Is gastric cancer part of the tumour spectrum of hereditary non-polyposis colorectal cancer? A molecular genetic study.

Abstract

Gastric cancer is the second most common extracolonic malignancy in individuals with hereditary non-polyposis colorectal cancer (HNPCC)/Lynch syndrome. As gastric cancer is relatively common in the general population as well, it is not clear whether or not gastric cancer is a true HNPCC spectrum malignancy.
To determine whether or not gastric cancer is a true HNPCC spectrum malignancy. Subjects and
The molecular and clinicopathological profiles of gastric cancers (n = 13) from HNPCC mutation carriers were evaluated and compared with the profiles of sporadic gastric cancers (n = 46) stratified by histology and microsatellite instability (MSI) status.
This study on sporadic and HNPCC gastric cancers revealed several important universal associations. Loss of heterozygosity in the adenomatous polyposis coli (APC) region was associated with intestinal histology regardless of the MSI (p = 0.007). KRAS-mutations (p = 0.019) and frameshift mutations in repeat tracts of growth-regulatory genes (p<0.001) were associated with MSI tumours being absent in microsatellite stable (MSS) tumours. The average number of methylated tumour suppressor gene loci among the 24 genes studied (methylation index) was higher in MSI than in MSS tumours regardless of histology (p<0.001). Gastric cancers from HNPCC mutation carriers resembled sporadic intestinal MSI gastric cancers, except that MLH1 promoter methylation was absent (p<0.001) and the general methylation index was lower (p = 0.038), suggesting similar, but not identical, developmental pathways. All these lacked the mismatch repair protein corresponding to the germline mutation and displayed high MSI.
The present molecular evidence, combined with the previous demonstration of an increased incidence relative to the general population, justify considering gastric cancers as true HNPCC spectrum malignancies.

Full text

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GASTRIC CANCER
Is gastric cancer part of the tumour spectrum of hereditary
non-polyposis colorectal cancer? A molecular genetic study
A Gylling, W M Abdel-Rahman, M Juhola, K Nuorva, E Hautala, H J Ja ¨rvinen, J-P Mecklin, M Aarnio,
P Peltoma ¨ki
............................................................... ............................................................... .....
See end of article for
authors’ affiliations
........................
Correspondence to:
Professor P Peltoma ¨ki,
Department of Medical
Genetics, University of
Helsinki, Biomedicum
Helsinki, P O Box 63
(Haartmaninkatu 8), Helsinki
00014 Finland; paivi.
peltomaki@helsinki.fi
Revised 15 January 2007
Accepted 16 January 2007
Published Online First
31 January 2007
........................
Gut 2007;56:926–933. doi: 10.1136/gut.2006.114876
Background: Gastric cancer is the second most common extracolonic malignancy in individuals with
hereditary non-polyposis colorectal cancer (HNPCC)/Lynch syndrome. As gastric cancer is relatively common
in the general population as well, it is not clear whether or not gastric cancer is a true HNPCC spectrum
malignancy.
Aim: To determine whether or not gastric cancer is a true HNPCC spectrum malignancy.
Subjects and methods: The molecular and clinicopathological profiles of gastric cancers (n=13) from
HNPCC mutation carriers were evaluated and compared with the profiles of sporadic gastric cancers (n=46)
stratified by histology and microsatellite instability (MSI) status.
Results: This study on sporadic and HNPCC gastric cancers revealed several important universal associations.
Loss of heterozygosity in the adenomatous polyposis coli (APC) region was associated with intestinal histology
regardless of the MSI (p=0.007). KRAS-mutations (p=0.019) and frameshift mutations in repeat tracts of
growth-regulatory genes (p,0.001) were associated with MSI tumours being absent in microsatellite stable
(MSS) tumours. The average number of methylated tumour suppressor gene loci among the 24 genes studied
(methylation index) was higher in MSI than in MSS tumours regardless of histology (p,0.001). Gastric
cancers from HNPCC mutation carriers resembled sporadic intestinal MSI gastric cancers, except that MLH1
promoter methylation was absent (p,0.001) and the general methylation index was lower (p=0.038),
suggesting similar, but not identical, developmental pathways. All these lacked the mismatch repair protein
corresponding to the germline mutation and displayed high MSI.
Conclusion: The present molecular evidence, combined with the previous demonstration of an increased
incidence relative to the general population, justify considering gastric cancers as true HNPCC spectrum
malignancies.
H
mutations in DNA mismatch repair (MMR) genes, usually
MLH1 or MSH2, and microsatellite instability (MSI) in tumour
tissue.1Besides cancer in the colon and rectum, individuals
with HNPCC develop cancers in several other organs. Among
these, cancer of the endometrium, small bowel, ureter and
renal pelvis are considered to have sufficiently high relative
risks in HNPCC compared with the average population, and
hence warrant their inclusion in the Amsterdam criteria II, the
clinical consensus criteria for the diagnosis of HNPCC.2
Despite the fact that gastric cancer is the second most
common extracolonic malignancy in HNPCC,3 4and in some
populations even the most common extracolonic cancer,5 6at
present it is not included in the Amsterdam criteria II. This is
because, despite the currently decreasing trends of this cancer
in the Western world, it is relatively common in the general
population making the specificity of the association of HNPCC
difficult to prove. Although the risk of extracolonic cancers,
including gastric cancer, could be higher in MSH2 than MLH1
mutation carriers,7the correlation between germline mutation
and clinical phenotype is generally poor in the case of HNPCC.
For a further assessment of the relationship between gastric
cancer and HNPCC, comprehensive molecular characterisation
of gastric carcinoma tumours from patients with HNPCC would
be necessary; however, the existing literature is surprisingly
scarce in this regard.
At the same time, the knowledge of the molecular path-
ogenesis of sporadic gastric cancers has increased enormously.
ereditary non-polyposis colorectal cancer (HNPCC, Lynch
syndrome) is among the most prevalent hereditary
cancers in humans and is associated with germline
Gastric carcinoma can histologically be classified into two
variants, an intestinal type and a diffuse type,8and these two
types seem to develop by distinct molecular pathways.9–12
Intestinal gastric carcinoma is often preceded by chronic
atrophic gastritis with intestinal metaplasia,13and is related to
environmental exposures such as diet, smoking, alcohol and
Helicobacter pylori infection.11High degree of MSI and, possibly,
increased loss of heterozygosity (LOH) frequencies have been
associated with intestinal-type differentiation.14 15Gastric can-
cers with MSI often display MLH1 promoter methylation and a
consequent lack of MLH1 protein.16–19The above-described
changes are rare in the diffuse type of gastric cancer, which is a
poorly differentiated tumour that often starts from the gastric
mucosa with normal appearance, and is seen only in the
stomach. Diffuse carcinomas occur more often among younger
patients. It could have a primary genetic aetiology, hereditary
diffuse gastric cancer, owing to germline mutations in the
E-cadherin gene.10Additionally, somatic E-cadherin mutations
have been detected in most diffuse-type tumours but not in
intestinal-type gastric cancers.20 21
We examined gastric carcinomas arising in families with
HNPCC for predisposing mutations and tumour alterations to
Abbreviations: APC, adenomatous polyposis coli; BRAF, B-Raf mutations;
CIMP, CpG island methylator phenotype; HNPCC, hereditary non-
polyposis colorectal cancer; IHC, immunohistochemistry; KRAS, K-ras
mutation; LOH, loss of heterozygosity; MLPA, multiplex ligation-dependent
probe amplification; MMR, DNA mismatch repair; MSI, microsatellite
instability; MS-MLPA, methylation-specific MLPA; MSS, microsatellite
stable; SNuPE, single nucleotide primer extension
926
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clarify whether gastric cancer is a true HNPCC spectrum
malignancy. The tumour profiles of HNPCC gastric cancers
were compared with those of sporadic gastric carcinomas,
stratified by histology and MSI status. Our findings shed light
on the molecular pathogenesis of gastric cancer in both HNPCC
and sporadic settings.
MATERIALS AND METHODS
Patients and specimens
The study material consisted of 61 gastric carcinoma samples.
Of these, 15 tumours were from patients belonging to families
with verified HNPCC, with HNPCC according to the Amsterdam
criteria2 22or the revised Bethesda guidelines.23All families were
known to have segregate germline mutations in MMR genes.
The other 46 tumours represented sporadic gastric cancers. To
obtain subgroups with defined histology and MSI status, a
larger cohort of sporadic late-onset gastric carcinomas was first
subjected to MSI screening, which made it possible to construct
three subgroups (not selected for any other characteristics):
intestinal MSI (n=10), intestinal microsatellite stable (MSS)
(n=20) and diffuse MSS (n=16). Due to the infrequent
occurrence of MSI among diffuse cancers, no ‘‘diffuse MSI’’
group could be assembled. The stage of tumours was
determined according to the TNM classification.24
Formalin-fixed paraffin-wax-embedded
tumours and matching normal tissues were collected from
the pathology departments of different hospitals in Finland.
Based on histological verification, areas with pure normal or
high tumour percentages were selected and subsequently
dissected out for DNA extraction. DNA was prepared from
archival tissue samples according to the method of Isola et al.25
In addition, 5 mm thick tissue sections were cut, mounted on
glass slides (Dako, Glostrup, Denmark) and air-dried overnight
at 37˚C for immunohistochemical (IHC) analysis. The relevant
institutional review boards of the Helsinki University Central
Hospital, Helsinki Finland and Jyva ¨skyla ¨, Finland, Central
Hospital, Jyva ¨skyla ¨ approved this study.
specimens of
Histology and H pylori status
Gastric carcinomas were classified according to Laure ´n8into
intestinal and diffuse types of tumours, and the differentiation
grade was determinedaccording
Organization classification.26Chronic gastritis, atrophy, intest-
inal metaplasia and H pylori infection were evaluated and
graded according to the Sydney classification.27H pylori status
was verified using hematoxylin and eosin (H&E) and Alcian-
blue/periodic acid Schiff stains added with modified Giemsa.
totheWorldHealth
IHC analysis
Formalin-fixed, paraffin-wax-embedded tissue sections were
stained by IHC methods using the following primary antibodies
of mouse: anti-MLH1 (clone G168-15, Pharmingen, San Diego,
California, USA), anti-MSH2 (clone FE-11, Calbiochem, San
Diego, California, USA /Oncogene Research, San Diego,
California,USA),anti-MSH6
Laboratories, San Diego, California, USA), anti-b-catenin anti-
body (clone 14, BD Transduction Laboratories) and anti-p53
(clone DO7, DakoCytomation, Dako).28The DAKO EnVision+
System (DakoCytomation, Dako) was applied according to the
manufacturer’s instructions with antigen-retrieval step by
microwave boiling for 15 min in citrate buffer pH 6.0. IHC
results for the MMR proteins were interpreted as described
previously.29b-Catenin expression was considered aberrant if
there was nuclear staining in .10% (not observed in the
matching normal tissue). In reporting p53 protein stabilisation
a cut-off level of .10% positive tumour cells was used.
(clone44,Transduction
MSI analysis
MSI status was determined by using the Bethesda panel
(BAT25, BAT26, D5S346, D2S123 and D17S250).30Tumours
with two or more unstable markers were considered to have
MSI-H, whereas those with no unstable markers were
considered as MSS.
Loss of heterozygosity
Single nucleotide primer extension (SNuPE) based on a
polymorphism in exon 8 was used to analyse the LOH at
MLH1.29In the carriers of a genomic deletion of MLH1 exon 16,
multiplex ligation-dependent probe amplification (MLPA) was
also applied to detect locus-restricted LOH.31LOH at adenoma-
tous polyposis coli (APC) was examined by SNuPE analysis
using a polymorphism in exon 11 as well as flanking
microsatellite markers, D5S1965 (200 kb upstream of APC)
and D5S346 (,100 kb downstream of APC).32In both SNuPE
and microsatellite marker LOH analyses, ratios of allelic peak
areas in normal tissue to tumour were calculated, and values
,0.6 or .1.67 (indicating that the transcript of one allele had
decreased .40% ) were considered to be strict LOH33and ratios
between 0.61–0.75 and 1.66–1.33 were considered to be
putative LOH.14 15 34
Mutation analysis
To examine whether patients with HNPCC gastric cancer
carried the mutations previously identified in their families,
the respective exons of MLH1 and MSH2 were screened by
direct sequencing of genomic PCR products using primers from
Chadwick et al.35For the genomic deletion affecting MLH1 exon
16, a direct assay described in Nystro ¨m-Lahti et al36was used.
KRAS, CTNNB1 and BRAF were screened for mutations by
single-strand conformation polymorphism analysis of genomic
DNA, followed by sequencing, to determine the exact nucleo-
tide changes. KRAS exon 2 was studied with primers from
Deng et al.37The primers for the CTNNB1 exon 3 were forward
59-TGCTAATACTGTTTCGTATTTATAGC-39
TGACTTTCAGTAAGGCAATGA-39. BRAF exon 15 was studied
using primers from Abdel-Rahman et al.28
Eight mononucleotide repeats in six frameshift-prone target
genes were studied using primers shown in table 1.
andreverse 59-
Methylation-specific MLPA
The SALSA methylation-specific (MS)-MLPA ME001 (MRC
Amsterdam, Holland) tumour suppressor probe mix detects
aberrant methylation of DNA using probes containing a
digestion site for the methylation-sensitive HhaI enzyme. All
reactions were carried out and the results were analysed
according to the manufacturer’s instructions (http://www.mrc-
holland.com). The 24 different tumour-suppressor genes whose
methylation status could be monitored are TP73, CASP8, VHL,
RARB, MLH1, RASSF1, FHIT, APC, ESR1, CDKN1B, CDKN2A/
p14ARF, CDKN2B, DAPK1, PTEN, CD44, GSTP1, ATM, IGSF4,
CHFR, BRCA1, BRCA2, CDH13, HIC1 and TIMP3. Normal DNA
specimens derived from the lymphocytes of healthy controls
and tumour cell lines (HCT116, HCT15, RKO, HEC59, LoVo,
SW48), with verified methylation status, were included in every
assay. For each MLPA reaction, 100–150 ng of DNA extracted
from paraffin-wax-embedded tissue was used.
The MS-MLPA technique was validated on tumour cell lines
by demonstrating high concordance relative to the results
obtained by an independent method (methylation-specific
PCR). On the basis of our titration experiments with cell lines
known to have full methylation or complete lack of methylation
of a given gene and, in the case of MLH1, correlating
methylation level and protein expression, a dosage ratio of
.0.15 (corresponding to 15% of methylated DNA) was
Gastric cancer and HNPCC927
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regarded to indicate promoter methylation. This threshold
value also provided the best discrimination of tumour DNA
relative to paired normal DNA where no methylation was
generally expected.
Statistical analysis
Significance level for the differences between groups (p value)
was determined using Fisher’s or Student’s t test as appro-
priate. All reported p values were two-tailed, and values ,0.05
were considered significant.
RESULTS
Clinicopathological characteristics
All gastric cancer specimens (n=15) from families with
HNPCC, with known mutations in MMR genes, wherever
adequate normal and tumour specimens were available, were
collected and subjected to molecular analyses, and 13 tumours
turned out to originate from germline mutation carriers (see
below). The average age at diagnosis for HNPCC gastric cancer
was 58 years. For comparison, a cohort of sporadic late-onset
gastric cancers assigned to the following three subgroups was
examined: intestinal MSI (n=10; average age at diagnosis
76 years), intestinal MSS (n=20; 73 years) and diffuse MSS
(n=16; 69 years). Table 2 gives the results from Sydney
classification27and the distribution of grades and stages.
HNPCC and the three sporadic subgroups did not reveal
essential differences in the Sydney classification. H pylori
infection was rare in all groups. Lymphocyte infiltration of
MSI colorectal cancers23was not common, being present in only
3/10, 1/10, 2/20 and 2/16 among HNPCC, intestinal MSI,
intestinal MSS and diffuse MSS groups, respectively (data not
shown). Poor differentiation was relatively common in HNPCC
(4/9) and was more frequent for MSI than for MSS cancers
among sporadic intestinal cancers (5/10 vs 1/20, p=0.009).
Among the MSI cancers (HNPCC and sporadic), the tumour
stages were relatively evenly distributed, whereas most sporadic
intestinal MSS gastric cancers were diagnosed at stages I and II
and most diffuse gastric cancers at stages III and IV.
MMR genes
Of the 15 examined patients with HNPCC, 13 were found to
have the predisposing MMR gene germline mutation of their
families, 11 in MLH1 and 2 in MSH2 (table 3). These cancers
were mainly of intestinal histology (12/13, 92% and 1 diffuse).
All 13 patients lacked the MMR protein corresponding to the
germline mutation (case 122:1 additionally lacked MSH6, as
expected for a MSH2 mutation carrier),38and displayed a high
degree of MSI. Among the initial 15 patients with tumours, 2
did not to have the predisposing MMR gene germline mutation
of their families, making a phenocopy frequency of 13%. These
tumours were MSS and showed no evidence of abnormal MMR
protein expression. The two phenocopies were omitted from
further calculations.
Among the sporadic gastric cancers that were studied for
comparison, all intestinal MSI gastric cancers showed the
absence of MLH1 protein by IHC (table 4). The mechanism of
MLH1 inactivation was different between HNPCC and sporadic
gastric cancers. Among sporadic MSI cancers, it was probably
due to MLH1 promoter methylation that was present in 7/10
(70%), and the degree of methylation (by MS-MLPA, see
Materials and methods section) suggested the methylation of
both alleles. HNPCC cancers showed no significant MLH1
promoter methylation (0/13; p,0.001 for differences relative to
the sporadic MSI tumours). Instead, by SNuPE and MLPA, LOH
at MLH1 seemed to be an important mechanism for somatic
inactivation of the wild-type allele in HNPCC gastric cancers (4/
8, 50%). Only two of the 10 sporadic intestinal MSI tumours
were informative (constitutionally heterozygotic) for the
intragenic MLH1 polymorphism used in SNuPE, and one
showed putative LOH. This tumour was one of those three
that did not show MLH1 promoter hypermethylation in this
group.
Wnt pathway genes
In the HNPCC group, strict or putative LOH at APC occurred in
5 (42%) of the 12 informative tumours. In general, APC-LOH
was typical of intestinal tumours, whether hereditary or
sporadic and whether MSI or MSS (20/39, 51%), and was
significantly less common in diffuse tumours (2/17, 12%;
p=0.001; tables 3 and 4). Nuclear localisation of b-catenin was
seen in 2/10 (20%) of gastric cancers from inherited MMR gene
mutation carriers and in sporadic cancers with comparable
frequencies (table 4). Among the 16 tumours with nuclear
localisation of b-catenin, APC-LOH co-occurred in 9 cases
(56%). No mutations in the b-catenin gene, CTNNB1, were
found in any group.
p53, KRAS/BRAF
The number of tumours with positive/stabilised p53 ranged
from 20–30% in all four groups (table 4). Somatic mutations in
Table 1
Target genes with intragenic mononucleotide repeats screened for mutations
GeneDescriptionRepeat (site)Primers (59–39)Reference
PTEN Phosphatase and tensin homologue
Mutated in multiple advanced cancers
1(MMAC1) (phosphatidylinositol 3-phosphatase)
A6 (exon 7)GACGGGAAGACAAGTTCAT
TTTGGATATTTCTCCCAATG
CAGAGGAAACCTCAGAAAAA
TTGGCTTTGTCTTTATTTGC
Kuismanen et al
62
A6 (exon 8)Kuismanen et al
62
ACVR2 Activin A type II receptor (ACTRII)
(serine-threonine kinase)
A8 (exon 3)AAAAACACTTGTTGTAGGGTCAG
CGCTGTGTGACTTCCATCTC
GTTGCCATTTGAGGAGGAAA
CCTCTGAAAAGTGTTTTATTGGAA
Jung et al
59
A8 (exon 10) Jung et al
59
BLMBloom’s syndrome protein (DNA helicase)A9 (exon 7) CACCAGGAAGAATCTTTTTGAA
TGCTTGTGAGAACATTTCCTG
This study
MBD4Methyl-CpG-binding endonuclease (MED1) A10 (exon 3)CTCAGTGTGACCAGTGAAGAAAA
TCTGAGTCTTTGGCTGAACAAA
This study
TGFb RII Tumour growth factor-b type II receptor
(serine-threonine kinase)
A10 (exon 3) CTAGAGACAGTTTGCCATGACC
TGTTGTCATTGCACTCATCAGA
This study
MRE11AMeiotic recombination 11 homologue A,
accessory splicing signal within intron 4
T11 (intron 4)AGTCAGTTTGCCTATGATTGC
TCTTGATAGTTCACCCATGGAA
This study
928 Gylling, Abdel-Rahman, Juhola, et al
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KRAS were only found in MSI tumours, 4/23 (17%), as opposed
to none among 36 MSS tumours (p=0.019). All KRAS
mutations consisted of G12D. No somatic mutations in BRAF
were found in any group.
Mononucleotide repeats within growth-regulatory
genes
The tumours were screened for mutations in six growth-
regulatory target genes known to be prone to inactivation by
frameshift mutations as they contain mononucleotide repeats
in their coding regions (PTEN, ACVR2, BLM, MBD4, TGFbRII)
or in an adjacent intronic splice site (MRE11A). All genes have
previously been implicated in the development of sporadic
gastric or colorectal cancers and the protein products of at least
three (BLM,39MBD4,40MRE11A41) interact with MLH1. As
expected, frameshift mutations were limited to MSI gastric
cancers only, whether hereditary or sporadic, and were absent
in MSS tumours (68/135 vs 0/97, p,0.001). ACVR2, TGFbRII
and MRE11A were highly mutable in MSI tumours, with
mutation frequencies ranging between 62% and 100%; MBD4
was mutated in 18–40%, and BLM and PTEN in ,10% (tables 3
and 4). The high rate of ACVR2 and TGFbRII frameshift
mutations might be explained by the similarity between the
activin and TGFb signalling systems: as both systems use the
same set of SMADs, they may share common regulatory
mechanisms.42
Promoter methylation of tumour-suppressor genes
MS-MLPA method was used to examine the methylation status
of the promoters for 24 tumour-suppressor genes, including
MLH1 (see Materials and methods section). Table 4 gives the
average numbers of methylated genes among these 24 for each
group of tumours as well as the methylation frequencies for
selected loci that showed frequent methylation in tumour DNA
(but not in paired normal DNA). As described above, MLH1
methylation was absent in HNPCC gastric cancers in contrast to
sporadic MSI gastric cancers (p,0.001). The average number of
genes with promoter methylation among all 24 genes studied
was significantly associated with MSI, being 4.9 for the MSI
tumours (HNPCC and sporadic intestinal) compared with 2.0
for the MSS tumours (sporadic intestinal and diffuse;
p,0.001). The present sporadic tumours, in particular, high-
lighted the fact that MSI, and not histology, determined the
general methylation profile, as among intestinal tumours, the
Table 2
Results from the Sydney classification in HNPCC and sporadic gastric cancers
Sydney classification
HNPCC Sporadic
Intestinal MSI
n=10*
Intestinal MSI
n=10
Intestinal MSS
n=20
Diffuse MSS
n=16
Chronic inflammation
Normal/none
Mild
Moderate
Marked
0
1
2
7
0
2
4
4
0
6
1
4
7
4
10
4
Neutrophils
Normal/none
Mild
Moderate
Marked
3
7
0
0
6
3
1
0
611
11
3
0
2
3
0
Atrophy
Normal/none
Mild
Moderate
Marked
3
2
5
0
5
2
3
0
4 10
10
5
1
2
4
0
Metaplasia
Normal/none
Mild
Moderate
Marked
4
4
2
0
3
5
2
0
7
8
2
3
14
2
0
0
H pylori
Normal/none
Mild
Moderate
Marked
8
2
0
0
5
3
2
0
9
5
3
3
12
4
0
0
Grade
Well differentiated (grade 1)
Moderately differentiated (grade 2)
Poorly differentiated (grade 3)
N/A
n=10*
n=10 n=20n=16
1
4
4
1
1
4
5
0
5–
–
–
14
1
0 16
Stage
I
II
III
IV
n=13n=10n=20n=16
2
5
5
1
1
4
3
2
8
6
5
1
3
1
6
6
HNPCC, hereditary non-polyposis colorectal cancer; MSI, microsatellite instability; MSS, microsatellite stable; N/A, not
available/not applicable.
*Of the 13 MMR gene mutation-positive gastric cancers, 10 were available for evaluation.
Differentiation grade and tumour stage are also shown.
Gastric cancer and HNPCC929
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average number of methylated genes was significantly higher
for MSI than MSS tumours (5.9 vs 2.3, p,0.001), whereas
among MSS tumours, intestinal and diffuse tumours showed
no difference (2.3 vs 1.6; table 4). Note that there was a
significant difference within MSI tumours depending on
whether the tumours represented the HNPCC or the sporadic
intestinal MSI group (4.1 vs 5.9; p=0.038). As the MLH1
methylation status was drastically different in these two groups
(table 4), one could conclude that the general methylation
profile did not entirely follow the methylation status of MLH1
even if the whole study series showed a strong correlation
between MLH1 methylation and methylation of other tumour-
suppressor-promoter loci (p,0.001).
DISCUSSION
Associations of molecular alterations with histology and
MSI status
The present study of 61 gastric cancer samples revealed several
important universal associations that increase the understand-
ing of gastric tumourigenesis. First, LOH in the APC region was
associated with intestinalhistology
(p=0.007). Other investigators have also noted an association
between APC-LOH and intestinal histology,43 44although con-
tradictory reports exist.45The Wnt pathway, in general, was
found to be involved in nearly half (27/59, 46%) of the present
gastric carcinomas, through either alterations in b-catenin
expression (nuclear localisation) or APC-LOH. We found no
mutations in CTNNB1, which is in agreement with many
published studies (eg, Sasaki et al46and Kim et al47), whereas
fairly high mutation frequencies (up to 38%) and variable
associations with histology have also been reported.48 49
Second, KRAS mutations (p=0.019) and frameshift muta-
tions in repeat-containing tumour-suppressor genes (p,0.001)
were limited to MSI tumours, being absent in MSS tumours,
whether intestinal or diffuse. Other investigators have also
described a preferential occurrence of KRAS mutations in MSI
gastric cancers.50 51Our data on instability in repeat-containing
target genes (association with MSI tumours, overall frequencies
of frameshift mutations) are broadly compatible with the
previous reports on sporadic gastric cancers.52–56The instability
profile we report for gastric cancer shares features with
gastrointestinal tumours in general, such as high frequency of
TGFbRII, ACVR2 and MRE11A mutations.57–60Specifically, the
instability profile for our HNPCC gastric cancers resembles that
reported for colorectal or small bowel tumours from patients
with HNPCC.61–64
Third, the average number of methylated tumour-suppressor-
gene loci among the 24 loci that were studied was significantly
higher in MSI versus MSS tumours, regardless of histology
(p,0.001). Our results are broadly compatible with some
previous reports, which found by studying a variable number
of indicator genes (typically ,10) that MSI gastric cancers are
associated with MLH1 hypermethylation and CpG island
methylator phenotype (CIMP).47 65Recent observations on
colorectal cancers suggest the existence of a CIMP+, non-MSI
group that shows different clinicopathological characteristics
relative to the CIMP+, MSI group.66Although the average
number of methylated loci in our sporadic MSS gastric cancers
was low (2 out of 24 loci studied), a small subgroup (3/36
tumours) did exist that had 6–7 methylated loci per tumour,
implying that a CIMP+, non-MSI phenotype also exists among
gastric cancers. Interestingly, two of these tumours were
diagnosed at stage I and one at stage II. MSS gastric cancers,
in general, showed an inverse trend between the overall
methylation index and tumour stage (with average methylation
indices of 2.5, 3.1, 1.6 and 0.71 for stages I, II, III and IV,
respectively). Among the MSI gastric cancers (HNPCC and
regardless ofMSI
Table 3
Histology and molecular features of gastric cancers from patients with hereditary non-polyposis colorectal cancer with germline mutations in DNA mismatch repair genes
ID # (criteria)
Histology
MMR gene germline mutation
MLH1 LOH
Immunohistochemistry
MSI status APC LOH
b-catenin IHC/
mutation
p53 IHC
KRAS/BRAF
mutation
Repeat containing genes
PTEN
ACVR2
BLM
MBD4
TGFbRII
MRE11A
MLH1
MSH2
MSH6
1:32 (A)
Intestinal
MLH1 exon 16 deletion
36
N
–
+
+
MSI
N
Mem/N
Negative
N/N
N
U
N
U
U
U
1:80 (A)
Intestinal
MLH1 exon 16 deletion
36
LOH
–
+
+
MSI
LOH?
Mem/N
Positive
(60%)
N/N
N
U
N
N
N
U
3:39 (A)
Intestinal
MLH1 exon 16 deletion
36
LOH
–
+
+
MSI
N
Nuc/N
Negative
N/N
N
U
N
U
U
U
4:4 (A)
Intestinal
MLH1 exon 3–5 deletion
73
N
–
+
+
MSI
LOH
Nuc/ND
Negative
N/N
N
U
ND
ND
N
U
7:18 (A)
Intestinal
MLH1 exon 17, GRC at 1976,
R659P
73
LOH?
ND
ND
ND
MSI
N
ND/N
ND
G12D/N
N
U
N
N
U
U
73:1 (A)
Intestinal
MLH1 exon 3, CRT at 298, R100X
29LOH
ND
ND
ND
MSI
LOH
ND/ND
ND
N/N
N
U
N
ND
U
U
83:18 (A)
Intestinal
MLH1 exon 17, CRT at 1975,
R659X
73
N
–
+
+
MSI
N
Mem/N
Negative
N/N
N
U
N
N
U
U
83:45 (A)
Intestinal
MLH1 exon 17, CRT at 1975,
R659X
73
NI
–
+
+
MSI
N
Mem/N
Negative
N/N
N
U
N
N
N
U
90:60 (A)
Intestinal
MLH1 intron 5, GRA at 454–1
36
NI
–
+
+
MSI
N
Mem/N
Negative
N/N
N
U
N
N
U
U
122:1 (B)
Intestinal
MSH2 exon 12, GRA at 1807,
D603N
33
NI
+
+
–
MSI
NI
Mem/ND
Positive
(20%)
N/N
N
U
N
N
U
U
130:1 (A)
Intestinal
MSH2 exon 11, CRT at 1720,
Q574X
NI
ND
ND
ND
MSI
LOH?
ND/N
ND
N/N
N
U
N
N
U
U
12:20 (A)
Intestinal/mucinous
MLH1 exon 16 deletion
36
N
–
+
+
MSI
N
Mem/N
Negative
N/N
N
U
N
N
N
U
67:15 (A)
Diffuse
MLH1 exon 4, T.G at 320, I107R
73
NI
–
+
+
MSI
LOH?
Mem/N
Negative
N/N
N
U
N
N
N
N
A, Amsterdam criteria I or II; APC, adenomatous polyposis coli; B, Bethesda criteria; IHC, immunohistochemistry; LOH, loss of heterozygosity; LOH?, putative LOH; MSI, microsatellite instability; MMR, DNA mismatch repair; MSS, microsatellite stable; Mem, membranous; N,
normal; ND, not done; NI, not informed; Nuc, nuclear; U, unstable.
930 Gylling, Abdel-Rahman, Juhola, et al
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sporadic), the methylation index was similar for all stages (being
4.3, 4.9, 5.3 and 4.3 according to increasing stage). Besides the
CIMP+ non-MSI group, the relatively high methylation index in
the present HNPCC gastric cancers, which was associated with
MSI but did not involve MLH1 promoter, may represent another
variant of CIMP. H pylori infection has been suggested to induce
methylation of CpG islands in gastric mucosa.67In our study, H
pylori infection was rare in all groups and therefore it is unlikely
to explain the observed methylation differences.
Differences and similarities between HNPCC and
sporadic gastric cancers
The present gastric cancers from germline carriers of MMR gene
mutations had mostly intestinal histology; most showed the
lack of MLH1 protein and all displayed a high-degree of MSI.
Thus, the closest sporadic counterpart was intestinal MSI
gastric cancer, most of which were also MLH1-deficient as
observed in this and in previous studies.68According to our
findings, the tumourigenic events in HNPCC gastric cancers
closely resembled those in sporadic intestinal MSI gastric
cancers, except for the mechanism of MLH1 inactivation and
overall epigenetic changes. Thus, although biallelic methylation
of the MLH1 promoter underlies MSI and the absence of MLH1
protein in most sporadic cancers, no significant MLH1 promoter
methylation was seen in HNPCC, and, instead, the wild-type
allele was inactivated by LOH. Even, more importantly, the
overall frequency of tumour-suppressor-gene loci with promo-
ter methylation was significantly (p=0.038) lower in HNPCC
than in sporadic intestinal MSI gastric cancers, let alone
sporadic MSS gastric cancers, which suggests that despite many
similarities, the tumourigenic mechanisms in HNPCC and
sporadic intestinal MSI gastric cancers are not identical. The
methylation rates of certain loci are known to increase with
age,69and as our patients with HNPCC gastric cancers were
diagnosed at a younger age than patients with sporadic
intestinal MSI gastric cancers (average 58 vs 76 years,
respectively) one might argue that age played a role. This
may partly be true as, for example, the oestrogen receptor a
locus whose methylation has typically been connected with age
was less often methylated in HNPCC than sporadic intestinal
MSI tumours; by contrast, the possible association with age did
not extend to sporadic MSS cancers (table 4).
Gastric cancers and HNPCC tumour spectrum
According to our study, HNPCC gastric cancer (MMR gene
germline mutation positive) has the following characteristics:
intestinal histology (92%), MSI-high (100%), absence of the
MMR protein corresponding to the germline mutation (100%),
frequent APC-LOH as typical of intestinal gastric cancer,
patterns of KRAS mutation and mutations in repeat-containing
target genes similar to sporadic MSI gastric cancers and a
unique pattern of promoter methylation of tumour-suppressor
genes. Additionally, our previous investigation shows rare
changes in DNA copy number by comparative genomic
hybridisation.70
At present, gastric cancer is not included in the Amsterdam II
criteria2but is included in the revised Bethesda criteria.23
Evidence to support the idea that a given type of tumour from
a MMR gene germline mutation carrier is part of the HNPCC
tumour spectrum would include the increased incidence in
HNPCC compared with the average population and the
demonstration of MMR deficiency as the driving force for
tumourigenesis.71The relative risk of gastric cancer in HNPCC
mutation carriers compared with the general population has
been reported to be higher by 4–19-fold3 4 72in populations of
the Western world and at least by twofold in endemic areas in
Asia.5Although all tissues of a MMR gene germline mutation
carrier are genetically predisposed to cancer as all cells carry the
‘‘first hit’’, tumour development additionally requires somatic
inactivation of the remaining wild-type allele (‘‘second hit’’) in
a target tissue. If a second hit does not occur, there will be no
loss of MMR protein, no MSI and no MMR deficiency-driven
tumourigenesis. This was not the case with the present HNPCC
gastric cancers, which showed MLH1-LOH as a second hit,
Table 4
positive) and sporadic gastric cancers
Summary of molecular data on hereditary non-polyposis colorectal cancer (mismatch repair gene germline mutation
HNPCC mut+
Sporadic
Intestinal*, MSI Intestinal, MSIIntestinal, MSSDiffuse, MSS
Decreased MMR protein expression:
MLH1
MSH2
MSH6
Nuclear b-catenin
CTNNB1 exon 3 mutation
APC LOH (strict)
APC LOH (strict + putative)
p53 positive IHC
KRAS exon 2 mutation
BRAF exon 15 mutation
Proportion of tumours with unstable loci:
PTEN (A6)
ACVR2 (A8)
BLM (A9)
MBD4 (A10)
TGFbRII (A10)
MRE11 (T11)
Average number of methylated genes?
MLH1 promoter methylation
TIMP3 promoter methylation
ESR1 promoter methylation
CHFR promoter methylation
n (%) n (%)
10/10 (100)
0/10 (0)
0/10 (0)
3/10 (30)
0/10 (0)
3/8 (38)
4/8 (50)
3/10 (30)
3/10 (30)
0/10 (0)
n (%) n (%)
9/10 (90)
1/10 (10)
1/10 (10)
2/10 (20)
0/10 (0)
2/12 (17)
5/12 (42)
2/10 (20)
1/13 (8)
0/13 (0)
ND
ND
ND
ND
ND
ND
8/20 (35)
0/12 (0)
8/20 (40)
12/20 (60)
6/20 (30)
0/20 (0)
0/20 (0)
3/16 (19)
0/16 (0)
0/16 (0)
1/16 (6)
4/16 (25)
0/16 (0)
0/16 (0)
0/13 (0)
13/13 (100)
0/12 (0)
2/11 (18)
8/13 (62)
12/13 (92)
1/10 (10)
10/10 (100)
1/10 (10)
4/10 (40)
9/10 (90)
8/10 (80)
0/20 (0)0/16 (0)
ND
ND
ND
ND
ND
ND
0/20 (0)
0/11 (0)
0/16 (0)
0/14 (0)
4,15,92,31,6
0/13 (0)
5/13 (38)
5/13 (38)
7/13 (54)
7/10 (70)
6/10 (60)
7/10 (70)
9/10 (90)
0/20 (0)
3/20 (15)
6/20 (30)
5/20 (25)
0/16 (0)
2/16 (13)
5/16 (31)
2/16 (13)
HNPCC, hereditary non-polyposis colorectal cancer; MMR, DNA mismatch repair; MSI, microsatellite instability; MSS, microsatellite stable; ND, not done.
*Twelve intestinal and one diffuse
? Of the 24 studied genes
Values are given as proportion of tumours with alterations (percentage) unless specified otherwise.
Gastric cancer and HNPCC931
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Page 7

MMR protein loss, MSI-high and a number of other tumouri-
genic events typical of MMR deficiency. Together, these data
justify considering gastric cancer as a true HNPCC spectrum
malignancy.
ACKNOWLEDGEMENTS
We thank Kirsi Pylva ¨na ¨inen, Tuula Lehtinen and Kaija Koivula for
sample collection and procurement, and Saila Saarinen for laboratory
analyses. This study was supported by the Academy of Finland, the
Finnish Cancer Foundation, the Sigrid Juselius Foundation, the Albin
K. Johansson Foundation, Helsinki University Science Foundation,
Jyva ¨skyla ¨ Central Hospital Science Foundation and the Cancer Society
of Middle-Finland.
Authors’ affiliations
A Gylling, W M Abdel-Rahman, E Hautala, P Peltoma ¨ki, Department of
Medical Genetics, University of Helsinki, Helsinki, Finland
M Juhola, K Nuorva, Department of Pathology, Jyva ¨skyla ¨ Central Hospital,
Jyva ¨skyla ¨, Finland
H J Ja ¨rvinen, Second Department of Surgery, Helsinki University Hospital,
Helsinki, Finland
J-P Mecklin, M Aarnio, Department of Surgery, Jyva ¨skyla ¨ Central Hospital,
Jyva ¨skyla ¨, Finland
.......................
Competing interests: None.
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