Abstract Gastric cancer is the second leading cause of cancerrelated

deaths worldwide and it seems that environmental and

lifestyle factors and infection with Helicobacter pylori (H.pylori)

have had a major role in the etiology of gastric cancer. The aim

of this study was to investigate the presence of H. pylori DNA in

archival gastric tissues of patients with gastric cancer disease by

rapid, sensitive and specific technique of Scorpion Realtime

PCR. This retrospective cross-sectional study was performed

on 285 paraffin embedded gastric specimens of patients who

were pathologically proved for gastric cancer admitted in Bou-

Ali, Shahid Rajaie and Dehkhoda hospitals and Bahar and

Farzam private laboratory in Qazvin city in Iran during 2009

and 150 paraffin embedded pathological specimens of patients

with other proved diagnosis other than gastric cancer. Results of

our Scorpion Realtime PCR analysis showed that DNA of H.

pylori DNA was present in 78.42 % of our total specimens.

Modified McMullen’s Staining of paraffin embedded sections

was positive in 210 patients. Also we were not able to finding

significant relationship between demographic characteristics of

our studied patients and presence of H. pylori DNA in their

formaldehyde fixed paraffin embedded gastric tissues samples.

Existence of H. pylori in gastric tissue samples of patients with

gastric cancer is controversial and our results indicated that in our

studied specimens prevalence of H.pylori was significantlymore

than recent published reports

Azimi, Akram

Bahrami, Mohammad

Farzam, Amir

Haj Manouchehri, Fatemeh

Jahanihashemi, Hassan

Johari, Pouran

Najafipour, Reza

Naserpour Farivar, Taghi

Nasirian, Neda

Qazvin University of Medical Sciences Repository

RESEARCHThe Relationship Between Gastric Cancer and Helicobacter Pyloriin Formaldehyde Fixed Paraffin Embedded Gastric Tissues of GastricCancer Patients-Scorpion Real-Time PCR Assay FindingsTaghi Naserpour Farivar & Pouran Johari & Reza Najafipour &Amir Farzam & Neda Nasirian & Fatemeh HajManouchehri &Hassan Jahani Hashemi & Akram Azimi & Mohammad BahramiReceived: 7 December 2012 /Accepted: 26 June 2013 /Published online: 1 November 2013# Arányi Lajos Foundation 2013Abstract Gastric cancer is the second leading cause of cancer-related deaths worldwide and it seems that environmental andlifestyle factors and infection withHelicobacter pylori (H.pylori)have had a major role in the etiology of gastric cancer. The aimof this study was to investigate the presence ofH. pyloriDNA inarchival gastric tissues of patients with gastric cancer disease byrapid, sensitive and specific technique of Scorpion RealtimePCR. This retrospective cross-sectional study was performedon 285 paraffin embedded gastric specimens of patients whowere pathologically proved for gastric cancer admitted in Bou-Ali, Shahid Rajaie and Dehkhoda hospitals and Bahar andFarzam private laboratory in Qazvin city in Iran during 2009and 150 paraffin embedded pathological specimens of patientswith other proved diagnosis other than gastric cancer. Results ofour Scorpion Realtime PCR analysis showed that DNA of H.pylori DNA was present in 78.42 % of our total specimens.Modified McMullen’s Staining of paraffin embedded sectionswas positive in 210 patients. Also we were not able to findingsignificant relationship between demographic characteristics ofour studied patients and presence of H. pylori DNA in theirformaldehyde fixed paraffin embedded gastric tissues samples.Existence of H. pylori in gastric tissue samples of patients withgastric cancer is controversial and our results indicated that in ourstudied specimens prevalence ofH.pyloriwas significantly morethan recent published reports.Keywords Scorpion . Real-time .Helicobacter pylori .Gastric . CancerIntroductionGastric cancer is the second leading cause of cancer-relateddeaths worldwide. International Agency for Research onCancer reported that in Asia malignancy of the stomach isthe most common cancer and nearly two-thirds of it occurs indeveloping countries [1]. Although the incidence and mortal-ity of distal gastric cancers has decreased in the Westernworld, the incidence of proximal tumors is increasing in themale population of those regions. In contrast, in Asian conti-nent distal tumors have increased while proximal tumors havedecreased [2]. This geographic variation accompanying withdifferences in lifestyle, time trends and migratory effectsassociated with the incidence of gastric cancer suggest thatenvironmental and lifestyle factors have a major role in theetiology of gastric cancer [3, 4].Gastric cancer is an outcome of a complex interactionbetween host and environmental factors and H. pylori infec-tion. Scientific evidence suggests the importance of host fac-tors and microbial agents in pathogenesis of cancer [5, 6].Helicobacter pylori (H. pylori) are Gram-negative, micro-aerophil and spiral-shaped bacteria. H. pylori infection affects50 % of the population, worldwide [7]. H. pylori infectioninitiate an inflammatory response which is influenced bygenetic polymorphisms which in turn can either accentuateT. Naserpour Farivar : P. Johari :R. Najafipour (*) :A. Farzam :N. Nasirian : F. HajManouchehri :H. Jahani Hashemi :A. Azimi :M. BahramiCellular and Molecular Reasearch Center, Qazvin Universityof Medical Sciences, Qazvin, Qazvin, Irane-mail: rnajafipour@gmail.comT. Naserpour Farivare-mail: t.naserpour@qums.ac.irPathol. Oncol. Res. (2014) 20:113–117DOI 10.1007/s12253-013-9669-2or attenuate the host’s response to inflammation and affectingthe interaction’s outcome. Recent advances in the study of theetiological backgrounds of non-cardia gastric adenocarcinomarevealed that H. pylori infection is an established importantcausal factor for this kind of cancer.In the process of gastric carcinogenesis, bacterial virulencefactors that have been implicated include the cytotoxin-associated gene A antigen (Cag A), vacuolating cytotoxin(VacA), and outer membrane proteins (OMP) [8]. The cagEgenotype has been associated with gastric cancer in somestudies, but contrary results have also been published [9, 10].On the basis of these differences of opinion about theprevalence of H. pylori association with gastric cancer existsin the literature [11–17] we planned this study to determine theoverall prevalence of H. pylori DNA in gastric carcinoma.Materials and MethodsPatients and Methods This retrospective cross-sectionalstudy was performed on 285 paraffin embedded gastric speci-mens of patients who were pathologically proved for gastriccancer admitted in Bou-Ali, Shahid Rajaie and Dehkhoda hos-pitals and Bahar and Farzam private laboratory in Qazvin city inIran during 2009. Ethical Committee of Qazvin University ofMedical Sciences approved our study. Control samples were 150paraffin embedded pathological specimens of patients with otherproved diagnosis other than gastric cancer.Clinical Samples Initially, the paraffin embedded gastric tis-sues were sectioned into 50 μm slices followed by DNA extrac-tion using DNA preparation protocol obtained from Section ofCancer Genomics, Genetics Branch, NCI, Institute of Health.Modified McMullen’s Staining of Gastric Biopsy The paraf-fin embedded sections was dewaxed, dehydrated and cov-ered by carbol-fuchsin for 2 min. The sections rinsed andstained malachite green for 2 min. Then slides rinsed in tapwater, air dried and observed under the light microscope.Magenta color particles are indicative of H. pylori.Scorpion Real-Time Scorpion real-time PCR was performedusing ABI Prism 7500 Sequence Detection System (AppliedBiosystem, USA). The tails of the Scorpion primers weredesigned as previously described byBurucoa [18]. All primersand probes used were synthesized by Metabion company(Germany) (Table 1). The total volume of the real-time PCRwas 20 μl containing 5 μl of DNA from a clinical sample orbacterial isolate, 0.1 μMof oligonucleotide primer 23SF2 and0.08 μM of 23SScWT, while the total volume of 20 μl wasachieved by addition of distilled water. The cycling conditionswere adjusted in a way suitable to be used by ABI Prism 7500Sequence Detection System (Applied Biosystem, USA) withan initial denaturation at 95 °C for 45 s, 50 cycles at 95 °C for15 s, 55 °C for 34 s, and 72 °C for 20 s. The acquisition of asignal was performed at 57 °C during each cycle. A negativecontrol for Scorpion real-time PCR obtained by observation ofno amplification signal except internal amplification byadding DDW instead of DNA to the prepared real-time PCRmaster mix. A positive control for Scorpion PCR was consti-tuted by using extracted DNA from H. pylori ATCC 26695.Positive extraction control was performed for each biopsyspecimen in a separate tube in a final volume of 25 μl withPremix Ex Taq (TaKaRa, Shiga, Japan), 5 μl of extractedDNA from biopsy specimen, 0.25 μM of BGLO1 andBGLO2 primers, and 0.5× Sybergreen 1 (Sigma Aldrich)and on the basis of the results of this test, 285 of the collectedbiopsies were entered in our study. The cycling program was1 cycle at 95 °C for 10 s and 40 cycles of 95 °C for 5 s, 55 °Cfor 34 s, and 72 °C for 10 s.Statistical Analysis Data were analyzed using SPSS11.5software with 95 % confidence intervals (95 % CI).ResultsIn this survey the prevalence of H. pylori infection in gastricsamples of patients with gastric cancer was determined usingthe data obtained by:Detection of H pylori by Scorpion Real-Time PCR:Initially, Scorpion real-time PCR was performed on H.pylori ATCC 26695. Of total 285 samples, 224 biopsiesfound to be positive for H. pylori by Scorpion real-timePCR (78.49 %) whereas 61 samples showed negativeresults. Among samples with positive Scorpion real-Table 1 Primers and probes se-quences for detection of H.pyloriin pathological specimens ofgastric patients and controlsOligonucleotide Sequence23SScWT 5′-FAM-AAGGTAGGTGAAAATTCCTCCTACC BHQ1 HEGGGACCACGGGGTCTTT-3′23SF2 5′-TGCGAACTGTGTGTTCACTAGC-3′BGLO1 5′-ACACAACTGTGTGTTCACTAGC-3′BGLO2 5′-CAACTTCATCCACGTTCACC-3′114 T. N. Farivar et al.time PCR test, 210 samples were also positive withModified McMullen’s Staining (93.75 %). From of total150 control samples 47 biopsies found to be positive forH. pylori by Scorpion real-time PCR 47 (31.3 %) where-as 103 samples showed negative results. Among sam-ples with positive Scorpion real-time PCR test,40(85.1 %) samples were also positive with ModifiedMcMullen’s Staining.Scorpion Real-Time PCR: Scorpion real-time PCR onDNA extracts of H. pylori ATCC 26695 as controlstrains produced expected signals. The standard curveused to calculate the concentration of H. pylori DNA inunknown samples is presented in Fig. 1a. The linearregression coefficient was 0.996 and the efficiency ofPCR 92.054 %. Figure 1b shows the fluorescent curves ofthe standard serial dilution from 1×104 to 1×102 copy permicroliter. Electrophoresis pattern of products in scorpionreal-time PCR assay in agarose gel is shown in Fig. 2. A140 bp fragment in the peptidytransferase gene of 23SrRNA showed a successful amplification. Of total patients(285 cases) with gastric cancer who entered the study, 224cases (78.24 %) were found to haveH. pyloriDNA in theirgastric biopsy specimens by Scorpion real-time PCRmeth-od. Our study showed that there is no significant associa-tion between demographic characteristics of our patients,history of gastric involvement, gastric disease, and thepresence of bacterium. Also we were not able to find anyrelationship between the sex, family income, and accom-modation variables and the colonization of H. pylori ingastric cancer tissues (data not shown).Fig. 1 a Standard curve used to calculate the concentration of H.pyloriDNA in unknown samples. The linear regression coefficient is 0.996and efficiency of PCR is 92.054 %; b Fluorescent curves of the standarddilution series. From the left to the right 104, 103, 102 and the negativecontrol is presented by the horizontal curve8 9Fig. 2 The Scorpion Realtime PCR products of tested samples. PCR products were electrophoresed in a 2 % agarose gel. Lane 8: Helicobacter pyoriATCC26695 with a positive 140 bp band; Lane 9:100 bp DNA ladder. Other lines: Scorpion PCR products of samplesHelicobacter pylori in gastric cancer patients 115DiscussionH. pylori infects human in childhood and at this time it hasinfected one half of the world’s population. It is the onlybacterium which is listed in the microbial agents which areinvolved in human cancer diseases.Our finding showed that from the total of 285 formalde-hyde fixed paraffin embedded samples of pathologicallyproved gastric cancer patients which enterd to our study, 224patients had H. pylori DNA while Hashemi and colleaguesreported that in their 161 studied normal mocusa specimens ofIranian population 54 had H.pylori (33.5 %) [19].The results of published researches in the medical litera-ture related to the involvement of H. pylori in gastric canceris controversial and confirmation of this involvement anddetermination of the level of this involvement in differentsocieties are also important issues.As H. pylori is a fastidious bacteria, microbiologicalmethods of culture and identification of these bacteria aredifficult and needs a long time and adequate sensitivity andspecificity of modern molecular methods in diagnosis ofthese bacteria make them attractive methods for studyingprevalence of H. pylori in different clinical specimens [6].Results of our study showed that from total 224 number ofspecimens with H.pylori DNA, all had CagA positive geno-type (Data not shown).Khanna et al. in a study on 50 proved cases of gastriccancer found that the prevalence rate ofH. pylori infection intheir patients was lower than in the control population,suggesting that H. pylori may not be responsible for gastriccarcinogenesis in this population [16]. In other study Rudiand colleagues in their study on the sera of 111 Caucasianpatients with histologically confirmed gastric cancer foundthat their data do not provide evidence that the contributionof H. pylori infection to the carcinogenesis of gastric canceris of major significance at least in a population with lowgastric cancer rates and with high socioeconomic status [17].On the other hand Babus and colleagues in a study onanalysis of 12 prospective case—control studies concluded thatbetween about 65 % and 80 % of non-cardia gastric cancerswere attributable to H. pylori infection [13, 19]. Uemura et al.studied 1,526 Japanese patients, of whom 1,246 had H. pyloriinfection and 280 were not infected. Over a mean follow-upperiod of 7.8 years, gastric cancer developed in 2.9% of patientswith H. pylori infection and none of the uninfected patientsdeveloped gastric cancer, giving a relative risk of 34.5 [14].Martínez and colleague in a case-control study including 46gastric cancer cases and 99 controls with non-atrophic gastritisfrom a high risk zone for gastric cancer showed that there is apositive association between Helicobacter pylori CagA posi-tive strains with non-cardial gastric cancer etiology [15].Although we studied a relatively large population and weused sensitive and specific method of scorpion real time PCRassay, our results indicated that in our studied specimensprevalence of H.pylori was many times more than recentpublished reports [20, 21] and is in consistent with the latterstudies indicating a possible role for H. pylori in gastriccancer and observed higher rate of prevalence of these bac-teria in our patients, may lay back in the polymorphism andgenetic diversity of isolated H.pylori strains which influencethe oncogenic potential of these bacteria [22]Acknowledgment The authors wish to thanks Deputy of Research inQazvin University of Medical Sciences for Financial support of this study.References1. Ferlay J, Bray F, Pisani P (2001) GLOBOCAN 2000: Cancerincidence, mortality and prevalence worldwide, version 1.0. IARCCancer Base No 5. IARC Pres, Lyon, 12. Crew KD, Neuget AI (2006) Epidemiology of gastric cancer. WorldJ Gastroenterol 12:354–3633. Tiwari SK, Manoj G, Vasanth Kumar G, Sivaram G, Hassan SI(2008) Prognostic significance of genotyping Helicobacter pyloriinfection in patients in younger age groups with gastric cancer.Postgrad Med J 84:193–1974. Ries LAG, Kosary CL, Hankey BF (1994) International Agency forResearch on Cancer Working Group on the Evaluation of CarcinogenicRisks to Humans. Schistosomes, liver flukes, and Helicobacter pylori.International Agency for Research on Cancer, Lyon, pp 177–2405. Fox JG, Wang TC (2007) Inflammation, atrophy, and gastric can-cer. J Clin Invest 117:60–696. Naserpour Farivar T, Pahlevan AK, Johari P (2012) Assessment ofHelicobacter pylori prevalence by scorpion Real-time PCR inchronic tonsillitis patients. J Global Infec Dis 4:38–427. Correa P, Piazuelo MB (2008) Natural history of Helicobacterpylori infection. Dig Liver Dis 40:490–4968. McNamara D, El-Omar E (2008) Helicobacter pylori infection andthe pathogenesis of gastric cancer: a paradigm for host-bacterialinteractions. Dig Liver Dis 40:504–5099. Fock KM, Dhamodaran S, Teo EK (2006) Is H. pylori genotypessignificant in Asian patients with gastric cancer and GERD. Gut55(Suppl. V):A25810. Erzin Y, Koksal V, Altun S (2006) Prevalence of Helicobacterpylori vacA, cagA, cagE, iceA, babA2 genotypes and correlationwith clinical outcome in Turkish patients with dyspepsia.Helicobacter 11:574–58011. Chomvarin C, Namwat W, Chaicumpar K (2008) Prevalence ofHelicobacter pylori vacA, cagA, cagE, iceA and babA2 genotypesin Thai dyspeptic patients. Int J Infect Dis 12:30–3612. Ghasemi A, Shirazi MH, Ranjbar R (2008) The prevalence of cagAand cagE genes in Helicobacter pylori strains isolated from differ-ent patient groups by polymerase chain reaction. Pak J Biol Sci11:2579–258313. Babus V, Strnad M, Presecki V (1998) Helicobacter pylori andgastric cancer in Croatia. Cancer Lett 125:9–1514. Uemura N, Okamoto S, Yamamoto S (2001) Helicobacter pyloriinfection and the development of gastric cancer. N Engl J Med345:784–789116 T. N. Farivar et al.15. Martínez T, Hernández-Suárez G, Mercedes Bravo M, Trujillo E,Quiroga A, Albis R (2011) Association of interleukin-1 ge-netic polymorphism and CagA positive Helicobacter pyloriwith gastric cancer in Colombia]. Rev Med Chil 139:1313–132116. Khanna AK, Seth P, Nath G (2002) Correlation of Helicobacterpylori and gastric carcinoma. J Postgrad Med 48:27–3517. Rudi J, Müller M, von Herbay A (1995) Lack of association ofHelicobacter pylori seroprevalence and gastric cancer in a popu-lation with low gastric cancer incidence. Scand J Gastroenterol30:958–96318. Burucoa C, Garnier M, Silvain C, Fauchère JL (2008) Quadruplexreal-time pcr assay using allele-specific scorpion primers fordetection of mutations conferring clarithromycin resistance tohelicobacter pylori. J Clin Microbiol 46:2320–232619. Hashemi MR, Rahnavardi M, Bikdeli B, Dehghani Zahedani M(2006) H pylori infection among 1000 southern Iranian dyspepticpatients. World J Gastroenterol 12(34):5479–548220. Bauer B, Meyer TF (2011) The human gastric pathogenhelicobacter pylori and its association with gastric cancer and ulcerdisease. Ulcers 2011, Article ID: 40157, 23 pages21. McColl K (2010) Helicobacter pylori infection. N Engl J Med362:1597–160422. Fuccio L, Eusebi LH, Bazzoli F (2010) Gastric cancer, Helicobacterpylori infection and other risk factors. World J Gastrointest Oncol2:342–347Helicobacter pylori in gastric cancer patients 117

The Relationship Between Gastric Cancer and Helicobacter Pylori

in Formaldehyde Fixed Paraffin Embedded Gastric Tissues of Gastric

Cancer Patients-Scorpion Real-Time PCR Assay Findings

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The Relationship Between Gastric Cancer and Helicobacter Pylori in Formaldehyde Fixed Paraffin Embedded Gastric Tissues of Gastric Cancer Patients-Scorpion Real-Time PCR Assay Findings

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