Inconclusive role of human papillomavirus infection in breast cancer

Molecular study

Of the 77 patients (age range: 39–74 years; mean: 49 years; SE?=?9.43) selected for our molecular study, 61 had been diagnosed with invasive ductal
carcinomas. The clinical characteristics of the breast cancer samples were shown in
Table 1. None of the patients reported a family history of any other cancer. DNA isolated
from all of these cases underwent detection of HPV infection by PCR. All samples were
positive for ?-globin gene amplification (Fig. 1a), indicating that DNA was available for molecular analysis. However, no HPV DNA was
detected using either mucosal consensus primers (MY09/11) or cutaneous consensus primers
(FAP59/64) in any of the 77 breast tumor tissues or 77 normal adjacent tissues (Fig. 1b and c). Weak bands between 400 and 500 bp appeared in two breast cancer samples, but the
results of three extra PCR runs of the first-cycle products or DNA from these specimens
were negative (data not shown).

Table 1. Clinical characteristics of breast cancer tissues

Fig. 1. Identification the quality of genomic DNA from breast cancer biopsy and detection
of HPV from them by PCR. a PCR amplification of a 268-bp fragment of human ?-globin gene. b PCR amplification of a 450-bp fragment in the HPV L1 region detected using MY09/11
primers. c PCR amplification of a 478-bp fragment in the HPV L1 region detected using FAP59/64
primers. Lane M: DL 1000 DNA marker (TaKaRa). Lane W: Sterile water (negtive control).
Lane c: DNA from HPV-16 positive cervical tissues. Lane p1: pBS322/HPV16 plasmid;
Lane p2: pBS322/HPV8 plasmid; Lane p3: pBS322/HPV11 plasmid (positive control). Lanes
1–14: Breast cancer biopsy DNA

The overall and type-specific prevalence of HPV in breast cancer and factors impacting
the prevalence of HPV in breast cancer

A total of 326 published records were retrieved using the key words mentioned previously
(Fig. 2). Among those publications, 38 studies 2]–4], 7]–40] were pooled to calculate the HPV prevalence in breast cancer. The selected studies
comprised a total of 2569 breast cancer cases, and the majority came from Asia (51.96 %)
and South America (20.32 %). The number of cases investigated in each study varied
from 17 to 228, and the prevalence of HPV ranged from 0 to 86 %.

Fig. 2. Flow chart of identifying the articles related to HPV and breast cancer

The pooled prevalence of HPV in breast cancer across the 38 studies was 30.30 % (95 %
CI?=?22.30–38.40 %). Stratified by region, Oceania yielded the highest HPV prevalence
in breast cancer, with 44.30 % (95 % CI?=?33.50–55.00 %), followed by Asia with 35.70 % (95 % CI?=?20.60–50.80 %), Europe with 32.60 % (95 % CI?=?6.50–58.70 %), South America with 14.60 % (95 % CI?=?5.80–23.50 %), and North America with 10.70 % (95 % CI?=?4.40–17.00 %). When the unconditional logit regression model was introduced to
compare HPV prevalence in breast cancer among different geographic regions, the result
remained consistent: Oceanian breast cancer patients exhibited the highest HPV prevalence,
and the priority of this prevalence was statistically significant (P??0.001) (Table 2). HPV prevalence was found to be higher when HPV DNA was extracted from fresh tissues
(37.30 %, 95 % CI?=?20.10–54.50 %) than when HPV DNA was extracted from paraffin-embedded tissues (27.20 %,
95 % CI?=?17.90–36.40 %), but this priority of prevalence was statistically insignificant
(p?=?0.08) (Table 2).

Table 2. HPV prevalence in breast cancer cases across region, HPV DNA source, and publication
period

A cumulative meta-analysis was conducted to investigate the impact of publication
period on HPV prevalence in breast cancer, and we found that the HPV detection rate
between 2007 and 2012 (22.60 %, 95 % CI?=?14.40–30.70 %) was dramatically lower than that between 1992 and 2006 (42.20 %,
95 % CI?=?25.60–58.80 %) (p??0.001) (Table 2).

PCR was the dominant method used to detect HPV in breast cancer; it was used in 31.30 %
(95 % CI?=?22.60–39.90 %) of the studies we reviewed, while HPV prevalence was 5.30 % (95 %
CI?=?2.40–8.30 %) using other methods, such as in situ hybridization and Southern blotting (Table 3). Multiple primers, including broad-spectrum PCR primers, type-specific PCR primers,
and a combination of these, were used in PCR-based methods of detecting HPV. The HPV
prevalence rate was 30.00 % (95 % CI?=?18.30–41.70 %) when broad-spectrum PCR primers were used; it was 31.90 % (95 %
CI?=?13.00–50.80 %) when type-specific PCR primers were used; and it was 35.30 % (95 %
CI?=?11.50 –59.00 %) when a combination of these two types of primers was used. Because
most studies dealt with invasive ductal cancer as well as non-invasive cancer, the
detection rates of HPV in invasive ductal cancer were pooled, yielding HPV prevalence
of 32.90 % (95 % CI?=?21.50–44.20 %) compared with non-invasive cancer, which showed a prevalence rate
of 22.00 % (95 % CI?=?12.40–31.50 %) (Table 3).

Table 3. HPV prevalence in breast cancer cases by detection method and histological type

Although multiple HPV types were determined in breast cancer cases across the 38 studies
in our meta-analysis, the five most common HPV types, in decreasing order of prevalence,
were the following: HPV-16 (30.70 %; 95 % CI?=?20.50–41.00 %), HPV-33 (25.60 %; 95 % CI?=?7.10–44.00 %), HPV-11 (17.20 %; 95 % CI?=?1.00–33.30 %), HPV-18 (9.90 %; 95 % CI?=?6.20–13.70 %), and HPV-6 (6.30 %; 95 % CI?=?2.00–10.50 %) (Table 4). Classified by HPV oncogenic features, the prevalence of high-risk HPV types was
35.50 % (95 % CI?=?25.00–46.10 %), much higher than the prevalence of low-risk HPV types, which was
11.70 % (95 % CI?=?5.80–17.70 %).

Table 4. Prevalence of Overall and Individual Human Papillomavirus (HPV) Types

The association between HPV infection and breast cancer risk

A total of 16 case–control studies were included in our evaluation of the association
between HPV infection and breast cancer risk (Fig. 1). Due to existing heterogeneity (p?=?0.000 for heterogeneity test, I ²
?=?63.9 %), a random model was chosen to calculate the combined OR and its 95 % CI
across the 16 studies. When these 16 studies were pooled in the meta-analysis, results
showed that HPV infection was associated with higher risk for breast cancer (OR =3.24,
95 % CI?=?1.59–6.57, p??0.001) (Fig. 3). Egger’s and Begg’s tests were performed to assess the publication bias; the results
of both indicated no statistical significance (p?=?0.16 and 0.45, respectively).

Fig. 3. Forest plots of studies evaluating odds ratio of HPV infection to breast cancer risk

Considering that substantial heterogeneity was observed among the 16 studies for HPV
infection and breast cancer, the prevalence of HPV infection in breast cancer was
further evaluated by subgroup analysis. The analyzed subgroups were defined according
to the main features of pooled studies: geographical region, DNA source, detection
method (i.e., PCR primer), and publication period (Table 5). In the subgroup of geographical regions, between-study heterogeneity was absent
(p?=?0.611 for heterogeneity test, I ²
?=?0.0 %) in Oceania and Europe, but pooled OR remained the same (OR?=?3.16, 95 % CI?=?1.27–7.90, p??0.001). When ORs were grouped by DNA source, however, significantly higher risk
for breast cancer was revealed among fresh-tissue methods (OR?=?7.88, 95% CI?=?3.99–15.60, p??0.001) without between-study heterogeneity. Similarly, breast cancer risk was greater
with HPV infection in studies published between 1992 and 2006 (OR?=?4.12, 95% CI?=?2.42–7.02, p??0.001) compared with studies published between 2007 and 2012. HPV infection detected
using broad-spectrum PCR primers was associated with higher risk for breast cancer
(OR?=?5.67, 95 % CI?=?3.40–9.45, p??0.001), while HPV infection detected using combined PCR primers showed the opposite
result (OR?=?0.68, 95 % CI?=?0.32–1.45, p??0.001).

Table 5. Associations Between HPV prevalence and breast cancer Grouped by Selected Factors