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jnci.oxfordjournals.org JNCI |Articles 291
Hereditary nonpolyposis colorectal cancer (HNPCC), also referred
to as Lynch syndrome, is characterized by an autosomal dominant
inheritance of early-onset colorectal cancer and an increased risk
of other cancers, including cancers of the endometrium, stomach,
ovary, urinary tract, hepatobiliary tract, pancreas, and small bowel
( 1 , 2 ). Males with HNPCC have a higher lifetime risk for colorec-
tal cancer (standardized incidence ratio [SIR] = 83 ) than females
(SIR = 48) ( 3 ), and surveillance with regular colonoscopy and
polypectomy has been shown to reduce disease morbidity and
mortality ( 4 ). HNPCC is caused by germline mutations in DNA
mismatch repair (MMR) genes, and the prevalence of this syn-
drome among unselected colorectal cancer patients in Sweden has
been estimated, based on family history, to be approximately 1%
( 5 , 6 ). Although seven genes have been associated with HNPCC
(MSH2, MLH1, MSH6, PMS1, PMS2, MLH3, and EXO1), mu -
tations in only three are currently considered to cause HNPCC:
MLH1, MSH2, and MSH6.
It is important to correctly diagnose all HNPCC patients and
to distinguish them from non-HNPCC patients so that all families
Affiliations of authors: Department of Clinical Genetics, Karolinska University
Hospital, Stockholm, Sweden (KLR, TL, JV, AL); Department of Oncology –
Pathology, Karolinska Institute, Stockholm, Sweden (TL); Department of
Pathology, Lund University Hospital, Lund, Sweden (BH); Department
of Pathology, Helsingborg Hospital, Helsingborg, Sweden (BH ); Human
Cancer Genetics Program, Comprehensive Cancer Center, The Ohio State
University, Columbus, OH (MC); Department of Genetics, Rouen University
Hospital, Rouen, France (TF); Howard Hughes Medical Institute and Sidney
Kimmel Comprehensive Cancer Center, Johns Hopkins Medical Institutions,
Baltimore, MD (NP, KWK, BV); Department of Medical Genetics, University of
Helsinki, Helsinki, Finland (PP); Ludwig Institute for Cancer Research,
University of California, San Diego, School of Medicine, La Jolla, CA (RDK);
Department of Oncology, Lund University, Lund, Sweden (MN) .
Correspondence to: Annika Lindblom, MD, PhD, Department of Clinical
Genetics, Karolinska University Hospital, S-17176 Stockholm, Sweden
(e-mail: annika.lindblom@ki.se ).
See “Notes” following “References.”
DOI: 10.1093/jnci/djk051
© 2007 The Author(s).
This is an Open Access article distributed under the terms of the Creative Com-
mons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/2.0/uk/), which permits unrestricted non-commercial use, distribution, and
reproduction in any medium, provided the original work is properly cited.
ARTICLE
Lynch Syndrome (Hereditary Nonpolyposis
Colorectal Cancer) Diagnostics
Kristina Lagerstedt Robinson , Tao Liu , Jana Vandrovcova , Britta Halvarsson , Mark Clendenning,
Thierry Frebourg, Nickolas Papadopoulos , Kenneth W . Kinzler , Bert Vogelstein , Päivi Peltomäki ,
Richard D . Kolodner , Mef Nilbert , Annika Lindblom
Background Preventive programs for individuals who have high lifetime risks of colorectal cancer may reduce disease
morbidity and mortality. Thus, it is important to identify the factors that are associated with hereditary colorec-
tal cancer and to monitor the effects of tailored surveillance. In particular, patients with Lynch syndrome,
hereditary nonpolyposis colorectal cancer (HNPCC), have an increased risk to develop colorectal cancer at an
early age. The syndrome is explained by germline mutations in DNA mismatch repair (MMR) genes, and there
is a need for diagnostic tools to preselect patients for genetic testing to diagnose those with HNPCC.
Methods Patients (n = 112) from 285 families who were counseled between 1990 and 2005 at a clinic for patients at
high risk for HNPCC were selected for screening to detect mutations in MMR genes MLH1, MSH2, MSH6,
and PMS2 based on family history, microsatellite instability (MSI), and immunohistochemical analysis of
MMR protein expression. Tumors were also screened for BRAF V600E mutations; patients with the muta-
tion were considered as non-HNPCC .
Results Among the 112 patients who were selected for screening, 69 had germline MMR mutations (58 pathogenic
and 11 of unknown biologic relevance). Sixteen of the 69 mutations (23%) were missense mutations.
Among patients with MSI-positive tumors, pathogenic MMR mutations were found in 38 of 43 (88%) of
patients in families who met Amsterdam criteria and in 13 of 22 (59%) of patients in families who did not.
Among patients with MSI-negative tumors, pathogenic MMR mutations were found in 5 of 17 (29%) of
families meeting Amsterdam criteria and in 1 of 30 (3%) of non-Amsterdam families with one patient
younger than age 50 years. In three patients with MSI-negative tumors who had pathogenic mutations in
MLH1 or MSH6, immunohistochemistry showed loss of the mutated protein.
Conclusion Our findings suggest that missense MMR gene mutations are common in HNPCC and that germline MMR
mutations are also found in patients with MSI-negative tumors.
J Natl Cancer Inst 2007;99: 291 – 9
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292 Articles |JNCI Vol. 99, Issue 4 | February 21, 2007
can be offered tailored counseling and surveillance and, when pos-
sible, genetic testing. Several attempts have been made to defi ne
clinical criteria that can be used to diagnose HNPCC. In the fi rst
studies, which aimed to defi ne the genes involved in HNPCC, the
International Collaborative Group on HNPCC defi ned and later
updated the so-called Amsterdam criteria for the classifi cation of
HNPCC ( 7 ). The Amsterdam criteria require that families have
three affected individuals over two generations — one being a fi rst-
degree relative of the other two and one younger than age 50 years.
MMR gene mutations have been identifi ed in up to 90% of fami-
lies who meet the original Amsterdam criteria, whereas application
of less stringent criteria results in lower sensitivity but higher
specifi city ( 8 , 9 ). Studies of young (<50 years) patients with nonfa-
milial colorectal cancer have shown that many have germline
mutations in MMR genes, and thus, the use of early age of onset
as a criterion in the selection of patients for genetic mutation
screening has been emphasized as being important for HNPCC
screening ( 10 ).
During the past 12 years, screening techniques for mutations in
MMR genes have been evaluated and optimized. Most early stud-
ies used various exon-by-exon polymerase chain reaction (PCR) –
based methods to screen subjects with a family history of cancer.
Mutations were found in 40% – 60% of patients of families who
met the Amsterdam criteria and in a smaller percentage of patients
in families who did not fulfi ll these criteria ( 11 ). Studies using a
combination of other mutation detection methods to detect larger
genomic rearrangements show higher mutation rates than those
fi rst used ( 12 – 17 ).
Tumor microsatellite instability (MSI), a hallmark of HNPCC
and a sign of an increased mutation rate in HNPCC tumors, is
also observed in approximately 15% of unselected colorectal
cancers ( 18 ). Although MSI in unselected colorectal cancer is
most often associated with somatic CpG island methylation of
the promoter of the MMR gene MLH1, a positive MSI test in
familial or early-onset colorectal cancer is highly associated with
HNPCC ( 19 , 20 ). Immunohistochemical analysis to detect loss of
expression of the MMR proteins MLH1, MSH2, MSH6, and
PMS2 can identify HNPCC-associated MMR-defective tumors
and potential mutations in these genes and can thus facilitate
mutation analysis ( 21 ). However, the inability of immunohisto-
chemistry to identify loss of MMR protein expression in all
HNPCC-associated MMR-defective tumors motivated the con-
tinuation of MSI analysis ( 22 , 23 ).
Recently, somatic mutations in the BRAF oncogene have
been used to distinguish HNPCC-associated tumors from sporadic
tumors with MSI. BRAF mutations were fi rst demonstrated in
melanomas and other types of cancers ( 24 ). Approximately 10% of
sporadic colorectal cancers have mutations in BRAF, and the
mutation is almost always V600E ( 25 , 26 ). There is a strong associ-
ation between BRAF mutation status and MSI in colorectal tumors
( 26 ). Moreover, BRAF is often mutated in association with meth-
ylation of the MLH1 promoter and is almost never mutated in
HNPCC tumors ( 26 , 27 ).
The number of HNPCC families whose genetic background
has been clarifi ed has gradually increased over time with the
improvement of diagnostic procedures. In this study, we evaluated
our current protocol to diagnose HNPCC. We reanalyzed all
families who were counseled for an increased risk of colorectal
cancer according to this updated protocol using family history,
MSI, and immunohistochemistry testing to preselect patients for
mutation screening of MLH1, MSH2, MSH6, and PMS2 genes.
We also tested for the BRAF V600E mutation ( Fig. 1 ).
Subjects and Methods
Patients
Families who were referred to the Karolinska University Hospital
for genetic counseling during 1990 – 2005 were included in
the study if they fulfilled the following criteria: 1) at least
one member was diagnosed with colorectal cancer before age
50 years or two or more first- or second-degree relatives were
diagnosed with colorectal cancer at any age, 2) colorectal tumor
tissue was available for MSI analysis, and 3) at least one member
was available for muta tion screening. In total, 285 families met
the criteria to be included in this study, including 37 patients
who had no family history of colorectal cancer and were
diagnosed when younger than age 50 years. A complete family
history was available from every family that was included in the
study. All diagnoses were verified through medical records or
death certificates.
The families were fi rst classifi ed as Amsterdam or as non-
Amsterdam according to family history. The Amsterdam criteria
require that families have three affected individuals over two
generations — one being a fi rst-degree relative of the other two
and one younger than age 50 years. Because HNPCC families
also have high risks for other tumor types, these criteria were
modifi ed in 1999 to include endometrial cancer and cancers of
the small bowel and the upper urothelial tract; the revised crite-
ria are referred to as the Amsterdam criteria II ( 7 ). Of the 285
families included in the study, 60 met the Amsterdam criteria.
The families that did not fi t the criteria (non-Amsterdam fami-
lies) could be divided into two groups — one group of 119 families
CONTEXT AND CAVEATS
Prior knowledge
Lynch syndrome, or HNPCC, is currently diagnosed by the detec-
tion of germline mutations in MMR genes.
Study design
A new protocol to select patients for screening for germline
mutations in MMR genes was tested on family members who had
undergone counseling for being at high risk for HNPCC.
Contribution
Overall, mutations in MMR genes were found in more than half of
the patients who were screened for mutations, including some
who would not have been screened using current protocols.
Implications
More true HNPCC cases were identified using the new protocol
than the previous protocol.
Study limitations
It is possible that some patients who were not selected for muta-
tion screening also had tumors with germline mutations in MMR
genes.
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jnci.oxfordjournals.org JNCI |Articles 293
each with two patients only (of which 21 had one patient younger
than age 50 years) and another group of 69 families each with at
least three patients affected with colorectal cancer. The second
group of 69 families did not fulfi ll the Amsterdam criteria for
the following reasons: 60 did not have any patient younger than
age 50 years, 23 did not have three patients with a fi rst-degree
relationship, and 11 did not fulfi ll the criteria across two genera-
tions. The study was undertaken in accordance with the decisions
in the Regional Ethical Review Board in Stockholm (97/205,
00/291, and 05/566).
Laboratory Diagnostic Procedures
This retrospective study used the protocol in Fig. 1 to diagnose
HNPCC patients. Dissection of tumor cells from paraffin-embedded
tissue for MSI and gene mutation assays were performed at the
Department of Clinical Genetics (Karolinska University Hospital).
The technique for MSI testing followed the international Bethesda
guidelines ( 28 ) and used at least one colorectal cancer patient from
each family. We preferred to test the youngest patient in the family
because of the risk that an older patient might have sporadic cancer.
If the preferred patient could not be used for MSI testing, if the
tumor was MSI negative and HNPCC was still suspected, or for
research purposes, at least one more tumor from the same patient
or another family member was tested, if possible. More than one
tumor was tested in a total of 35 families. In 19 families, three or
more tumors were tested. A pathologist at the hospital where the
specimens were archived provided the tumor tissue to be used for
MSI testing. None of the MSI tests was performed using adenoma
tissue. MSI testing was performed as described previously ( 19 ) or
using a commercial kit (Microsatellite Instability Multiplex System
Kit, Promega Corp, Madison, WI) according to the manufacturer’s
instructions. Briefly, genomic DNA prepared from tumor tissue
was amplified by PCR in a multiplex PCR, amplifying selected
microsatellite markers. Microsatellite analysis was performed
using an ABI310 (Applied Biosystems, Foster City, CA) with the
GeneScan 3.1 software (Applied Biosystems). MSIH (MSI high)
was considered MSI positive and MSS (MS stable) and MSIL
(MSI low) were considered MSI negative. The classification of
MSIH, MSS, and MSIL were according to the Bethesda/National
Institutes of Health (NIH) guidelines ( 28 ).
Immunohistochemistry for MLH1, MSH2, MSH6, and PMS2
was performed on tumors from MSI-positive patients only because
our previous study ( 23 ) showed that 90% of MSI-positive tumors
and none of 70 MSI-negative familial colorectal tumors lacked
immunostaining pinpointing a mutated MMR gene ( 23 ). In 37
families, mutations had been identifi ed before the introduction
of immunohistochemistry analysis to the clinical protocol. Thus,
tumor samples from 38 families were subjected to immunohisto-
chemical analysis. MSI-positive samples were selected, analyzed,
and evaluated as described by Halvarsson et al. ( 23 ).
Samples from the youngest colorectal cancer patient available
from each Amsterdam family, MSI-positive family, and MSI-
negative family with one patient younger than age 50 years were
screened for mutations in MLH1, MSH2, MSH6, and PMS2.
Screening was also performed in all single patients with early-
onset disease and MSI-positive tumors. Exon-by-exon screening
was performed using genomic DNA – based methods, such as
Positive
Negative No
Yes
2Sequence
MSI test
IHC assay
MSI
IHC
3Alternative
screening
method
Yes
No
1 CRC with at least one 1° or 2° relative with CRC or
1 CRC with age of onset before age 50
Mutation?
No
1Sequence
Mutation? -
Full expression
Lack of expression
Two
CRC, one
<50
4BRAF
5Counseling
173 cases
Yes
Fig. 1 . Flow diagram of the hereditary nonpolyposis colorectal cancer
(HNPCC) diagnostic procedure used in this study. Top , inclusion criteria.
1 ) Microsatellite instability (MSI) – positive samples expressing all four mis-
match repair (MMR) genes (MLH1, MSH2, MSH6, and PMS2 ) and MSI-
negative samples from familial cases, among which one patient was
younger than age 50 years, were sequenced for MLH1, MSH2, and MSH6.
2 ) MSI-positive samples were sequenced according to which protein had
low or no expression by immunohistochemistry (IHC), e.g., MLH1 if loss of
MLH1 (with or without simultaneous loss of PMS2), MSH2 if loss of MSH2
(with or without simultaneous loss of MSH6), MSH6 if loss of MSH6, PMS2
if loss of PMS2 only . 3 ) Alternative screening methods were quantitative
multiplex polymerase chain reaction of short fl uorescent fragments or
multiplex ligation-dependent probe amplifi cation. 4 ) BRAF was sequenced
only to verify a potential V600E mutation when no germline MMR gene
mutation was found. 5 ) Counseling for HNPCC if a MMR gene germline
mutation was found and considered pathogenic. Disease was considered
non-HNPCC if no pathogenic germline mutation was found. CRC =
colorectal cancer.
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294 Articles |JNCI Vol. 99, Issue 4 | February 21, 2007
denaturant gradient gel electrophoresis or sequencing. Screening
for rearrangements used RNA- or DNA-based methods, such as
protein truncation test, monoallelic mutation analysis, quantitative
multiplex PCR of short fl uorescent fragments, or multiplex
ligation-dependent probe amplifi cation. All nonsense mutations,
splice mutations, and genomic rearrangements (deletions and
insertions) were considered pathogenic. The pathogenicity of mis-
sense mutations was defi ned based on published data, based on the
segregation of the mutation in at least three affected subjects in the
family, and by comparing the frequency in 80 – 200 normal control
subjects.
The mutation status of BRAF was assayed only to rule out
HNPCC in MSI-positive patients for whom no MMR gene muta-
tion had been identifi ed or for whom mutations were found but
considered to be of unclear biologic relevance. In brief, genomic
DNA prepared from tumor tissue was amplifi ed using PCR. The
PCR product was then used for direct sequencing using the
BigDye Terminator v1.1 Sequencing Kit (Applied Biosystems)
according to the manufacturer’s recommendation. Sequences were
analyzed using an ABI Prism 3730 Sequencer (Applied Biosystems).
The chromatograms were evaluated manually by eye or with
SeqScape v2.5 (Applied Biosystems). The tumor was considered
V600E positive if the mutation could be detected on the chro-
matograms. The rationale for using BRAF V600E mutations to
rule out HNPCC was based on fi ndings that this mutation almost
never occurs in HNPCC ( 26 , 27 ).
Results
Patients from 285 families were evaluated using a new diagnostic
protocol ( Fig. 1 ). The study included 60 families who met the
Amsterdam criteria, 188 non-Amsterdam families, and 37 single
patients with early-onset (younger than age 50 years at diagno-
sis) colorectal cancer. Patients who had an MSI-positive tumor
were identified in 70% of the Amsterdam families, 9% of the
non-Amsterdam families, and 13% of the early-onset patients
( Table 1 ).
A total of 112 patients were screened for mutations in MLH1,
MSH2, MSH6, and PMS2; 173 were not. Patients were screened
if they were from an Amsterdam family, an MSI-positive family,
or an MSI-negative family with one patient younger than age
50 years. Single patients with early-onset disease and MSI-positive
tumors were also screened.
Mutations in Patients in Amsterdam Families With
MSI-Positive Tumors
In total, 43 germline mutations in MLH1, MSH2, MSH6, or
PMS2 were found in 42 of 43 patients in Amsterdam families with
MSI-positive tumors; 38 of the mutations were considered patho-
genic (88%) ( Table 2 ). Nine missense mutations were found, as
discussed below. Among families with a mixed pattern of MSI (both
MSI-positive and MSI-negative tumors in the same family), MMR
mutations were either not found (family 722) or did not segregate
with disease within the family (families 19, 110, and 119). MMR
mutations that did not segregate with disease were defined as
having unknown biologic relevance.
Mutations in Patients of Non-Amsterdam Families and
Early-Onset Single Patients With MSI-Positive Tumors
A germline mutation in one of the MMR genes was found in 15 of
22 MSI-positive patients in non-Amsterdam families ( Table 2 ).
Thirteen of these mutations, including two missense mutations,
were considered pathogenic (59%). In two patients from different
families, the same truncating PMS2 mutation was found that was
considered to be of unclear biologic relevance. We considered the
seven MSI-positive non-Amsterdam families with no MMR gene
germline mutations as non-HNPCC families. Within these seven
families, tumors of patients in five families showed lack of MLH1
protein staining and had the BRAF V600E mutation (families 237,
289, 350, 374, 409) and/or had a mixed MSI pattern (families 237,
350, 409, 301, 399). Finally, all five of the single patients with
MSI-positive early-onset colorectal cancer and who were included
in the non-Amsterdam group had germline mutations in one of
the MMR genes ( Table 2 ). Of those mutations, four were consid-
ered pathogenic. Thus, we found evidence for or against HNPCC
in 20 of 22 patients with MSI-positive tumors in non-Amsterdam
families.
Mutations in Patients With MSI-Negative Tumors
A germline mutation in one of the MMR genes was found in 8
of 17 MSI-negative Amsterdam families ( Table 2 ). Five of the
eight mutations were considered pathogenic (in total 29%) — one in
Table 1 . Hereditary nonpolyposis colorectal cancer screening of patients from 285 families *
Criteria MSI status
No. of patients with
MMR mutation/total
MMR gene mutation type
Pathogenic Unknown
Amsterdam family Positive 43/43 38 5
Negative 8/17 5 3
Total 51/60 43 8
Non-Amsterdam family Positive 10/17 9 1
Negative 2/171 † 11
Total 12/188 10 2
Single patients Positive 5/5 4 1
Negative 0/32 0 0
Total 5/37 4 1
All patients 68/285 57 11
* MSI = microsatellite instability; MMR = mismatch repair.
† Of these, 30 were screened for mutations because one patient in the family was younger than age 50 years.
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Table 2 . Germline or somatic mutations identified during hereditary nonpolyposis colorectal cancer (HNPCC) screening
Family No. Type * MSI † IHC ‡ BRAF § E-age || M-age || Gene ¶ Mutation Type HNPCC # Reference
Amsterdam families with MSI-positive tumors **
1 Amsterdam + − 24 29 MLH1 [del exon 1 – 15] Del Yes This study
2 Amsterdam + − 34 51 MLH1 [c.131C>T] Missense Yes ( 29 )
3 Amsterdam + − 42 50 MLH1 [c.104T>G] Missense Yes ( 30 )
4 Amsterdam + − − 32 45 MLH1 [del exon 11] Del Yes ( 15 )
5 Amsterdam + 29 50 MSH2 [c.201del G] Del Yes ( 31 )
6 Amsterdam + − 44 48 MSH2 [c.892C>T] Nonsense Yes ( 32 )
7 Amsterdam + + − 27 36 MLH1 [del exon 16] Del Yes ( 33 )
10 Amsterdam + 35 46 MLH1 [c.208 − 2A>G] Splice Yes ( 30 )
11 Amsterdam + − − 37 43 MSH2 [c.2038C>T] Nonsense Yes This study
14 Amsterdam + − 36 45 MLH1 [del exon 16] Del Yes ( 33 )
19 Amsterdam M M − 44 54 MLH1 [c.2059C>T] Missense This study, ( 34 )
24 Amsterdam + − 24 47 MSH2 [c.1216C>T] Nonsense Yes ( 32 )
27 Amsterdam + 27 41 MLH1 [del exon 16] Del Yes ( 33 )
30 Amsterdam + 33 34 MSH2 [c.1097 del A] Del Yes ( 35 )
58 Amsterdam + 31 39 MLH1 [del exon 16] Del Yes ( 32 )
63 Amsterdam + 45 58 MLH1 [c.1769 del T] Del Yes ( 35 )
66 Amsterdam + − − 41 42 MLH1 [del exon 6] Del Yes ( 15 )
69 Amsterdam + 34 48 MLH1 [c.199G>A] Missense Yes ( 30 )
98 Amsterdam + 32 41 MLH1 [del exon 16] Del Yes ( 32 )
110 Amsterdam M − 33 50 MLH1 [c.2104-10_11del
GT ins A]
Intronic This study
113 Amsterdam + − − 31 48 MSH2 [c.2131C>T] Nonsense Yes This study
119 Amsterdam M − − 24 57 PMS2 [c.2113G>A] Missense ( 36 )
124 Amsterdam + 40 42 MSH2 [c.942+3A>T] Nonsense Yes ( 32 )
136 Amsterdam + 38 43 MLH1 [del exon 16] Del Yes This study
149 Amsterdam + − − 40 45 MSH2 [del exon 8] Del Yes ( 15 )
163 Amsterdam + − 40 46 MLH1 [c.454 − 13A>G] Splice Yes This study
187 Amsterdam + 45 49 MSH2 [c.942+3A>T] Splice Yes This study
198 Amsterdam + − 38 48 MLH1 [c.793C>T] Missense Yes ( 32 )
228 Amsterdam + 35 42 MLH1 [c.131C>T] Missense Yes This study
246 Amsterdam + 38 59 MLH1 [c.1772_1775 del
ATAG]
Del Yes This study
262 Amsterdam + − − 31 39 MSH2 [del exon 13 – 15] Del Yes ( 15 )
269 Amsterdam + − 41 52 MLH1 [c.665 del A] Del Yes This study
292 Amsterdam + 46 51 MSH2 [c.2228 del CATT] Del Yes This study
294 Amsterdam + 34 50 MSH2 [c.1447G>T] Nonsense Yes This study
302 Amsterdam + − 39 49 MSH2 [c.811_814 del
TCTG]
Del Yes This study
362 Amsterdam + − 44 44 MLH1 [c.1225C>T] Nonsense Yes This study
368 Amsterdam + − 35 44 MSH2 [del exon 1 – 6] Del Yes This study
388 Amsterdam + − 40 51 MLH1 [del exon 16] Del Yes This study
435 Amsterdam + − 56 57 MLH1 [c.306+3A>C] Splice Yes This study
475 Amsterdam + − 44 44 MSH2 [c.1447_1448
del AG]
Del Yes This study
546 Amsterdam + − 39 47 MLH1 [c.1459C>T] Nonsense Yes This study
722 Amsterdam M − 44 65
765 Amsterdam + − − 36 46 MLH1 [c.2059C>T] Missense This study
765 PMS2 [c.1866G>A] Missense This study
Non-Amsterdam families with MSI-positive tumors
15 Non-Amsterdam + − 36 51 MLH1 [c.131C>T] Missense Yes ( 30 )
16 Non-Amsterdam + 30 36 MLH1 [c.546 − 2A>G] Splice Yes ( 30 )
28 Non-Amsterdam + − 35 39 MLH1 [c.1373T>A] Nonsense Yes ( 32 )
34 Non-Amsterdam + − 22 28 MLH1 [del exon 4 – 11] Del Yes ( 15 , 31 ) † †
88 Non-Amsterdam + − 41 42 MLH1 [del exon 14 – 15] Del Yes ( 15 , 31 ) † †
204 Non-Amsterdam + − 52 63 MSH2 [del exon 1 – 8] Del Yes ( 15 )
291 Non-Amsterdam + − 42 43 MLH1 [c.677+1G>T] Splice Yes This study
347 Non-Amsterdam + − 38 44 MSH2 [del exon 1 – 7] Del Yes ( 15 )
400 Non-Amsterdam + − 45 47 MSH2 [dup ex 7 – 8] Duplication Yes ( 15 )
391 Non-Amsterdam + − + 54 77 PMS2 [c.736 del 6 ins11] Del ( 36 )
237 Non-Amsterdam M − + 55 66 BRAF V600E
289 Non-Amsterdam + − + 53 64 BRAF V600E
350 Non-Amsterdam M + 57 72 BRAF V600E
(Table continues)
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MLH1, one in MSH2, and three in MSH6. Four of the eight
mutations were missense mutations ( Table 2 ).
Mutation screening was also performed in the 30 MSI-negative
non-Amsterdam families among whom one member was younger
than age 50 years at diagnosis. From the 30 families screened,
two mutations were found ( Table 2 ). One was a splice mutation in
MLH1; the other was a missense mutation in MSH6. To confi rm
MSI status, additional MSI analyses as well as immunohistochem-
istry analysis were performed on a subset of tumors from 38 fami-
lies. Tumors from two patients in family 145 were tested for MSI
and both were negative. Tumors from two patients in family 424
tested MSI negative, but one showed lack of MLH1 protein by
immunohistochemistry. The MSI test was repeated once using a
new section of this tumor after the immunohistochemistry result
and was still negative. A tumor from a patient in family 534 was
MSIL but lacked expression of MSH6 by immunohistochemistry.
Two patients in family 341 had the (c.454 − 1G>A) MLH1 splice
mutation, and both had metachronous colorectal cancers at ages
51 and 68 and 35 and 51 years, respectively. Both the tumor at
age 35 years and the metachronous tumor were MSI negative.
How ever, immunohistochemistry showed loss of MLH1 protein,
and therefore, the mutation was considered to be pathogenic.
Missense Mutations
In total, we found 16 missense mutations. Missense MLH1 muta-
tion (c.131C>T) in families 2, 15, and 228 is a Swedish founder
mutation that segregated with affected status in families and was
not found in normal control subjects ( 29 , 30 ). The MLH1 mutation
(c.2059C>T) in families 19 and 765 has been observed in Polish
families ( 34 ) and was not found in normal control subjects. This
mutation did not segregate consistently in the families in this study
and thus was considered of unclear biologic relevance. A PMS2
missense mutation (c.1866G>A), which was previously published
as a polymorphism ( 39 ), was also found in family 765. The MLH1
mutation in family 198 (c.793C>T) segregated with some MSI-
positive tumors and was not found in 96 control subjects. In family
198, one patient had MSI-negative colorectal cancer at 46 years
of age and two metachronous tumors at 79 and 82 years of age.
Family No. Type * MSI † IHC ‡ BRAF § E-age || M-age || Gene ¶ Mutation Type HNPCC # Reference
374 Non-Amsterdam + − + 55 63 BRAF V600E
409 Non-Amsterdam M − + 40 58 BRAF V600E
301 Non-Amsterdam M − − 69 75 BRAF not
informative
399 Non-Amsterdam M + − 53 60 BRAF wild type
Single early-onset patients with MSI-positive tumors
59 1 CRC < 50 + MSH2 [c.1226_1227
del AG]
Del Yes ( 35 )
166 1 CRC < 50 + − − MSH2 [del exon 2] Del Yes ( 15 )
331 1 CRC < 50 + − MSH2 [del exon 1 – 6] Del Yes ( 15 )
480 1 CRC < 50 + − + PMS2 [c.736_741 del 6
ins11]
Del ( 36 )
669 1 CRC < 50 + − MSH2 [c.1906C>T] Missense Yes This study
Amsterdam families with MSI-negative tumors
145 Amsterdam − 50 65 MSH6 [c.3052_3053
del CT]
Del Yes ( 37 )
175 Amsterdam − − 50 66 MLH1 [c.1733A>G] Missense ( 38 )
181 Amsterdam − − 40 56 MLH1 [c.1733A>G] Missense ( 38 )
199 Amsterdam − + − 29 53 MSH2 [c.593A>G] Missense Yes ( 32 )
241 Amsterdam − − 45 65 MLH1 [c.2146G>A] Missense This study
340 Amsterdam − 41 53 MSH6 [c.2303 del CCT] Del Yes This study
424 Amsterdam − − 41 45 MLH1 [c.298C>T] Nonsense Yes This study
534 Amsterdam − − 49 52 MSH6 [c.2851_2858
del 8]
Del Yes This study
Non-Amsterdam families with MSI-negative tumors
341 Non-Amsterdam − − 35 43 MLH1 [c.454 − 1G>A] Splice Yes This study
487 Non-Amsterdam − + − 49 49 MSH6 [c.3674C>T] Missense This study
* Single early-onset patients do not meet Amsterdam criteria (7).
† MSI = microsatellite instability; + = positive; − = negative; M = more than one tumor were studied and showed different results.
‡ IHC = immunohistochemistry; + = the mutated gene expressed the protein; − = the mutated gene did not express the protein; M = results differed between
tumors; missing values indicate that immunohistochemistry was not done.
§ BRAF: + = V600E; − = wild-type missing values indicate that the test was not done.
|| E/M = early/mean; missing values for single patients younger than age 50 years.
¶ Gene names according to GenBank.
# Yes indicates that the mutation was considered pathogenic and causes HNPCC. Missing values indicate unclear biologic relevance.
** In family 1, the mutation was found using the monoallelic mutation analysis technique, performed as in (14).
† † Mutation results in this study differ from the two studies because different techniques were used to detect them.
Table 2 (continued).
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However, this patient did not inherit the mutation; thus, another
predisposing factor may contribute to colorectal cancer in this
family. The MLH1 mutations in families 3 (c.104T>G) and 69
(c.199G>A) segregated with disease in the families and were not
found in normal control subjects. The mutation in family 69 has
been observed in numerous other families ( 40 ) . The MSH2 mis-
sense mutation c.1906C>T in family 669 is a founder mutation in
Ashkenazi Jews that is considered to cause HNPCC ( 41 ) . A germ-
line missense MSH2 mutation (c.593A>G) segregated in family 199
with three MSI-negative tumors and was not found in control sub-
jects. We consider this MSH2 mutation to be pathogenic, although
it is possible that a coexisting MLH3 mutation contributed to dis-
ease in this family ( 42 ) . In family 241, a MLH1 missense mutation
(c.2146G>A) segregated with disease in affected family members
and was not found in normal control subjects. This mutation has
been published in other HNPCC families but is still considered to
be of uncertain biologic relevance ( 39 ). The same MLH1 missense
variant (c.1733A>G) was found in families 175 and 181. This vari-
ant did not segregate with disease and was considered to be of
unclear biologic relevance. However, this variant may serve as a
low-risk/modifying allele ( 38 ). The missense MSH6 mutation
(c.3674C>T), not found in control subjects, was found in family
487, in which both parent and child had MSI-negative colon
tumors at age 49 years. Because segregation analysis was not possi-
ble, this missense mutation was considered as having unclear bio-
logic relevance. The PMS2 missense mutation (c.2113G>A) in
family 119 did not segregate well with disease in the family, which
had a mixed pattern of MSI, and was thus considered as having an
unclear biologic relevance.
Discussion
As expected, the MMR gene mutation rate was high (88%) among
HNPCC patients in Amsterdam families with MSI-positive tumors.
We also observed that 59% of patients in non-Amsterdam families
with MSI-positive tumors had MMR gene mutations. The Bethesda
criteria would have selected all mutation-positive patients to be
tested for MSI or immunohistochemistry ( 28 ). If we had only
screened MSI-positive patients for mutations, the 10 mutations
in MSI-negative patients would have been missed. If we had used
immunohistochemistry instead of MSI testing, we would have
found some of the mutations in MSI-negative patients but might
have missed some of the missense mutations.
We found fi ve deleterious MMR gene mutations in tumors
from MSI-negative patients (in families 145, 340, 341, 424, and
534). Three of these were in MSH6, associated with a lower
degree of MSI-positive tumors ( 43 ), and two were in MLH1. MSI
associated with MSH6 mutations preferentially shows mutations
in mononucleotide repeats ( 43 ). The MSI test used four mononu-
cleotide repeats, and only one patient (family 534) had a tumor
that was MSIL. For three of the fi ve MSI-negative tumors with
MMR gene mutations, immunohistochemistry results showed lack
of the mutated protein in all tumors, suggesting that immunohis-
tochemistry can sometimes be more informative than MSI test-
ing in predicting a germline predisposing mutation. The negative
MSI test was confi rmed in a second analysis after the immuno-
histochemistry result (in families 145, 341, 424, and 534). For
MSI-negative tumors with MMR missense mutations, it is diffi cult
to evaluate the risk of disease. In this study, we considered four
(175, 181, 241, and 487) of fi ve mutations to be of unknown
biologic relevance.
Families among whom a full genetic investigation did not reveal
a pathogenic MMR gene mutation were considered to be non-
HNPCC. Because we did not continue the search for deleterious
mutations in all patients using all current methods known, it is still
possible that a deleterious mutation could have been undetected
in patients with no mutation and even in patients with missense
mutations. Mutations in at least seven genes have been associated
with HNPCC: MSH2, MLH1, MSH6, PMS1, PMS2, MLH3,
and EXO1. The former three gene mutations are associated with
the vast majority of HNPCC ( 5 , 40 ). Mutations in PMS1 are no
longer considered to be associated with colorectal cancer, and the
consequences of germline PMS2 mutations, including the three
mutations identifi ed in our study, are still unclear ( 36 , 39 , 44 , 45 ).
The MLH3 and the EXO1 genes have been studied in families
fulfi lling or not fulfi lling the Amsterdam criteria, and several mis-
sense mutations of unclear biologic relevance have been identifi ed
( 42 , 46 ). Because so many MSI-negative patients in our study had
MMR gene mutations, it is still possible that we missed HNPCC
families by not screening the 173 patients with MSI-negative
tumors who were not selected by our new protocol ( Fig. 1 ). Based
on this protocol, we selected 30 families with at least one patient
younger than age 50 years. This criterion resulted in mutations
being found in two families, both having a pedigree that is highly
suggestive of HNPCC. Thus, it might be possible to improve
our protocol by defi ning new criteria for selecting MSI-negative
patients for mutation screening. The new criteria should include
the age of onset in the entire family as well as penetrance and
what type of tumors should be considered. Immunohistochemistry
could also be used to preselect MSI-negative patients for mutation
screening.
When a germline MMR gene mutation was not detected in a
family with MSI-positive tumors, we tried to obtain evidence for
or against HNPCC. Five such families showed a mixed pattern of
MSI. The presence of the BRAF V600E excluded HNPCC in
fi ve families. A syndrome involving a mixed pattern of MSI as well
as BRAF mutations in tumors and precursor lesions has been sug-
gested and is supported by our data ( 47 ). A new syndrome X was
also recently suggested to occur in Amsterdam families with MSI-
negative tumors ( 48 ). Thus, both the BRAF-associated syndrome
and the syndrome X could explain some of the non-HNPCC fami-
lies in our study.
For now, we consider the vast majority of the remaining 173
unscreened patients in our cohort of non-Amsterdam patients to
have non-HNPCC. We hypothesize that most non-HNPCC
patients are at risk for disease due to other genetic factors that
act as monogenic or additive/modifying factors in a complex
disease, and in the Karolinska University Hospital, we offer
also non-HNPCC families tailored surveillance programs based
on empiric risk fi gures. We also offer subjects at risk for disease
who are members of families with known or suspected MMR
gene mutations colonoscopy screening at 2-year intervals begin-
ning at age 25 years. We offer all non-HNPCC risk individuals
a screening colonoscopy every 3 – 5 years, depending on the
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298 Articles |JNCI Vol. 99, Issue 4 | February 21, 2007
risk, starting at least 10 years before the earliest age of onset in
the family.
Although it is critical to identify all individuals who are at
increased risk for colorectal cancer to offer them surveillance pro-
grams, it is equally important to accurately exclude individuals
in a specifi c family who are not at increased risk. Thus, it is very
important to fi nd the gene mutations that are associated with
HNPCC. Different criteria to select patients for mutation screen-
ing have been outlined and evaluated. The Centre for Reviews and
Dissemination ( 49 ) concluded that a mixed strategy, similar to our
protocol, showed the lowest incremental cost-effectiveness ratio. A
recent study ( 50 ) used a population-based strategy and performed
MSI testing on 1066 unselected colorectal cancer patients, screened
208 patients’ samples for mutations, and identifi ed 23 patients with
HNPCC. Our current family-based screening protocol used
family history and tumor MSI within a selected cohort of 285
patients suspected of HNPCC that led to mutation screening of
112 samples and the identifi cation of at least 57 HNPCC patients.
Although a population-based mutation-screening strategy has the
potential to also identify nonfamilial HNPCC patients, more
patients will need to be screened, and some mutations in patients
with MSI-negative tumors will be missed. The most important
advantage with the family history – based selection process we used
is that non-HNPCC families are also identifi ed and can be offered
preventive programs.
We present our current protocol using a family-based strategy
for HNPCC detection. We estimate that this protocol detected the
majority of DNA MMR gene mutations. However, the unexpected
fi ndings of several mutations also in MSI-negative patients suggest
that it might be worthwhile to modify our protocol and to fi nd new
criteria to detect HNPCC in MSI-negative patients. The high
number of MMR gene missense mutations in both MSI-positive
and MSI-negative patients emphasizes the need for functional
assays to be used in genetic counseling. For now, these mutations
will have to be interpreted using clinically relevant available infor-
mation in each family.
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Notes
Supported by grants from the Swedish Cancer Society, the Stockholm Cancer
Society, the Finnish Cancer Society, Academy of Finland, Sigrid Juselius
Foundation, NIH CA82282, NIH CA67941, and the State of Ohio Biomedical
Research and Technology Transfer Commission. The funding agencies had no
role in the study design, data collection and analysis, the interpretation of the
results, or the preparation of the manuscript.
Under licensing agreements between the Johns Hopkins University and
Genzyme Molecular Oncology, N. Papadopoulos, K. W. Kinzler, and B.
Vogelstein are entitled to a share of royalties received by the University on
sales of products related to this article. K. W. Kinzler and B. Vogelstein own
Genzyme Molecular Oncology stock, which is subject to certain restrictions
under University policy. R. D. Kolodner is an inventor on issued patents
and patent applications covering a number of MMR genes, including MSH2,
MLH1, and PMS2, which are owned by Dana-Farber Cancer Institute, and he
receives royalties as the result of these efforts.
We are grateful to Albert de la Chapelle for critical reading of the manu-
script and to Cecilia Österman, Inger Svensson, Ulla Platten, Norma Lundberg,
Yvonne Borg-Lavesson, Johanna Rantala, and Sam Ghazi for valuable techni-
cal contribution.
Mutations in this article are described according to recommendations by
den Dunnen and Antonarakis ( 51 ). The following GenBank reference sequen-
ces have been used — MLH1: NM_000249, MSH2: NM_000251, MSH6:
NM_000179, PMS2: NM_000535, and BRAF: NM_004333.
Funding to pay the Open Access publication charges for this article was pro-
vided by The Swedish Cancer Society.
Manuscript received August 14 , 2006 ; revised October 18 , 2006 ; accepted
December 28 , 2006 .
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