Iranian Journal of Medical Microbiology

Sample Selection and Tumor Classification

 In this study, 101 formalin-fixed paraffin-embedded biopsy samples of different grades of the cervical lesion, including LSIL, high-grade squamous intraepithelial cell lesion (HSIL), SCC and Adenocarcinoma (ADC), were collected from archives of the department of pathology, Motahari Clinic of Shiraz University of Medical Sciences, Shiraz, Iran, during 2014-2015. Hematoxylin and eosin slides were retrieved, and the classification of different histologic subtypes of the cervix, including cervical intraepithelial neoplasia (CIN) I, II, III, and SCC, was reconfirmed by the pathologist according to the WHO Classification of Tumors of the Female Genital Tract classification system (18).

Sample Preparation and DNA Extraction

Ten μm-thick sections of Formalin-fixed, paraffin-embedded (FFPE) tissue blocks were subjected to deparaffinization as described previously (19). In brief, xylene was added to microtubes containing the samples, vortexed vigorously, incubated, and centrifuged (two times). Then the pellet was washed twice with 96% ethanol, vortexed, and centrifuged. The tubes were incubated at 42°C for evaporation of residual ethanol. After deparaffinization, genomic DNA was extracted by using the QIAamp FFPE Tissue Kit (QIAGEN-Hilden, Germany) according to the manufacturer's instructions. The extracted DNA was stored at -20°C until analysis.

Evaluation of the quality of extracted DNA

After DNA extraction, to check the quality of extracted DNA, a PCR test was carried out to detect the 111 bp product of the human β-globin as described previously (7).

Duplex Taq-Man Real-time PCR for Detection of HPV 16 and 18

Positive β-globin samples were processed to identify high-prevalence HPV genotypes, including HPV 16/18, using an in-house duplex real-time PCR described previously (20). Positive β-globin samples were processed to identify high-prevalence HPV genotypes, including HPV 16/18, using an in-house duplex real-time PCR, as described previously. Briefly, real-time PCR was performed using Ampliqon real Q Plus 2x Master Mix for the probe and each HPV genotype probe and primer. In each run, HPV16 and HPV18 standard curve uses a plasmid containing the HPV16 or HPV18 E7 gene, with 10-fold serial dilutions (ranging from 3×10⁷ to 30 copies for HPV16 and 3×10⁶ to 30 copies of HPV18) used.

Nested PCR Assays and Sequencing

An in-house nested PCR assay using HPV general MY09/11 primers followed by GP5+/6+ primers was done on negative samples for HPV16/18 by duplex real-time assay. PCR amplification was performed as previously described (7). Briefly, PCR amplification was performed in a reaction containing 100-200 ng of DNA template, 2.5 mM of MgCl2, 10 pmol of each primer (GP5+/6+), and 50 mM of each dNTP and 2 U of Taq DNA polymerase. The PCR amplification cycles were included as follows: initial 3-min denaturation at 94°C, followed by 45 cycles of 94°C for 30 s, 50°C for 45 s, and 72°C for 1 min, and a final extension period of 5 min at 72°C. The PCR products were run on a 1.5% agarose gel. In order to determine the genotype, a 150 bp PCR product indicating the partial L1 gene fragment amplification was introduced into bidirectional sanger sequencing with the help of GP5+ and GP6+ primers

Genotype Determination by Sequence Analysis 

The sequenced fragments from samples were aligned with confirmed HPV reference sequences that were available in Gene Bank databases, and their similarity was investigated by using the NCBI BLAST program (http://www.ncbi.nlm.nih.gov/blast/).

Statistical Analysis

The results were analyzed using SPSS 16 (SPSS Inc., Chicago, IL., USA) software. Fisher's exact test was used for data analysis, and a P-value less than 0.05 was considered statistically significant.

Results of molecular epidemiology of HPV genotypes are necessary to devise the appropriate preventive or diagnostic strategies for cervical cancer in different areas. This study determined the distribution of different HPV genotypes among patients with different grades of cervical lesions of LSIL, HSIL, SCC, and ADC in southwest Iran.
The results showed that overall HPV prevalence among patients with LSIL, HSIL, SCC, and ADC is 90%. Following this study's results, HPV DNA has been reported in 100% of the premalignant and malignant lesions in Brazil (21), 95% in the United States (22), 94% in Zambia (23) and 88% in Pakistan (24). Moreover, in Iran, HPV DNA was detected in 87.1% of cervical cancer samples (25). Also, in Shiraz, Iran, in two separate studies, 79.5% (26) and 73% (27) of CINs and SCC samples were infected with different types of HPV.
We also found that with the progression and severity of cervix lesions, the frequency of HPV increases from 78% in LSIL to 93% in HSIL and 100% in SCC/ADC. In agreement with our results, Heydari et al. (28) reported the prevalence of HPV was 44.5% in LSIL, 92.3% in HSIL, and 98.2% in invasive cervical cancer. Moreover, Jalilvand et al. reported that HPV prevalence in Iranian women with LSIL, HSIL, and invasive cervical cancer was 65.3%, 71.8%, and 77.4%, respectively (29). The lower prevalence of HPV in all grades of their study might be due to the lower sensitivity of the molecular method used for HPV detection.
The results of our study also showed that HPV 16 with 72 % (24% mono-infection and 48.5% co-infections with HPV18) frequency was the most prevalent HPV genotype in all grades of the cervical lesion. The frequency of HPV 16 in LSIL was 63.7% (15.2% mono-infection and 48.5% co-infection with HPV18),74% in HSIL patients (31% mono-infection and 42.9% co-infection with HPV18),78.3% in SCC (26.1% mono-infection and 52.2% co-infection with HPV 18) and 100% in ADC patients (all have HPV 16 and 18 co-infection). The second rank in frequency was HPV 18 with 10% mono-infection and 48.5 co-infections with HPV16. Research done by Ciapponi et al. (30) revealed that 46.5% and 8.9% of  HSIL cases harbored HPV 16 and HPV18, and 53.2% and 13.2% of invasive cervical lesions harbored HPV16 and HPV18.
The investigation of non-HPV16/18 presence in samples showed that HPV 31(3%), HPV 53(2%), HPV 6 (2%) and HPV 51(1%) were detected by sequencing. Jalilvand et al. (29) and Ghaffari et al. (27) Reported that HPV -16, 18, 6/11, 31, and 33 are the most frequent types in all grades of cervical lesions in Iran. Moreover, in another study from Iran, Ahmadi et al. showed that the most frequent HPV types in LSIL and HSIL specimens were HPV-18 and 6, but in SCC grade, HPV-16 was the most frequent genotype (16). Furthermore, according to a meta-analysis, about 70% of invasive cervical cancer are caused by HPV 16 and/or 18 worldwide (29).
In contrast to our results, a study in shiraz reported that HPV18 (41%), 16 (29.49%), 45 (24.36%), and 39(5.13%) were the most common HPV genotypes (26). On the other hand, Jalilvand et al., in a meta-analysis, reported that the frequency of HPV types in different grades of cervical lesions is low. They reported that 37.5% and 16.3% of LSIL cases, 47.6% and 16.9% of HSIL cases, and 50.1% and 14.4% of SCC cases were infected with HPV type 16 and 18 were lower than our findings. They assumed these types must be more prevalent than extrapolated from past reports based on the low sensitivity of molecular assays.
Regarding the HPVs co-infection, we found that 48.5% (49/101) of all positive HPV samples were infected with HPV 16 and 18, simultaneously. Similar to this finding, de Jesus et al. reported that 75.4%  (43/57) of cervical biopsies had HPV 16/18 co-infection in Brazil (21). Ghaffari et al. also reported this co-infection in 29% (2/7) of SCC samples (27). The importance of HPV co-infections, particularly oncogenic types, is the increased risk of progressing to cervical cancer compared with mono-infection (31). Besides, we see a higher prevalence of co-infections in all grades than mono infections. Therefore, in the single genotype detection PCR method, co-infections of high-risk HPV might be missed, samples with lower virus load might be neglected, and a false negative result may be reported.
Regarding low frequent HPV genotypes, two samples with HSIL lesions were infected with HPV53. In a study in Korea, Yung-Taek et al. found that HPV 53 was the most prevalent genotype in healthy women (1). Halec et al. reported that mono-infection of HPV 53 in the cervical lesion could activate the same cellular pathways as carcinogenic HR-HPV types; therefore, those women infected with this genotype may finally progress to cervical cancer (32). Also, we found that 2.4% of HSIL grade and 4.2% of SCC grade are infected with HPV type 6 as a low-risk HPV type. In this accordance, Heydari et al. reported that 1.9% of SCC grade were infected with HPV6 (28).

 

Our results show that HPV 16 and 18 were the most prevalent genotype among different grades. HPV genotypes 31, 51, and 53 were also detected in a few cases, which might need more attention in HPV screening and prevention programs. Besides, our study demonstrated that more sensitive molecular methods could give more accurate results about HPV genotype prevalence, especially in multiple HPV infections. Furthermore, a higher frequency of co-infections in all grades may imply the importance of using duplex real-time PCR assay

 

The limitations of our study were the relatively small sample size for different studied groups, particularly the ADC group, which can be overcome in future studies by considering a larger sample size.

 

The Medical Ethics Committee of the Shiraz University of Medical Sciences has approved the study (IR.sums.med.rec.1397.306).
 

None.

 

Conflicts of Interest

The authors declared no conflict of interest.