Immunochemical and molecular-Genetic markers in diagnostics of gastric cancer

ISSN 1990-7508, Biochemistry (Moscow) Supplement Series B: Biomedical Chemistry, 2009, Vol. 3, No. 1, pp. 33–43. © Pleiades Publishing, Ltd., 2009.Original Russian Text © E.V. Elistratova, P.P. Laktionov, P.I. Shelestuk, S.A. Tuzikov, V.V. Vlassov, E.Y. Rykova, 2009, published in Biomeditsinskaya Khimiya.

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INTRODUCTION

Gastric cancer is the fourth most frequent malignanttumor (about 8.6% in the structure of tumor morbidityin the world) and the second leading cause of cancerdeath (700000 deaths every year) [1].

The highest 5-year survival rate in patients with gas-tric cancer (52%) was regisreted in Japan; since 1960this country introduced the method of fluorography intoa common program of medical examination. However,this methods is not widely used in other countries dueto its high costs and this explains lower 5-year survivalrate (27%) in Western Europe [2]. Among 45 worldcountries Russia is on the first place by lethality of thisdisease [3].

Such disappointing statistical data are explained bylate diagnostics of gastric cancer, when surgical opera-tion (the main radical method of treatment of this dis-ease) becomes basically ineffective. Proportions ofpatients with stage III and stage IV gastric cancer is31.4% and 42.6%, respectively, and the 5-year survivalrate after the radical treatment decreases is the follow-ing: 80–100% at stage I, 40–65% at stage II, 15–35% atstage III, and 0% at stage IV [4].

Early diagnostics of gastric cancer is very difficultbecause tumor may be clinically recognized after

reaching certain size and therefore existing methods fordetection of tumor process (e.g., gastroscopy, as well ashistology, roentgenology and tomography methods) arepoorly applicable for cancer detection at preclinicalstages and basically inapplicable during formation ofrisk groups at the stage of tumor transformation ofcells.

1. PROTEIN MARKERS

Currently used immunological and biochemicalmethods based on detection of tumor markers (the pro-teins, which blood concentrations are changed in tumordiseases) are low informative for early diagnostics ofgastric cancer. In most cases, the protein tumor markersare of diagnostic value, when the tumor has alreadybeen formed; this explains rather late appearance ofsuch markers in blood. Thing is that the increase inblood tumor markers above a detection limit requiressufficient number of tumor transformed cells, express-ing such markers. On the other hand, tumor trans-formed cells which do not express increased levels ofparticular markers are not detected. There are severalprotein markers, which are most frequently used inmodern diagnostics of gastric cancer: carcinoembry-onic antigen (CEA), alpha-fetoprotein (AFP), cancerantigen CA 72.4 (CA 72.4), carbohydrate antigen CA19.9 (CA 19.9).

Immunochemical and Molecular-Genetic Markers in Diagnostics of Gastric Cancer

E. V. Elistratova

a

*, P. P. Laktionov

a

, P. I. Shelestuk

b

, S. A. Tuzikov

c

, V. V. Vlassov

a

, and E. Y. Rykova

a

a

Institute of Chemical Biology and Fundamental Medicine, Siberian Branch, Russian Academy of Sciences, pr. Lavrent’eva 8, Novosibirsk, 630090 Russia; tel.: (383)3304654, fax: (383)3333677;

*e-mail: [email protected]

b

Novosibirsk Oncological Dispensary, ul. Plachotnogo 2, Novosibirsk, 630108 Russia

c

Cancer Research Institute, Siberian Branch, Russian Academy of Medical Sciences, per. Kooperativnyi 5, Tomsk, 634009 Russia

Received February 6, 2008

Abstract

—Intensive studies of molecular mechanisms responsible for tumor transformation results in identifi-cation of new proteins and their genes involved into tumor development. These proteins may be used as markersof tumor transformation of cells and the level of their expression may be evaluated by means of modern highlysensitive and technological methods of analysis. This review summarized literature data on currently usedimmunohistochemical and molecular genetic markers of gastric cancer. It highlights genetic and epigeneticchanges detected in nucleic acids of tumor tissue cells in malignant and benign gastric diseases as well as in thelevel of DNA circulating in blood of patients with gastric cancer.

Key words

: gastric cancer, diagnostics, tumor markers, circulating DNA.

DOI:

10.1134/S1990750809010041

REVIEWS

*To whom correspondence should be addressed.

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Carcinoembryonic antigen (CEA)

is the mostknown and widely used tumor marker. This glycopro-tein is produced by embryonic and fetal gastrointestinaltract tissues. After birth CEA synthesis is suppressedand in blood (and other biological body fluids) of adulthealthy individuals it is not detected [5]. The increasein blood serum CEA concentration (above 5 ng/ml) isdetected in various malignant tumors of digestive andrespiratory systems, in breast, head, and neck carcino-mas. Increased level of CEA was also observed inbenign liver and lung diseases, as well as in autoim-mune diseases (20–50% of cases).

In the case of CEA sensitivity and specificity of pri-mary diagnostics of gastric cancer do not exceed 57.6and 79.0%, respectively [6–11]. It was shown that theCEA level correlated with the stage of gastric cancer,depths of invasion, involvement of lymph nodes intothe tumor process, and with distant metastases [12–15].CEA has prognostic value in gastric cancer. In gastriccancer patients with the increased level of CEA the5-year survival rate was 31.7%, whereas in patientswith an undetectable level of this marker this parameterwas 77.3% [12]. According to Marelli et al. [16], inrecurrent gastric cancer sensitivity of diagnostics basedon the CEA level was 44%, whereas in the case of pri-mary diagnostics it was just 16%. This is consistentwith other observations [7]. Using data of numerousstudies CEA is used in diagnostics of gastric cancermainly for evaluation of tumor distribution and moni-toring of this disease.

AFP

was originally identified in fetal serum in 1956[17]. This glycoprotein is normally secreted by yolk saccells and at later stages by embryonic liver and gas-trointestinal tract cells. In adult humans plasma concen-trations of AFP do not exceed 15 ng/ml. Increased AFPcontent was registered during development of hepato-cellular carcinoma, yolk sac tumors, liver cirrhosis, andhepatites [5]. AFP-producing tumors are characterizedby various localizations [15]. It was shown thatincreased level of blood serum AFP was registered in1.3–15.0% of patients with gastric cancer and this formwas denominated as gastric cancer producing AFP(AFP-producing gastric cancer) [18]. The AFP-produc-ing gastric cancer is characterized by more aggressivebehavior. Kono et al. [19] reported that more than 63%of patients with AFP-producing gastric cancer had livermetastases and in this group of patients the 5-year sur-vival rate was just 28.4% compared with the group ofpatients with non-AFP-producing gastric cancer. In thelatter group only 9% of patients had liver metastasesand the 5-year survival rate was 62%. Thus, detectionof AFP level in blood serum may be used for differen-tiation diagnostics of AFP-producing gastric cancer andnon-AFP-producing gastric cancer.

Glycoprotein CA 72.4

(cancer antigen) is deter-mined in blood serum by means of B72.3 and CC-49monoclonal antibodies to CA 72.4 antigen, which is theantigenic determinant of tumor-associated glycoprotein

72 [14]. In blood serum of healthy donors concentrationof this antigen is 0–6 U/ml. The increase in blood CA72.4 was found in patients with breast cancer, coloncancer, endometrial cancer, lung cancer, and ovariancarcinoma. In some cases increased level of CA 72.4was also registered in benign and inflammatory pro-cesses of various localization [5].

Although sensitivity of primary diagnostics of gas-tric cancer based on immunodetection of CA 72.4 doesnot exceed 58% specificity is about 98% [6, 10, 11].Numerous studies have shown that increased level ofCA 72.4 is registered in patients with advanced tumorprocess, which involves lymph nodes, peritoneum, liver[6, 8, 10, 13]. Increased level of CA 72.4 in gastric can-cer patients indicates a greater risk of death in thesepatients. Gaspar [13] reported the 3-year survival rateof 34% in patients with increased level of CA 72.4whereas in patients with normal level of CA 72.4 thisparameter was 69%. Marrelli reported 51% of sensitiv-ity and 97% of specificity of diagnostics of recurrentgastric cancer based on CA 72.4 [16]. Thus, changes inthe level of CA 72.4 may be used for evaluation of stageof tumor process, its distribution and prognosis of thisdisease.

Carbohydrate antigen CA 19.9

is a glycoproteinsynthesized by epithelial cells of pancreas, stomach,endometrium and salivary gland [5, 15], where its lowconcentrations have been detected. Increased level ofCA 19.9 (exceeding 37 U/ml) is registered in pancreatictumors (with sensitivity and specificity of 70–90 and68–91%, respectively) [20], tumors of gastrointestinaltract (colorectal tumors, gastric tumors, tumors of thehepatobiliary system, and liver benign diseases [15]. Ingastric cancer sensitivity of primary diagnostics basedon the ELISA assay of carbohydrate antigen CA 19.9does not exceed 50% [6, 7, 10, 11]. This rather low sen-sitivity of primary diagnostics of gastric cancer basedon CA 19.9 increases in diagnostics of recurrent gastriccancer up to 55–56% and this analysis is characterizedby high specificity (ranged from 74 to 94%) [7, 15, 16,21]. Measuring CA 19.9 level it is possible to evaluatetumor distribution, invasion depths, involvement ofperitoneum, serous layer into tumor process, andmetastases in lymph nodes [6, 8, 13, 15].

Tocchi et al. [6] investigated CEA, CA 72.4, CA19.9 in blood and gastric juice from patients with gas-tric cancer, benign gastric diseases and clinicallyhealthy individuals. Statistically significant changesbetween these groups were found only in the level ofgastric juice CEA and blood CA 19.9. Sensitivity ofdiagnostics of gastric cancer by means of combinedassay of all three markers was 81.4%. However, in sim-ilar study Duraker et al. concluded that determinationof the markers CEA and CA 19.9 in gastric juice has nodiagnostic and prognostic importance [22].

Some studies have demonstrated that combinedassay of all tumor markers in blood does not result insignificant increase of sensitivity and specificity of pri-


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mary diagnostics of gastric cancer [7, 10, 11]. In thisconnection the main use of these protein markers con-sists in monitoring of the disease, evaluation of botheffectiveness of medical treatment and appearance ofrecurrent cancer.

2. MOLECULAR GENETIC MARKERS

Transformation of normal cells into cancer cells andprogression of tumor process are related to accumula-tion of genetic and epigenetic changes appearing in thegenome as the result of its impaired functioning [23,24]. The genetic changes include changes in nucleotidesequences: point mutations and chromosomal rear-rangements such as aneuploidy, chromosomal aberra-tions, loss of heterozygosity, microsatellite DNA insta-bility [23]. Most of epigenetic changes in tumor dis-eases are characterized by impairments of DNAmethylation resulted in changes of gene expression [25,26]. Employment of modern molecular genetic meth-ods for analysis of genetic and epigenetic changesobserved during appearance of tumor cells is especiallyperspective for early diagnostics of tumors. Indeed,using modern methods of molecular biology it is possi-ble to detect changes (of high sensitivity and selectiv-ity) in genome typical for cancer cells. This is impor-tant for the development of methods for screening,early diagnostics, and monitoring of cancer diseases.

2.1. Genetic Changes

Mutations in genes are one of the main causes ofcarcinogenesis that results in activation protoonco-genes (K-Ras,

β

-catenin) or inactivation of tumor sup-pression genes (p53 APC, p16). These mutationsappear due to substitution, insertion, and deletion ofnucleotides. There are some genes, which contribute tothe development of gastric cancer due to their muta-tions. These include p53, APC,

β

-catenin, K-Ras, andBRAF genes [27].

p53 gene is a tumor suppressor gene.

Product ofthis gene modulates transcription; it specifically inter-acts with DNA and causes transactivation of the cyclin-dependent kinase inhibitor gene and proapoptoticgenes. Mutations in p53 gene are one of the most fre-quent in gastric cancer. Most mutations have beenfound preferentially in 4–11 exons with the hot pointsin codons 175, 248, 273, 282, 245, 213. Most fre-quently mutations result in base pair substitution (gua-nine-cytosine for adenine-thymine) at CpG sites [28].The reported frequency of mutations in p53 gene intumor tissue from patients with gastric cancer variesfrom 0 to 77% [27–34]. There are contradictory reportson correlation between frequency of p53 gene muta-tions and clinicopathological characteristics. Accordingto results of many studies frequency of mutations in p53gene increases in the case of tumor localization in thecardiac part compared with the distal parts of stomach[28, 31]. Some researchers reported about predomi-

nance of p53 mutations in intestinal type gastric cancer[27, 35–37], whereas others failed to find such correla-tion [33, 38]. There are observations that mutations inp53 are detected in metastases more frequently than inprimary tumors. High percent of p53 gene mutations(up to 67%) was found at high-grade dysplasia andmetaplasia of gastric epithelium [28, 38]. On the otherhand, some authors point to p53 mutations preferen-tially at early stage of dysplasia [36, 39] and at chronicgastritis associated with

H. pylori

(52%) [40]. Such dis-crepancies decrease specificity of the method of diag-nostics of gastric cancer based on detection of muta-tions in p53 gene.

APC gene.

Product of this gene is involved intodegradation of the proto-oncoprotein

β

-catenin. Inacti-vation of APC gene results in the increase of transcrip-tion and lifetime of the

β

-catenin-regulated transcrip-tion factor; this results in activation of cyclin-dependentkinases determining cell entry into S-phase of the cellcycle [41]. Changes in APC gene are the key momentin the development of colon cancer (more then 90% ofcolon tumors are characterized by changes in this gene)[38]. In patients with gastric diseases mutations of APCgene are more frequent in benign diseases (gastric pol-yps) (76%) than in adenocarcinoma (up to 32.4%) [27,30–32, 38, 42]. It should be noted that there was no cor-relation between frequency of APC mutations anddegree of gastric dysplasia/polyps (as well as betweenother clinicopathological characteristics).

In gastric cancer the frequency of mutations in

β

-catenin gene (which is coupled to APC gene) variedfrom 0 to 27% [42–45] and these mutations were spe-cific for tumor process. Park et al. found correlationbetween mutations in this gene and the interstitial typeof cancer [44].

K-ras gene

belongs to the family of RAS genes[46]. Proteins of the RAS family exhibit GTPase activ-ity and are an important link in regulation of signaltransduction. During stimulation of cells with growthfactors activated RAS protein stimulates mitogen-acti-vated proteins of the kinase pathway (RAS-RAF-MEK-ERK-MAP kinase pathway), which plays animportant role in cell proliferation (and which is activein a tumor cell) [46]. Mutations in K-ras gene mayresults in appearance of constitutively activatedGTP-bound form of the protein. High percent of pointmutations in K-ras gene was observed in pancreaticcancer (70–100%), colon cancer (7–80%), and lungcancer (10–50%) [47, 48]. In gastric cancer frequencyof point mutations of K-ras gene varies from 0 to 28%in tumor tissue [30, 46–52]. In chronic gastritis associ-ated with

H. pylori

mutations of K-ras gene were foundin tissues specimens taken from a pathological alteredregion. Hiyama et al. found that the frequency ofmutations of this gene is higher in the group ofpatients with

H. pylori

-associated chronic gastritiscomplicated with cancer than in cancer free patients(with

H. pylori

-associated chronic gastritis) [50]. There

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was correlation between frequency in K-ras gene muta-tions with histologic type of tumor (high at differenti-ated cancer) [50].

E-cadherin gene

(CDH1) encodes a protein, whichbelongs to the cadherin family of transmembrane cal-cium-dependent glycoproteins. The external domain ofE-cadherin is involved into intercellular contacts, medi-ating calcium-dependent intercellular adhesion. Itsinternal domain binds

β

-catenin and in the bound form

β

-catenin does not function as the transactivator tran-scription factor [41]. This results in stimulation of celldivisions, impairments of intercellular contacts and theincrease of invasion capacity of cells. Relatively highfrequency of mutations (28–56%) was found in E-cad-herin gene in hereditary diffuse gastric cancer [36].However, mutations of this gene do not play a key rolein the development of sporadic gastric cancer.

It was demonstrated that mutations in B-raf, BAD,EPHB2 genes frequently found in melanoma, colorec-tal cancer are basically not detected in patients withgastric cancer [46, 51, 53–55].

DNA sequence copy number changes

are one ofthe widespread phenomena resulted in malignant trans-formation of cells. This may be determined by themethod of comparative genomic hybridization (CGH).This method is based on simultaneous hybridization ofDNA probes (one isolated from examined patient’s tis-sues and the other one from normal tissues) with humanchromosomes. Differences in the results of hybridiza-tion may reveal increase or decrease in number of cop-ies of chromosomal regions in an investigated speci-men. This method may detect losses of genome regionsof about 10 million base pairs [56]. Amplification (i.e.,the increase in copy number) may be detected inaffected genome regions of smaller size, provided thatthere is large number of repeats in these regions. Sepa-rate genes may bee seen if they have been amplified by40 and more times.

It was demonstrated that the increase in the numberof tandem-repeat copies in DNA (due to amplification,translocation) stimulates activation of correspondingprotooncogen. For example, in gastric cancer DNAamplification in the chromosome regions 17q12,10q26, 7q31, 8q23-24, 20q13 increases activity ofERBB2, K-SAM, C-MET, c-MYC, STK6, respectively[37, 57].

Detection of amplified regions of various chromo-somes may detect impairments in new genes responsi-ble for tumor transformation in gastric cancer. Forexample, Kang et al. found that amplification of thechromosome region 1p36.11-p33, including p73 geneand the increase in expression of p73 protein correlatedwith tumor progression [58]. In gastric cancer numer-ous changes in the DNA sequence copy number werefound in almost all chromosomes. The highest percentof the increase in the copy number was observed in var-ious chromosome regions: 1q (40%), 6p (85%), 8q(35%), 17 (35%), 19q (55%), 20q (40%) [58–64]. Data

on correlation of these changes with histological typeof tumors, age, and gender of patients with gastric can-cer are not unequivocal. Further studies are needed fordetection of genes corresponding to these regions andtheir role in the development of tumor process.

Loss of heterozygosity (LOH) is a particular phe-nomenon of the decrease in DNA sequence copy num-ber due to deletion of one of alleles. LOH detectionemploys polymorphic microsatellite markers (micro-satellite analysis). Knudson originally proposed thatdeletion of one of alleles increases probability of tumortransformation due to the development and manifesta-tion of chromosomal changes in the other allele [65].For example, loss of the chromosome regions 9p21,5q21, 17p13, which correspond to the tumor suppres-sion gene p16INK, APC, and p53, respectively, resultsin loss of functions of these genes [37, 58, 66].

Loss of heterozygosity in tumor cells allows tosearch tumor suppressor genes in the lost regions. Forexample, search for tumor suppressor genes involvesthe region 8p21-23 [64, 67]. Loss of the genetic mate-rial in the chromosome regions 1p (80%), 4q (75%), 5q(65%), 15q21 (65%), 21q (65%), 16q21 (45%) repre-sents a wide field for search of genes, which are lostduring the development of gastric cancer [58–61, 63,64]. There is correlation between such chromosomalchanges and clinicopathological parameters of patientswith gastric cancer and survival. It was found that lossof various sites in various regions of chromosomes 4q,14q correlated with the development of regional anddistant metastases. Loss of genetic material at 4q21.1and 18q21 was associated with poor prognosis of thisdisease [58–60].

It was reported that in gastric cancer LOH causedloss in functioning of p53 gene (26–83%), APC gene(20%), TFF1 (13–28%), CDH1 (10%) [28, 37, 68].Attempts to find correlation between LOH and clinico-pathological parameters gave contradictory results.Some researchers failed to find such correlations,whereas others found loss of heterozygosity of p53gene observed at late stages of gastric cancer, exophytegrowth and interstitial type of tumor [33] and loss ofheterozygosity of p73 gene at highly differentiated ade-nocarcinoma [69].

As it has been mentioned above loss of heterozygos-ity is detected by means of polymorphic microsatellitemarkers. However, first of all microsatellite analysismay detect changes in microsatellites, which are wide-spread in tumor diseases. Microsatellites are highlypolymorphic multiple repeats of short (1–6 base pairnucleotides) sequence repeats. Biological role of mic-rosatellites is not yet well understood; it is only knownthat human genome contains several thousand micro-satellites and most of them are located within the non-coding DNA sequence [48].

The first suggestion on involvement of microsatel-lite DNA in the development of tumor diseasesappeared after discovery of widespread changes in


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polyA regions in genome of tumor cells of sporadiccolon cancer. Subsequent studies have shown that othermicrosatellites including polyCA repeats were charac-terized by similar changes [23]. This phenomenon wasnamed as microsatellite instability (MSI); this termdefines changes in the number of one of several micro-satellite repeats due to their deletion or amplification.Good evidence now exists that microsatellite instabilityis associated with impaired functioning of specificrepair system. In 1993 Strand et al. [70] based on obser-vation of microsatellite instability in bacteria with defi-cient genes of the reparation system mutS or mutL pro-posed a hypothesis that this phenotype appears due todamages in the repair system. This hypothesis was thenconfirmed after identification of mutS homologues(hMSH2 and hMLH1) on human chromosomes 2 and 3and detection of inactivating mutation in these genesfound in close relatives at hereditary nonpolyposis col-orectal cancer. Now 6 homologues of mutS and mutLare known; inactivating mutations in their genes resultin appearance of the microsatellite instability pheno-type in cancer patients [23].

Microsatellite instability is subdivided into twotypes: low frequency MSI is associated with replicationerror in one of five microsatellite loci, and high fre-quency MSI is associated with replication errors inmore than one microsatellite loci.

Low and high frequency MSI was observed in 13–45% of patients with gastric cancer [52, 55, 71–73].Data on correlation of MSI with clinicopathologicaland demographic parameters of patients with gastriccancer are not unequivocal [37, 55]. Analyzing micro-satellite instability Vauhkonen et al. demonstrated thatgenome is more stable in diffuse-type gastric cancerthan in the interstitial type [61]. This is supported byother observations [37, 42, 74]. In some studies low fre-quency MSI was typical for tumor localized in gastricantrum and for other tumors characterized by low inva-sive and metastatic potential into lymph nodes [33, 52].On the other hand, An et al. [72] did not find statisti-cally significant difference between MSI status andclinicopathological and demographic parameters ofpatients including age, gender and races of patients.Lee et al. concluded that the high frequency MSI phe-notype is more frequent in gastric polyp/dysplasia asso-ciated with carcinoma (17%) than gastric polyp/dyspla-sia unassociated with carcinoma (3%) [74]. Theseauthors did not find such correlation for low frequencyMSI.

Summarizing results of numerous studies it shouldbe noted that genetic changes currently detected in gas-tric cancer are not specific and results obtained in thisfield are inconsistent and often contradictory. However,the development of such modern methods as DNAhybridization on oligonucleotide chips, completegenome sequencing [75, 76], which may significantlyextent and intensify comparative analysis of geneticchanges in norm and pathology, inspire optimism in

discovery of tissue specific DNA markers which wouldbe used in diagnostics of malignant tumors in the recentfuture.

2.2. Epigenetic Changes

In cancer a phenomenon of hypermethylation ofCpG islets of tumor suppressor gene was originallydescribed in 1989 for retinoblastome (Rb) gene [77].However, only in 1994 after discovery of the mecha-nism of VHL gene inactivation by methylation the phe-nomenon of hypermethylation of promoter regions ofgenes as the cause of gene inactivation in malignanttumors had attracted much attention of scientists. Thediscovery of the main pathway of gene inactivation dueto hypermethylation of CpG islets of promoter regions(investigated using tumor suppressor gene p16 as anexample) stimulated large-scale studies on the role ofthis epigenetic process in tumor transformation. Thedevelopment of the main methodological approaches,the method of bisulfite modification and methylation-specific polymerase chain reaction, made substantialcontribution in the development of studies of DNAmethylation; these methods are simple and are charac-terized by high sensitivity and specificity in detectionof methylated regions of DNA [77].

Use of aberrantly methylated DNA as markers of atumor disease has several advantages compared withemployment of genetic changes of DNA describedabove. First, aberrant DNA methylation is now consid-ered as the most widespread and earliest events result-ing in tumor transformation of a cell [77]. Second, sen-sitivity of detection of aberrantly methylated geneunder conditions of excess of non-methylated allele ishigher that for mutant allele [78]. Third, there are manygenes inactivated by means of changes in the methyla-tion status of CpG islets of promoter regions typical fortumor transformed cells [77, 79].

In tissue specimens from patients with malignanttumors and benign gastric diseases the methylation pro-file was investigated for genes involved into control ofcell cycle (p16, p15, APC, COX2, RASSF1A, CyclinD2), regulation of angiogenesis (THBS1), enzymes ofDNA repaid (MGMT, GSTP1, HMLH1), receptors,involved into transduction of external signals (MDR1,RAR beta2), as well as intercellular interactions andadhesion (E- and H-cadherins) [80].

p16 gene

(CDKN2A, MTS1, INK4A) localized onthe chromosome 9p21 encodes protein inhibitor ofcyclin dependent kinase; it has a major role in cell cyclecontrol by phosphorylation of Rb protein. In theabsence of p16 protein cyclin dependent kinase phos-phorylates Rb retinoblastoma protein; this results inrelease of transcription factors E2F from an inactivecomplex and activation of genes determining cell entryinto S phase of cell cycle [81]. It is known that changein methylation profile of p16 gene is observed in 20–67% of solid human tumors of various localizations

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[81]. Methylated form of p16 gene was found in 10–80% specimens of gastric cancer tissue [71, 82–94].Percent of hypermethylation of the promoter region ofthis gene investigated in other gastric diseases (chronicgastritis, gastric polyps) varies from 0 to 15% [82, 85,88–90%]. Evaluating diagnostic importance of thehypermethylated form of p16 one should take into con-sideration that aberrant methylation is also detected inhealthy gastric tissue of 29% of humans above 45 years[95].

p15 gene (CDKN2B)

is located on the chromosomeregion 9p21 as p16 and shares high homology with it.Protein p15 suppresses activity of cyclin dependentkinase by inhibiting catalytic activity of complexesformed by cyclin D and cyclin dependent kinases.Studies of gastric adenocarcinoma biopsy specimensrevealed altered methylation profile of this gene in 48–73.1% of patients [82, 84, 86, 87, 92], whereas in tissuespecimens of intestinal metaplasia associated and non-associated with gastric cancer this parameter waschanges in 14 and 7%, respectively [82]. In other solidtumors hypermethylation of the promoter region of p15gene is rather rare event. In cancers of the blood systemit is the major cause of p15 gene inactivation; for exam-ple, methylation of the promoter region was found in70% of cases of acute myeloid leukemias [96]. Relativeeasiness of differential diagnostics of gastric cancer andblood cancer as well as high percent of altered status ofp15 gene methylation in gastric cancer allow to con-sider the aberrantly methylated promoter (locus AF513858, region 1359–1375) of p15 gene as potentialspecific tumor marker of gastric cancer.

APC

Inactivation of this gene by hypermethylationof the promoter region was observed in 61–84% ofcases of gastric cancer [68, 86, 88, 92, 94, 97]. It shouldbe noted that hypermethylated form of this gene wasfound not only in tumor tissue but also in benign gastricdiseases [88, 92]. Kang et al. reported about age-relatedincrease in hypermethylation of the promoter region ofAPC gene [88]. In that study 15% of gastric mucosaspecimens from children with chronic gastritis showedaltered methylation status of CpG islets of the promoterregion of APC gene; in adults under 50 years thisparameter was 65%. Evidently, additional studies areneeded for estimation of the role of altered status ofAPC gene methylation in the development of benignand malignant gastric tumors.

COX-2 gene

encodes cyclooxygenase 2 (COX-2),which is involved into synthesis of prostaglandins fromarachidonic acid. Expression of COX-2 gene is inducedin all tissues (except kidney cortex and brain cortex) bycytokines and growth factors inducing tumor growth.Some studies of colon cancer and gastric cancerrevealed decreased expression of COX-2 due to alteredstatus of methylation of both CpG islets of the promoreregion of COX-2 gene and also its exon 1 [98]. Hyper-methylated form of COX-2 gene was found in 4.1–46.3% of patients with gastric cancer [73, 88, 89, 92,

99]. In benign gastric diseases this parameter waschanged in 1.4–8.8% of cases [88]. One can see thatfrequency of methylated form of COX-2 gene widelyvaries and therefore subsequent studies are needed forevaluation of contribution of this event in gastric car-cinogenesis.

RASSF1A gene

is a gene encoding protein of theRAS associated domain family. Numerous studies onbiological functions of RASSF1A gene have shownthat its protein product is involved into regulation ofcell cycle and apoptosis; it also decreases carcinogenic-ity of tumor cell lines. Changes in this gene are oftendetected in tumor diseases (lung cancer, breast cancer,prostate cancer, bladder cancer, cervical cancer) [100].In gastric cancer methylated form of RASSF1A genewas detected in 26% of cases and it was not found inhealthy individuals [82, 88, 89, 92, 100]. Relativelyhigh specificity but low sensitivity of diagnostics ofgastric cancer based on detection of aberrantly methy-lated form of RASSF1A gene imply that it may be usedas an additional marker in combination of other mark-ers increasing sensitivity of diagnostics.

MGMT gene

encodes enzyme O

6

-methylguanineDNA methyl transferase); removing alkyl group at theO

6

position of guanine moiety this enzyme is involvedinto DNA repair. DNA alkylation at the O

6

position ofguanine is of the most important events in tumor trans-formation of cells: after DNA replication this results inbase pair substitution (guanine-cytosine for adenine-thymine) [101]. In most human tumor cells inactivationof MGMT gene is associated with hypermethylation of(normally non-methylated) CpG islets of a promoterregion of this gene [102]. Studies of gastric cancer tis-sue have shown that hypermethylation of CpG islets ofMGMT gene was observed in 18–61% of cases [34, 52,73, 88, 92, 93, 101–104]. Methylation of MGMT geneis more frequent in interstitial than in diffuse cancer[103] and there is correlation between this parameterand tumor expansion (involvement of lymph nodes andtumor stage) [105]. Hypermethylation of the promoterregion of MGMT gene was also found in patients withbenign gastric diseases [88, 92]. In healthy individualsthis parameter ranged from 10.9 to 25.0% [89, 92].Thus, aberrant methylation of the promoter region(locus AL355531 46931–46953) of this gene has poorperspectives for diagnostics of gastric cancer.

hMLH1 gene

encodes a protein involved into repairof unpaired bases, which are frequently formed duringreplication of microsatellite DNA sequences [23]. Inac-tivation of genes of the repair system due to mutationsor hypermethylation increases frequency of mutationsin microsatellite sequences and probably in DNA cod-ing sequences. hMLH1 gene mutations are responsiblefor hereditary nonpolyposis colorectal cancer and mic-rosatellite instability seen at sporadic cases of colorec-tal cancer, endometrial cancer, and gastric cancer [79].In gastric cancer frequency of changes in the methyla-tion status of hMLH1 gene varies from 8.1 to 42.0%


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[34, 52, 68, 71–73, 82, 85–88, 90, 92, 93, 95, 97, 103,104, 106]. In benign gastric diseases changes in themethylation status of this changes are characterized bylower frequency [82, 85, 88–90]. In biopsy specimensof healthy gastric tissue and in chronic gastritis methy-lated form of hMLH1 gene was not found [34, 95, 97],and therefore analysis of methylation of hMLH1 genemay be used in diagnostics of gastric cancer.

GSTP1 gene

encodes class pi glutathione S-trans-ferase (GST). GSTs are involved into metabolism ofxenobiotics. These enzymes catalyze conjugation ofreduced glutathione with electrophilic substratesaccompanied by their detoxification. Impairments inthis process may be one of the main reasons for tumortransformation of cells. Hypermethylation of CpGislets of the promoter region of this gene in somaticcells stops GSTP1 transcription and this almost alwaysaccompanies prostate cancer (90% of cases). Inendometrial cancer changes in the methylation of thisgene were found in 80% of patients [107]. Irrespec-tively to age hypermethylation of the promoter regionof GSTP1 was found in 7.0–37.5% of patients with gas-tric cancer no changes in this parameter were found inbiopsy specimens obtained from patients with intestinalmetaplasia and chronic gastritis [68, 82, 84, 86–89,104]. Thus analysis of methylation of GSTP1 gene maybe used for diagnostics of gastric cancer only in combi-nation with other markers increasing specificity of thismethod.

TIMP-3 gene

encodes one of tissue inhibitor ofmetalloproteinase (TIMP). Matrix metalloproteinases(MMP) are enzymes hydrolyzing extracellular matrix.MMP-3 catalyzes cleavage of matrix decorin; this resultsin release of biologically active form of growth factor

β

,which activates cell proliferation and promotes forma-tion and growth of tumor. Inactivation of TIMP-3 gene isan important for tumor progression and metastases [108,109]. Hypermethylation of the promoter region of thisgene was found in 12.5–65.0% of patients with gastriccancer [68, 86, 88, 90, 92]. This marker was also foundin chronic gastritis, metaplasia, gastric polyps and inhealthy persons; there is age-related increase in fre-quency of aberrantly methylated TIMP-3 [89–91]. Thesedata argue against possible use of the methylated form ofTIMP-3 gene as a marker of gastric cancer.

DAP kinase gene

encodes calmdulin-dependentkinase of serine/threonine type (160 kDa). DAP kinasecontains cell death domain; in the presence of

γ

-inter-feron this domain triggers cascade reactions leading toapoptosis. The decrease in DAP kinase expression dueto hypermethylation of promoter region increases met-astatic potential of tumors [110]. In gastric canceraltered methylation status of this gene was found in 34–70% [68, 82, 84, 88, 90, 111, 112], however, this wasalso determined in norm, chronic gastritis, polyps, andintestinal metaplasia [88–90]. There was correlationbetween altered methylation of this gene and age [112],tumor distribution and a 5-year survival rate. In the

presence of metastases (stages III–IV) percent ofhypermethylated form of DAP kinase gene increasedand so DAP kinase gene may be considered as a poten-tial marker for distribution and prognosis of this disease[111].

E-cadherin gene.

As it has already been mentionedabove this gene encodes E-cadherin, protein of the cad-herin family of transmembrane calcium-dependent gly-coproteins. E-cadherin gene expression is suppresseddue to mutations and methylation of promoter regionsof this gene. In gastric cancer frequency of methylatedform of E-cadherin gene varies from 15 to 80% [71, 73,82, 84, 86–89, 92, 93, 113, 114]. It should be noted thatin chronic and benign gastric diseases this parameter is58.2–85.0% [82, 88, 92], and age-related increase infrequency of methylated form of this gene [95] makesthis marker inapplicable in diagnostics of gastric cancer.

THBS1 gene

encodes an angiogenesis inhibitor,known as thrombospondin 1 (TSP). TSP is glycopro-tein located in extracellular matrix of various tissues. Itis involved into platelet aggregation and regulation ofadhesion and proliferation of endothelial cells. Thedecrease in THBS expression promotes tumor progres-sion by influencing angiogenesis, invasion, and migra-tion of tumor cells [115]. In patients with gastric cancerpromoter region of this gene is methylated in 30–60%of cases [68, 88, 90, 92], in patients with chronic gastri-tis this parameter represents 10–18% [88, 90, 92]. Inbiopsy specimens of normal gastric tissue frequency ofthe methylated form of this gene varies from 0 to 37%[89, 92]. Evidently, changes in the methylation status ofTHBS1 gene do not represent a key event in the devel-opment of gastric cancer.

Thus, data presented here demonstrate that results ofvarious studies on frequency of the same methylatedmarkers significantly differ. This may be attributed todifferent sensitivity and specificity of variants of PCRanalysis used in each study (qualitative, different ver-sions of quantitative analysis, employment ofrestrictases, etc.). Differences may be also associatedwith individual variations methylation profile of CGpairs in promoters of tumor suppressor genes both invarious patients and in tumors from various organs. Forexample, in prostate cancer and breast cancer methyla-tion involves promoter region of RAR

β

2, whereas inlung cancer methylation occurs in the first exon of thisgene [116]. Consequently, successful development oftissue specific analyses for methylation markers oftumor process requires identification of particularmethylation sites of DNA isolated from tumors of vari-ous patients. Such analysis will specify selection ofanalyzed gene regions for PCR diagnostics of gastrictumors. Employment of quantitative analysis of methy-lation level by means of quantitative PCR would makesubstantial contribution in solution of this problem.Using this method it will become possible to evaluatebasal methylation levels and to elucidate differencesbetween malignant and benign tumors.

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3. CIRCULATING DNA

Studies on molecular genetic markers described inthe previous section were carried out using specimensof tumors tissues obtained by means of biopsy or afteroperation. However, biopsy is possible only in the caseof visually detected tumor and thus this method is inap-plicable for detection of early stage of tumor develop-ment or monitoring of recurrent cancer. The most per-spective modern direction is noninvasive detection oftumor markers by analysis of biological liquids such asblood, urine, bronchoalveolar lavage, duct liquids, etc.[48, 107]. The development of such methods becamepossible only recently after detection of free circulatingDNA (cirDNA) in blood plasma of healthy individualsand demonstration of increased plasma DNA content insome disease (e.g., autoimmune pathologies, diabetes,traumas, tumors) [48, 117]. It was especially importantthat changes in blood cirDNA (genetic and epigenetic)were identical to those seen in DNA from tumor cellsof the same patients [117]. However, in most cases fre-quency of detection of molecular genetic markers inblood plasma of cancer patients was significantly lowerthan that in tumor tissue specimens [48].

Certain attempts were undertaken to find moleculargenetic and epigenetic markers within blood cirDNA ofpatients with gastric cancer. Wang et al. found muta-tions of p53 and APC genes in 59% of blood serumspecimens of the patients with gastric cancer andwith the same mutations detected in tumor tissue [32].Lee et al. [84] compared frequency of detection ofmethylated p15 gene in tumor tissue and in a DNA poolisolated from blood serum. Hypermethylation of thepromoter region of this gene was found in 68% of tissuespecimens from examined patients and similar changeswere also detected in 81% of serum DNA.

Comparison of methylation status of p16 gene inbiopsy specimens and serum revealed serum methyla-tion only in 26% of patients, which had methylation ofp16 gene determined in biopsy specimens [118].

Based on comparative analysis of methylation ofseveral genes (APC, E-cadherin, hMLH1, TIMP-3) inserum and corresponding specimens of adenocarci-noma Leung et al. have concluded that detection ofhypermethylated gene in serum gives 100% confidencefor the presence of the change in corresponding tumortissue [86].

In has recently been shown that extracellular DNAsnot only freely circulate in blood plasma, but they maybe bound to surface of blood cells. cirDNAs bound tosurface of blood cells represent the major proportion ofcirDNA (more than 90%) in blood of healthy donors,whereas in the cases of tumors of various localizationthere is redistribution between free plasma cirDNA andcirDNA bound to cell surface [119]. It should be notedthat there are tumor specific sequences of DNAdetected in both cell surface bound cirDNA and plasmaDNA [120]. Use of total extracellular blood DNA inPCR significantly increases sensitivity of analysis of

methylation of tumor suppressor genes. It was demon-strated that hypermethylated forms of RAR

β

2,RASSF1A, HIC-1 genes were detected in blood plasmaDNA in 15–55% of patients with breast cancer and intotal DNA in 65–90% of patients [121]. This suggeststhat the problem of lower frequency of detection ofmolecular genetic markers in blood compared with cor-responding analysis of gastric cancer tissue may be suc-cessfully overcome.

Now tumor specific sequences are successfullyinvestigated in extracellular DNAs of other biologicalbody fluids from patients with tumor diseases of vari-ous localizations. Tumors originating from epitheliallining of human body lumens represent a major propor-tion of all malignant tumors in man. Desquamatedtumor cells appear in a lumen of corresponding organand they are then excreted from the body. Thus, detec-tion of specific DNA markers in natural excrements is asensitive mode of diagnostics and monitoring of tumorsof corresponding organs. In colorectal cancer the fecesanalysis is used, in pancreatic cancer DNA studyemploys a pancreatic secrete, in prostate cancer andbladder cancer urinary DNA is investigated [48]. How-ever, in gastric cancer such studies are not widely used.This may be associated with difficulties of specimencollection and limited excretion of material (requiredfor such examination) through biological fluids.

CONCLUSIONS

In spite of evident achievements in the developmentof molecular diagnostic methods for tumor diagnostics,there are many unsolved problems in diagnostics ofgastric cancer. These include need in the increase ofsensitivity and specificity of these methods. Mostgenetic and epigenetic changes found in genes consid-ered in this review take place not only in gastric cancerbut also in non-malignant diseases. It still remainsunclear whether molecular genetic (and epigenetic)markers found in benign tumors suggest possibility ofmalignant transformation of these tumors. Data on agerelated changes require detailed correlation analysis ofgenetic/epigenetic modification and age of examinedpatients. Obviously, subsequent search of genes selec-tively involved into the development of gastric cancer isneeded.

Nevertheless, there are clear perspectives in thedevelopment of molecular genetic methods of analysisand directions increasing diagnostic values of thesemethods. Use of total circulating blood DNA, theincrease in sensitivity and specificity of PCR analysisdue to real-time detection of a reaction product, combi-nation of several markers, long-term studies on detec-tion of correlations between identified markers andtumor development may probably culminate in thedevelopment of diagnostic tests required for practicalmedicine.


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