In 1992, Di et al. [37] detected HPV-16 DNA sequences in 29.

4% of the 40 paraffin embedded

breast cancer specimens and in 17.1% of the lymph nodes containing breast cancer metastases
using polymerase chain reaction (PCR). However, HPV DNA could not be detected in any of the
samples using in situ hybridisation (ISH), suggesting that PCR is more sensitive than ISH.
Hennig et al. [38] detected HPV-16 DNA in 19 out of 41 (46%) women who had a history of
high grade cervical intraepithelial neoplasia (CIN III) and breast carcinoma using PCR, but only
one positive case was confirmed using ISH. They postulated that the oncogenic HPV DNA might
be transported from the original infection site to other organs via a haematological or lymphatic
route, and may be responsible for the development of cancer in various organs. They further
compared the expression of p53, p21, and c-erbB-2 proteins between the HPV-16-positive and
HPV-16-negative breast carcinomas using immunohistochemistry (IHC) and found no significant
differences [39]. HPV-33-derived DNA was found in 14 cases (43.8%) of invasive breast ductal
carcinoma by Yu et al. [40], but HPV-16 and HPV-18 DNA were not detected in any cases. This
was the first report detecting HPV-33 in human breast carcinomas.

HPV DNA was detected in 25 (24.75%) breast cancer specimens by Damin et al. [42], but none
of the benign breast tissue specimens tested positive (P\0.001). Whilst a higher HPV prevalence
was detected in human breast cancer tissue, a causative rather than an associative relationship
was difficult to prove, as it was not clear if HPV was present before or after the development of
cancer. de Villiers et al. [44] analysed HPV subtypes in areolar and tumour tissue from patients
with breast cancer. HPV DNA was present in 25 of 29 breast cancer specimens and in 20 of 29
samples from the corresponding mammilla. Multiple HPV types were detected in seven cancer
tissues and ten nipple tissue specimens. These results demonstrate the occurrence of HPV in
nipple and areolar tissue in patients with breast carcinoma and suggest that HPV may infect
breast tissue though the nipple. Kan et al. [45] screened 50 unselected invasive ductal breast
cancer specimens using PCR for HPV subtypes 16, 18 or 33 gene sequences.

Overall, 24 (48%) of the 50 samples were HPV-18 positive, and the authors postulated that HPV
could infect breast tissue through contaminated hands. Kroupis et al. [46] utilised four PCR
methods to verify the presence of genital HPV in frozen breast cancer specimens, and 17 samples
out of 107 were tested positive (15.9%).
Results of this study found one sample with a double infection and two samples with triple
infections. Fourteen samples were tested positive for HPV-16 (67% of all detected HPV types),
whilst three tested positive for HPV-59, two for HPV-58, and one each for HPV-73 and HPV-82.
This study further indicated the presence of high-risk HPV DNA sequences in breast cancer
tissue. Gumus et al. [47] postoperatively obtained malignant and normal breast tissue samples
from 50 patients with breast cancer and tested these samples for the presence of HPV-11, HPV-
16, HPV-18, and HPV-33. Thirty-seven malignant breast tissue samples (74%) contained HPV
DNA. Additionally, sixteen normal breast tissue samples (32%) were also shown to be positive.
HPV-18 was detected in 20 of the HPV DNApositive malignant tissue samples (54.4%) and in 9
of the HPV DNApositive normal tissue samples (56.3%). HPV-33 was detected in 35 (94.6%)
of the HPV DNApositive malignant tissue samples and in 14 (87.5%) of the HPV DNA
positive normal tissue samples. These results contradict with the findings from the study
conducted by Damin et al. [48], which indicated that normal breast tissue from patients
diagnosed with breast cancer does not carry HPV DNA.

Sarkola et al. [51] tested 223 breast milk samples obtained 3 days postpartum for HPV-16 DNA
using PCR, ISH and sequencing, and found that 4.0% of the milk samples were positive. The
presence of HPV DNA in the breast milk was not correlated to the mothers oral or cervical HPV
status or any demographic data. Surprisingly, oral HPV infection of the spouse 6 and 12 months
postpartum was significantly associated with the presence of HPV in the breast milk. Akil et al.
[52] determined that 69 (61.06%) of 113 breast cancer tissue samples were HPV positive.
Amongst these specimens, 24 tissues (34.78%) were co-infected with more than one HPV type.
Furthermore, the authors reported that the expression of the E6 oncoprotein of these high-risk
HPV subtypes was correlated with Id-1 overexpression in the majority of invasive breast cancer
tissues.

De Leon et al. [36] measured HPV DNA in paraffin embedded specimens selected from 5
patients with breast cancer using PCR and sequencing techniques. These findings were compared
with 43 cases of non-malignant breast lesions (e.g., fibroadenoma, fibrocystic disease or
phyllodes tumour) matched according to patient age and tumour size.

They found that 15 (29.4%) of the paraffin-embedded specimens from patients with breast
cancer were positive for HPV DNA. Amongst these positive patients, ten were positive for HPV-
16, three were positive for HPV-18, and two were positive for both types. In contrast, all of the
samples from the benign samples were negative for HPV DNA. These results supported the
hypothesis proposed by Damin et al. [42] that normal breast tissue does not harbor HPV. Heng et
al. [53] used ISH and standard PCR to assess the presence of high-risk HPV in the cells of breast
cancer specimens and in breast cancer cell lines and found that the oncogenic characteristics of
HPV-associated breast cancer were very similar to HPV-associated cervical cancer, such as HPV-
16 and HPV-18. These observations further support a role for high-risk HPV subtypes in the
pathogenesis of human breast cancer and offer the possibility of primary prevention of breast
cancer using an HPV vaccine. Evidence against HPV involvement in the aetiology of human
breast cancer In 1992, Bratthauer et al. [54] tested for the presence of HPV-6, HPV-11, HPV-16,
and HPV-18 in 15 papillomas, 15 papillary carcinomas, and 13 infiltrating ductal carcinomas of
the breast using a Southern blot of PCR products and found no HPV-related DNA sequences in
any of the samples assayed. In the same year, Wrede et al. [55] were also unable to demonstrate
the presence of HPV DNA in a series of 80 breast carcinomas. Gopalkrishna et al. [56] analysed
fine needle aspirate cell samples from 26 patients with breast cancer and 4 breast cancer biopsies
for the presence of HPV-16 and HPV-18 DNA sequences using PCR and Southern blot
hybridisation. No positive results were found using either method. The authors concluded that a
causal role for high-risk genital HPV subtypes, most notably subtypes 16 and 18, in the
pathogenesis of human breast carcinoma seems to be remote. Though these viral genomes can
putatively transform breast epithelial cells in vitro, they were not able to do so in vivo. Lindel et
al. [57] analysed paraffin-embedded sections from 81 patients with breast cancer for HPV DNA
using PCR with the SPF1/2 primers that targeted a conserved domain in L1 and covered
approximately 40 different low-risk, intermediate-risk, and high-risk types. They found that all of
the samples were negative for HPV DNA. However, as Damin et al.

[48] highlighted, there are two main reasons why the L1 primers should not be used in isolation:
the L1/E1 regions, rather than the E6/E7 regions, are lost during integration of the viral DNA
into the host genome, a process that is an essential component of progression from HPV
infection to development of the malignant phenotype. In addition, the E6/E7 region exhibits less
nucleotide variation.
Khan et al. [59] examined samples from 124 female Japanese patients with breast carcinoma
using PCR with the SPF10 primers that target the E6 region of HPV-16, HPV-18, and HPV-33.
HPV DNA was detected in 26 (21%) breast carcinomas. The most frequently detected HPV
genotype was HPV-16 (92%), followed by HPV-6 (46%), HPV-18 (12%), and HPV-33 (4%). The
estimated viral load was low with a geometric mean of 5.4 copies per 10 cells. The authors
concluded that although HPV DNA was detected in 21% of breast carcinomas and that in all
HPV-16-positive cases the HPV genome was considered integrated into the host genome, the low
viral loads suggest that it is unlikely that the integrated HPV is etiologically involved in the
development of breast carcinoma in Japanese patients. Choi et al. [61] examined DNA from 154
patients, including 123 breast cancer samples and 31 intraductal papillomas and nipple tissue
from 27 patients with cancer using DNA microarrays. HPV DNA was detected in eight breast
carcinomas (6.5%), but not in any intraductal papilloma specimens. All of the detected HPV
genotypes were high-risk subtypes. There was a slightly increased prevalence of HPV in the
papillary carcinomas (11.5%) and invasive ductal carcinomas with adjacent intraductal
papillomas (11.8%) compared to the other histological subtypes (3.24.3%). However, these
results could not demonstrate whether the HPV infection directly contributed to the development
of breast cancer.

Geographic differences

The reported prevalence of HPV infection and HPV types in breast cancer shows great variation
worldwide, ranging from 0 to 86% (supplement Table 1), suggesting that demographic features
and genetic backgrounds may contribute to geographic differences in HPV infection in breast
carcinomas. European studies [39] have mainly focused on HPV-11, HPV-16, HPV-18, and HPV-
33, whereas studies examining Chinese and Japanese women have demonstrated that breast
cancer can be associated with rare HPV subtypes, such as 33 and 56 [40]. HPV-18 was the major
subtype detected in Australian patients with breast cancer (positive rate 36%) [45]. A Brazilian
study found that 25 out of 101 (24.8%) breast carcinoma samples contained HPV DNA
sequences, and HPV-16 was detected in 56% of these cases (16/25), HPV-18 was detected in
40% (10/25) and HPV-16 and HPV-18 were detected in 4% (1/25) [42].
These variations based on the different geographic areas could be due to the differential
susceptibility of the populations or various HPV detection methods or PCR primers used in the
assays.

Conclusions and future directions

There has been substantial evidence presented in this review that suggests HPV may play a role
in the development of breast cancers. However, data linking breast cancer with chronic HPV
infection have been contradictory, resulting in a lack of a consensus. There are criteria developed
to test the validity of the evidence for specific viruses to cause specific cancers [64]. These
criteria include the presence of viral genetic material in the cancer whilst being absent in normal
tissue; the ability of the virus to transform or immortalise normal cells in culture virus-specific
antibodies being more frequently detected in patients when compared to control subjects; the
presence of the virus, virus-specific antibodies or both preceding the development of the cancer;
induction of specific malignant phenotypes and characteristic histological changes; evidence of
oncogenicity of the virus in laboratory animals; and the positive effect of an intervention (such as
preventative vaccines) against the virus on the incidence of a specific cancer. With respect to
their possible role in breast cancer, HPV meets virtually all of the above criteria, with the
important exceptions of evidence of a positive effect of an intervention and consistent
immunologic evidence.

However, the evidence presented to date seems inconclusive for a definitive role of HPV in
breast cancer because the strength of any study has been too low [22, 6568].

We can image this scene: HPV transmission occurs between a woman and her spouse around the
age of 20 or even younger through their mothers milk, body fluid, blood or even the air. The
viruses are located in the mammary calculus, entering into the nucleus at a cellular level to
destroy DNA. During breast tissue repair and regeneration, abnormal processes may occur.

In light of the above findings, we postulate that prophylactic HPV vaccines for cervical cancer
may also reduce the development of breast cancer in women. Long-term follow up of women
who receive this vaccine at a young age may determine the validity of this hypothesis. In
addition, prevention of high-risk HPV infection by prophylactic vaccination is likely to prevent
many HPV-associated cancers.
HPV-associated cancers maintain and express HPV viral genes even during the advanced stages
of the disease, and the repression of viral oncogene expression can prevent the growth or survival
of breast cancer cells.