DOI: 10.18585/inabj.v17i1.3400

HPV18 E6 and E7 Mutation in Cervical Cancer (Mahendra INB, et al.)
Indones Biomed J. 2025; 17(1): 51-9

RESEARCH ARTICLE

HPV18 E6/E7 Mutation and Their Association with The Expression Level of
Tumor Suppressor Proteins p53 and pRb among Indonesian Women with
Cervical Cancer
I Nyoman Bayu Mahendra1, Pande Kadek Aditya Prayudi1,, Ida Bagus Nyoman Putra Dwija2
Division of Gynecologic Oncology, Department of Obstetrics and Gynecology Faculty of Medicine, Universitas Udayana/
Prof. dr. I Gusti Ngoerah Gede Ngoerah General Hospital, Jl. P.B. Sudirman, Denpasar 80232, Indonesia
2
Department of Clinical Microbiology, Faculty of Medicine, Universitas Udayana, Jl. P.B. Sudirman, Denpasar 80232, Indonesia.
1

*Corresponding author. Email: prayudipande@yahoo.com
Received date: Oct 28, 2024; Revised date: Jan 12, 2025; Accepted date: Jan 14, 2025

Abstract

B

ACKGROUND: The E6/E7 mutation contributes to the intra-typic variant of HPV18 which may differ in their
oncogenic potential. E6 and E7 target the tumour suppressor protein p53 and pRb, respectively, and their degradation
play a crucial role in cervical carcinogenesis. However, the prevalence of HPV18 E6/E7 variants among Indonesian
women with cervical cancer has not been elucidated. Therefore, this study was conducted to characterize the HPV18 variants
among Indonesian women with cervical cancer and their association with tumor suppressor protein p53 and pRb.
METHODS: A hundred Indonesian women with pathologically proven cervical cancer were consecutively recruited into
the study. Polymerase chain reaction (PCR) was performed to detect HPV18 DNA E6 and E7 oncogenes using specific
primers and the variants was determined through nucleotide sequencing. Expressions of p53 and pRb were analyzed through
immunohistochemistry by using specific antibodies targeting p53 and pRB.
RESULTS: The rate of HPV18 positivity was 24%. The rate of E6 and E7 mutation was 45.4% and 59.1%, respectively.
Those with E6 mutation had significantly higher expression of p53 and pRb as compared to those with wildtype E6 (p<0.05).
Subjects with E7 mutation only had higher expression of pRb (p<0.05). Phylogenetic analysis revealed that 54.5% subjects
had genetic sequences closely related to Asian lineages, particularly A1, A4, and A5 sublineage. Interestingly, 3 subjects had
genetic sequences closely related to MK813921, a newly identified sequences. However, 45.5% subjects had distinct genetic
sequences that did not related to the reference sequence used in this study.
CONCLUSION: E6 and E7 mutation was common among Indonesian women with HPV18 cervical cancer and associated
with the level of tissue p53 and pRb expression.
KEYWORDS: HPV18 E6/E7, mutation, epidemiology, Indonesian women
Indones Biomed J. 2025; 17(1): 51-9

Introduction

Cervical cancer remains as one of the most significant
health problem for women all over the world. In 2020,
approximately 604,127 new cases of cervical cancer and
341,831 related deaths were reported worldwide, with an
age-standardized incidence rate of 13.3 cases per 100,000

woman-years and a mortality rate of 7.2 deaths per 100,000
woman-years.(1) The incidence of cervical cancer in
Indonesia is 100 cases per 100,000 women.(2) It is well
established that persistent infection with high-risk human
papillomavirus (HPV-HR) is the primary cause of cervical
cancer. HPV18 is the one of the most common genotypes
that causes cervical cancer.(3,4) It contributes to around
12% of cervical squamous cell carcinoma (SCC) cases and

Copyright © 2025 The Prodia Education and Research Institute.
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International (CC-BY-NC) License.

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The Indonesian Biomedical Journal, Vol.17, No.1, February 2025, p.1-108

37% of adenocarcinoma (ADC) cases.(5) In Indonesia,
the overall prevalence of HPV18 infection was reported to
be 16.1%, which was the third commonest HPV-HR after
HPV52 (23.2%) and HPV16 (18%).(6)
As in other HPV-HR genotypes, E6 and E7 also play
vital role in driving carcinogenesis after persistent HPV18
infection. E6 oncoprotein acts specifically in driving
carcinogenesis by inducing the proteasomal degradation
of p53, while E7 induce pRb inactivation.(7) Primarily a
transcription factor, the p53 protein controls a multitude
of processes, including cell cycle arrest, DNA repair, cell
apoptosis, autophagy, and metabolism, and it also decides
whether cells die under stress. By recruiting transcriptional
corepressors and/or chromatin remodelling protein factors,
such as Histone deacetylase (HDAC), Swi-independent
(Sin3), C-terminal binding proteins (CtBP), and SWItch/
Sucrose Non-Fermentable (SWI/SNF), to promoter regions,
pRB suppresses the transcription of the E2F-target gene,
thereby halting cell cycle progression.(7) A recent study
reported that inactivation of HPV18 E6/E7 oncogene
by Clustered Regularly Interspaced Short Palindromic
Repeats/CRISPR-associated protein 9 (CRISPR/Cas9)
vector increase the p53 and p21 protein levels, inhibit
cell proliferation, induced apoptosis and subsequently,
inhibit tumour growth.(8) Another study demonstrated that
knockout of HPV18 E7 expression resulted in decreased E6
expression with activation of pRb/p21 pathway.(9) However,
the genetic sequences of HPV18 E6/E7 oncogenes are
prone to mutation which result from the interaction between
HPV genome and the host cells.(10) A study in Korea had
identified 15 new nucleotides substitution within the E6,
E7 and L1 oncogene, and 6 of those mutations resulted in
amino acid changes.(11) While a study in China reported
that 81.1% HPV18 variants that were identified were novel
variants.(12) An international study involving HPV18positive cervical samples from 39 countries had identified
a total of 209 unique HPV18 sequence variants which form
three distinct phylogenetic lineages.(13)
The E6/E7 mutation contributes to the intra-typic
variant of HPV18 which may differ in their oncogenic
potential. One study reported the association between
certain HPV18 variant lineages and the risk for higher grade
cervical lesions. Non-European variants were more strongly
associated with risk of high-grade cervical lesions as
compared to European variants.(14) On the contrary, several
studies had reported that HPV18 variants had no significant
effect on the degree and progression of the cervical lesions.
(11,13,15) A study conducted in Japan found no significant
difference in the prevalence of specific HPV18 sub-lineages

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between cervical cancer/precancer cases and controls,
nor between cases of SCC and ADC.(16) However, one
study showed that Non-European HPV18 variants were
commonly seen in ADC.(17) These conflicting findings
may indicate that HPV18 genetic diversity exists within
the context of geographic or ethnical variation. Thus,
knowledge of the HPV18 variation on certain geographic or
ethnic population is important and serves as a potential tool
in developing strategies of combating cervical cancer for
targeted populations. In Indonesia, data about the prevalence
and pattern of HPV18 variants based on E6 and E7 gene
sequence is still lacking. Moreover, their association with the
target tumor suppressor protein p53 and pRb is a novel and
interesting topic to study. Thus, this study was conducted to
characterize the HPV18 variants among Indonesian women
with cervical cancer and their association with the tumor
suppressor protein p53 and pRb.

Methods

Study Design and Subject Recruitment
A cross-sectional study was conducted involving 100
Indonesian women with pathologically proven cervical
cancer who attended the Gynecologic Oncology Outpatient
Clinic, Prof. dr. I G. N. G. Ngoerah General Hospital,
Denpasar, Bali, Indonesia during June 2019 to December
2020. Tissue sample from cervical cancer was taken to
analyse the HPV18 E6/E7 variants and the expression of
p53 and pRb protein. The inclusion criteria were women
with pathologically proven cervical cancer and HPV18
positive with clear quality of sample. The exclusion
criteria were women who had underwent any form of
treatment (surgery, radiation, or chemotherapy) before the
commencement of this study. Before the study, all subjects
or their legal surrogate signed a written informed consent to
participate in this study. With the prevalence of 0.25 (16),
the the adequate sample size in this study was calculated
following the formula as mentioned in previous publication
(18), and the total number of samples required was 22. This
study was approved by the Ethical Committee of Faculty of
Medicine, Udayana University/Prof. dr. I G. N. G. Ngoerah
General Hospital, Denpasar, Bali, Indonesia (No. 2381/
UN14.2.2.VII.14/LT/2022).
Clinical Characteristics
Data about the clinical characteristics of the study population
(age, parity, histologic type, and stage) were all obtained
from the medical records. Stages were classified according

DOI: 10.18585/inabj.v17i1.3400

to 2018 FIGO Classification.(19) Histologic types were
classified according to WHO Classification, i.e., squamous
carcinoma and adenocarcinoma of the cervix.(20)
DNA Isolation
Cervical cancer tissue was biopsied from the study
participants and embedded in PBS 1X/NaCl 0.9% solution.
DNA isolation was performed using High Pure PCR Template
Preparation kit (Cat.No. 11796828001; Roche, Basel,
Switzerland). A 25-50 mg tissue was placed into a mortar
and 200 µL tissue lysis buffer was added before grinding.
The tissue was then transferred into 1.5 mL centrifuge tube
and incubated at 55°C for 60 minutes. A 200 µL lysis buffer
and 40 µL proteinase K were added to inactivate the DNAse
and the tissue was incubated subsequently at 70°C for 10
minutes. A 100 µL isopropanol was added to precipitate the
DNA. The sample was then transferred into a filter tube and
centrifuged at 8000 rpm for 1 minute. A 500 µL inhibitor
removal buffer was added to remove all the residues. After
centrifugation, a 500 µL wash buffer was added. The sample
was centrifuged again at 8000 rpm for 1 minutes. Elution
was added before another round of centrifugation at 8000
rpm for 1 minute. The solution of sample was then used for
further analysis.
Polymerase Chain Reaction (PCR)
Before proceeding with HPV18 detection, identification
of the universal HPV DNA in collected samples were
performed. The sample which were positive for universal
HPV were then considered eligible for HPV18 detection.
One hundred cervical cancer tissue sample were screened
and the positivity rate for HPV18 was found to be 24%.
Subsequently, the sample that was positive for HPV18
was included into the study population. PCR was used to
detect HPV DNA with primers My09 (5’-CGT CCM ARR
GGA WAC TGA TC-3’) and My11 (5’-GCM CAG GGW
CAT AAY AAT GG-3’).(21) The PCR protocol included
an initial denaturation at 95°C for 3 minutes, followed by
35 cycles of denaturation at 95°C for 1 minute, annealing
at 55°C for 1 minute, extension at 72°C for 1 minute, and
a final extension at 72°C for 5 minutes. HPV18 DNA was
amplified using specific primer for E1 gene, as the surrogate
gene.(22) (Forward: 5’-ATA GCA ATT TTG ATT TGT
C-3’; nucleotide position: 1989–2007; Reverse: 5’-AAA
CTC ATT CCA AAA TAT G-3’; reverse primers position:
2385–2403; amplified DNA size 415 bp). The PCR protocol
included an initial denaturation at 95°C for 3 minutes,
followed by 35 cycles of denaturation at 95°C for 1 minute,
annealing at 45°C for 1 minute, extension at 72°C for 1

HPV18 E6 and E7 Mutation in Cervical Cancer (Mahendra INB, et al.)
Indones Biomed J. 2025; 17(1): 51-9

minute, and a final extension at 72°C for 5 minutes. The
25 μL reaction mixture consisted of 2.5 μL DNA template,
1.25 μM of each primer, 12.5 μL Go2Green Master Mix
(Promega, Madison, WI, USA), and 7.5 μL of water. DNA
amplification was performed using a MiniAmp Thermal
Cycler (Applied BioSystems, Waltham, MA, USA).
E6 and E7 Gene Amplification
HPV18 E6 and E7 oncogenes were amplified using
specific primers as follows: HPV18‐E6 F: 5′‐AGA AAC
ACA CCA CAA TAC TAT GGC G‐3′; HPV18-E6 R : 5’GTC GGG CTG GTA AAT GTT GAT-3’; HPV18-E7 F:
5’-CGACAGGAACGACTCCAACGA-3’; HPV18-E7 R:
5’‐ATA AAA CCA GCC GTT ACA ACC CGT G‐3’.(11)
The 25 μL amplification mixture included 2.5 μL DNA
template, 2 μM of each primer, 12.5 μL Go2Green Master
Mix (Promega), and 6 μL of water. DNA was amplified on
a MiniAmp Thermal Cycler (Applied BioSystems) using
the following settings: initial denaturation at 95°C for 3
minutes, 35 cycles of denaturation at 95°C for 1 minute,
annealing at 57°C for 1 minute, extension at 72°C for 1
minute, and a final extension at 72°C for 5 minutes. After
PCR, samples were analyzed by electrophoresis on a 1.5%
agarose gel.
E6 and E7 Gene Sequencing
The amplified E6 and E7 sequences were aligned with the
prototype sequence from NCBI (http://blast.ncbi.nlm.nih.
gov/Blast) to create alignments. The HPV 18 prototype
E6/E7 gene sequence (GenBank: NC_001357) served as
the reference standard for comparisons.(23) Sequencing
was performed using software program MEGA10 (Mega
Software, Pennsylvania State University, University Park,
PA, USA). The sequence of nucleotide and amino acid
was aligned using software program BioEdit (Informer
Technologies, Los Angeles, CA, USA). Phylogenetic
analysis was performed using MEGA10 software to classify
the HPV variants into their respective lineages (A, B and
C) and sublineages (A1-8, B1-3 and C).The distinction
into lineages and sublineages was based on their rate of
nucleotide variations, i.e., the lineages and sublineages
vary by 1–10% and 0.5–1%, respectively. The accession
numbers used for phylogenetic analysis were as follows.
A1 sublineage: EF202143-EF202145, A2 sublineage:
EF202146, A3 sublineage: EF202147- EF202149,
A4 sublineage: EF202150-EF202151, A5 sublineage:
GQ180787, A6 sublineage: KY457833- KY457836,
A7 sublineage: KY45737-KY45840, A8 sublineage:
KY457826-KY457827; B1 sublineage: EF202153-

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The Indonesian Biomedical Journal, Vol.17, No.1, February 2025, p.1-108

EF202155, B2 sublineage: KC470224-KC470225, B3 sublineage: EF202152; and C lineage: KC470229-KC470230.
(11)
Immunohistochemistry (IHC)
Cervical cancer tissue was immersed in a 10% phosphatebuffered formalin solution for 24 hours. It was then soaked
in graded alcohol successively (30%, 40%, 50%, 70%,
80%, and 96%) 3 times each for 25 minutes. The tissue was
placed in xylene clearing agent for 1 hour each 3 times until
transparent. After infiltrating 3 times for 1 hour each with
pure paraffin, the tissue was embedded in liquid paraffin,
left to form blocks (± 3 hours) so that it could be easily
sliced with a microtome. The tissue was then cut using a
microtome for 5 µm thick, and attached to a glass object that
has been coated with an adhesive such as Poly-Lysine, then
incubated at 60°C for 2 hours.
p53 staining was performed using rabbit polyclonal
antibody that target nuclear p53 protein (Cat. No. bs-0033R;
Bioss, Woburn, MA, USA) with the concentration of 1 μg/
μL, and dilution 1: 50 in PBS pH 7.4, following the protocol
stated by the manufacturer. pRb staining was performed
using rabbit polyclonal antibody Rb/P105 RB (Ser807 +
Ser811) that targets nuclear pRb protein (Cat. No. bs-3380R;
Bioss) with the concentration of 1 μg/μL and dilution 1: 100
in PBS pH 7.4, with the same protocol as p53 staining. The
expression of p53 and pRB was examined in one preparation
and one area was selected randomly. The expression was
examined in high field magnification (40x Objective).
The immune-reactive score (IRS), which was based on
the proportion of positive cells and the staining intensity,
was used to evaluate the IHC staining of p53 and pRb. The
cells were considered to be positive if an intranuclear DAB
staining was observed (brown hue with mild, moderate, and
intense staining). The labeling index was used to calculate
the proportion of positive cells (Labeling Index = Number of
IHC Positive Cells × 100/Total Number of Cells Observed).
The IRS score, which ranged from 0 to 12 and represented
groupings of p53/pRb expression with ≤6 low and >6 high,

Print ISSN: 2085-3297, Online ISSN: 2355-9179

was calculated by multiplying the two scores (Table 1). Two
observers completed the counting, with the final count being
determined by taking the mean.(24)

Results

Baseline Characteristics
The baseline characteristics of the study population are
summarized in Table 2. There was no difference in age,
FIGO stage, and histologic subtype of cervical cancer
among wildtype and mutant E6/E7 group.
Mutation of HPV18 E6 and E7 Oncogene
One hundred cervical cancer samples during the study
period were collected. The rate of HPV18 positivity was
24%. Among 24 samples which were HPV18 positive, 22
were eligible for E6/E7 gene sequencing and subsequent
mutation analysis. Two subjects were not eligible for gene
sequencing due to fragmentation of DNA material before
the analysis. The length of E6 gene and amino acids that had
been successfully sequenced were 477 base pairs and 158
amino acids, respectively. Meanwhile, the length of E7 gene
and amino acids that had been successfully sequenced were
318 base pairs and 105 amino acids, respectively.
The rate of E6 mutation was 36.4% (8/22 subjects)
(Table 3). E6 C445A, which was a synonymous mutation,
was the most prevalent mutation (8/22 subjects; 36.4%).
The rate of non-synonymous mutation was only 18.2%
(4/22 subjects). Two subjects (9.1%) present with more
than one type of E6 mutation (C28T or T10C co-exist with
C33A+C445A).
The rate of E7 mutation was 59.1% (13/22 subjects)
(Table 3). E7 C162T, which was a synonymous mutation,
was the most prevalent mutation (6/22 subjects; 27.3%).
The rate of non-synonymous mutation was 40.9% (9/22
subjects). Two subjects (9.1%) present with more than one
type of E7 mutation (G241A+C276G and C162T+G312C).
Eight subjects (36.4%) had co-existing E6 and E7 mutation.

Table 1. The Immunoreactive score.
A (Percentage of Positive Cells)

54

B (Intensity of Staining)

IRS Score (Multiplication of A and B)

0 : no positive cells

0 : no color reaction

0-1 : negative

1 : <10% positive cells

1 : mild reaction

2-3 : mild

2 : 10-50% positive cells

2 : moderate reaction

4-8 : moderate

3 : 51-80% positive cells

3 : intense reaction

9-12 : strongly positive

4 : >80% positive cells

Final IRS score (AxB) : 0-12

HPV18 E6 and E7 Mutation in Cervical Cancer (Mahendra INB, et al.)
Indones Biomed J. 2025; 17(1): 51-9

DOI: 10.18585/inabj.v17i1.3400

Table 2. Baseline characteristics of the study population (n=22).
Variable

All Subjects

Mutant E6

Wildtype E6

p -value

HPV18 E6 gene
Age (years), mean±SD

49.7±11.8

49.0±15.4

50.1±9.7

0.833a

Early
LACC
Histologic subtype, n (%)

8 (36.4)
14 (63.6)

3 (37.5)
5 (62.5)

5 (35.7)
9 (64.3)

0.642

Squamous
Non-squamous
HPV18 E7 gene
Age (years), mean±SD

14 (63.6)
8 (36.4)

6 (75)
2 (25)

8 (57.1)
6 (42.9)

0.649b

49.7±11.8

48.7±13.4

51.2±9.3

0.632a

8 (36.4)
14 (63.6)

4 (30.7)
9 (69.3)

4 (44.4)
5 (55.6)

0.662b

Squamous

14 (63.6)

9 (69.3)

5 (55.6)

0.662b

Non-squamous

8 (36.4)

4 (30.7)

4 (44.4)

Stage, n (%)

Stage, n (%)
Early
LACC
Histologic subtype, n (%)

b

t-test was used to obtain p-value, bchi square test was used to obtain p-value.

a

Mutation of E6 C445A/E7 C162T, which was synonymous
mutation, was the most prevalent co-existing mutation (6/8
subjects; 75%). Two subjects present with non-synonymous
co-existing mutation E6 T449G/E7 T269G, which translates
into amino acid changes E6 L150R/E7 F90C (Table 3).
Expression of p53 and pRb
All subjects had low expression of p53 and pRB (<5%
positive cells) (Figure 1). However, those with E6 mutation
had significantly higher expression of p53 and pRb as
compared to those with wildtype E6 (p<0.05). Subjects with
E7 mutation only had higher expression of pRb (p<0.05)
(Table 4).

Phylogenetic Analysis
The phylogenetic tree analysis of the 22 samples were
summarized in Figure 2. These variants formed 5 major
cluster. Four samples in the first cluster had close similarity
to sublineage A4 Two samples in the second cluster had
distinct genetic sequences that did not match any reference
sequences. Two samples in the third cluster were close to
sublineage A5. Three samples in the fourth cluster had close
similarity to MK813921. The fifth cluster was diverged into
two sub-cluster: the first subcluster had close similarity to
sublineage A1; the second sub-cluster had distinct genetic
sequence that did not resemble the reference sequence.
Three samples also had distinct genetic

Table 3. Mutation of HPV18 E6 and E7 gene.
Nucleotide Position

Prototype

Variant

N(%)*

Amino Acid Position

Prototype

Variant

N (%)

10
28
33
432

T
C
C
A

C
T
A
C

1
1
2
2

4
10
11
144

F
R
P
R

S
#
P
R

1
1
-

445
449

C
T

A
G

8
2

149
150

R
L

R
R

2

158

G

A

4

53

R

C

4

162
241
269
276
312

C
G
T
C
G

T
A
G
G
C

6
1
2
1
1

54
81
90
92
104

A
D
F
N
C

A
N
C
K
H

1
2
1
1

E6

E7

*Mutation rate was among all subjects with HPV-18 positive cervical cancer, #unidentified amino acid.

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Figure 1. Immunohistochemical staining for p53 (left) and pRB (right), showing positive cells stained brown. Orange arrow: p53
positive cells; Green arrow: pRB positive cells. White bar: 50 mm.

Discussion

In this study, the rate of HPV18 positivity among cervical
cancer samples was 24%. The rate of HPV18 positivity in
Indonesia range from 24% to 40%.(7) This is in accordance
to the rate reported from other South East Asian countries.
(25) However, the rate was lower in European countries
such as Portugal (4.8%), UK (19.5%) or in Africa (18%).
(26-28) These findings indicate that HPV18 positivity in
cervical cancer was higher in South-East Asian region as
compared to Europe and Africa. Among asymptomatic
Indonesian women, a population-based study reported the
prevalence of HPV18 infection was 16.1%.(6)
HPV18 genome is relatively conserved and the
occurrence of variants is considered a rare event.(29)
Among Indonesian women with Balinese ethnicity, as much
as 36.4% and 59.1% rate of HPV18 E6 and E7 mutation
were identified in this study, respectively. A study involving
Indonesian, Surinamese, and Dutch women found the rate
of HPV 18 variants was 18.2%.(30) In a study involving
10 HPV18 positive cervical cancer samples from Southwest
Chinese women, the rate of HPV18 E6 and E7 mutation
was 30% and 10%, respectively.(23) E6 C287G was the

most frequent mutation of HPV18 found among Chinese
women.(31,32) A study in Mexico reported the rate of
HPV18 variants based on LCR gene sequences in cervical
cancer samples was 15.5%.(33) In another study conducted
in Portugal, based on E6 and LCR gene sequences, the rate
of HPV18 variants was 37.2%.(34) None of the nucleotide
changes found in studies involving Chinese women was
similar with nucleotide changes observes in current study.
(23,31,32) A study involving 12 HPV18 positive cervical
cancer samples from Korean women also identified
nucleotides changes that differed than our results (E6:
C287G, T485C, C549A and C554T; E7:C751T).(11)
A study involving 25 Taiwanese women with cervical
cancer identified C183G was the most common HPV18 E6
mutation.(35) Thus, mutations found in our study may be
unique to Indonesian population. To our knowledge, the
current study is the first study to identify HPV18 mutations
based on E6 and E7 gene sequence among Indonesian
women. The mutation patterns were also different as
compared to mutation pattern found in other studies.
HPV18 variants were initially categorized into
European (E), Asian-Amerindian (AA), or African (AFR)
lineages based on the E6-E7, L1, and/or LCR sequences.
(13) In this study, we found that 54.5% (12/22) subjects

Table 4. Expression of p53 and pRb among subjects with mutant and wildtype E6/E7.
IRS Score

All Subjects

Mutant E6

Wildtype E6

p- value

p53

1.4±0.8

1.9±0.7

1.1±0.8

0.044*,a

pRb

1.1 (0.7-1.6)

1.4 (1.1-1.9)

0.9 (0.5-1.1)

0.026*

All Subjects

Mutant E7

Wildtype E7

p -value

p53

1.4±0.8

1.6±0.7

1.1±0.9

0.256a

pRb

1.1 (0.7-1.6)

1.4 (0.9-1.7)

1.0 (0.5-1.1)

0.030*,b

,b

t-test was used to obtain p-value, bMann-Whitney test was used to obtain p-value. *p<0.05 is considered
significant.
a

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Indones Biomed J. 2025; 17(1): 51-9

DOI: 10.18585/inabj.v17i1.3400

29

29

31

32

EF202151
EF202147
EF202150
Bali 55
Bali 33
Bali 103

29
24
55

Bali 20

60

62

58

29
24

42

94

Bali 97
Bali 74
KY457826
GQ180787
Bali 95
Bali 101
KY457833

14

Bali 23
Bali 63

4

3

2

Bali 64
Bali 24
Bali 70
8

12

45
65

0
12
62

62

62

Bali 100
MK813921
Bali 10
EF202143
Bali 91
Bali 31
Bali 35
Bali 41
Bali 61
Bali 45
Bali 62

Figure 2. Phylogenetic tree of the HPV18 variants.

had genetic sequences closely related to Asian lineages,
particularly A1 (3 subjects), A4 (4 subjects), and A5
sublineage (2 subjects). Interestingly, 3 subjects had genetic
sequences closely related to MK813921, a newly identified
sequences in one Korean study.(11) However, 45.5%
(10/22) subjects had distinct genetic sequences that did not
related to the reference sequence used in this study.
One study reported that HPV18 variants among Indonesian
sample were classified into Asian Amerindian and European
phylogenetic branches.(30) The distribution of HPV18
variants have been found to be varied geographically, with a
predominance of the A lineage in most regions except subSaharan Africa.(13) However, studies investigating the
association between HPV18 variants and the progression
of cervical lesions reported conflicting results. The nonEuropean HPV variants are associated with HPV persistence
and progression of intraepithelial abnormalities.(36)
Another study report that HPV18 variants did not associate
with the risk of cervical cancer.(13)
In this study, subject with mutant E6 had higher
level of p53 expression as compared to wildtype E6.
One study in Mexico also demonstrated that E6 variants
distinctively affects p53 levels.(37) pRb expression was also

significantly higher among patients with mutant E6 and E7
as compared to their wildtype counterparts. No study has
ever demonstrated the association between mutation of E6/
E7 and pRb expression in cervical cancer. We hypothesize
that E6/E7 variants will differ in their capacity to degrade
p53/pRb, as compared to the wild-type E6/E7. Furthermore,
the results of this study indicate that mutation of E6 and
E7 gene could affect the expression of p53 and pRb. It was
observed that subjects with E6 mutation had higher p53 and
pRb expression while subjects with E7 mutation had higher
pRb expression. E6 directly affects the expression of p53
while E7 affects the expression of pRB.(38) It's logical to
think that altered expression of E6 and E7 due to mutation
will also affect the expression pattern of p53 and pRb.
However, studies investigating mutation or variants of
HPV18 E6 and E7 and their association with the biologic
behaviour of cervical cancer cells are still lacking. We
have been aware that HPV variants can be classified into
3 lineages (A, B and C), and sublineages (A1-8, B1-3 and
C) which differ in their rate of nucleotide variations. The
nucleotide sequences of HPV intratype variant lineages
and sublineages vary by 1–10% and 0.5–1%, respectively.
HPV18 variants from A lineage has been reported to
exhibit more oncogenic potential than the B lineage.
Immortalized primary human keratinocytes (PHK) with
E6/E7 from A1 lineage (PHK18A1) showed significantly
faster immortalization, generated more colonies in both
monolayer and 3D cultures, exhibited enhanced invasion
capabilities, and demonstrated increased resistance to
apoptosis triggered by actinomycin-D, as compared to cells
infected with E6/E7 from B1 lineage (PHK18B1).(39)
Number of subjects recruited in this study is relatively
small and only include Indonesian women of Balinese
ethnicity. Future study with larger size of participants and
multi-centre approach involving various ethnic group to
enhance the power of the study and to fully understand the
nature of HPV18 E6/E7 mutation in Indonesia is suggested.
In this study, semi-quantitative methods of detection for
p53 and pRb activity was used, which limits the accuracy.
For this issue, employing the use of quantitative methods to
detect protein or mRNA expression level for p53 and pRb,
such as enzyme-linked immunosorbent assay (ELISA) or
reverse transcription-polymerase chain reaction (RT-PCR)
should be further studied. Since this study is a pilot study
using cross-sectional design, future study with prospective
design to evaluate the temporal association between HPV18
E6/E7 mutation with the clinical behaviour of cervical
cancer, such as progression, metastasis, recurrence, or
response to treatment are also suggested.

57

The Indonesian Biomedical Journal, Vol.17, No.1, February 2025, p.1-108

Conclusion

Print ISSN: 2085-3297, Online ISSN: 2355-9179
5.

In conclusion, we demonstrate that mutation of HPV18
E6 and E7 genes are common among Indonesian women,
with higher prevalence of E7 mutation as compared to E6
mutation. Mutation of E6 and E7 also affect the expression
of tumour suppressor protein p53 and pRb.

6.

Acknowledgments

8.

We would like to thank the Laboratorium Biomedik Terbaru
(LBT) FK UNUD for providing technical services. We also
thank Alisza Novrita Sari and Daniel Victor Harrista for
their excellent technical assistance.
This research has received funding from The Ministry
of Education and Culture Republic of Indonesia (Hibah
PNBP 2021).

Authors Contribution

INBM and PKAP were involved in the conception and
design of the study. PKAP and IBNPD were involved in
the acquisition of data. INBM, PKAP and IBNPD analyzed
and interpretated the data. PKAP drafted the manuscript,
while INBM and PKAP revised the manuscript critically
for important intellectual content. All authors have given
approval of the final version of manuscript to be published.

1.

2.

3.

4.

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