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Human Papilloma Virus Prevalence among Women with Cervical Intraepithelial Neoplasia III and Invasive Cervical Cancer from 2001 till 2006 in BandarAbas,Iran

A. Sobhani (1) ,Z.Etaati (2), A.Sadeghi (1),  A.SH Jahanlou(3), M.Shiroodi (4)

Correspondence author:A.Sadeghi

Abstract : To estimate the risk of Human Papilloma Virus (HPV) infection for cervical malignancies we conducted a case-control study in Iran.52 paraffin embedded blocks with exact diagnosis of cervical carcinoma from years 2001 till 2006 and 52 praffin embedded blocks of cervical tissue specimens with normal histopathology as the control group were tested for the presence of HPV DNA using PCR based assay.HPV DNA was found in 16 out of 52 patients (30.7%) while it was not detected in any of the control group.

Introduction: 

Cervical cancer is one of the most prevalent forms of carcinoma among women world wide and accounts for about 12% of all cancer cases among women with an incidence of more than 400/000 cases per year.[1]Yet some studies have shown that cervical cancer is the fifth most frequently seen cancer and the second most common cancer in women world wide.[2]

The association between certain Human Papilloma Viruses (HPVs) and cervical cancer is well documented and research over the past 2 decades has convincingly revealed that HPVs are etiologically related to the development of most cases of cervical cancer.[3-5] There is no exact statistical data of cervical cancer prevalence among Iranian women however cervical cancer like other forms of genital cancer is more prevalent in the northern part of Iran including Mazandaran province.[6],but generally the association between HPVs and cervical cancer has not been studied among Iranian women except for Mazandaran province.[7]

The main goal of our case-control study was PCR based assay of the association of HPV DNA with cervical cancer in patients admitted to BandarAbas Shariati hospital in Hormozgan province (south of Iran) since years 2001 till 2006.

Methods :

Specimens: We extracted paraffin embedded blocks of cervical tissue specimens with histopathologic diagnosis of cervical carcinoma in  the years 2001 till 2006 from archive of the pathology department of BandarAbas Shariati hospital.After revision of corresponding slides and assurance of consistency ,2 drops out occurred and finally 52 paraffin embedded blocks with exact diagnosis of cervical carcinoma (50 carcinomas and 2 carcinoma in situs)were entered in our study.In parallel we selected 52 blocks of paraffin embedded cervical tissue specimens with normal histopathology as the control group.We tried to age match the control group with the study group.

1-Department of pathology,BandarAbas University of Medical sciences 2-Department of gynecology and obstetrics,BandarAbas University of Medical Sciences3-Department of Educational medicine,BandarAbas University of Medical Sciences 4-Department of genetics,BandarAbas University of Medical Sciences

DNA extraction: Sections of 5-10 micrometer wide were prepared from each specimen,avoiding any cross contamination between samples (using separate disposable items such as gloves,blades and tubes).Sections were subsequently deparaffinized by xylene and digested using digestion buffer containing proteinase K.[8],followed by extensive extraction with phenol/chloroform[9].The extracted DNA was stored at 4 C until tested.

PCR: DNA quality was evaluated by PCR using primers G73/G74 that amplify a 268 bp product from the human b-globin gene.Then b-globin positive samples were subjected to HPV PCR by GP5+/GP6+ primers for L1 open reading frame (ORF) that amplifies a 150 bp product from the HPV L1 ORF.[10]

-G 73: 5'-GAAGAGCCAAGGACAGGTAC

-G 74: 5'-CAACTTCATCCACGTTCACC

-GP5+: 5'-TTGTTACTGTGGTAGATACTAC

-GP6+: 5'-GAAAAATAAACTGTAAATCATATTC

PCR was performed according to the procedure described by Yi Ting et al.[10] Samples were subsequently subjected to agarose gel electrophoresis (2% agarose),and stained with ethidium bromide.

Results:

HPV DNA was found in 16 out of 52 cases (30.7%) while HPV DNA was not found in any of the control group.(table-1)Out of 52 cases of cervical carcinoma 38 cases (73%) were squamous cell carcinomas,4 cases (7.6%) were adenocarcinomas, 6 cases (11.5%) were poorly differentiated carcinomas and 4 cases (7.6%) were carcinoma in situs (CIN III).At the same time 10 out of 38 (26.3%) squamous cell carcinomas, 2 out of 4 (50%) adenocarcinomas,2 out of 6 (33.3%) poorly differentiated carcinomas and 2 out of 4 (50%) carcinoma in situs were positive for HPV DNA.(table-2)

TABLE-1: PCR RESULTS IN STUDY AND CONTROL

 

TOTAL

NEGATIVE (%)

POSITIVE (%)

 

52

36(69.2)

16(30.7)

CASES

52

52(100)

0

CONTROLS

 

TOTAL

NEGATIVE

(%)

POSITIVE (%)

 

38

28(73.6)

10(26.3)

SCC

4

2(50)

2(50)

ADENOCARCINOMA

6

4(66.6)

2(33.3)

POORLY DIFFERENTIATED CARCINOMA

4

2(50)

2(50)

CARCINOMA IN SITU

    Table-2:PCR RESULTS IN VARIOUS CARCINOMA TYPES

 

 

 

Discussion:

Many epidemiological studies indicate that human cancer is a multifactorial disease,which can develop through different molecular biologic pathways.Currently there is compelling evidence that the development of human cervical cancer without the involvment of specific human papilloma viruses is exceptional or impossible.[11]The international biological study on cervical cancer demonstrated that 92.9% of cervical cancers from 22 countries contained HPV DNA ranging from 75 to 100%.[5] In our study  we found that the frequency of HPV DNA in our cervical carcinoma patients was 30.7%.This figure is much lower than what was found in Mazandaran province(78.6%) in Iran.[7]Also it is lower than what was reported from Brazil(76%),[12],Mozambique(92%),[13],and several other countries in latin America.[14&15]

Since studies have reported difficulties in reproducing PCR results with formalin fixed and paraffin embedded tumor tissues,therefore it is possible that if fresh frozen tumor tissue had been examined,HPV positive cases might have been higher because amplification could be hampered by formalin.[12] On the other hand our finding is much closer to the study performed in an Islamic country like Malaysia inwhich 45.1% of specimens were positive for HPV.[16]Hence we conclude that before generalizing the concept of low frequency of HPV infection in women of Islamic countries larger number of samples from different regions need be examined.

References:

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3- Franco E.Viral etiology of cervical cancer : a critique of the evidence .Reviews of infectious diseases,1991,13:195-206

4- Human papillomaviruses. Lyon,International Agency for Research on cancer, 1995 (IARC monographs on the Evaluation Carcinogenic Risks to Human,vol.64)

5-Bosch FXetal . prevalence of HPV DNA in cervical cancer in 22 countres.Journal of the National cancer Institute,1995,87:796-802.

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9-Snijders PJ et al.The use of general primers in the polymerase chain reaction permits the detection of a broad spectrum of human papilloma virus genotypes.Journal of general virology,1990,71:173-81

10-Innis MA etal . eds . PCR protocol . A guide to methods and applications . London , Academic Press , 1990.

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12-SH . Rabelo – Santos , L zeferino , I.L.Villa , JP Sobrinho , RG Amarial , AV Magalhaes . Human papilloma virus prevalence among women with cervical intraepithelial neoplasia III and invasive cervical cancer from Goiania , Brazil . Mem . Inst . Oswaldo Cruz . Mar.2003 ; Vo198

13-Pontus Noucler , Flora Mabota da Costa , Otto L Jungberg , Antonio Bugalho and Joakim Dillner . Human papilloma Virus genotypes in cervical cancers in Mozambique.J GenVirol.2004.85:2189-90

14-Marta Torroella – Kouri , Steven Morsberger , Adella Carrillo , Alejandro Mohar , Abelardo Meneses , Margarita Ibarra , et al . HPV prevalence among Mexican Women with neoplastic and normal cervixes . Gynecologic oncology . 1998 , 70:115-120 .

15-P Hindryckx , A Garcia , P Claeys , C Gonzalez , R Velasquez , J Bogers , et al. Prevalence of highrisk human papilloma virus types among Nicaraguan women with histological proved Preneoplastic and neoplastic lesions of the cervix. Sexually Transmitted Infections, 2006; 82: 334 -336

16-Tiang YP, Ngeow YF , Yap SF , Ngkp , Por P. HPV genotypes in cervical biopsies from Malaysian Women. The Malaysian Jourrnal of Pathology. Aug.2007 , 29:222-223.