Can HNPCC Be Diagnosed in Presymptomatic Patients?

April 1, 1997

This special series on cancer and genetics is compiled and edited by Henry T. Lynch, MD, director of the Hereditary Cancer Institute and professor of medicine and chairman of the Department of Preventive Medicine and Public

ABSTRACT: This special series on cancer and genetics is compiled and editedby Henry T. Lynch, MD, director of the Hereditary Cancer Institute andprofessor of medicine and chairman of the Department of Preventive Medicineand Public Health, Creighton University School of Medicine, and directorof the Creighton Cancer Center, Omaha, Nebraska.

Colon cancer will affect approximately 140,000 persons and kill an estimated55,000 people in 1997,[1] making colon cancer the second deadliest cancerin the United States. Hereditary nonpoly-posis colon cancer (HNPCC) accountsfor approximately 3% to 10% of colon cancers.[2]

This autosomal dominant condition is characterized by an earlier ageof onset of colon cancer (in the 40s versus about age 65 for sporadic coloncancer), a tendency for multiple tumors to form either simultaneously orover time, and a tendency for tumors to occur proximal to the splenic flexure.[3]

The pathology of the tumors is more likely to be mucinous and poorlydifferentiated, but in contrast to this histologic grade, which would connotea more aggressive tumor, HNPCC colon cancers have a better clinical outcomethan sporadic tumors matched for stage.[3] [This will be discussed by Dr.Risto Sankila of the Finnish Cancer Registry in the next article in thisseries.]

In addition, there is an increased incidence of certain noncolonic tumorsin some HNPCC families, including those of the female reproductive tract,stomach, and urinary tract.[3]

Criteria for Studies of HNPCC

In 1991 in Amsterdam, the International Collaborative Group on HNPCCdeveloped uniform criteria for collaborative studies on HNPCC. (These criteriawere not designed for the diagnosis of HNPCC.) The requirements are:

  • The family includes three or more relatives with histologically verifiedcolorectal cancer, one of whom is a first-degree relative of the othertwo, and familial adenomatous polyposis has been excluded.
  • The colorectal cancers involve at least two successive generations.
  • At least one of the colorectal cancers has been diagnosed before theage of 50 years.[4]

However, these criteria are restrictive in that they exclude patientswith extra-colonic tumors and late-onset variants, thus overlooking someHNPCC families by this definition.

The heritable nature of HNPCC (and its variant, the Muir-Torre syndrome)is caused by germline mutations in one of the several genes involved inthe DNA mismatch repair (MMR) system, an editing mechanism for polymeraseerrors that occur during DNA replication.

The MMR genes are so named for their gene products' ability to recognizeand direct repair of nucleotide mispairs and misalignment at short repetitivese-quences of DNA (called microsatellites) whose length was not accuratelycopied during DNA replication. Repair by the MMR system occurs on the newlysynthesized DNA strand, as documented by in vitro repair of mispairs onthe strand containing nicks.[5,6]

Base mispairing can lead to nucleo-tide transitions or transversions,altering the authentic genetic sequence. If the mismatch occurs in thecoding region for a particular gene, the newly introduced point mutationmay affect the expression and/or function of that particular gene.

Perhaps more importantly, some of the approximately 100,000 DNA micro-satellitesequences scattered throughout the human genome may be altered in lengthwhen this system is defective. A microsatellite may become lengthened ina daughter cell if there is nucleotide-pairing slippage (looping) alongthe newly synthesized strand during DNA synthesis, or it may become shortenedif the template strand microsatellite has slippage during DNA replication.

This alteration in microsatellite length is termed microsatellite instability(MIN)[7-9] and can be identified by electrophoretic resolution of amplifiedmicrosatellite DNA sequences. MIN is relatively easy to detect in the laboratory,and when found in the DNA of a tumor, it indicates the presence of a hypermutablephenotype.

Occasionally, microsatellites are present in the coding region (exons)of critical growth regulatory genes. This has been best demonstrated withthe transforming growth factor-beta type II receptor (TGF-beta RII); whenthis receptor is bound by TGF-beta in the colonic epithelium, cellularproliferation is inhibited.

Mutation of TGF-beta RII occurs with defective MMR, commonly resultingin length changes in the polyadenine mononucleotide repeat microsatellite(A10) within the gene, producing a frameshift mutation and renderingthe receptor inactive.[10,11] This mutation removes the growth brake providedby TGF-beta, allowing the colon cells to undergo clonal expansion.

The evolutionarily conserved genes that comprise the MMR system andthat have been found mutated in HNPCC families include hMSH2[12,13], hMLH1[14,15],hPMS1, and hPMS2.[16] Although other components of the MMR system havebeen identified, germline mutations of these components have yet to befound in HNPCC families.

When a physical deformation remains in the newly replicated DNA doublehelix (caused by the mispairing of nucleo-tides or by slippage and loopingat microsatellite loci), a complex termed hMutS-alpha (a heterodimer ofhMSH2 and hMSH6 proteins) identifies the error and binds the DNA at thissite.[17-20]

Subsequently, hMutS-alpha recruits the hMutL-alpha complex, a heterodimerof hMLH1 and hPMS2 proteins, which targets the newly synthesized daughterDNA strand for "long patch" excision repair.

It remains unclear how hPMS1 interacts within the complex. Loss of anyof the four components of the MMR system will inactivate or attenuate repair.Germline mutations in the hMLH1 and hMSH2 genes account for the majority(approximately 90%) of HNPCC families identified to date.[21]

One wild-type allele of an MMR gene is generally sufficient to maintainnormal MMR function. For colon cancer to develop in HNPCC patients, a secondsomatic event (in addition to the vertically transmitted mutant allele)must occur in the wild-type allele of a colonocyte. This completely inactivatesboth of the MMR genes[22] and causes the hypermutable phenotype seen withHNPCC tumors.

Methods of Diagnosing HNPCC

Identification of genes involved in HNPCC has prompted efforts to diagnosethis condition in presymptomatic patients (see table).There is no premor-bid clinical phenotype that identifies an HNPCC patientantecedent to the development of cancer.

Nonneoplastic cells in HNPCC patients have a normal phenotype, sincethe inactivation of only one allele of an MMR gene is not permissive ofhypermutability.[23] The presence of inactivating mutations in both allelesof MMR genes in tumors confirms Knudson's "two-hit" hypothesisfor tumor-suppressor genes.[24]

Although MIN should be a necessary finding for this condition, in fact,only 92% of HNPCC tumors show MIN.[25] Furthermore, it has been demonstratedthat only a minority of tumors with MIN actually come from HNPCC families.[26,27]

Adenomas in HNPCC patients often manifest MIN,[28,29] indicating thatinactivation of the second allele of an MMR gene occurs as an early eventin colorectal tumorigenesis. Unfortunately, the finding of MIN in a tumoris not perfectly sensitive and is quite nonspecific, so it is not a practicalscreening strategy for HNPCC.

Obtaining peripheral blood is the least invasive method to diagnoseHNPCC. In HNPCC, the lymphocytes will have one mutated allele and one wild-typeallele in one of the above-mentioned MMR genes.

Direct sequencing of the four MMR genes involved in HNPCC is a possiblediagnostic strategy, but the number of genes involved and the number ofexons in each gene make this clinically impractical at this time.

These problems extend to common mutation detection methods like single-strandedconformational polymorphism (SSCP) and denaturing gradient gel electrophoresis(DGGE).

Nevertheless, direct DNA sequencing of hMLH1 and hMSH2, the two mostcommon genes involved, is the most reliable approach to diagnosis at thistime. Interpretation of the sequence data, however, may prove difficult,since the full spectrum of disease-producing mutations and innocuous polymorphismshas not been catalogued.

The reported mutations in hMLH1 and hMSH2 include insertions, deletions,nonsense mutations, and some missense mutations.[21,25] Many variationsin the DNA sequences of these genes are not associated with an increasedrisk for tumor formation. A large fraction of these mutations yield a truncatedprotein after translation, more so with hMSH2 than hMLH1.[21]

This fact has led to the application of an in vitro transcription/translationassay (IVTT) for these genes.[30] With the IVTT approach, RNA is extractedfrom the peripheral blood, reverse transcribed into complementary DNA,and expressed as protein.

The altered migration of a truncated protein on a polyacrylamide gelcan provide a means of identifying which MMR gene contains an inactivatingmutation. A study assaying hMLH1 and hMSH2 by the IVTT method was about50% sensitive in patients who fulfilled the Amster-dam criteria for HNPCC.[30]

Linkage analysis can identify current or future family members as carriersof a mutated MMR gene (and thus distinguishes the carrier as an HNPCC patient).

The mutated allele may be identified from affected family members (andexcluded from unaffected family members) using microsatellites or restrictionfragment length polymorphisms (RFLPs) that are linked to the MMR gene.This approach, however, requires obtaining blood from more than one generationof first-degree relatives, including the affected and unaffected members.

The MAMA Technique

Another novel but time-consuming approach for certain mutations missedby conventional techniques is the isolation of each allele from peripheralblood lymphocytes into a somatic cell hybrid, termed MAMA (monoallelicmutational analysis).[31]

The MAMA method fuses individual chromosomes from human cells from apatient with an appropriate hamster cell line to individually analyze eachallele for mutations that might be masked by the wild-type allele. However,the time required to perform this assay makes its use impractical outsideof the research setting.

At this time, the use of the IVTT assay combined with direct DNA sequencingmay be the most appropriate approach for finding germline mutations inHNPCC.

These tests can be applied to families who fit the Amsterdam criteria,as well as to the young patient (ie, less that 45 years of age) who developscolon cancer that has characteristics typical of HNPCC tumors (ie, right-sided,mucinous, poorly differentiated adenocarcinomas).

When available, neoplastic tissue, including paraffin-embedded archivalblocks, can be valuable for MIN analysis, since patients with HNPCC willusually have microsatellite alterations at multiple loci. Supporting informationat the time of microsatellite analysis includes loss of heterozygosityof an MMR gene, which can be identified using appropriate microsatellitetargets.

The aim of identifying the germline lesion in the patient is for screeningremaining family members. There is no substitute for assessing the familyhistory, including the ages of affected family members and the occurrenceof extra-colonic tumors.

In clinically defined HNPCC families in which a mutation cannot be identifiedby current genetic techniques, or in family members who do not wish tobe genetically tested, a regular screening program consisting of colonoscopyor barium enema and sigmoidoscopy significantly reduces the rate of tumordevelopment and death.[32,33]


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This work has been supported by NIH grants DK 02433 and CA 72851,the Research Service of the Department of Veterans Affairs, and the JohnsonFamily Fund.