Evaluation frequency of Human Herpes Virus type 8 in Patients with Breast Cancer

Breast cancer is one of the most common malignancies and the most common cause of death women around worldwide. Recently, viral etiology theory has been proposed on physiopathology of the breast cancer. Method: In a retrospective study, to study of non-familial agents and a viral factors breast cancer, the Real time PCR were used to detect Kaposi Herpes virus or Human herpes virus type8(HHV8) in 138 patients with breast cancer. Result Out of the 138 embedded paran breast cancer tissues, 17(12.3%) were positive for HHV8. Among the viral positive breast cancer samples, 8 samples (10.95%) were positive in intermediate grade and 10 samples (11.9%) were positive in 40–60 years old age group. There was no signicant relation between grades of tumors and there was a signicant relation between age and breast cancer.


Introduction
Breast cancer is one of the most common cancers in women [1], with more than 2.1 million people diagnosed with breast cancer annually [2]. Breast Cancer is the second most common cancer in women in the United States, with 268,600 new cases in the year. After lung cancer, breast cancer is the second deadliest cancer in the world and is responsible for 627,000 and 41760 deaths in the world and the United States respectively [2]. About 15% of women's cancer deaths are due to Breast cancer [1]. Until 2015, the incidence of breast cancer had an upward trend, but today the incidence of breast cancer has a steady trend [1]. However, the exact cause of breast cancer is unknown, and several factors such as lifestyle, smoking, obesity, aging, and infectious agents are considered as risk factors for breast cancer [2,3].
The HHV-8 is the eighth human virus of the Herpesviridae and is also known as Kaposi's Sarcomaassociated Herpes Virus (KSHV) [11]. The HHV-8 virus causes Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease [12,13]. Kaposi's sarcoma is more common among immunocompromised individuals such as AIDS patients [14]. Studies have shown that this virus is tumorigenic and is closely related to bro adenoma tumors [9,15]. However, the virus is not associated with cervical and oral tumors [16,17]. A study by PAUL et al. Showed that the HHV-8 virus has the potential to invade and persist in the brain tissue [18]. Several studies have also shown that the HHV-8 virus has been detected in breast cancer patients. The study by Tsai showed that the prevalence of HHV-8 in breast cancer patients is higher than other viruses [9]. Also, the Amira S. Mohamed study showed, 28.8% blood samples of breast cancer cases were positive for HHV-8 DNA [10].
The HHV-8 virus has the ability to produce cytokine homologs such as interleukin-6 (IL-6). Studies have shown that an increase in IL-6 is associated with metastasis and the progression of breast cancer [19][20][21]. Increased IL-6 expression activates the virus lytic cycle and increases the expression of genes involved in pathogenesis, leading to the development of malignancy [22]. The ability to infect and proliferate in epithelial cells, the ability to produce interleukin homologs, and the detection of the hhv-8 virus genome in breast cancer tissue are factors that suggest that the hhv-8 virus may be associated with breast cancer [23].
We aimed to investigate the presence of HHV-8 genome in breast tissues in women with breast cancer.

Sample collection and preparation
A thin 10-μm tissue section was obtained from the blocks, and depara nization was performed with xylene. The, a series of distilled water and graded ethanol solutions were used for rehydration, according to previous studies.

DNA Extraction
The DNA extraction was performed by using viral nucleic acid extraction kit (North Korea), according to the manufactures instruction. Spectrophotometry Nano drop ND-1000 (thermo Fisher Scienti c Inc. Waltham, MA) was used for evaluation of the extracted DNA. The extracted DNA was stored at -20°C until molecular tests were performed.

DNA ampli cation
For the detection of HHV-8-DNA, a Taq Man real-time PCR approach was applied. About 40 ng of extracted DNA was used as the template in a reaction including 10μl of 2x-PCR master mix (amplicon, Austria), 10 pmol/ml of probe (FAM-TGCAGCAGYTGTTGGTGTACCACAT-BHQ1), 30 pmol/ml of each following primers (5′-AGCCGAAAGGATTCCACCATT-3′) and (5′-TCCGTGTTGTCTACGTCCAGA-3′), then brought to 20μl using sterile distilled water. The test was performed in 45 cycles, started by one cycle 10 min at 94°c, followed by 45 cycles of ampli cation, consisting 10 s at 94°c and 40 s at 60°c (Table 1).   Tumor-infected lymph nodes were divided into four categories: <3, 3-6, 6-9, and > 9 (Table 4).    The relationship between the number of lymph nodes involved in the tumor and viral infection was also examined. In 14 cases between 6 and 9 lymph nodes were involved and in 1 case more than 9 lymph nodes were involved. Statistical analysis showed that there was a signi cant relationship between the number of lymph nodes involved in the tumor and the frequency of HHV-8 virus infection (P-value = 0.016) ( Table 8)

Discussion
Breast cancer is one of the most common and deadly diseases that has an unknown cause [24]. There are several factors that increase the risk of breast cancer, including infectious agents, especially viruses [25].
Various studies have shown that the prevalence of viral infections in breast cancer patients is higher compared to the control group. A study by Amira S. Mohamed and colleagues showed that the HHV-8 virus genome was found in the blood of 28.8% of breast cancer patients [10]. While our results showed that the frequency of HHV-8 virus genome in breast cancer tissue samples was 12.38%. In the present study, we did not nd a signi cant relationship between tumor grade and the frequency of viral infection, while in the Amira study, the prevalence of viral infection also increased with increasing tumor grade.
Similar to Amira study, our results showed that there is a signi cant relationship between tumor-infected lymph nodes and the prevalence of viral infection, so that with the increase of cancer-infected lymph nodes, the prevalence of viral infection has also increased (P-value < 0.05). Therefore, it is likely that the lymphocytes infected with HHV-8 virus may be the source of cancer cells and may also cause infected cells to spread to other organs.  [15].
The HHV-8 virus is associated with a variety of tumors, such as prostate tumors [28], endothelial cell tumors [29], and B cell lymphocytes [30]. Studies have also shown that the HHV-8 has the ability to immortalize and transform breast cells by inhibiting apoptosis, inducing cell proliferation, cell survival, increasing angiogenesis, and modulating the immune system. In addition, the virus can induce tumor genesis by producing interleukin homologs [10].
The results of this study showed that the prevalence of HHV-8 infection in patients with breast cancer is high and may be associated with an increased risk of breast cancer. In addition, it is likely that the lymphocytes infected with HHV-8 virus may be the source of cancer cells and may also cause infected cells to spread to other organs, as well as the treatment of these patients is di cult.