth and division, some 5. In the majotity ofpatients with FAP, one mutant or are integrally involved in programmed cell death (apop- allele of APC has been identified in fue constitucional tosis). Oncogenic activity requires mutation, inactivation, (gerrnline) DNA. In most cases,the defect is either a non- or loss of both alleles (on the paremally and matemally sense mutation or an insertion or deletion resulting in a derived chromosomes), and results in a complete loss-of- truncated and thus inactive APC producto A second so- function and presumably unregulated cellular growth and matic event involving mutation or 105s(allelic deletion) of proliferation. Most recently, loss-of-function mutations in the remaining APC allele within a somatic cell results in a third category of genes that normally function in DNA tumor formarían. damage recognition and r~pair (DNA mismatch repair The APC gene encodesa 2,843-amino acid product that genes)have been associatedwith colorectal and other can- is present in the cytoplasm bound to the adherens junction cers in the hereditary nonpolyposis colorectal cancer proteins a- and ,B-catenin. It has been postulated that loss- (HNPCC) syndrome,as well as a significant number of spo- of-function mutations in both APC genes mar disrupt nor- radic colorectal cancers. mal epithelial cell-to-cell interaction. Familiarity with the mechanisms of tumorigenesis and Importantly, somatic mutations of APC can be detected known genetic defects in human neoplasms assumesin- in tumor DNA from more than two~thirds of sporadic co- creasing importance for the surgical oncologist. Molecular lorectal neoplasms.Therefore, in addition to causing a rafe familial syndrome when inherited as a germline mutation, AmJSurg.1997;173:37-41. the APC gene is an important target for oncogenic muta- From the Endocrine and Oncologic Surgery Section, Department tions in the common sporadic variety of colorectal cancer. of Surgery, Washington University School of Medicine, Sto Louis, Missouri. Supported by an American Cancer Society Instiutional Research Hereditary Nonpolyposis Colorectal Cancer (HNPCC) Grant. T umors arising in patienrs with the hereditary nonpoly- Requests for reprints should be addressed to Jeffrey A. Norton, posis colorectal cancer syndromes mar account for 5-10% MD, Cytokine and Metabolism Section, Laboratory of Biological of cancers of the colon and rectum. Affected patients do Therapy, Department of Surgery, Washington University, Sto Louis, Missouri 63110. not develop polyposis, but carry a high risk of developing colorecral cancers as well as other neoplasms (eg, endo- metrial carcinoma). Colon cancers in these individuals are

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cancer cells harboring a K-RAS mutation! It is likely that tations of the samegene result in different, although similar, molecular genetic analysis will play an increasingly impor- diseases. tant role in diagnosisand treatment of salid human tumors in the,future. Early Thyroidectomy in Patients with MEN 2A Based on Genetic T esting GENETICS OF THE MULTIPLE The detection of elevated plasma levels of calcitonin after ENDOCRINE NEOPLASIA (MEN) TYPE 2 stimulation with intravenous calcium and pentagastrin pro- SYNDROMES vides a sensitive method for the diagnosis of MTC in in- MEN 2A is inherited as an autosomal dominant disorder dividuals from kindreds with MEN 2A who are at risk for and is composed of medullary thyroid carcinoma (MTC), the development of MTC. However, the biochemical pheochromocytoma(s), and parathyroid hyperplasia. Vir- screening of kindred members at risk for familial MTC re- tually 100% of individuals who have this disorder develop quires annual provocative testing, which is associatedwith MTC that subsequently can be lethal. The MEN 2A syn- considerable unpleasantside effects. Direct DNA testing of drome is associated with germline mutations in the RET peripheralleukocytes for mutations in RET of individuals proto-oncogene, a receptor tyrosine kinase that maps to the at risk for one of the MEN 2 syndromes mar now be rou- centromere region of chromosome 10. These misSensemu; tinely performed at any age, providing an opportunity for tations involve one of five codons in either exon 10 or 11 definitive and potentially curable surgical intervention (codons 609, 611, 618, 620,634), each of which specifies based on a molecular genetic diagnostic test. Patients who a highly conservedcysteine residue in the extracellular por- have inherited a mutation in RET mar be treated by early tion of the molecule immediately adjacent to the trans- thyroidectomy at an age when the MTC is confined to the membrane domain (Figure). The mutations result in re- thyroid and likely curable. Our group has reported the re- placement of a cysteine residue with one of several other sults of 13 patients undergoing early thyroidectomy based amino acids. Mulligan and coworkers5have studied the re- on the results of direct DNA testing for MEN lA! AIl lationship between genotype and phenotype in patients patients inheriting a RET mutation had evidence of C-cell with MEN 2A and have recognized that a significant cor- neoplasia (C-cell hyperplasia, MTC, or both) in the re- relation exists between any mutation at codon 634 and the sected thyroid gland. In this groupof 13 patients, there were development of pheochromocytomas. In addi~ion, a strong no metastasesto regionallymph nodes, and postoperative correlation is present between the development of parathy- stimulated plasma CT levels were normal in each patient. raid diseaseand the specific mutation TGC > CGC (cys- Thus, the surgerywas not only prophylactic, but therapeu- teine to arginine) at codon 634. tic, because it was able to remove carcinoma confined to Familial medullary thyroid carcinoma (FMTC) is only as- the thyroid gland for cure. Further, individuals at risk who sociated with the autosomal dominant inheritance of med- do not inherit a mutation of RET do not need additional ullary thyroid carcinoma and no other endocrinopathies. testing as they will not develop the disease. The MTC in this diseaseis most indolent and patients sel- dom die from it. It is associatedwith missencemutations in PANCREATIC CANCER both the extracellular and intracellular domain of RET Pancreaticcancer is a highly lethal neoplasm.Few, if any, (Figure). individuals are currently cured. Each year in the United MEN 2B is algo inherited as an autosomal dominant dis- States approximately 28,000 patients die from pancreatic arder and is causedby a mutation of RET, but this disorder cancer. Three different gene mutations have been associ- is composedof a slightly different complex of diseasesthan ated with pancreatic cancer: K-RAS, DPC4, and CDKN2. MEN 2A and FMTC. Individuals with MEN 2B have a K-RAS is an oncogene that is mutated in approximately characteristic appearance that includes mucosal neuromas, 80% of pancreatic cancersand is associatedwith tumor pro- prognathism, Marfanoid habitus, bony abnormalities, intes- gression in adenocarcinomas. DPC4, deleted in pancreas tinal ganglioneuromatosis,and corneal nerve hypertrophy. cancer locus 4, is a tumor suppressorgene that is located These patients all deveIop an aggressiveform of MTC; most on the long arm of chromosome 18. This gene is missing in develop pheochromocytomas,while none develop parathy- approximately 30% of pancreatic cancer specimens. The raid disease. Virtually all patients with the rafe MEN 2B exact mechanism of DPC4's effect on cell growth is un- syndrome have been shown to have an identical mutation known, but sequencehomology to other genessuggests that at codon 918 in the tyrosine kinase catalyticdomain ofRET it works through the TGF-B signaling pathway.8 CDKN2, that results in substitution of a threonine for a methionine also known as MTS 1, is a tumor suppressorgene that is (Figure). known to be mutated or lost in 80% of pancreatic cancer The RET -MEN 2A mutant allele results in receptor di- celllinesand 40% of primary pancreatic cancers. lts exact merization and autophosphorylation of the protein with ty- function is unknown, but it is associatedwith one of the rosine kinase activation,6 which presumablycan confer tis- ryrosine kinase membrane receptors. CDKN2 mutations sue-specificneoplasia of the thyroid C-cells, the chromaffin mar be able to identify individuals with familial melanoma cells of the adrenal medulla, and more variably of the para- who are at risk for the development of pancreatic cancer. thyroid glands. The MEN 2B mutation in the catalytic do- lnherited CDKN2 mutations have been reponed in a family main of RET has beenpostulated to alter the catalytic prop- with both melanoma and pancreatic cancer.9CDKN2 mu- erties or substrate specificity of the tyrosine kinase portion tations in pancreatic cancer specimensare also assoéiated of the receptor, which has been shown to result in receptor with a poorer prognosisand decreasedsurvivaUo Thus, be- autophosphorylation approximately one-third to one-fifth side tumor size and the detection of positive lymph nade that of RET -MEN 2A mutant allele.6 Thus different mu- metastases,which are known classic poor prognostic vari-

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codons609. 611. 618.620. 634 mutated in :\IEN 2A codons 768, 883 codon 918 mutated and Fl\ITC Inutated in FMTC in i\IIEN 2B

Cadherin homology regIon reglan

EXTRACELLL'LAR TRANSMEMBRANE TYROSINE KINASE DOMAIN DOMAIN DOMA IN

Figure. Different germline mutations in the RET proto-oncogene are assocíated with the distinct clinical syndromes MEN 2A, MEN 28, and FMTC. The RET protein product is a receptor tyrosine kinase, schematically depicted by the salid line in the figure. The majar domains 01 the RET product are represented by the shaded figures. Missense mutations that result in the substitution 01 a different amino acid 10r one 01 five highly conserved cysteines in the extracellular domain immediately adjacent to the transmembrane portion 01 the proteín are associated with MEN 2A and some FMTC families. Some FMTC 1amilies have been shown to have mutations in codons 768 and 883 in the intracellular partían 01 the protein. Virtually all patients with MEN 28 have an identical mutation in codon 918 of the putative tyrosine kinase catalytic domain that results in the substitution of a threonine for a methionine.

ables, molecular changes wirhin the tumor can be another laryngeal cancer, leukemia, and adrenal cancer. LFS can be detrimental variable. 10 caused by a germline mutation of the TP53 gene.l1 How- ever, not all individuals with LFS have mutations ofTP53, Diagnosis and Prognosis and other mechanismshave been implicated such as post- These studies provide important new findings for surgeons translation protein modifications. TP53 encodes a nuclear involved in the management of patients with pancreatic phosphoprotein whose normal function is to respond to cancer. Since a family history of melanoma can be associ- DNA damage by blocking the cell cycle or inducing apop- ated with pancreatic cancer,9 patients with pancreatitis tosis. The absence of an inhibitory effect of p53 protein have a greater risk of pancreatic cancer,and nearly all pan- following DNA injury contributes to cellular transforma- creatic tumors will have a mutation in either K-RAS, tion by allowing accumulation of unrepaired DNA muta- DPC4, or CDKN2, it may be possibleto make the diagnosis tiOflS. earlier in individuals at risk by screening staol or panc~eatic drainage specimens for the presenceof specific gene muta- Hereditary Early-onset Breast Cancer and Breast- tions. In many centers studies are currently underway to Ovarian Cancer Syndrome assessthe detection of genetic changes for early diagnosis. Hereditary early-onset breast cancer syndrome is associ- Further, in patients with a diagnosis of pancreatic cancer, ated with a genetic abnormality on the long arrn of chro- important prognostic information can be ascertainedby the mosome 17. The diseasegene locus was further refined to detection of a mutation in CDKN2. the 17q21 chromosomal region and the gene was subsequently cloned and sequenced as BRCAI.12 BRCAI is a BREAST CANCER large protejo with 1,863 amino acids and includes a zinc Approximately 5-10% (9-18,000 cases per year in the finger motif that appearsto be invo1ved with transcriptional United States) of breast cancer casesare causedby the au- regulation. However, the exact function of this gene is not tosomal dominant inheritance of a mutated gene. Forty-five yet known. There have been a large number of mutations percent of inherited breast cancers are due to BRCA1. in different alleles of BRCAl identified. Different alleles are Thirty-five percent of inherited breast cancers are due to associatedwith a different risk for the development ofbreast BRCA2. Breast cancer is a feature of the Li-Fraumeni syn- and ovarian cancer. Subsequently, it has been shown that drome (LFS), hereditary early onset breast cancer syn- only 45% of hereditary early onset breast cancers are asso- drome, breast-ovarian cancer syndrome,Cowden's disease, ciated with BRCAl mutations. In 1994 linkage studies Muir- Torre syndrome, and Peutz-Jegherssyndrome. demonstrated that another gene is involved that maps to Li-Fraumeni Syndrome 13q12-13.13 Seventy percent of hereditary breast cancers Breast éancer is the most frequent manifestation of me not 1inked to BRCAl are linked to this other site, so called LFS. Individuals may algo develop sarcomas,brain tumors, BRCAl. Cloning of the BRCA2 gene has allowed direct

40 THE AMERICAN JOURNAL OF SURGERY~ VOLUME 173 JANUAR'( 1997 I I ADVANCES IN MOL.ECULAR GENETICS/LAIRMORE AND NORTONf

genetic testing for individuals at risk.14Women with either defects in DNA mismatch repair genes. Examples of each a BRCAl or BRCA2 mutation have a significantly in- are included to substantiate me import~nce of understand- creased risk for the development of breast cancer: approx- ing mese mechanisms. RET is a proto-oncogene thai is fun:- it¡atety .50% by age 45, 85% lifetime, risk of second breast damental to the pathogenesis,and in the current era, mo; cancer 65%, and 30% of women who develop breastcancer lec~lar diagnosis of MEN 2 syndromes. TP53 is a tumor under the age of 35 will have inherited susceptibility. Fur- suppressorgene that ismutated in individuals with Li-Frau- ther, women with BRCAl mutation have a 40-60% risk of meni syndrome. CDKN2 is a tumor suppressorgene tha~ is ovarian cancer, while women with BRCA2 mutation have mutated in pancreatic icancers and is associated with a a 15-20% risk. Menwith BRCA2 mutation have a 5-10% poorer prognosis and th~ development of melanoma. MSH2 lifetime risk of breastcancer. is a mismatch repair gene that is important in the patho- genesisof HNPCC and Muir- Torre syndrome.Altered gene Muir-Torre Syndrome and Other Rare Syndromes function such as loss of DCC in colon cancers mar affect Muir- Torre syndrome (MTS) comprises an associationof cell adhesion properties and promote metastases.As we be- skin tumors and adenocarcinomas of the gastrointestinal gin to better define and understandthe mechanismsof neo- and genitourinary tract. The risk of breastcancer in woman plasia, we will be able to improve current diagnosis and with MTS is 12%. It has been hypothesized that MTS is a treatment. variant of HNPCC. Patients from two families with MTS have been found to have tight linkage to the mismatch REFERENCES repair gene MSH2.15 Cowden's diseaseis a rafe autosomal 1. 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Malkin D, Li FP, Strong LC, et al. Germline p53 mutations in Prophylactic mastectomy mar be a reasonable option for a familial syndrome ofbreast cancer. sarcomaand other neoplasms. these individuals. Long-term prospective studies will be Sdence. 1990;250:1233-1238. needed to better define the optimal management.The true 12. Miki Y, SwensenJ,Shattuck-Eidens D, et al. A strong candidate incidence of breast cancer in individuals with BRCAl and for the breast and ovarian cancer susceptibility gene BRCA1. Sd- 2 mutations who have undergone prophylactic mastectomy roce. 1994;266:66-71. will need to be determined as well as the psychological im- 13. Wooster R, Neuhausen SL, Mangion J, et al. Localization of breast cancer susceptibility gene,BRCA2, to chromosome 13q12- pact of this procedure. 13. Science.1994;265:2088-2090. 14. Wooster R, Bignall G, LancasterJ. et al. 1dentification of the SUMMAR y breast cancer susceptibility geneBRCA2. 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