Gastric Cancer (2001) 4: 113–121

 2001 by
International and
Japanese Gastric
Cancer Associations

Review article

Molecular diagnosis of gastric cancer: present and future

Wataru Yasui1, Naohide Oue1, Hiroki Kuniyasu1,2, Reiko Ito1, Eiichi Tahara3, and Hiroshi Yokozaki1
1
First Department of Pathology, Hiroshima University School of Medicine, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan
2
Department of Oncological Pathology, Cancer Center, Nara Medical University, Nara, Japan
3
Hiroshima Cancer Seminar Foundation, Hiroshima, Japan

Abstract Introduction
Although histopathological diagnosis is extremely useful for
the definitive as well as the supportive diagnosis of gastric According to the Hiroshima Tumor Registration
cancer in clinical practice, it is limited in certain respects. Over Committee, which registers tumors that have been
the past 15 years, integrated research in molecular pathology diagnosed by pathologists, the number of cases regis-
has clarified the details of genetic and epigenetic abnormali-
tered has increased threefold for gastric cancer and
ties of cancer-related genes in the course of the development
20-fold for gastric adenoma over the past 20 years.
and progression of gastric cancer. These abnormalities, which
include telomerase activation, genetic instability, and abnor- This suggests that, recently, many tumors have been
malities in oncogenes, tumor suppressor genes, cell-cycle biopsied or dissected endoscopically, while tumor tis-
regulators, cell adhesion molecules, and DNA repair genes, sues were collected only by surgery in earlier periods.
could be effective markers in the molecular diagnosis of gas- Although histopathological diagnosis is, thus, extremely
tric cancer. It is possible that the molecular analysis of these important for obtaining definitive and supportive diag-
alterations in histopathology specimens may overcome defi- noses, diagnosis that depends only on histopathol-
ciencies in diagnoses that depend only on histomorphology, ogy has certain limitations. Many lesions have a
and, consequently, we may be able to improve the differential morphology which is between that of benign and
diagnosis of cancer, obtain information on the grade of malig- malignant lesions, and differential diagnosis is there-
nancy, and identify patients at high risk of developing multiple
fore difficult. Variations between pathologists may
primary cancers. In Hiroshima, we have established a system
lead to changes in assessments of the lesion. Differences
of molecular-pathological diagnosis as a routine service; about
5000 lesions of the stomach have been subjected to this diag- in diagnostic criteria, such as those for early gastro-
nosis, and much useful information has been obtained. In the intestinal cancer diagnosed by Western and Japanese
near future, genetic analysis by means of DNA microarray pathologists, lead to misunderstanding by clinicians.
may become routine in the diagnosis of gastric cancer. Genetic Furthermore, information derived from morphology
analysis of histopathology specimens may make clear the is of limited use in determining the degree of malig-
characteristics of individual cancers; indicating the common nancy and the patient’s prognosis. No genetic infor-
and specific features of molecular pathogenesis that may be mation can be obtained in hereditary cancers, such as
directly connected with gene therapy or molecular-targeted hereditary non-polyposis colorectal cancer (HNPCC)
therapy. By analyzing the relationship between single- kindreds.
nucleotide polymorphisms and cancer susceptibility, we will
Integrated research in molecular pathology over the
be able to obtain information on cancer prevention from his-
past 15 years has clarified details of the genetic and
topathology samples.
epigenetic abnormalities of cancer-related genes in the
Key words Molecular diagnosis · Gastric cancer · Histopathol- course of the development and progression of gastric
ogy · Genetic alterations · Epigenetic alterations cancer [1–3]. Because most of these alterations can be
analyzed in the formalin-fixed specimens that are taken
for the purpose of histopathological diagnosis, these
alternations are good targets for molecular diagnosis
[4–7]. It is possible to apply molecular findings in tissue
samples to diagnosis, together with the results of mor-
Offprint requests to: W. Yasui phology. Thus, molecular diagnosis can overcome the
Received: May 21, 2001 / Accepted: July 3, 2001 drawbacks of histopathological diagnosis and can make
114 W. Yasui et al.: Molecular diagnosis of gastric cancer

a great contribution to the understanding of cancer tion of the c-erbB2 gene, inactivation of the APC gene,
histopathology. and loss of pS2 expression preferentially occur in the
This review outlines the procedure for molecular well differentiated type. Precancerous lesions, such as
diagnosis in gastric specimens that has been routinely intestinal metaplasia and adenoma, share various alter-
implemented in Hiroshima. An overview of the genetic ations similar to those seen in well differentiated carci-
and epigenetic alterations in gastric cancer is given, to nomas. Loss of heterozygosity (LOH) of the p73 gene
show the significance of these changes as biological occurs specifically in well differentiated carcinomas
markers in diagnosis. We also discuss future prospects with foveolar epithelial phenotype [8]. Inactivation of
for the molecular diagnosis of gastric cancer. cadherins and amplification of the K-sam gene and
c-met gene are frequently associated with poorly differ-
entiated or scirrhous type carcinomas. A review of
Molecular basis of multistep stomach carcinogenesis representative abnormalities of individual genes and
molecules, and their significance as markers for molecu-
In the course of multistep carcinogenesis in the stom- lar diagnosis, is given below.
ach, various genetic alterations of oncogenes, tumor Telomere maintenance by telomerase activation in-
suppressor genes, DNA repair genes, cell-cycle regula- duces cellular immortalization [9]. Strong telomerase
tors, and cell adhesion molecules are accumulated (Fig. activity is detected in most gastric carcinomas, regard-
1) [1–3]. Of the various genetic and epigenetic alter- less of histological type and tumor stage [10]. Relatively
ations, some are found in both well and poorly differen- good correlation was found between telomerase activi-
tiated types of carcinoma, while others are found in only ties and levels of catalytic subunit of telomerase
one particular histological type. Genetic instability, (TERT) expression in gastric carcinomas. Some in-
inactivation of tumor suppressor genes by CpG island testinal metaplasias and gastric adenomas express
methylation, and telomerase activation commonly par- telomerase activity at certain levels, suggesting partici-
ticipate in the initial step of stomach carcinogenesis. pation in an early stage of stomach carcinogenesis [11].
While p53 gene loss and mutation occurs at an early Telomerase activity is found in approximately half of
stage of carcinogenesis, amplification of the c-met gastric adenomas, whose levels of the activity are about
and cyclin E genes is frequently associated with 10% of those in gastric carcinomas. TERT protein is
an advanced stage. Reduced expression of p27Kip1, a expressed in the nuclei of the adenoma cells at moder-
cyclin-dependent kinase (CDK) inhibitor, participates ate levels that are not necessarily comparable with the
in both the development and the progression of gastric telomerase activity. Therefore, TERT expression may
cancer. Overexpression of growth factors and cytokines be a prerequisite for telomerase activation in the early
partipates in progression. K-ras mutations, amplifica- stage of stomach carcinogenesis; thus, the expression of

Fig. 1. Genetic and epigenetic altera-

tions during multistep stomach carcino-
genesis. LOH, Loss of heterozygosity;
HP, Helicobacter pylori; TERT, catalytic
subunit of telomerase; Chr., chromo-
some; GF, growth factors
W. Yasui et al.: Molecular diagnosis of gastric cancer 115

TERT is a useful marker for detecting “true precancer- p16MTS1/INK4A is detected in 20%–30% of gastric cancers
ous lesions”. [24–26]. Reduced expression of the gene products is
Genetic instability causes an accumulation of genetic well correlated with CpG island hypermethylation.
alterations, and participates in the early stage of stom- Hypermethylation of these three genes is more fre-
ach carcinogenesis [3]. Cases in which two or more of quently found in well differentiated adenocarcinomas
five microsatellite loci show replication error are con- than in poorly differentiated adenocarcinomas. How-
sidered as showing high-frequency microsatellite insta- ever, cases with hMLH1 hypermethylation do not
bility (MSI-H), and those with only one locus showing coincide with cases with MGMT hypermethylation, as
replication error are considered to show low-frequency reported in colorectal cancer. Furthermore, hMLH1
MSI (MSI-L) [12]. MSI-H is observed in only 4% of hypermethylation occurs in intestinal metaplasia,
gastric carcinomas, in particular in the well differenti- suggesting that hMLH1 expression reduced by
ated type in aged patients. The frequency of MSI-L is promoter methylation may be an initial event in the
about 30% in primary gastric cancers, including early accumulation of genetic abnormalities in stomach car-
cancers [3]. Some intestinal metaplasias and adenomas cinogenesis. On the other hand, CpG island methyla-
also show MSI-L, and these should be considered “true tion of CDH-1 (E-cadherin) and consistently reduced
precancerous lesions” [13]. MSI-H is used as an indica- E-cadherin expression is commonly observed in scir-
tor of HNPCC, which is caused by the germline muta- rhous gastric cancers. Although the enzymes DNA
tion of mismatch repair genes, including hMLH1 and methyltransferase and demethylase potentially affect
hMSH2 [14]. In sporadic gastric cancers with MSI-H, promoter methylation status, the tumor-specific hyper-
CpG island hypermethylation of hMLH1 is associated methylation of hMLH1, p16MTS1/INK4A, and CDH-1 does
with loss of hMLH1 protein [15,16]. Another important not simply depend on the expression levels of pro-
aspect of MSI is that multiple primary cancers, includ- methylating (DNMT1, DNMT3A, DNMT3B) and anti-
ing stomach, colon, and gallbladder carcinomas, arising methylating (MBD2) enzymes [24,26].
in a single individual frequently display replication er- Nucleosomes consist of core histones, wrapped with
rors at multiple microsatellite loci [17]. This indicates turns of double-stranded DNA [20]. The acetylation
that the detection of MSI in a cancer may serve as a of histones disrupts nucleosome structure, leading to
good molecular marker for the assessment of the risk of DNA relaxation, and a subsequent increase in accessi-
a second cancer in the same patient. bility for transcription factors. On Western blotting,
Abnormalities in cell-cycle regulators are involved with anti-acetylated histone H4, the level of acetylated
in the development and progression of gastric cancers histone H4 expression was significantly reduced in 70%
through unbridled cell proliferation [2,3]. The cyclin E of gastric carcinomas in comparison with findings in
gene is amplified in 15%–20% of gastric carcinomas. normal mucosa, indicating a reduced acetylation status
The overexpression of cyclin E tends to be correlated in gastric cancers. The histone deacetylase inhibitor,
with the aggressiveness of gastric carcinomas, such as trichostatin A (TSA), inhibits cell growth and induces
deep invasion and advanced tumor stage. On the other apoptosis in gastric carcinoma cell lines [27]. TSA
hand, reduction in the expression of the CDK inhibitor, induces the expression of p21WAF1/cip1, CBP, Bak, and
p27Kip1, is frequently associated with advanced gastric cyclin E, while it reduces the expression of E2F-1, E2F-
carcinomas [18]. Gastric adenomas with reduced p27Kip1 4, HDAC-1, and the phosphorylated form of Rb protein
expression tend to progress to carcinomas [7]. Reduced [27]. These findings suggest that histone deacetyla-
expression of p27Kip1 also significantly correlates with tion may participate in unbridled cell proliferation by
depth of tumor invasion and the presence of lymph- modulating the expression of cell-cycle regulators and
node metastasis. Therefore, gene amplification and the apoptosis-related molecules. The expression of acety-
overexpression of cyclin E, and the reduced expression lated histone H4 is also reduced in 40% of gastric
of p27Kip1, are good markers for the diagnosis of high- adenomas and some intestinal metaplasias adjacent to
grade malignancy. E2F-1, a target of cyclins/CDKs at carcinoma, suggesting that reduced histone acetylation
G1/S transition, is overexpressed in about 40% of gas- may occur even in precancerous cells, and may be a
tric carcinomas and may participate in the development candidate molecular marker to indicate such cells.
of gastric carcinomas [19].
Abnormalities in transcriptional regulation by CpG
island methylation and histone deacetylation are be- Molecular diagnosis of gastric cancer on
lieved to be the most important of various epigenetic histopathology specimens
alterations [20–23]. DNA hypermethylation is known
to be accompanied by allelic loss at the same locus. Based on the molecular pathological findings men-
CpG island hypermethylation of the genes hMLH1, tioned above, it is clear that we can improve the quality
MGMT (O6-methylguanine methyltransferase), and of histopathological diagnosis by analyzing genetic and
116 W. Yasui et al.: Molecular diagnosis of gastric cancer

epigenetic alterations. We established a molecular H&E staining is marked with a felt pen under the micro-
diagnosis system for histopathological samples of gas- scope by the pathologist. A technician collects the tis-
trointestinal tract, in cooperation with Hiroshima City sues on the marked area into an Eppendorf tube, using
Medical Association Clinical Laboratory, and we have the tip of the needle, and extracts DNA using a conven-
been performing this as a routine service [6,7]. This tional method with proteinase K and sodium dode-
system is designed mainly for the differential diagnosis cylsulfate (SDS). For this extracted DNA, mutation or
of benignancy or malignancy, diagnosis of the degree of deletion of the genes of interest is examined by noniso-
malignancy, identification of susceptibility to multiple topic polymerase chain reaction single-strand confor-
primary cancers, and identification of HNPCC. mation polymorphism (PCR-SSCP) analysis and PCR
restriction fragment length polymorphism (RFLP). For
the detection of genetic instabilities, microsatellite loci
Procedures for molecular diagnosis
are amplified with PCR, using fluorescent stain-labeled
The procedures used, from sample receipt to the final primers, and analyzed with a PRISM 310 autosequencer
diagnosis in the pathology laboratory, are outlined in (Applied Biosystems, Foster City, CA, USA). The final
Fig. 2. The samples are formalin-fixed tissue materials molecular-pathological diagnosis, made by combining
obtained by biopsy or dissection (endoscopic mucosal the results of genetic analysis, immunostaining, and
resection or surgical removal) that are submitted for histopathological findings, is reported to the clinicians.
routine histopathological diagnosis. Routine paraffin The immunostaining, DNA extraction, PCR-SSCP,
sections are prepared, stained, and subjected to histo- and PCR-RFLP are conducted by technicians in the
pathological observation. A pathologist indicates the clinical laboratory center of Hiroshima City Medical
cases to be examined for molecular diagnosis (cancer, Association, and microsatellite analysis is performed
adenoma [dysplasia] or borderline lesion) at micro- at our department (First Department of Pathology,
scopic examination. A laboratory technician performs Hiroshima University School of Medicine). Most ex-
immunostaining with various molecular and genetic penses are covered by a donation from the Hiroshima
markers, described below, and at that time prepares City Medical Association and by research funds from
another five sections with H&E staining that are not our department. The molecular pathologists who are in
included by covering glass for genetic analysis. The charge of the molecular pathological diagnosis system
pathologist combines the results of immunostaining work voluntarily.
and the histopathological findings, adds the molecular
pathological findings and their significance if new infor-
Molecular and genetic markers
mation is obtained, and makes a molecular-pathological
report for the attending clinicians. As the significance of each genetic and epigenetic ab-
Some cases are further analyzed using DNA ex- normality differs in esophageal, gastric, and colorectal
tracted from paraffin sections. In order to assure that cancers, the roles of individual abnormalities should
only the histologically doubtful portion is examined ac- be sufficiently understood before these abnormalities
curately, the position to be analyzed on the section with are used in diagnosis. In gastric cancer, p53, APC, and

Fig. 2. Procedures for the molecular-

pathological diagnosis of histopathology
samples of the stomach. EMR, Endo-
scopic mucosal resection; TGF-α, trans-
forming growth factor alpha; EGFR,
epidermal growth factor receptor; PCR,
polymerase chain reaction; SSCP, single-
strand conformation polymorphism;
RFLP, restriction fragment length poly-
morphism; MS, microsatellite
W. Yasui et al.: Molecular diagnosis of gastric cancer 117

Fig. 3. Changes in molecular and genetic

markers for molecular-pathological diag-
nosis of the stomach during the 7 years
that our system has been operating.
PCNA, Proliferating cell nuclear antigen

Table 1. Molecular-pathological diagnosis of gastric lesions

Histological diagnosis Lesions Molecular diagnosis

Adenoma 1154 Adenoma with malignant potential 119 (10%)

s/o Adenocarcinoma 19 (2%)
Borderline 469 Adenoma with malignant potential 6 (1%)
s/o Adenocarcinoma 45 (10%)
Adenocarcinoma 53 (11%)
s/o Adenocarcinoma 250 Adenocarcinoma 63 (25%)
High grade malignancy 3 (1%)
Adenocarcinoma 2969 High-grade malignancy 357 (12%)
s/o, Suspected of

CD44 have been used as markers for differential diag- nosis was begun with immunostaining alone, with only 6
nosis, and transforming growth factor alpha (TGFα), markers; genetic analysis of the p53 and APC genes by
epidermal growth factor receptor (EGFR), c-met, c- PCR-SSCP and PCR–RFLP was introduced in 1995;
erbB2, cyclin E, p27Kip1, and CDC25B for degree of and, since 1996, microsatellite analysis has been
malignancy. For the screening of genetic instability, performed with an autosequencer. At present, we ana-
hMLH1 expression is examined. With PCR-SSCP and lyze 14 molecular and 3 genetic markers.
PCR-RFLP, deletions or mutations of the APC gene
and the p53 gene are examined. Mutations of the p53
Results and evaluation
gene are analyzed in exons 5 to 8, and LOH is analyzed
using polymorphism at the BamH1 site of the 3⬘- Approximately 5000 lesions in histopathological
untranslated region of the gene. Genetic instability is samples of the stomach have been analyzed. The histo-
monitored by microsatellite assay. Four loci of two CA pathological diagnoses are adenoma, dysplasia, bor-
repeats (D1S191, D17S855; BRCA1 locus) and two derline lesion, carcinoma, and lesion suspected of
poly A tracts (BATRII; TGFβ type II receptor and carcinoma. The results of molecular-pathological diag-
BAT40) are examined, and if two or more loci with nosis of gastric lesions are summarized in Table 1. As
replication errors are detected, MSI-H is considered mentioned above, molecular diagnosis is made by com-
to be present. For MSI-H, the presence or absence of bining the results of molecular and genetic analyses and
mutation of hMLH1 and hMSH2 is determined by the histopathological findings. The definitions we use in
PCR-SSCP methods. molecular diagnosis are as follows: (1) “adenoma with
The assessment of biomarkers is reviewed periodi- malignant potential” is an adenoma with a high prob-
cally, and we always study new markers retrospectively ability of becoming carcinoma. These tumors contain
to verify their usefulness for diagnosis prior to use in some molecular and genetic abnormalities, including
clinical practice. Figure 3 shows the history of molecular p53, but morphological aberrations are within the range
markers for stomach specimens during the 7 years that for benign tumor. (2) “Suspected of (s/o) adenocarci-
our system has been operating. In 1993, molecular diag- noma” is a tumor that has genetic abnormalities of p53
118 W. Yasui et al.: Molecular diagnosis of gastric cancer

Fig. 4. A representative case of double

cancer (gastric and colon cancers) with
high-frequency microsatellite instability
(MSI-H). DNA was extracted and sub-
jected to microsatellite assay, as described
in the text. Three loci (D17S855, D1S191,
and BAT-40) were shown in both the gas-
tric and the colon cancers, but not in the
normal counterparts

and/or APC, as well as abnormal expression of many mal epithelia, such as in signet ring cell carcinoma, or
cancer-related molecules, such as c-erbB2, cyclin E, when cancer cells are infiltrated diffusely in fibrous
EGFR, and c-met, while histological atypia is not severe stroma, such as in scirrhous carcinoma. To deal with
enough to be regarded as carcinoma. (3) “Adenocarci- these problems, we are introducing laser microdissection
noma” is a tumor whose genetic and molecular abnor- [28] for routine use. With this technique, tumor cells can
malities are the same degree as those in (2) above, that be distinctly separated from normal mucosal epithelia.
can be also regarded as malignant histopathologically if Another problem with our system is that we do not
such molecular findings are available. Ten percent of have enough molecular markers; we need additional
histologically diagnosed adenomas were diagnosed as markers, e.g., for differential diagnosis, grade of malig-
adenoma with malignant potential, while 2% were sus- nancy, and drug sensitivity, to make diagnosis more
pected of adenocarcinoma. Adenocarcinoma or s/o efficient. The DNA microarray technique is a powerful
adenocarcinoma was identified in more than 20% of tool to identify novel genes participating in the devel-
histologically diagnosed borderline lesions. Twelve per- opment and progression of gastric cancer. Figure 5
cent of histologically diagnosed adenocarcinomas were shows the representative result of a comparative gene
regarded as showing high-grade malignancy because of expression profile of well differentiated adenocarci-
abnormalities of growth factors or growth factor recep- noma and poorly differentiated scirrhous gastric carci-
tors, cell-cycle regulators, and so on, such as c-erbB2, noma obtained using the DNA microarray technique.
EGFR, cyclin E, and p27Kip1. The prognosis of patients Strong expression of cadherin, cell-cycle regulators, and
diagnosed with these abnormalities tended to be poor mitogen-activated protein (MAP) kinases was observed
on follow-up observation. About 4% of gastric cancers in the well differentiated type, while survivin, growth
exhibited MSI-H by microsatellite analysis (Fig. 4). In factors (TGFβ, platelet-derived growth factor [PDGF],
half of the patients who showed MSI-H, clinically syn- insulin-like growth factor [IGF]), and proteases (matrix
chronous or asynchronous multiple primary cancers metalloproteinase [MMP]-2, MMP-3, plasminogen
were confirmed. We found germline as well as somatic activator [PA]) were found to be strongly expressed in
mutations of DNA mismatch repair genes in some scirrhous type carcinoma. Large numbers of genes are
cases, suggesting a kindred of HNPCC, but most cases differentially expressed in various tumors, but their
showed epigenetic inactivation of hMLH1, possibly role in stomach carcinogenesis is not known. Further
caused by CpG island methylation. studies should be performed to verify the value of such
genes in molecular diagnosis, both retrospectively and
prospectively.
Points at issue
With our diagnosis system, the portion of the section
Another contribution of molecular analysis to
used for DNA extraction is marked with a felt pen, and
clinicical findings
tissue in the marked area is collected as accurately as
possible. One of the technical problems with this system
Super-early diagnosis of gastric cancer
is that accurate sample collection is sometimes difficult,
and genetic analysis cannot be done in cases in which a As described above, many genetic and epigenetic
small number of cancer cells are distributed in the nor- abnormalities are found in incomplete type intestinal
W. Yasui et al.: Molecular diagnosis of gastric cancer 119

Fig. 6. CpG island hypermethylation of the hMLH1 gene in

intestinal metaplasia. The methylation status of the CpG is-
land in the promoter of the hMLH1 gene was examined in
intestinal metaplastic mucosa (IMM) and non-metaplastic
mucosa (NMM) by a methylation-specific PCR method

Fig. 5.A,B. Differential such as the mRNA for keratin 19 or carcinoembryonic

gene expression of well
differentiated adenocarci- antigen (CEA) is useful for detecting micrometastasis
noma (A), detected by Cy5, [30,31]. The expression of these genes can be found by
and poorly differentiated reverse transcriptase (RT)-PCR in many lymph nodes
scirrhous carcinoma (B) that are histologically negative for metastasis. Histologi-
detected by Cy3, using cally node-negative patients with micrometastasis de-
cDNA microarray
tected by means of cytokeratin expression show worse
prognosis than those without micrometastasis [32].
The detection of cancer cells in the peritoneal cavity
metaplasia without obvious morphological aberration. has important prognostic implications. Routine cyto-
Therefore, if molecular analysis is extended to intestinal logical examinations, however, are not sensitive, and
metaplasia, super-early diagnosis of gastric cancer can gastric cancer patients with negative peritoneal cytology
be made. For instance, mutations of the p53, APC, or K- sometimes show peritoneal recurrence. The analysis
ras genes have been detected in intestinal metaplasia, of CEA mRNA expression by RT-PCR is sensitive
although at low frequency [29]. The intestinal metapla- enough to detect free cancer cells in peritoneal washes
sia in 30% of gastric cancer cases shows weak but sig- that are negative for abnormal cytology, indicating that
nificant telomerase activity, suggesting the existence of such analysis is a powerful technique for predicting the
“stem-cell hyperplasia” or “already immortalized so- risk of peritoneal dissemination. Real-time RT-PCR is
matic cells” in intestinal metaplasia [3]. TERT protein is more rapid and quantitative than the conventional one,
expressed in some incomplete-type intestinal metapla- and is applicable to decision-making during surgery
sias adjacent to carcinomas [11]. The expression of [33]. A similar strategy can be employed for diagnosing
acetylated histone is reduced in intestinal metaplasia. the presence of cancer cells in peripheral blood [34].
Examinations of foci with genetic instability in stomach CEA mRNA from circulating cancer cells is associated
resected because of gastric cancer have shown, replica- with an advanced disease stage, and is a useful diagnos-
tion error-positive foci located both in the cancer and in tic modality to determine patient selection for chemo-
the intestinal metaplasia around the cancer [13]. Some therapy after surgery and to determine the patient’s
intestinal metaplasias show CpG island hypermethy- prognosis.
lation of the hMLH1 gene and its reduced expression
(Fig. 6). Foci with replication error and portions with
reduced hMLH1 expression in nonneoplastic mucosa Future prospects for molecular diagnosis
are lesions that are susceptible to cancer progression
(true precancerous lesions), and genetic instability is a The human genome sequence, determined by both the
powerful genetic marker to predict such a condition. International Human Genome Sequencing Consortium
and Celera Genomics of Rockville, was published in
Nature of 15 February 2001 [35], and in Science of 16
Diagnosis of presence of cancer
February 2001 [36], respectively. Although the project
It is needless to state that lymph node metastasis is the is not yet completed, approximately 3 billion base pairs
most important determinant of patient outcome. How- were sequenced, covering 90% of the euchromatic
ever, with routine histopathological examination of rep- portion of the human genome. The number of protein-
resentative sections of the cut surface of lymph nodes, coding genes is estimated to be about 30 000. We believe
there is a chance that micrometastasis may be over- that this determination of the human genome sequence
looked. The molecular detection of mRNAs expressed is the greatest scientific achievement made in the past
in cancer cells but not in other cells in lymph nodes, decade. Because one principle of the Human Genome
120 W. Yasui et al.: Molecular diagnosis of gastric cancer

Project, reflected in the Universal Declaration of the The examination of morphological changes in rela-
Human Genome and Human Rights, is unlimited access tion to genetic and epigenetic abnormalities is the main
to the sequence, the entire information on the human benefit of the genetic analysis of histopathological
genome in Nature is available without restriction specimens, and this will improve the quality of histo-
(http://www.nature.com.genomics). This is the beginning pathological diagnosis. “Histopathology” has discov-
of the post-sequence era. DNA microarray technology ered minute histological abnormalities, and has closely
has been established, and with this technology, informa- classified pathological conditions by accumulating
tion on the mutation and expression of many genes in knowledge obtained from morphological observations.
human cancers can be quickly detected, stored, and Another important mission for genetic diagnosis in the
analyzed. It is a time of evolution in that molecular field of pathology is to clarify how abnormalities in the
diagnosis in the field of pathology must correspond to functions of the genes and molecules are reflected in
this new scientific age. The findings that have already morphology. The ultimate objective of molecular pa-
accumulated on the molecular mechanisms of the devel- thology is that the morphological abnormalities in all
opment and progression of gastric cancer, and findings diseases described in the pathology literature will be
that will be systematically clarified in the future will be found to correspond to genetic and epigenetic alter-
combined, and genetic diagnosis by means of DNA ations. In the field of carcinogenesis, molecular pathol-
microarray will surely become routine in the diagnosis ogy must evolve into morphological genomics.
of gastric cancer. If essential abnormalities and second-
ary changes can be clarified in the many genetic and Acknowledgments This work was supported, in part, by
epigenetic alterations identified in cancers, examina- Grants-in-Aid for Cancer Research from the Ministry
tions of a hundred to several hundred genes should be of Education, Culture, Sports, Science, and Technology
sufficient for the diagnosis of gastric cancer. It is impor- of Japan; and from the Ministry of Health, Labor, and
tant that the characteristics of each cancer, that is, the Welfare of Japan. We would like to thank Dr. S. Usui
common features and specific features in the develop- and Dr. T. Yamagata (Hiroshima City Medical Associa-
ment and progression of each cancer, are clarified so tion) for their continuous support on this project. Spe-
that gene therapy or molecular-targeted therapy can be cial thanks are given to Mr. M. Matsumoto, Mr. T.
directly related to the results of molecular diagnosis of Kabuto, and other staff members of the Pathology
histopathological samples. We plan to create DNA and Cytology Division, Hiroshima City Medical Asso-
miniarrays for the diagnosis of gastric cancer to predict ciation Clinical Laboratory, for their skillful technical
the patient’s susceptibility and the grade of malignancy assistance.
and chemosensitivity of the cancer.
Most genetic variation between individual humans is
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