Development of In-House Multiplex Real Time PCR for Human Papillomavirus Genotyping in Iranian Women with Cervical Cancer and Cervical Intraepithelial Neoplasia

Human Papillomaviruses (HPVs) that comprise a large genus belonging to the Papillomaviridae, is a non enveloped virus with a circular double stranded DNA (ds DNA) genome approximately 7.9 kb in size. The genome encodes 6 early genes (E1, E2, E4, E5, E6, and E7) and 2 late genes (L1 and L2). L1 is the major of viral capsid that targeted at a conserved region is used for phylogenetic classification and molecular diagnosis. It has more than 100 subtypes that are capable of infecting the epithelial cells. Depending on the oncogenic potential, its genotypes are categorized into High Risk (HR) and Low Risk (LR) groups. HPVs can cause a variety of clinical manifestations such as warts, anogenital malignancies, cervical cancer and etc. Cervical cancer is the third most common cancer in women worldwide (Laudadido, 2013; Shayanfar et al., 2013). Globally, cervical cancer accounts for more than 529,000 new cancer cases and more than 275,000 cancer deaths in women annually (Jemal et al.,


Introduction
Human Papillomaviruses (HPVs) that comprise a large genus belonging to the Papillomaviridae, is a non enveloped virus with a circular double stranded DNA (ds DNA) genome approximately 7.9 kb in size.The genome encodes 6 early genes (E1, E2, E4, E5, E6, and E7) and 2 late genes (L1 and L2).L1 is the major of viral capsid that targeted at a conserved region is used for phylogenetic classification and molecular diagnosis.It has more than 100 subtypes that are capable of infecting the epithelial cells.Depending on the oncogenic potential, its genotypes are categorized into High Risk (HR) and Low Risk (LR) groups.HPVs can cause a variety of clinical manifestations such as warts, anogenital malignancies, cervical cancer and etc. Cervical cancer is the third most common cancer in women worldwide (Laudadido, 2013;Shayanfar et al., 2013).Globally, cervical cancer accounts for more than 529,000 new cancer cases and more than 275,000 cancer deaths in women annually (Jemal et al.,

Development of In-House Multiplex Real Time PCR for Human Papillomavirus Genotyping in Iranian Women with Cervical Cancer and Cervical Intraepithelial Neoplasia
Amir Sohrabi 1 , Siamak Mirab-Samiee 2 , Mohammad Hossein Modarresi 3 , Narges Izadimood 4 , Kayhan Azadmanesh 5 , Marjan Rahnamaye-Farzami 6 * 2011), with more than 80% of these cases occurring in developing countries (Li et al., 2013).Different methods are available for the HPVs screening, detection and genotyping such as cytologic evaluations (e.g.Pap Tests), PCR Hybridizations, PCR and Real Time PCR.HPV vaccination with cervical cancer screening (via both cytology and HPV molecular assays) can be effective in preventing cervical cancer and decrease its mortality (Jemal et al., 2011;de Freitas et al., 2012;Adams and Carnright, 2013).Based on the data reported by WHO, the crude incidence and mortality rates of HPV related cervical cancer until 2010 in Iran are about 1.8 and 0.8 per 100,000 per year, respectively.HPVs 16(HR), 18(HR), 31(HR), 6(LR), 11(LR), 33(HR) are the most common HPV genotypes in Iranian women with and without cervical dysplasia.Comparing the alternate national cancer registry reports in Iran shows a slow increase in rate of cervical cancer in country and health authorities have a great concern to conduct country wide programs for estimating the latest prevalence of HPV infection in general population (WHO, 2010).In parallel, there is an increasing request for HPV detection and genotyping by clinicians that has led to considerable rise in new introduced commercial and in-house molecular assays for HPV diagnosis.Regarding the above mentioned explanation access to accurate diagnostic methods with a reasonable cost is a necessity in Iran as developing country.This survey was conducted to design and develop a HPV genotyping assay by In-House Multiplex Real Time PCR in Iran.The quality and performance of developed assay is assessed based on an evaluation protocol provided in national reference laboratory.

Patients' Specimens
From March 2012 to April 2013, a total of 112 samples (48 ThinPrep and 64 Archival Formalin-Fixed Paraffin-Embedded Tissue (FFPE) specimens) from women with known cervical precancerous and cancer referred to Tehran Women Central hospital, Imam Khomeini Hospital and Imam Hossein Hospital in Tehran, were collected.All the diagnoses were confirmed in histopathology examinations.A questionnaire was designed and filled out for each patient and necessary clinical data was recorded.In order to meet ethical considerations, each patient was informed about the objectives of study and a signed a consent form before entering the study.After cytologic examination of specimens and confirming the histopathologic diagnoses, collected Liquid Based Cytology (LBC) and FFPE blocks were transported to Molecular Biology department in Health Reference Laboratory of Ministry of Health and Medical Education, Iran.The LBCs were stored at -20̊ C and FFPE tissue blocks in a proper storage area until experimental phase.

Extraction of HPV DNA
Viral DNA from Thin Prep preservatives and 20-30 mg FFPE specimens was extracted using the QIAamp DNA Mini kit and QIAamp DNA FFPE Tissue kit (Qiagen, Germany), according to the manufacturer's instructions.

Design of primers and probes
We developed and designed 15 set of specific primers and TaqMan probes on the partial region of L1 HPV genotypes sequences by AlleleID 7.5 software.All of sequences comprehensive aligned by Mega5 software and blasted in NCBI database (www.ncbi.nlm.nih.gov/nucleotide).Analysis of extensive bioinformatics was done on primers and probes designed by Oligo7, Primer Express, Generunner, CLC Main Workbench 5 software's and internet online sites.So, β globin endogenous gene as internal control primers and probe were considered in Multiplex Real Time PCR for ruling out possible PCR inhibition and quality of extracted DNA.

Analytical sensitivity, specificity and accuracy
The quality and performance of developed assay was evaluated focusing on analytical sensitivity (detection limit), specificity and accuracy.
Analytical specificity: the extracted genome of HBV, HCV, HSV1 and 2, VZV, BKV, CMV, Streptococcus pneumonia, Klebsiella pneumonia and Ecoli, 10 HPV negative and 20 HPV positive samples (both as single specimens and mixed cocktails with different copy numbers of each genotype per micro liter from 1 to 1000) were selected.There was no non specific amplification signal in all samples.
Analytical sensitivity: 20 HPV positive samples (15 high risks and low risks HPV genotypes) were tested and only one sample (genotype 52) was falsely negative.
Detection limit: serial dilutions of HPV plasmids were prepared (from 1 to 1000 copy /µl) and the assay could detect 5 copy/µl of each HPV genotypes in single and mixed cocktails.Data for these evaluations are not shown.
Standard curves obtained from HPV plasmids serial dilutions in HPV Mix 1 to HPV Mix 4 showing dynamic ranges from 10 Copies/µl to 1000 Copies/µl are indicated in Figure 1.

Human papillomaviruses genotyping
HPV genotyping was assessed by testing 112 samples (48 Liquid Based Cytology and 64 Archival FFPE) collected from patients with confirmed diagnoses of cervical dysplasia and cancer.105 out of 112 specimens (93.7 %) were positive for different Human Papillomavirus genotypes and 7 samples (6.3 %) were negative.Demographic data of selected patients consist     , 68; 1/105 (0.96%)), respectively.Among samples with the HPV mixed genotypes, HPV 16, 18 (13/105 (12.4%) were the prevalent combination genotypes.We also participated in 2013 WHO HPV LabNet proficiency study with our developed assay.The panel of specimens in this program consists of different single and mixed cocktails of HPV plasmids containing HPV genotypes 6,11,16,18,31,33,35,39,45,51,52,56,58,59, 66 and 68b.To be considered as proficient in this program, the assay shall detect 50 International Units (IU)/5 µl of HPV 16 and HPV 18 DNA, and 500 genome equivalents (GE)/5 µl of the other HPV types included in the panel both in samples contains single and multiple plasmids and no more than one false positive result is accepted.Accordingly, our data set using the In-House Multiplex Real Time PCR genotyping assay is recognized as proficient for detection of HPV 6,11,16,18,31,33,35,39,45,51,52,56,58,59, 66 and 68b with no false positive result.

Discussion
To identify HPV genotypes in positive samples detected in cervical screening (i.e., Pap tests), molecular based methods are the most common tests and they can differentiate various high and low risk genotypes.Real Time PCR is one of the revolutionized molecular diagnostic methods that is used as a routine diagnostic assay for rapid detection and genotyping of clinical samples.
In Conclusions, It seems that molecular methods e.g.PCR and Real Time PCR as high throughput, reliable methods with capability of fast detection and genotyping must be developed and used in combination of pap smear in order to proper screening and diagnosis in women with cervical epithelial lesions.Certainly, reliable results obtained from valid diagnostic methods have an important role in selecting best national policies particularly for immunization and prophylactic programs.

Figure 1 .
Figure 1.A) Standard Curves of HPV Plasmids Mix 1 (HPV 16, 18, 31, 45) as Positive Controls by Serial Dilutions from 10 copies/µl to 1000 copies/µl in Order to Analysis of the Assay LOD and Analysis of Linearity Range.B) Standard Curves of HPV plasmids Mix 2 (HPV 6, 11, 52, 68)  as positive controls by serial dilutions from 10 copies/µl to 1000 copies/µl in order to analysis of the assay LOD and analysis of linearity range.C) Standard Curves of HPV plasmids Mix 3 (β Globin, HPV 56, 58, 59) as positive controls by serial dilutions from 10 copies/µl to 1000 copies/µl in order to analysis of the assay LOD and analysis of linearity range.D) Standard Curves of HPV plasmids Mix 4(HPV 33, 35, 39, 51)  as positive controls by serial dilutions from 10 copies/µl to 1000 copies/µl in order to analysis of the assay LOD and analysis of linearity range

Table 2 . Analysis of Relationship between the HPV Genotypes and Cytopathology Reports
Detected HPV genotypes are shown in Figure2.Among detected HPV genotypes, HPV 18 (69/61.6%)andHPV16(48/42.9%)weredominantones.Demographic data of selected patients consist of Age, Marital status, Educational level and history of abortion in previous pregnancies are shown in Table1 and Figure 3. Furthermore, distribution of HPV genotypes in comparison to histopathology diagnoses are mentioned in Table2.From the 105 HPV genotypes and 7 samples (6.3 %) were negative.