Human papillomavirus and breast cancer: Examining the evidence of a connection

INTRODUCTION

Breast cancer (BC) is the most common cause of cancer-related mortality among women globally.^[1] Approximately 2.3 million new cases and 685,000 deaths were reported among women in 2020.^[2] The burden of BC morbidity and mortality has increased by more than 20% since 2008 and is projected to reach over 3 million new cases and 1 million deaths by 2040.^[3] With the highest incidence rate across Asian countries, one in nine Pakistani women have BC. The incidence of BC in Pakistan is reported to be 4.5 times higher than that of any other cancer type.^[4] Women with BC are at a higher risk of developing other comorbidities, such as osteoporosis, cardiovascular diseases,^[5] and diabetes,^[6] as well as human papillomavirus (HPV).^[7] This increasing incidence and its association with other comorbidities necessitate research on these etiological factors across diverse geographical regions.

Various factors contribute to the etiology of BC among women, including genetic mutations, lifestyle, reproductive history, and viral infections.^[8,9] Viruses have been reported to play a significant role in cancer development, and approximately 18-20% of human malignancies are attributed to them.^[8] Several viruses, particularly hepatitis B virus, hepatitis C virus, Epstein-Barr virus, and human papillomavirus (HPV), upregulate oncogenes, accelerate cell cycles and induce chronic inflammation, leading to cellular damage that triggers carcinogenesis.^[10,11] This study focuses on determining the associations between HPV and BC. As a sexually transmitted virus, HPV typically damages cutaneous and mucosal epithelia.^[12] HPV initiation occurs following direct skin-to-skin mucosal contact, and studies suggest that HPV can spread between an individual’s hands and a partner’s hands and genitalia. Studies show that 80% of people worldwide have contracted at least one type of HPV at some point in their lives.^[13,14]

Recent studies propose that HPV may increase BC risk.^[15] Viruses such as HPV can integrate their viral deoxyribonucleic acid (DNA) into the host genome, causing dysregulation of cellular proliferation and leading to tumor development. A study reported that HPV infection increases the risk of BC incidence 3-6 times among women.^[16] This association between HPV and BC is crucial for several reasons. The architecture of the mammary ducts increases the risk of HPV infection since the ductal openings are exposed to the external environment. The mammary duct epithelium is the site for the majority of breast tumors, which progress to in situ and invasive ductal carcinomas following ductal hyperproliferation.^[17]

Previous cross-sectional studies conducted in the United Kingdom,^[17] Qatar,^[18] and Taiwan,^[7] have identified HPV infection as a significant risk factor for BC progression across different geographical regions. These findings imply that HPV in breast tissue may influence BC development in some cases. A recent study reported a significant distribution of high-risk HPV genotypes among Pakistani women with BC, supporting the hypothesis that HPV may contribute to breast carcinogenesis in this region.^[19] However, data remain limited, and to date, no study has explored the expression of HPV E6 antigen or its clinicopathological correlations among BC patients, specifically in Khyber Pakhtunkhwa. Therefore, this cross-sectional study aimed to determine the association between HPV and BC progression in this population.

MATERIALS AND METHODS

This descriptive cross-sectional study was conducted in Nowshera Medical College, Khyber Medical University and Khyber Teaching Hospital, Peshawar, over 6 months from January 2023 to June 2023. The sample size was determined using the World Health Organization (WHO) sample size

Here, Z = 1.96 at a 95% confidence interval, P = 8.2% (the frequency of HPV in BC), q = 1−p, and e = 5% (margin of error). The minimum required sample size for the study was 118, as calculated using the WHO formula. However, to enhance the reliability and generalizability of the findings, 311 patients were included in the study. This larger sample size enabled a more comprehensive analysis and increased the statistical power of the results. Patients were selected using convenience sampling during the study period based on eligibility criteria outlined below. Potential confounders such as age, tumor grade, and hormone receptor status were analyzed in stratified subgroups to minimize bias.

Biopsy specimens were selected based on the following inclusion criteria: Confirmed cases of BC reported by a consultant histopathologist without age restrictions. Exclusion criteria included male patients with BC and cases of benign breast disease.

Ethical approval for the study was obtained from the Nowshera Medical College, Advanced Studies and Research Board and the Ethical Board of Khyber Medical University, Peshawar. Paraffin-embedded blocks of BC tissues meeting the inclusion and exclusion criteria were retrieved from the respective laboratories with proper departmental consent. Detailed data were collected on patient demographics, including age and residence, and clinical findings, such as the presence of a mass and pain. Morphological features were also recorded, including tumor size, overlying skin and nipple-areola complex status, consistency, and mobility. Histological data included tumor grade and lymph node involvement.

Immunohistochemistry was performed on 3-5 mm-thick sections prepared using a microtome and placed on glass slides. The sections were dewaxed using xylene and rehydration in a graded ethanol series (100-50%). Antigen retrieval was carried out using ethylenediaminetetraacetic acid (pH 9.0) for 10 min at boiling temperature, then cooling for 20 min. Sections were incubated in a 0.3% hydrogen peroxide solution for 20 min to block endogenous peroxidase activity. The primary antibody (E6 antibody, 1:500 dilution in phosphate-buffered saline, Catalogue #MA1-46057 by Thermo Fisher) was applied and incubated in a humidified chamber at room temperature.

After three washes with phosphate buffered saline (PBS), the sections were treated with a biotinylated horseradish peroxidase-conjugated secondary antibody. 3,3’-Diaminobenzidine (DAB) substrate solution was then applied to visualize the antibody staining. The sections were counterstained with hematoxylin for 30 s, washed, dehydrated, cleared in xylene, and mounted with glass coverslips. The slides were examined under a microscope, and each slide’s immunoreactive scores (IRSs) were calculated.

Data were recorded and analyzed using the Statistical Package for the Social Sciences version 26. Continuous variables, such as age, were presented as mean ± standard deviation, while categorical variables, including the presence of HPV, were summarized as frequencies and percentages. HPV presence was stratified by age, E6 antigen expression, human epidermal growth factor receptor 2 (HER2)/neu, and estrogen and progesterone receptor (ER/PR) status. A Chi-square test was applied post-stratification, with P ≤ 0.05 considered statistically significant. The results were illustrated through tables and graphs.

RESULTS

We recruited 311 diagnosed cases of breast carcinoma. All participants were female, aged 25-80, with a mean age of 45.24 ± 9.75 years. The majority of the patients, 107 (34.41%), were between 36 and 45 years, followed by 75 (24.12%) between 46 and 55 years, 74 (23.79%) >55 years, and only 55 (17.68%) between 25 and 35 years. Almost two-thirds, 236 (75.88%), were cases of invasive ductal carcinoma. About 153 (49.20%) were classified as Grade 2 tumors. The demographic data are shown in Table 1.

HPV was detected in 53 (17.04%) cases, with expression noted in 34 (10.93%) out of 311 cases. The majority of cases, 277 (89.07%), received an IRS score of 0, followed by an IRS score of 1 (<10% of the cells) in 21 cases (6.75%). ER/PR expression was positive in 197 (63.34%) cases. The findings are illustrated in Table 2 [Figures 1-3].

Close

Figure 1:

Detection of human papillomavirus (HPV) (black arrow). Immunohistochemistry (a) 10× and (b) 40×. (Stains used: Eosin, hematoxylin).

Export to PPT

Close

Figure 2:

Detection of human papillomavirus (HPV) (black arrow). Immunohistochemistry (a) 10× and (b) 40×. (Stains used: Eosin, hematoxylin).

Export to PPT

Close

Figure 3:

Detection of human papillomavirus (HPV) (black arrow). Immunohistochemistry (a) 10× and (b) 40×. (Stains used: Eosin, hematoxylin).

Export to PPT

Table 3 presents a comparative analysis of the HPV status across various demographic and clinical characteristics among the 311 participants. When stratified by age group, the data did not indicate statistically significant differences in HPV presence (P = 0.308). Similarly, the analysis of HPV status across different cancer diagnoses did not reveal significant associations (P = 0.956). There was no significant difference in HPV presence between Grade 1, Grade 2, and Grade 3 tumors (P = 0.202). Furthermore, ER/PR and HER2/neu expression status did not significantly correlate with HPV, with P = 0.577 and 0.381, respectively.

Comparative analysis of HPV detection and expression showed a statistically significant association with P < 0.001, as shown in Table 4.

DISCUSSION

Several studies have suggested a potential carcinogenic role for HPV in breast carcinoma progression, as it transforms virus-infected cells into a malignant phenotype.^[20,21] Some studies have reported a strong association between BC incidence and HPV infection, with prevalence rates ranging from 4% to 86%, likely reflecting differences in detection methods, geographic populations, and sample types. HPV infections can cause hypergranulosis, epithelial hyperplasia, koilocytic changes, and parakeratosis, which are hallmark changes in HPV-associated epithelial lesions. However, their significance in breast tissue remains uncertain.^[22] The HPV provirus triggers the synthesis of viral oncoproteins that impact carcinogenic mechanisms.^[23] Recent research suggests that HPV oncogenicity in breast tissue may be similar to other types of carcinogenesis, where the viral infection may be an early event leading to cumulative changes throughout a chronic viral infection. These findings complicate establishing a viral etiological role in human BC development based solely on detecting viral DNA in breast tissue.^[23,24]

This study analyzed 311 female patients diagnosed with breast carcinoma (mean age: 45.24 ± 9.75 years). Approximately 75.88% of the included population was diagnosed with invasive ductal carcinoma, consistent with the global incidence levels of this subtype.^[25] Among the diagnosed cases, those with advanced tumor stages (Grade 2) were greater in number, with 49.20%, followed by 44.37% Grade 3 cases. These findings indicated a predominance of more advanced tumor stages, likely due to a lack of early diagnosis and aggressive tumor behavior in this study population. However, the HPV incidence was relatively low, with only 17.04% positive cases in the study population. In addition, 10.93% of cases were detected with HPV expression, suggesting a potential role in BC in this cohort. The IRS for most patients was 0 (89.07%), indicating that most tumors lacked detectable HPV staining. These results align with previous studies, which remain ambiguous regarding HPV involvement in BC. Approximately 63.34% of the patients showed positive ER/PR expression, suggesting that a significant percentage of this group may benefit from hormone therapy. Furthermore, 22.51% of patients exhibited positive HER2/neu expression, indicating a potential for targeted treatments such as trastuzumab. No statistically significant differences were found when comparing HPV status across age groups, cancer diagnoses, tumor grades, and receptor expression. HPV may not significantly impact tumor behavior or advancement in this cohort, as evidenced by the lack of correlation (P > 0.05) between HPV presence and these clinical traits. Nonetheless, a substantial correlation was discovered between HPV detection and its expression (P < 0.001), suggesting that individuals who tested positive for HPV had a higher likelihood of HPV expression. This highlights the necessity for further research into the molecular pathways involved in HPV-associated BC.

This study is subject to several limitations, including its cross-sectional design, which prevents us from making causal inferences. In addition, the lack of longitudinal follow-up limits our ability to assess the temporal relationship between HPV infection and BC. Detection bias may also have influenced the results, as HPV presence was detected based on tissue samples that may not represent all patients. These factors should be considered when interpreting the findings

CONCLUSION

Overall, the findings indicated that invasive ductal carcinoma is the most commonly diagnosed subtype of BC, with a higher proportion of Grade 2-3 diagnoses, highlighting the predominance of advanced-stage disease, which may reflect delayed diagnosis or aggressive tumor biology’s. The study’s findings also reported a high proportion of ER/PR receptor positivity, suggesting hormone-targeted therapies as a potential treatment strategy for these patients. Although the detection of HPV-linked expressions among BC patients was relatively low, these findings may predict a significant association between BC incidence and the presence of HPV in this cohort. However, the significant association between HPV detection and expression warrants further research into the role of HPV in BC pathogenesis and progression.