（ 様 式 ３ ）論文要旨THE PREVALENCE OF THE HUMAN PAPILLOMAVIRUS
AND ITS COFACTORS IN BREAST CARCINOMAS TO
EVALUATE ITS ROLE IN BREAST CARCINOGENESIS

乳がんにおけるヒトパピローマウイルスの分布および発がん過程におけるその役割と関連要因に関する検討
Noureen Afsar Khan
ABSTRACT
Purpose of this study
Recent studies have revealed a possible association with human papillomavirus (HPV) in the pathogenesis ofbreast cancer. To investigate the etiological role of HPV in breast cancer, we examined the presence, genotype,viral load, and physical status of HPV in breast carcinomas (BCs) collected from different countries.

Subjects and methods
The present study examined 307 BCs collected from Japan (n=124), Pakistan (n=61), India (n=11), Mexico(n=63), and Chile (n=48). Cervical carcinoma (CC) samples from Japan and Pakistan were also used forcomparison. HPV presence was examined by PCR using SPF10 primers, and primer sets targeting the E6 regionof HPV-16, -18, and -33. The INNO-LiPA HPV genotyping kit was used to determine genotype. Real-time PCR
analysis was used to examine viral load and physical status of HPV DNA. The p16INK4a and p53 expressions
were analyzed by immunohistochemistry assays.

Results
1. HPV detection rate
HPV DNA was detected in 26 (21%), 17 (28%), 4 (36%), 8 (13%), and 5 (10%) BCs from Japan, Pakistan,India, Mexico and Chile, respectively. The difference of HPV detection rate among these countries wasstatistically significant (P=0.048). In CCs, HPV was detected in 72 (85%) Japanese and 77 (94%) Pakistanicases. The presence of HPV was significantly different between the breast and CC (P < 0.001). The mostfrequently detected HPV genotype was HPV-16 in both sites, and its frequency was 54 (90%) and 125 (84%)
carcinomas of breast and cervix, respectively. The detection rate of HPV-16 in all HPV-positive cases was notdifferent between breast and CC.

2. Distribution of HPV genotypes
In all HPV-positive cases with BCs, the frequency of low-risk HPV genotype (P=0.021) and HPV multiple
infection (P=0.032) was significantly different among the five countries. Most cases with low-risk HPV
infection were revealed to harbor multiple HPV types (93%), and the highest frequencies of low-risk types andmultiple infections of HPV were observed in Japanese series (46% for both). Multiple infections were also
found in 2 (12%) and 1 (13%) BCs of Pakistan and Mexico, respectively. In CC, multiple infection was more
frequently found when compared to BCs (P < 0.001): 91% in Pakistan and 15% in Japan. The observed
difference between the two countries was statistically significant (P < 0.001).
3. HPV detection in normal tissue and breast milk
In 19 normal epithelium specimens adjacent to 19 HPV-16-positive BCs, 10 (53%) were HPV-16-positive.
However, three (5%) of the normal breast tissue specimens adjacent to HPV-negative BCs were also
HPV-positive. Nipple specimens adjacent to eight HPV-16-positive Mexican BCs were also examined, and one
nipple specimen was HPV-16-positive. In addition, ten clostrum and 25 breast milk samples from Japan werealso examined for HPV presence. One (10%) clostrum specimen was positive for HPV-16 and two (8%) breast
milk samples were positive for HPV. However, only low-risk types of HPV were detected in milk samples.
4. Viral load and physical status
To clarify the etiological involvement of HPV in carcinogenesis of the breast, further analyses were conducted.
Real-time PCR analysis suggested the presence of integrated-form of viral DNA in all HPV-16-positive BCs
from all countries, and estimated viral load was low with geometric mean of 5.4, 1595, 158, 5.3 and 6402 copies
per 104 cells in carcinomas of Japan, Pakistan, India, Mexico and Chile, respectively. The geometric mean ofHPV-16 viral load was significantly different among these countries (P < 0.001). All HPV-16 positive CCs
were found integrated into the host genome except two cases, which were suspected to have only episomal
HPV-16. The geometric mean of HPV-16 viral load in cervical cancers of Japan and Pakistan was 410,000 and
2,370,000 copies per 104 cells, respectively.

The geometric mean of viral load in the CC was higher than BC

and the difference was statistically significant.
5. Immunostainning
The p16INK4a expression was significantly high in HPV-positive CCs (P = 0.031). Although a similar
association was found in BC, their relationship was not statistically significant. On the other hand, p53
expression tended to decrease in HPV-positive CCs (P = 0.110), while there was no trend in BC. These
differences of p16INK4a and p53 expressions between cervical and BCs were statistically significant (P = 0.013
and P < 0.001, respectively).

Conclusion
In conclusion, the relatively low HPV copy number and infection rate in breast cancer suggest that HPV is
unlikely to play as an essential role in the carcinogenesis of breast cancer as in genital neoplasia. However, since
oncogenic HPVs were consistently detected in BC and most of the detected HPV-16 DNA was considered
integrated into the host genome, HPV infection may play some roles in the carcinogenesis of a subset of BCs.