Association between mutant E6 and E7 Human Papilloma Virus Type 16 oncogenes with cyclin D1 expression in cervical cancer: Observational study in Bali

Introduction

The estimated incidence of cervical cancer in the world is 13.1 every 100,000 women. The incidence of cervical cancer is found to be higher in lower- and middle-income countries like Indonesia. In 2018, Indonesia ranked second in the incidence and mortality rate of cervical cancer in Asia^1,2

One of the known risk factors of cervical cancer is the Human Papilloma Virus (HPV). HPV types 16 and 18 are the most common types found in cases of cervical squamous cell carcinoma. The HPV oncoproteins that play a role in the development of cervical cancer are E6 and E7.^3–5

E6 and E7 initiate a series of processes leading to chronic infection and transformation into cervical cancer. E6 causes degradation of p53, and p21 inhibition leads to a decrease in cellular apoptosis. E7 inactivates pRb so that infected cells experience increased cell proliferation, causes an aberrant synthesis phase of the cell cycle, and increases transcription factor E2F. The sequences carried by E6 and E7 lead to an increase in cyclin D1 expression.^5–7

Increase in cyclin D1 expression is found in cervical cancer cells, including those resistant to chemotherapy.^8–10 Cyclin D1 overexpression can augment cell cycle and increase cellular proliferation. The expression of Cyclin D1 also affects the function of apoptosis, hypertrophy and differentiation. Cyclin D1 is thought to have mitogenic activity so as to stimulate hypertrophic growth.¹¹

Determination of E6 and E7 mutations for HPV type 16 can be useful in the development of HPV molecular diagnostics, prevention, and therapeutic efforts to control or eliminate cervical cancer. Moreover, the development of agents to target cyclin D1 activity, directly or indirectly, by targeting cyclin-dependent kinase (CDK) has also been studied. These agents are used concurrently with chemotherapy and have been shown to reduce resistance to chemotherapy and increase patient survival rates^12–14 when patients are referred for chemotherapy or radiotherapy.^15,16 Given their significance the association between cylin D1 expression and the E6 and E7 oncogene mutation of HPV type 16 infection as cervical cancer risk factor, they are crucial in understanding cervical cancer. Thus, this research was carried out.

Methods

An observational analytic, cross-sectional study was conducted from August 2020 until August 2021 at the Gynecology polyclinic of RSUP Prof. Dr I.G.N.G. Ngoerah, Denpasar laboratory of microbiology, faculty of medicine Universitas Udayana (FK UNUD), Denpasar and laboratory of histology FK UNUD, Denpasar. This study was approved by the Ethical Board for Research of FK UNUD, Denpasar on 21 December 2021 with protocol number 2021.02.1.1247.

The sample size was calculated with the following formula:

N=Minimum sample size

Zα=Confidence interval with α score of 5% and Zα is 1.960

P=Proportion of oncogene E6 and E7 mutation of HPV 16 was around 70% based on a study by Zhe et al. (2019)¹⁷

Q=1-p (proportion of oncogene E6 and E7 of HPV 16 which have compability with the prototype), around 30%

d=Error limitation or absolute precision, 18%

Data was collected from cervical cancer patients who visited the polyclinic between August 2020 until August 2021. The sampling method used was consecutive sampling of the population and included patients who fulfilled the eligibility criteria. Patient eligibility was assessed with inclusion and exclusion criteria and provided written informed consent as seen in Table 1. Patient eligibility was assessed with inclusion criteria such as first-time cervical cancer diagnosis during the study setting; not having received surgery, chemotherapy, or radiotherapy. The exclusion criteria were cervical cancer that was not caused by HPV type-16 and if the cervical cancer tissue samples were damaged or not representative for DNA PCR or immunohistochemistry examination. Eligible patients were asked for and provided written informed consent.

Eligible patients were asked for demographic characteristics, clinical history, received physical examination, and pathological examination of cervical cancer cells. Patient characteristics such as age, parturition history, body mass index (BMI), cervical cancer clinical stage, and pathological findings were recorded. Cervical cancer tissue specimens were tested for oncogene E6 or E7 mutation by HPV DNA PCR The PCR analyses were conducted at our institution laboratory, DNA isolation was carried out with Roche DNA extraction kit following the product standard protocol. After extraction, a PCR was performed to determine positive and negative HPV with primers My09 (5′-CGT CCM ARR GGA WAC TGA TC-3′) and My11 (5′-GCM CAG GGW CAT AAY AAT GG-3′). Meanwhile, DNA PCR for HR-HPV type-16 were performed with primers (Forward: 5′-GAC CCA GAA AGT TAC CAC AG-3′ and reverse: 5′-CAC AAC GGT TTG TTG TAT TG-3′). The HPV HR Type 16 PCR program is the same as the PCR program for universal HPV. To determine the quality of the extracted DNA, a PCR was performed for the betaglobin gene with the primers (Forward 5-CAA CTT CAT CCA CGT TCA CC-3 and reverse: 5-GAA GAG CCA AGG ACA GGT AC-3); The PCR results were then compared with the wild type gene on GenBank according to sequence K02718/HPV16R. Amplification of E6 and E7 gene was done with primer sequences as follow: upstream, 5′AAG GGC GTA ACC GAA AT3′; downstream, 5′TCC ATT ACA TCC CGT ACC CTC3′ (Primer 2 OD; 1 OD=33 μg). The primer was dissolved up to 100 mmol/L at a concentration of 10 μM (M=mol/L). The PCR reaction mixture of E6 and E7 genes (25 μL) wasas follows: 1 μL DNA template (1:20), 1 μL upstream primer (10 μM), 1 μL downstream primer (10 μM), 12.5 μL of 2× Taq PCR MasterMix, and double distilled water. The PCR cycle conditions were as follows: pre-denaturation at 94°C for 5 minutes; 30 cycles of denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 60 seconds; and final extension at 72°C for 5 minutes. After PCR was completed, 5 mL of sample were analyzed by electrophoresis using 1.5% agarose gel; The MEGA6 software was utilized for the sequencing of the E6 and E7 genes. BLAST software was used to compare the nucleotide sequences of the E6 and E7 genes to the HPV 16 prototype (HPv16.P, GenBank access code: NC 001526) from the European variation; cyclin D1 expression was measured by immunohistochemistry with rabbit primary ERβ polyclonal antibodies (BY-02101, Yueyan Biotechnology, Shanghai, China) and rabbit secondary polyclonal antibodies tagged with horseradish peroxidase (K500711, Gene Biotechnology, Shanghai, China) and categorized by Immunoreactive Scoring System (IRS); diagnosis of cervical cancer was made based on histopathological findings with histological classification such as squamous cell carcinoma, adenocarcinoma, adenosquamous carcinoma.

After the patient was diagnosed with cervical cancer, they were grouped according to E6 or E7 oncogene mutation and each group was tested for cyclin D1 expression. The data was analyzed with IBM SPSS for Windows version 22.0. The correlation between oncogene E6/E7 mutation and cyclic D1 expression was analyzed with a contingency coefficient correlation test. The result ranges from 0 until 1, 0 indicating no correlation and 1 a very strong correlation.

Results

From August 2020 until August 2021, a total of 100 cervical cancer patients was tested for HPV 16. 31 samples tested positive for HPV 16 with oncogene E6/E7 mutation distribution of wild type of n=19; 61%, and mutated oncogene n=12; 39%. Table 2 summarizes the demographics and characteristics of sample including age, parturition history, BMI, cervical cancer histological classification, and cancer stage which were distributed normally. Mean age for wildtype and mutant type were 50 and 54.5 years old, respectively. Mean body mass index for wildtype was 23.7 and 22.1 for the mutant. Squamous cell carcinoma was the most common histological findings, and cancer clinical stage II was the most common in both types.

Table 3 summarize the characteristic and proportion of E6 and E7 mutation on this study. The proportion of E6 mutants in this study group was 25.8% (8/31), while in the E7 group it was 12.9% (4/31). The population characteristics of the E6 mutation group were dominated by the nucleotide position of 27 prototype T variant C (n=5; 16.1%) followed by position 360 prototype A variant G (n=2; 6.4%) and position 371 prototype G variant A (n=1; 3.2%). The mutation in strain E6 is a synonymous mutation and G371A/R124K the only non-synonymous mutation. In the E7 group, the same proportion was obtained, namely one sample (3.2%) for each nucleotide position, prototype and variant. At E7 the majority of mutations were non-synonymous 9.6% (3/31) in N29T; N29S; R77C.

The association of mutations in the E6 and E7 HPV type 16 oncogenes with cyclin D1 expression in patients with cervical cancer is summarized in Table 4. There was no association between mutations in the E6/E7 HPV type 16 oncogene with cyclin D1 expression (c=0.283; p value=0.1). The correlation coefficient between the mutations of the E6 and E7 HPV type 16 oncogenes with Cyclin D1 expression was weak, as seen in Table 4.

Discussion

In this study, we described the association of mutations in the E6 and E7 HPV type 16 oncogenes with cyclin D1 expression in patients with cervical cancer. Our study included 31 patients with cervical cancer and HPV type 16 infection based on pathological examination and HPV DNA PCR. The samples median age was 54.5 (21-58) years old, in accordance with a study in Denmark and Sweden with a higher incidence of cervical cancer in patients aged 50 years or older.^18,19 Women aged 50 years and over are usually in the perimenopausal or postmenopausal phase; therefore, their physiological and pathological characteristics differ significantly from women of childbearing age.²⁰

The mean BMI was 22.1 kg/m² in cervical cancer patients with mutant oncogenes E6 and E7 with a BMI range of 19.5-28.2 kg/m². In this study, patients were found to be in the ideal BMI and overweight categories. The effect of body mass index (BMI) on precancerous cervical lesions (PCL) and cervical cancer is not clear. There are claims that underweight women may have an increased risk of cervical lesions while overweight women have a reduced risk of PCL compared to women with a normal BMI.²¹ One of the identified risk factors for PCL is being underweight (BMI<18.5 kg/m²), so the probability of being positive for PCL was fourfold higher among underweight women than women with a normal BMI. This relationship can be explained by the possible association between low BMI, poor nutritional status and low immunity. Therefore, participants with a low BMI tend to be malnourished, which in turn can compromise their immunity.²²

Conclusions

This study concluded that there is no association of mutation in the E6/E7 HPV type 16 oncogenes with cyclin D1 expression in patients with cervical cancer.