Human papillomavirus, gene mutation and estrogen and progesterone receptors in breast cancer: a cross-sectional study
Abdallah Mohammed Elagali1,2,&, Ahmed Abdelbadie Suliman3,
Mohammed Altayeb1,4, Anas Ibrahim Dannoun4, Narasimha
Reddy Parine5, Hader Ibrahim Sakr6, Howayda Saeed Suliman7,
Moustafa Elsaeid Motawee8
1Faculty of Medicine, Batterjee Medical College of Science
and Technology, Jeddah, KSA, 2Histopathology and Cytology Department,
Faculty of Graduate Study and Scientific Research, National Ribat University,
Khartoum, Sudan, 3Pathology Department, Faculty of Medicine, Taibah
Almadinah Almonawarah, Saudi Arabia, 4Molecular Genetics Department,
Faculty of Medicine, Umm Al-Qura University, Mecca, KSA, 5Medical
Genetics Department, Genome Research Chair, Department of Biochemistry, College
King Saud University, Riyadh, KSA, 6Physiology Department, Kasr Al-Ainy
Faculty of Medicine, Cairo University, Cairo Governorate, Egypt, 7Department
of Biochemistry, Faculty of Medicine, Alexandria University, Alexandria, Egypt, 8Department
of Histology and Cytology, Faculty of Medicine, Al-Azhar University, Cairo,
Abdallah Mohammed Elagali, Histopathology and Cytology Department, Faculty of Graduate Study and Scientific Research, National Ribat University, Khartoum, Sudan
recent studies show a good relationship between breast cancer (BC) and
human papillomaviruses (HPV) wich is responsible for about 18% of BC
cases. This study aimed to assess the relationship between different genotypes
of HPV and
expression of P53 and retinoblastoma (RB) genes and estrogen and progesterone
receptors in BC among Sudanese women.
one hundred and fifty tissue blocks were obtained from females diagnosed with BC. Positive samples were used to determine genotypes with an applied biosystem (ABI 3730XL) genetic analyzer for sequencing and immunohistochemistry.
13/150 samples showed HPV DNA. High-risk HPV-16 was detected in 5 cases, high-risk-HPV-58
was found in four cases, and HPV-18 was detected in three cases. Low-risk-HPV-11
was detected in a single invasive lobular carcinoma (ILC) case. P53 and RB gene
mutations were detected in 35 and 30 BC cases, respectively. P53 gene mutation
was frequently identified in grade (III) BC while RB gene mutation was positive
in grade (II). Grade (II) BC had a higher incidence of HPV-16 and 58. On the
hand, HPV-18 had a higher incidence in grade (III). Estrogen and progesterone
were expressed in 94 and 79 HPV cases among the study group, respectively.
this study elucidates the associations between HPV genotypes and BC. A statistically
significant association was observed among p53 and RB gene mutations and different
BC histological types. On the other hand, there was a statistically insignificant
association between HPV genotyping and different BC gradings, BC histological
types, P53 and RB genes mutations, and estrogen and progesterone receptor expression.
Also, there was a statistically insignificant association among estrogen and
progesterone receptors expression and BC grading. RB gene mutation was significantly
associated with different BC grades. On the other
hand, there was a statistically insignificant
association between progesterone
receptor expression and BC.
Breast cancer (BC) is the most common cancer in women and is the number
one cause of cancer mortality in women worldwide. In 2008, about 1.38
million new BC cases were diagnosed in lower-income countries, representing
half of all BC cases and nearly 60% of deaths . BC's
survival rates are widely variable, with an estimated 5-year survival
of 80% in high-income countries to below 40% for low-income countries
According to Globocan, about two million new cases were diagnosed
in the United States in 2018 with expected deaths of 626,679 .
BC incidence increases in North Sudan tribes, with 16.3% in Galyean,
6.6% in Shygea, and 5.3% in Danagala . Women living
in rural areas in Sudan constituted 58% of individuals presented with
those in urban areas .
Many risk factors are associated with BC, mostly related to lifestyle and environmental
considerations. Other factors include gender, age, genetic factors,
dense breast tissue, and positive family history .
In Africa, women under the age of forty are most likely to succumb
to BC .
The chance of a BC diagnosis after menopause is higher in women who
are overweight or obese. Also, the use of diethylstilbestrol may increase
incidence of BC . Early diagnosis is essential for
effective treatment. Self-examination is the best technique for detecting
breast inflammation or nodules and explaining sickness signs .
Radiologic studies, such as computed tomography, magnetic resonance
imaging, or positron emission tomography, are crucial for preliminary
Breast tissue biopsy and microscopic examination can confirm the diagnosis.
Fine needle aspiration cytology facilitates and accelerates clinical
and histopathological recognition of BC . Many
techniques can also help diagnose, such as in situ hybridization
and immunohistochemistry .
Some viruses, such as Epstein-Barr virus, mouse mammary tumor virus, and human
papillomavirus (HPV), have a role in BC development and progression .
HPV is linked to other types of cancers, such as head and neck, cervical, vulvar,
vaginal, anal, and penile carcinomas . The association
between HPV and BC is not yet well established. The prevalence of HPV in BC
ranges from 0 to 86% . Several studies support HPV
involvement in BC, but other studies could not detect HPV subtypes in BC tissues
. Clarification of the roles and mechanisms of HPV
involvement in BC is needed. HPV is a small, circular, double-stranded DNA
virus. Approximately 200 different HPV strains have now been identified and
classified into mucosal and cutaneous forms. The mucosal HPV can be classified
(Lr) and high-risk (Hr) based on the propensity for malignant progression of
lesions . LrHPV subtypes include HPV-6 and HPV-11.
These subtypes cause more than 90% of genital warts. HrHPV subtypes (e.g.,
16, 18, and 58) cause squamous intra-epithelial lesions that can progress to
invasive squamous cell carcinoma . Contaminated hands
can transmit HPV from the female perineum to the breasts .
HPV exhibits three oncogenes, E5, E6, and E7; two regulatory proteins, E1 and
E2; and two structural proteins, L1 and L2 . HPV E6
and E7 proteins are known for blocking pRb and p53 tumor suppressor genes,
thus contributing to oncogenesis . E6 binds to and
degrades p53. E7 binds to and causes dysfunction of pRb. Significantly, hrHPV
is integrated early into the host genome, and the structure of genes is identified.
P53 possesses an N-terminal transactivation domain (TAD), a proline-rich region
(PRR), a central DNA binding domain (p53C), a tetramerization domain (TET),
and an extreme C terminus. The DNA binding domain is the location of most cancer-associated
p53 mutations. Several human tumors show mutations and deletions of the RB
gene (13q14), with inherited allelic loss of RB conferring increased predisposition
to cancer formation .
Our study explored the association between HrHPV, p53, and RB gene mutation
in BC among Sudanese patients. The prevalence of HrHPV was assessed using PCR,
genotyping, and sequencing. Expression of p53 and RB gene mutations used immunohistochemical
methods to examine correlations between HPV genotypes and the expression of
mutant p53 and RB gene and tumor grades.
Study design: this cross-sectional study design initially recruited 2000 patients with breast tumors. One thousand patients declined, 500 did not meet study criteria, sampling was insufficient for 100 patients, 50 were benign, and the remaining 150 participants were successfully included in the study after pathologist approval.
Sampling method and recruitment: formalin-fixed paraffin wax processed blocks were investigated. The sample collection technique was based on a randomization formula.
N = (2 x Joint Success rate x Joint Failure rate) x (ZÞ+ Zß)2/(Diff)2
Study site: the samples were collected from histopathology laboratories
at the Radiation Isotope Center, Bahri and Al-Ribat Hospital, Khartoum State,
Sudan. This facility is the central referral system for the diagnosis and treatment
of patients with breast tumors.
Data collection: the study was conducted between January 2015 and December
The average monthly rate of newly diagnosed cases is about 90%. Interpretation
of BC grading was reported, according to the Bloom-Richardson (Nottingham)
Grading System: 1) Grade 1 or well-differentiated cells (scores 3, 4, or 5):
grow slower and look more like normal breast tissue. 2) Grade 2 or moderately
differentiated cells (score 6, 7): between Grades 1 and 3. 3) Grade 3 or poorly
differentiated cells (score 8, 9): grow rapidly and look very different from
normal cells .
Data were collected for age, histological type and tumor type (malignant or benign),
and grade. Master sheets were used to record all PCR and immunohistochemistry
Immunohistochemistry (IHC): followed the method of Pu et al. .
Paraffin-embedded blocks of breast tissues were retrieved from histopathology
laboratories. The sections were mounted on poly-L-lysine-coated slides and dried
in a hot air oven at 60°C for 1 hour. Sections were dewaxed in xylene and
rehydrated in descending grades of ethyl alcohol. Sections were then washed three
times with 1% phosphate buffer saline (PBS). Sections were boiled in Target Retrieval
Solution of Dako
(Real Envision Detection Kit) in a water bath at 95°c for 30 min. Sections
were then blocked with 3% hydrogen peroxide in the dark. The following antibodies
(Abs) were used: 50-100 microliters of primary mouse monoclonal mutant (RB Ab-1)
antibody (Catalog no MS-107-B) or primary mouse monoclonal mutant (p53 Ab-8)
antibody (Catalog no MS-738-P0, Thermo Scientific Company) at working dilutions
of 1/100 for 30 min at room temperature; 50: 100 microliters of a secondary antibody
(Thermo Scientific Company) were added. Immune reactivity was detected using
diaminobenzidine (DAB) (Thermo scientific Company); 50: 100 microliters of DAB
working solution was added and incubated at room temperature for 10 minutes.
Finally, sections were counterstained with Mayer's Hematoxylin, dehydrated and
in xylene, and mounted with DPX. For each IHC assay, proof slides were used for
negative and positive control. IHC stained sections were examined under light
microscopy (Olympus CHT, Optical. Co.Ltd, Japan) using 4×, 10×, 40× 100×,
objective and eyepieces of 10×, giving a maximum magnification of 1000.
Mutated P53 and RB were observed only as nuclear staining of epithelial
cells, and nuclei with clear brown color were scored as positive. Two investigators
scored the intensity of IHC staining for each marker based on subjective evaluation
of color exhibited by antigen, antibody, and chromogen complex. Scores were 0
for negative (no color), 1+ for weak (light brown color), 2+ for moderate (dark
brown color), 3+ for strong staining (dark brown color), and 4+ for overexpressing
(very strong dark brown color). Scores of 0 or 1 were defined as negative,
2, 3 or 4 as positive.
DNA extraction was performed using a DNA FFPE Tissue Kit .
Amplification (Table 1 and Figure 1) and interpretation (Table 2) used the HPV kit from Sacace Technologies . The length of specific amplified DNA fragments was HPV- 267-325 bp.
Statistical analyses: data were coded and entered using the statistical package for the Social Sciences version 25 (IBM Corp., Armonk, NY, USA). Data were summarized using frequency and relative frequency for categorical data. For comparing categorical data, Chi-square (2) tests were used. Exact tests were used instead when the expected frequency was less than 5 . P-values less than 0.05 were considered statistically significant.
Ethical consideration: the institutional research board of Ribat University,
Sudan, approved this study on May 9, 2015. Before data collection, study objectives
were discussed thoroughly with the local authorities and staff. Data were collected
anonymously, and confidentiality was guaranteed. The study protocol was compliant
with the declaration of Helsinki.
A total of 150 BC samples were analyzed for the presence of HPV DNA with PCR. All negative specimens were considered as baseline characteristics. Cases were further subdivided into medullary carcinoma, invasive lobular carcinoma (ILC), mucinous carcinoma, and invasive ductal carcinoma (IDC). The association of HPV DNA with BC cases was statistically insignificant.
Association between HPV genotypes and different histological types of BC among the study groups:
the association of HPV-DNA with BC cases was statistically insignificant (p = 0.7308) in the form of thirteen HPV-positive cases out of one hundred and fifty BC cases. We can notice from (Figure 2 A) that HPV-positive cases were distributed as follows: 1) five high-risk HPV-16-positive cases in the form of IDC. 2) Four high-risk HPV-58-positive cases in the form of one mucinous Carcinoma and three IDC. 3) Three high-risk HPV-18-positive cases in the form of ILC. 4) Only one low-risk HPV-11-positive case in the form of ILC.
Association among P53 and RB genes mutations and different histological types
BC among the study groups:
P53 gene mutation was detected in thirty-five patients, twelve ILC, twenty-one
IDC, and two medullary carcinoma cases. No P53 gene mutations were found in mucinous
carcinoma tissues. On the other hand, RB gene mutation was detected in thirty
patients, nineteen IDC, eight ILC, two mucinous carcinomas, and a single medullary
carcinoma case. This reflects a statistically significant association between
P53 (p = 0.0055*) and RB (p = 0.0154*) genes mutations and BC histological types
(Figure 2 B).
Associations among HPV genotypes and BC grading among the study groups:
two, six, and five HPV positive cases in grades (I), (II), and (III) were found
among 150 BC tissues, respectively. A higher incidence of HPV-16 was found in
grade (II) (three cases) than in grades (I) and (III) (a single case in each).
HPV-58 showed a similar profile with three positive cases in grade (II) and a
single case in grade (III). On the other hand, HPV-18 showed a higher
incidence in grade (III), with two positive cases, than in grade (I), with a
single positive case, while no positive cases were found in grade (II). HPV-11
was positive only in a single grade (III) case. The association between different
HPV genotypes and different BC grades was statistically insignificant
(Figure 3, p = 0.580).
Associations among P53 and RB genes mutations and BC grading among the study
P53 gene mutation was positive in thirty-five BC cases, distributed as two grade
(I), eleven grade (II), and twenty-two grade (III) cases. The association between
P53 gene mutation and BC cases appeared to be statistically insignificant (p
= 0.144). RB gene mutation was positive in a total of thirty BC cases and was
distributed as six grade (I) cases, thirteen grade (II) cases, and eleven grade
(III) cases. The association between BC grading and RB genes mutation appeared
to be statistically insignificant (p = 0.0599) (Figure
4, Figure 5, Figure
Associations among estrogen and progesterone receptor expression and BC grading among the study groups: estrogen receptors were expressed in a total of ninety-four BC cases, including eight grade (I), thirty-four grade (II), and fifty-two grade (III) cases. Such expression appeared to be statistically insignificant (p = 0.449). Progesterone receptors were expressed in a total of seventy-nine BC cases, including eight grade (I) cases, thirty-six grade (II) cases, and thirty-five grade (III) cases. The association between progesterone receptor expression and BC grade was also statistically insignificant (p-value = 0.247) (Figure 4).
Associations among P53 and RB genes mutations, estrogen and progesterone receptor
expression, and HPV positive cases among the study groups:
P53 and RB gene mutations were detected in six and three HPV positive BC cases,
respectively, which was statistically insignificant with p-value = 0.089 for
P53 and 0.568 for RB gene mutations. Also, both estrogen and progesterone receptors
were positively expressed in nine HPV positive BC cases and statistically insignificant
correlation (p = 0.522 and p = 0.129, respectively) (Figure
BC is a serious health problem worldwide. Viral pathogens are extensively
explored for BC, but a moderate association between HPV and BC
suggests a potential to improve early diagnosis, prevention, treatment,
Many studies have investigated HPV genotypes in BC in different
parts of the world, but no such studies are available for Sudan.
This study examined
the association of variables HPV integrations, P53 and RB gene
mutations, and estrogen and progesterone receptor (ER/PR) expression
with BC among
Sudanese women. In the current study, a statistically insignificant
association was observed between HPV infection and women diagnosed
with BC; with an
8.67% incidence of HPV. Previous studies showed 0 to 86% HPV prevalence
in BC worldwide [28,29]. Similar
studies succeeded in detecting HPV in BC in Thailand 
and China . Conversely, studies in Spain 
and India  failed to detect HPV in BC due to several
factors related to the used techniques or a different genetic population.
Our study provides additional evidence that HPV may play a role
in the development of BC . Our results showed an
HPV incidence of 8.69% for IDC, which is much lower than 21%, 29%,
and 35.41% incidences
in the studies conducted by Heng et al. ,
Salman et al. , and Francis et al.
respectively. HPV was also found in 14% of ILC cases in the Salman et
al. study  compared to 9.09% in our study.
This higher incidence is affected by the number of samples among
UK patients and fresh samples.
HPV-16 is the most common genotype in breast tumors .
Our study showed a statistically insignificant association between HPV-16 and
the five cases of IDC-BC with an incidence of 3.33%. A study conducted in Iraq
in 2017 found a 25.6% incidence of HPV-16 in BC . Lawson et
al. conducted a study on twenty cases of BC found a 10% incidence of HPV-16
. The present study showed no significant association
among BC samples and the three ILC cases exhibiting HPV-18. Al-Awany et al.
found a 27.1% association of HPV-18 and BC . Our study
shows no statistically significant association (2.67%) with HPV-58. A study conducted
by Ling et al. showed a higher incidence . Our
study found no statistically significant association (0.67%) with HPV-11, in
of a single ILC case. In Kuwait, the study delivered by Francis et al.
a 13.7% association of HPV-11 in IDC .
Regarding IDC, this study found that HPV-16 and HPV-58 had the highest incidence
of 5.43% and 3.26%, respectively. Hong and Tang reported a 51.1% incidence
of HPV-16 . In another study by Wang et al. the
incidence of HPV-58 was 35.6% . Moreover, the current
study demonstrated that HPV-18 and HPV-11 were presented more in ILC with percentages
of 6.82% and 2.27%, respectively. In Syrian women, HPV-18 was found in 37.16%
of ILC cases . However, Wang et al. found HPV-18
DNA more often in IDC than ILC, with a prevalence of 46% .
A third study in Isfahan showed that HPV-11 was the most common LrHPV in IDC
and not ILC, with a prevalence of 3.6% .
A statistically significant association was observed among p53 and retinoblastoma
(RB) gene mutation, and different BC histological types, with percentages
of 23.33% and 20%, respectively. Bertheau and colleagues found that p53 gene
are the most frequent
genetic alterations in and different BC histological types, with an incidence
of 30% . Conversely, Masri et al. did not find
any association between p53 gene mutation and BC samples obtained from 20 BC
patients . Our study's findings are consistent with
Anderson et al. who found the RB gene mutation in 21% of cases .
The present study indicates that p53 gene mutation was 27.3%, 22.8%, and 20.7%
for ILC, DLC, and medullary carcinoma, respectively. RB gene mutation incidences
were 40%, 20.7%, 18.22%, and 11.1% for mucinous carcinoma, IDC, ILC, and medullary
carcinoma, respectively. A study performed by Hong and Tang 
found the expression of p53 gene mutation 46.7% in IDC. Our findings for RB
gene mutation are consistent with an Italian study performed in 2009 .
No statistically significant association was identified between HPV genotypes
and BC grading. HPV-16 had a higher incidence in grade I (7.69%), HPV-58 had
a higher incidence in grade II (5.08%), HPV-18 showed relatively high incidence
in grade I (7.69%), and HPV-11 was found only in grade III cases (1.28%).
P53 gene mutation was not significantly related to different BC grades, with
twenty-two positive cases in grade (III), eleven cases in grade (II), and two
cases in grade (I), out of thirty-five P35 gene mutation-positive cases. This
finding is slightly different from a study conducted by von Deimling et
al. who observed p53 gene mutation in eleven cases out of twenty-two grade
(II) and (III) BC cases . Also, the RB gene mutation
was statistically insignificantly associated with different BC grades. ER and
were not statistically significantly associated with BC grades. ER incidence
was 61.54%, 57.63%, and 66.67% for BC grades II, III, and I, respectively,
in ninety-four positive cases. The PR incidence was 38.46%, 42.37%, and 33.34%
for BC grades II, III, and I, respectively, in seventy-nine positive cases.
The role of ER/PR in deciding the management and assessing the prognosis of
BC is well-established .
Our study also found that P53 and RB gene mutations were positive in six and
three out of thirteen HPV positive BC cases, respectively. Both ER/PR were
positive in nine out of thirteen HPV positive BC cases. Two studies by Hong
and Tang  and Wang et al. 
reported statistically significant expression of HPV-DNA associated with expression
of p53 gene mutations. The primary transforming capacities of HPV originate
from the E6 and E7 proteins. The tumor suppressor p53 initiates checkpoints
causing cell cycle arrest or induces apoptosis. HPV E6 oncoprotein initiates
degradation of p53 . Direct binding of E7 to pRB impairs
its function . Moreover, the E7 can impair p53 function
even in the absence of E6 .
A statistically insignificant association among HPV, both generally and
for different genotypes, and estrogen and progesterone
receptors was observed for different histological types and grades
of BC among the study group of Sudanese women. A statistically significant
association among p53 and RB genes mutation with different histological
types of BC was found.
Further studies with larger sample sizes are recommended to measure
the real burden of HPV in BC etiology in Sudan.
What is known about this topic
- Breast cancer is the most common cancer among Sudanese women;
- HPV is the most common in Sudan linked to other types of cancers;
- Gene mutation, one of the causal agents of cancer.
What this study adds
- There is a high-risk HPV 16, 18, 58, and low-risk HPV11 sequencing with breast
cancer and grades in Sudan;
- We identified in high-risk populations, p53 and RB gene mutations can be used to screen breast cancer is particularly noteworthy and have important implications for screening programs in Sudan.
The authors declare no competing interests.
All the authors had contributed to this work (preparation, discussion, and therapeutic decision). All the authors of the manuscript have read and agreed to its content. The manuscript is freely available to any scientist wishing to use them for non-commercial purposes.
The authors would like to thank the staff at the radiation isotope center
and different histopathology laboratories in the national Ribat and Bahri
A special thanks to Dr. Ihab Abo-Ali, the associate professor at the
Community Medicine Department, Faculty of Medicine, Tanta University,
Egypt, for his valuable contribution to this work.
Tables and figures
Table 1: PCR results for controls from the HPV kit
different HPV Genotype sequences (5´-3´) detected in BC cases using an ABI
3730xL DNA analyzer (Applied Biosystems, Foster City, CA, USA)
Figure 1: PCR amplification of HPV in breast cancer samples with a gel documentation system
Figure 2: association between different histological types of BC and (A) HPV genotypes and (B) P53 and RB gene mutations
Figure 3: association between different HPV genotypes and BC grading in the study groups
Figure 4: association among P53 and RB gene mutations, estrogen, progesterone receptor expression and BC grading
Figure 5: p53 gene mutation
immunohistochemistry staining of BC tissue (40 X) showing; (A) strong positive
(4+), (B) moderate positive (3+), (C) weak positive (2+), and (D) negative (1+)
Figure 6: RB gene mutation
immunohistochemistry staining of BC (40 X) tissue showing; (A) strong positive
(4+), (B) moderate positive (3+), (C) weak positive (2+), and (D) negative (1+)
Figure 7: association
between P53 and RB genes mutations, estrogen, and progesterone receptor expression
and HPV positive cases among the study groups Human Papilloma Virus (HPV),
base paring (bp), Invasive Ductal Carcinoma (IDC), Invasive Lobular Carcinoma
(ILC), Retinoblastoma (RB), strong positive (A), moderate positive (B), weak
negative (D), Statistically significant (p < 0.05)
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