William Check, PhD
Of the approximately 150,000 people who develop colorectal
cancer each year, the vast majority are sporadic cases. But a few percent
of colorectal cancer cases occur in people who have a genetic predisposition.
Most of those with a genetic predisposition don't know they are carrying
a genetic defect that greatly increases their risk of colorectal cancer
and a few other selected cancers. "Until recently, there has not been
a way, other than family history, to readily identify these individuals,"
says Stephen N. Thibodeau, PhD. "Now there is." Dr. Thibodeau, who is
co-director of the clinical molecular genetics laboratory at the Mayo
Clinic and professor of laboratory medicine and pathology at Mayo Medical
School, gave a presentation on this procedure, called microsatellite instability,
or MSI, testing, at the 2006 Association for Molecular Pathology meeting.
Hereditary nonpolyposis colon cancer, or HNPCC, is
one of several hereditary forms of colorectal cancer. HNPCC is an autosomal
dominantly inherited tendency to develop colorectal cancer and other tumors
such as endometrial cancer, gastric cancer, and urothelial carcinoma of
the upper urinary tract. Colorectal cancer and other tumors generally
occur at a significantly younger age than is observed among patients with
sporadic tumor of the same type. Historically, the diagnosis has been
made clinically based on a pedigree that shows a strong family history
of colorectal cancer or other HNPCC-associated tumor type. However, smaller
families, incomplete histories, and incomplete penetrance often complicate
a clinical diagnosis of HNPCC.
Accordingly, in 1991 an international consortium held
in Amsterdam established the following "Amsterdam criteria" for HNPCC:
- Historically verified colorectal cancer in three
or more relatives, one of whom is a first-degree relative of the other
- Presence of disease in two or more successive generations.
- Age of colorectal cancer onset of less than 50
years in at least one person.
- Exclude familial adenomatous polyposis.
The Amsterdam criteria were expanded in 1999 (Amsterdam
criteria II) to recognize the importance of all HNPCC-associated tumors,
and not just colon cancer, in the syndrome.
With the use of such highly selected families, that
is, those meeting the Amsterdam criteria, the genes involved for a substantial
fraction of clinically diagnosed HNPCC were eventually identified. These
genes are those involved in one particular pathway: DNA mismatch repair.
Thanks to these early findings and subsequent research, a clear story
has emerged over the past decade that has important research and clinical
Based on the presence or absence of functional DNA
mismatch repair, or MMR, colon cancer is now divided into at least two
broad categories. Tumors with defective MMR (dMMR) are characterized by
the presence of a particular tumor phenotype, termed microsatellite instability,
and by the absence of protein expression for any one of a number of genes
involved in DNA mismatch repair, including hMLH1, hMSH2, hMSH6,
or PMS2. Tumors with dMMR have been identified in approximately
15 percent to 20 percent of sporadic colon cancer and in a subset of patients
with HNPCC (~two-thirds), now referred to as Lynch syndrome. Tumors with
dMMR have a distinct MSI phenotype termed MSI-H (MSI at ?30 percent of
In sporadic colon cancer, the MSI-H phenotype is associated
with distinct clinicopathologic features, including proximal tumor site,
high grade, early stage, and diploidy. This phenotype has consistently
been associated with a more favorable outcome. Among sporadic colon cancer,
the majority of MSI-H cases are due to inactivation of hMLH1
(~95 percent), with hMSH2 and hMSH6 accounting for a
smaller percentage, approximately five percent and less than one percent
respectively. Germline mutations in these same mismatch repair genes are
also responsible for Lynch syndrome, with hMLH1 and hMSH2
accounting for the majority of cases (approximately 40 percent each) and
hMSH6 and PMS2 again accounting for a smaller percentage, about 10 percent
and five percent respectively. Among all cases involving hMSH2
and hMSH6 (sporadic or inherited), the presence of a germline
mutation appears to be the most frequent mechanism of gene inactivation.
For hMLH1, however, current data suggest that the most common
mechanism of gene inactivation among unselected cases (~90 percent of
cases) is promoter hypermethylation and, less frequently, by mutations
in the gene itself. Thus, the molecular etiology of those tumors exhibiting
dMMR is very heterogeneous, involving several different genes and numerous
mechanisms of gene inactivation, including epigenetic (promoter hypermethylation),
somatic, and germline alterations.
Identifying HNPCC patients is useful because colonoscopic
and other organ screening measures of mutation carriers offer the chance
to reduce cancer morbidity and mortality in such individuals. Identifying
other family members with the gene defect is also important for their
screening. The overall goal is to prevent death from metastatic disease,
so preventive screening of HNPCC patients is essential.
Dr. Thibodeau says the initial purpose of the Amsterdam
criteria was to help identify a homogeneous group of families with hereditary
colon cancer syndrome for research purposes, such as linkage studies.
"But these criteria are not sufficient to identify all cases of HNPCC,
and as a result, these criteria have limitations for clinical purposes,"
he says. Many cases would be missed if these were the only criteria used,
and "it is important to recognize," Dr. Thibodeau says, "that not all
patients who fulfill the Amsterdam criteria have evidence of defective
MMR." It has become increasingly important to distinguish families who
meet the Amsterdam criteria for HNPCC and have defective MMR genes (Lynch
syndrome) from those who meet the Amsterdam criteria but do not have defective
MMR genes. The latter group has a lower incidence of colorectal cancer
and perhaps other cancers as well, which has important implications for
surveillance and counseling (Lindor NM, et al.
JAMA. 2005; 293:1979-1985).
The Amsterdam criteria for a diagnosis of HNPCC were
established before the discovery of MSI and the identification of the
DNA MMR genes that are mutated in most patients with HNPCC. The greater
understanding of HNPCC and its etiology led to a revised set of criteria—the
Bethesda criteria—to help identify which patients are most likely
to have Lynch syndrome and would benefit most from MSI testing. These
criteria have since been revised (Umar A, et al. J
Natl Cancer Inst. 2004;96:261-268) and include the following:
- Colorectal cancer in patients under 50 years of
- Synchronous or metachronous colorectal or other
HNPCC-associated tumors, regardless of age.
- Colorectal cancer with MSI-H [a high level of MSI]
morphology in patients under 60 years of age.
- Colorectal cancer with one or more first-degree
relatives with HNPCC tumors, one diagnosed under age 50.
- Colorectal cancer in two or more first- or second-degree
relatives with HNPCC-related tumor, regardless of age.
With the discovery of a genetic cause for a substantial
subset of HNPCC, "testing of tumors, especially colorectal tumors, for
MSI by PCR can now be utilized as a useful phenotypic marker to help identify
patients with this syndrome," Dr. Thibodeau says. Defective MMR can also
be assessed with immunohistochemistry, or IHC. This generally involves
evaluating tumors for expression of the protein products of the four DNA
MMR genes—hMLH1, hMSH2, hMSH6, and PMS2. More
than 95 percent of tumors with defective DNA MMR show loss of expression
of one or more of these four genes. "The real value" of these two screening
tests, Dr. Thibodeau says, "is to be able to better define the risk of
a patient for having a hereditary cancer syndrome or Lynch syndrome."
Given a patient whose family history suggests hereditary
nonpolyposis colorectal cancer syndrome or a patient who has other risk
factors (such as young age of onset or multiple tumors), the method at
Mayo Clinic is to screen the patient's tumor to identify whether it has
evidence of defective DNA MMR (based on MSI) and which gene is most likely
involved (based on IHC). "If there is no evidence of dMMR," Dr. Thibodeau
says, "subsequent gene testing for the presence of a germline DNA MMR
mutation is not likely to be very productive." If MSI or immunohistochemistry
is abnormal, that is very strong evidence for the possibility of a genetic
defect in one of the MMR genes, and the IHC results point to which gene
is most likely affected. Sequencing of the MMR gene of interest would
follow if the patient is interested in this testing after appropriate
genetic counseling. In at-risk patients, Dr. Thibodeau cautions that a
negative tumor test for defective MMR (MSI and IHC) does not rule out
other hereditary causes of colorectal cancer. (Mayo Clinic's algorithm
has been published: Baudhuin LM, et al. Fam
Several issues are generally considered when testing
for Lynch syndrome. The first is the relative value of MSI versus IHC
and whether both should be performed or only one or the other. At Mayo,
testing for defective MMR status by MSI and immunohistochemistry is generally
performed simultaneously. "For most cases, we do both of these tests in
our laboratory," says Kevin C. Halling, MD, PhD, assistant professor of
laboratory medicine and pathology at Mayo Medical School and co-director
of the clinical molecular genetics laboratory. "We find them complementary.
Each provides important information. MSI testing provides for a more global
view of whether or not the tumor exhibits defective DNA MMR, while IHC
provides a very valuable bit of information—which mismatch repair
gene is defective." And because the results of MSI and IHC testing are
highly associated, Dr. Thibodeau says, they provide "valuable quality
control information when performed together."
The second issue is when to perform the tumor testing.
"Should one go directly to gene testing or should one utilize tumor screening
first to identify those who are at highest risk? The approaches taken
by various groups are complicated and differ based on a number of factors,"
Dr. Thibodeau says, "including the availability of laboratory testing,
availability of tumor material, level of risk the patient presents with
to begin with, etc. There clearly are pros and cons with both approaches."
James Eshleman, MD, PhD, associate professor of pathology
and oncology and associate director of the molecular diagnostics laboratory
at Johns Hopkins Medical Institutions, agrees this is a complicated area.
"I don't think that there is a simple algorithm that fits every patient
scenario," he says. Dr. Eshleman sees the approach differing with the
patient's pre-test probability of having Lynch syndrome. "If someone comes
in with an extraordinarily strong family history and colon cancer at a
young age, it may make sense to go straight to gene sequencing," he says.
"With a less strong family history, most people would do one of the functional
Whether the first functional test should be MSI or
IHC depends to some degree on local expertise, in Dr. Eshleman's view.
"Some places are very comfortable doing immunohistochemistry," he says,
"while their molecular pathology laboratory may not have taken on MSI
testing. In other places perhaps MSI is easy and routine to do." Dr. Eshleman
cites one advantage to MSI testing: It has a good correlation with prognosis
and possibly 5-fluorouracil responsiveness and is "essentially a nearly
perfect functional assay for MMR defects." On the other hand, he adds,
"Immunohistochemistry is widely available. It also provides some guidance
as to which gene is likely defective. Almost every pathology lab does
it. It takes some experience to validate the antibodies, but in general
labs have the expertise to do it. I would argue that it is still in flux
what the best approach would be."
At ACL Laboratory in Rosemont, Ill., MSI testing is
done but not immunohistochemistry. "It would be useful to have IHC assays,"
says Jan A. Nowak, MD, PhD, a pathologist at Advocate Lutheran General
Hospital in Park Ridge, Ill., and medical director of the Rosemont ACL
Laboratory. "If one decides to go that route, you would need to test for
all four MMR proteins and have pretty good validation that you are doing
it well." Dr. Nowak wonders about the cost of doing IHC in addition to
MSI testing. "It's also beginning to sound like HER2," he says, "leaving
me with a sense of déjà vu. Will someone soon be telling us what to do?"
Whatever happens, Dr. Nowak believes that the "CAP and pathologists should
be ahead of the game."
Testing for MSI has increased in the past few years,
Dr. Nowak says. The number of laboratories subscribing to MSI proficiency
testing went from 35 three years ago to 72 in the CAP's 2007 MSI-A Survey.
"Performance on that Survey has been good," Dr. Nowak says. About half
of the participants also do immunohistochemistry.
Dr. Halling, vice chair of the CAP Molecular Oncology
Committee (and chair as of Jan. 1), foresees subscriptions to this Survey
approaching 100. The chief reason for the ongoing increase in MSI testing,
he says, may be the greater understanding that pathologists and other
physicians have of the existence of HNPCC and the value of doing the test
to identify which patients may have it. "It takes a while for word to
filter out to everybody," Dr. Halling says. "Increasingly clinicians understand
that they should order this test in a colon cancer patient under age 50
or if the patient has a family history of colon cancer." No Survey exists
yet for MMR protein immunostaining. "That is something we want to do.
We're hoping the [CAP] committee that covers that area will come up with
something and we may make a joint Survey," he says.
As with much testing, volume is one of the drivers
that influences whether MSI testing is adopted in a particular diagnostic
laboratory. "The complexity of MSI testing is well within the realm of
what most molecular diagnostics labs can do," Dr. Thibodeau says. "And,
as with all tests, proficiency in performing and interpreting the test
results is critical."
While many laboratories now perform MSI testing, immunohistochemistry,
or both, "relatively few perform gene sequencing," Dr. Halling says. "Many
groups will send to a larger laboratory like Mayo or Myriad Genetics for
that." With only about 10 laboratories in the U.S. now doing sequencing
for defective MMR genes, there would not be much demand for proficiency
testing. "But it will come," Dr. Halling predicts.
The possible need for guidelines for MSI testing is
an "active topic for discussion" in the Clinical Practice Committee of
the Association for Molecular Pathology, says Dr. Nowak, who is past chair
of the committee. A start to establishing such guidelines was made under
Dr. Nowak's chairmanship. Now, he says, "there may be some utility in
publishing those guidelines."
Microsatellites are repetitive DNA elements with unit
lengths from one to six nucleotides and repeat numbers of 10 to 60 that
are found abundantly throughout the genome. The majority of microsatellite
repeats are found in noncoding regions of the genome; they occur occasionally
within coding regions of genes. (Interestingly, instability of microsatellite
repeats within coding regions of some tumor suppressor genes such as TGF-beta
have been implicated in colorectal tumorigenesis.) "Microsatellite loci
are incredibly prone to a process called slippage," Dr. Eshleman says.
"Because of their repetitive nature, during replication when DNA is somewhat
fluid you get realignment. A CA pair on one strand may bind to a GT upstream
or downstream on the other strand and form a small loop." The result is
a single cell that has a mismatched pair. "In an MMR functional cell,"
Dr. Eshleman continues, "repair enzymes fix that. If MMR proteins are
lacking or dysfunctional, slipped intermediates are maintained and get
transmitted to daughter cells." Usually the altered microsatellite is
shorter. This mechanism explains why MSI and defective MMR genes are so
In clinical practice, testing for suspected Lynch syndrome
is done on paraffin-embedded tissue using one or more 5-µm thick
sections. In addition to testing tumor tissue, it is important to test
normal tissue (usually adjacent tissue from the same organ, but any normal
tissue such as peripheral blood will suffice). Tissue from colon cancer
is preferred since most work has been done on that. "Although adenomas
can be useful in some circumstances, colon adenocarcinomas are the tissue
of choice and most often used," Dr. Thibodeau says.
For MSI testing, marker selection is important. Most
but not all labs assess for MSI by analyzing five (Umar A, et al. J
Natl Cancer Inst. 2004;96:261-268) to 10 microsatellites weighted
toward mononucleotide markers, since these are more sensitive and specific
for the type of MSI found in DNA MMR defective tumors. Mononucleotide
repeats are especially important for identifying patients with germline
hMSH6 mutations since tumors in these patients frequently show
lower levels of instability and a predilection to show instability in
mononucleotide compared with dinucleotide markers. In a validation study
done in Dr. Thibodeau's laboratory, 10 specific microsatellite loci markers
were used: BAT 25, BAT 26, BAT 34c4, BAT40, D5S346, mycL, ACTC, D10S197,
D18S55, and D17S250. Microsatellite instability is defined as a variation
in the size of PCR amplicons for the specific microsatellite loci as seen
in tumor compared with normal tissue. Three MSI phenotypes have been defined:
microsatellite stable (MSS, no instability), low-frequency instability
(MSI-L, <30 percent of markers affected), and high-frequency instability
(MSI-H, >30 percent of markers) (J
Natl Cancer Inst. 2004;96:261-268).
Dr. Thibodeau and his colleagues have investigated
the reproducibility between laboratories of MSI testing for Lynch syndrome.
They recently reported the experience of a six-center multinational consortium
with MSI quality control activities, as laboratories developed competency
with MSI testing and interpretation (Lindor NM, et al. Cancer
Biomark. 2006;2:5-9). "Each group used the same set of markers
while trying to set up the assay," Dr. Thibodeau says. "It was proficiency-type
testing with experienced and inexperienced labs in a consortium. In the
first round, experienced labs did well and inexperienced labs did generally
less well," he says. "Then we had a training session and after that repeated
the evaluation." In the second there was substantial improvement. "The
point of the paper is that there is a bit of a learning curve," Dr. Thibodeau
says. Laboratorians have to learn how to score, what to look for, and
how to optimize the assays. The good news: "Under the best conditions,
this is a very robust assay," he says.
For IHC scoring, protein staining must be nuclear,
and non-tumor cells (for example, normal colonic mucosa or lymphocytes)
must be present and demonstrate normal staining. Among cases that show
a loss of staining for one or more of the DNA MMR proteins (and thus evidence
of defective DNA MMR), typical IHC patterns are loss of both hMLH1 and
PMS2 with normal hMSH2 and hMSH6 staining, loss of hMSH2 and hMSH6 with
normal hMLH1 and PMS2 staining, or loss of either hMSH6 or PMS2 alone.
Mismatch repair proteins exist as heterodimers, Dr. Eshleman points out,
which explains the IHC patterns. For instance, hMSH2 and hMSH6 are partners,
with hMSH2 being dominant; hMLH1 and PMS2 are partners, with hMLH1 dominant.
"To be stable in the cell, both partners have to be present," he says.
"When you lose hMSH2 expression, you should also lose hMSH6 expression.
But if you lose hMSH6 expression, you shouldn't lose hMSH2 expression.
So by doing IHC tests for both partners, you have a kind of built-in-control."
Now let's say you are evaluating a patient with colon
cancer and a suggestive family history. The patient's tumor tests abnormal
by MSI and IHC. Can you now say that this patient has Lynch syndrome?
No, because MSI and concomitant loss of DNA MMR protein expression are
found in many colorectal cancer cases not due to Lynch syndrome. "Defective
DNA MMR occurs in both sporadic and inherited colon cases," Dr. Thibodeau
cautions. Defective MMR is observed in 15 percent to 20 percent of sporadic
cases. In these non-hereditary cases, defective MMR is almost always due
to somatic (nonheritable) changes, rather than heritable germline mutations.
When hMSH2/ hMSH6 expression is lost by IHC, germline hMSH2 mutations
are found in a very high proportion of cases. However, if hMLH1/PMS2 expression
is lost, Dr. Thibodeau says, "it is a different story." In this case,
there is a fairly high frequency of somatic hypermethylation of the hMLH1
gene promoter, which indicates that the colorectal tumor is likely a sporadic
tumor and not inherited.
The probability of a patient having Lynch syndrome
is strongly influenced by his or her family history, other risk factors,
or both. It is here that the Amsterdam or Bethesda criteria provide valuable
information, as demonstrated by results from two sets of patients—one
unselected, the other selected to be moderate or high risk for HNPCC.
Among the 788 unselected patients, Dr. Thibodeau showed, 14 percent were
MSI-H and 99 percent of them had abnormal IHC findings. However, in 84
percent of these patients with defective MMR, loss of hMLH1/PMS2 was found.
Based on current literature, the vast majority of these (approximately
90 to 95 percent) will be due to promoter hypermethylation, a somatic
and epigenetic mutation. Only about 10 percent of the overall cases demonstrated
loss of hMSH2/hMSH6.
In contrast, the results in 675 cases selected by multiple
criteria, including the Amsterdam and Bethesda criteria, to be of moderate
or high risk, showed that twice as many, 27 percent, were MSI-H, of which
94 percent were IHC positive. In these patients, 32 percent were found
to have loss of hMSH2/ hMSH6 and another 10 percent had loss for hMSH6
alone by IHC. As indicated earlier, the loss of hMSH2 or hMSH6, or both,
is highly associated with the presence of a germline mutation (Lynch syndrome).
Thus, by using some selection criteria, there was a substantial enrichment
for those cases most likely to have Lynch syndrome.
Dr. Thibodeau summarizes these findings: "As you increase
the level of risk that a patient will have Lynch syndrome by selection,
the proportion of colorectal cancer cases with defective DNA MMR due to
hMLH1 hypermethylation decreases and the proportion due to germline
mutation increases." It is important to distinguish these two types of
colorectal cancer because they have different implications for the patient
and the patient's family (one is heritable in an autosomal dominant fashion,
one is not).
When hMLH1 is implicated in a case of apparently hereditary
colorectal cancer, both promoter hypermethylation and germline mutation
must be considered, especially if a germline mutation is not found in
the hMLH1 gene. "You have to ask," Dr. Thibodeau says, "is there
germline hMLH1 mutation and I didn't find it? Or is the test
negative because the loss of hMLH1 expression is due to hypermethylation
of the hMLH1 promoter?"
One way to help with this problem is to test directly
for the presence of hMLH1 promoter methylation. For this, a number
of quantitative and non-quantitative methods are available. For example,
a study from the Institute of Pathology and Molecular Diagnostics at the
University of Regensburg in Germany showed that, in MSI-H colorectal cancer,
"[Q]uantitative MLH1 methylation analysis is a valuable molecular tool
to distinguish between HNPCC and sporadic MSI-H CRC" (Bettstetter M, et
Cancer Res. 2007;13:3221-3228).
Dr. Thibodeau says another way to discriminate such
cases is to assess the colorectal tumor for the presence of a V600E mutation
of the BRAF oncogene. To date, BRAF V600E mutations have been found frequently
in cases with hMLH1 promoter hypermethylation but not in germline
cases. Thus, the finding of a BRAF mutation in the tumor strongly
suggests that the patient does not have a germline mutation. "Our data
suggest that using both tests—hMLH1 promoter methylation
and the BRAF V600E mutation-provides the best discrimination,"
Dr. Thibodeau says.
In typical clinical practice, patients are selected
for MSI testing by gastrointestinal, oncologic, or medical genetics practitioners
as being at high risk. Focusing on colon cancer patients with a family
history will detect many, but not all, cases of heritable cancer. For
this reason, many institutions are instituting limited screening in selected
patients with a variety of protocols. At Mayo Clinic, reflex testing of
selected surgical patients by pathology is practiced. "Reflex testing
is a bit different," Dr. Thibodeau says. "It is done as part of surgical
pathology practice when a patient has a newly diagnosed colon cancer."
Indications of higher risk for MMR, such as age of onset under 50 years,
are used. When a patient has surgery, the pathologist looks at the tumor.
"If it is a young onset patient, we automatically reflex to MSI testing,"
Dr. Thibodeau says. "That allows us at the time of surgery to see whether
the patient is at risk for having Lynch syndrome." Tumor is tested for
MSI only and results are reported to the surgeon and colorectal neoplasia
clinic. If the test result is abnormal, the patient is approached to see
if additional testing is desired. "We don't do IHC until the patient consents,"
Dr. Thibodeau says. This is related to the fact that IHC reveals more
information with respect to the likelihood of having a germline mutation.
William Check is a medical
writer in Wilmette, Ill.