In developing and developed countries, including Iraq, breast cancer is a widespread malignant disease among women [1, 2]. Studies in Iraq show that most women with breast tumors have a potential prevalence of more aggressive types of tumors in progressive stages [3, 4]. Several risk factors, including possible viral etiology, have been related to breast tumor advancement and development [5]. Several dangerous influences have been found, such as the age of the patient, case history, and long exposure to estrogen hormones. Sometimes, an apparent danger influence may be present in 50%-80% of patients [6]. So, current studies have been achieved to determine further dangerous influences related to humor.

Some studies have reported a causative association between breast tumors and viral infection, such as Epstein-Barr virus, mouse mammary tumor virus, human papillomavirus, and human cytomegalovirus [7].

HCMV is one of the herpes virus families that infected the women 70%-90% of the world’s people. It is reactivated frequently after a dormant infection in the host [8].

Nucleic acids and proteins of HCMV have been detected in a variety of tumors such as colon, kidney, breast tumor, glioblastoma, medulloblasoma, muco epidermoid salivary gland cancer, and rhabdomyosarcoma [9].

One research indicated HCMV was identified by immunohistochemistry test in normal breast tissue epithelial cells and in malignant breast carcinoma epithelial cells, but was higher at a later stage [10] On the other hand; current research did not detect HCMV in breast tumor tissue [11].

HPV is a DNA virus that is often related to cervical cancer in women who are particularly great danger types 16 and 18. It was also observed in anogenital and oral cancer and categorized as an onco virus by IARC [12]. When the combination of the virus to the host cell genome, viral proteins such as E6 and E7 are expressed, and the tumor suppressor proteins P53 and Rb are inactivated [13]. Identification of HPV in breast cancer has demonstrated contradictory findings ranging from 0 to 86 percent in the various studies [14].

Several studies have investigated HCMV, EBV, and HPV in breast tumors with PCR that cannot distinguish viruses in cancer cells from non-epithelial cells. Immunohistochemistry testing will then find viral capsules either in malignant epithelial cells or in non-epithelial cells, giving exact results [15].

Various molecular techniques are obtainable for the detection of CMV nucleic acids. Of these, in situ hybridization (ISH) can be used with freezing cells and tissues using radio-labeled probes or probes with non-radioactive labeling such as fluorescent moieties-biotin- digoxigenin-or enzyme-conjugated probes and cytological preparations and fixed tissues [16].

Design and Setting

This retrospective study used 140 selected breast tissue . The study that carried out in hospitals and private laboratories in Kirkuk and Tikrit city from 15th January 2022 to 15th of december2022 by using in situ hybridization (ISH) technique.

Study participants and sampling

In this study used 140 selected breast tissue formalin-fixed, paraffin-embedded pieces from 100 patients (70 cases ductal mammary carcinoma, 15 lobular cancer, 5 combination (ductal and lobular cancer) ,10 medullary carcinoma and 20 patients benign mammary epithelial lesions. A further 20 pieces as control groups. 16-72 years the patients’ age range. A research carried out by the in situ hybridization (ISH) technique in hospitals and private laboratories in the Kirkuk and Tikrit city from 15 January 2022 to 15 December 2022.

Laboratory methods

Sections of thick tissue (4 mm) have been prepared and fixed on positively charged slides. For digoxigenin-labelled cock tail HPVDNA probes for a extensive variety of HPV genotypes including genotypes 16, 18 and 31, an in situ hybridization discovery system (Zyto Gmb)). At the site of sequence complementarity, ISH signals were differentiated as red discoloration as nuclear signs by good use of the Zyto Gmb system.

In tissue specimens, another ISH discovery system (Maxim Biot Inc.) was used to goal DNA sequences using a long biotinylated DNA probe for HPV genotypes 16, 18 and 31. The techniques were carried out according to the manufacturing company’s data. By exchanging the probe with a biotinylated house kept gene probe, positive responses were obtained. Both substances were inserted for negative regulation, excluding the dilute probe. Proper using of this ISH recognition system provides a concentrated blue sign in positive test tissues at particular positions of the hybridization probe (depended to definition of the second difference kit giving the blue color). , the sign was measured in microscopy. The ISH results given ratio grade according to positive signs and the numeral of cells that made these signs.

One piece was stable on the normal slide and hematoxyline and eosin stain, while another slide fixed on the charged slide to be used for CMV identification for ISH. The ISH kit (US Biolol. USA) detection of CMV-DNA was done on 4\(\mu\)m paraffin fixed tissue pieces using biotyinlated-labeled oligo-nucleotide probe targeting CMV-DNA.

This retrospective study used 140 selected formalin-fixed, paraffin-embedded pieces 100 patients with breast tumor, 20 patients with benign epithelial mammary lesions, and 20 blocks as control groups from 15 January 2020 to 15 December 2020 in hospitals and private laboratories in Kirkuk and Tikrit governorates, aged 60-73 years, using in situ hybridization.as showed in Table 1.
At the site of complementary sequences, the nuclear signals of genotyping HPV and HCMV- ISH were observed as blue discoloration as seen in Figure 1.

Breast cancer is more shared in the developed populations and is 100 times more in females than in males [16] . Opinion of the increased risk factors of breast cancer are positive family histories, age, the primary pregnancy following 25 years old, menarche, delayed menopause, null parity, long-standing utilization of exogenous estrogens, obesity following menopause, as well as encountering ionizing ray [17]. Other factors that important in the mechanism of increased risk factors are estrogen receptors and estrogen levels, and the adipokinesleptin and adiponectin [18].

In situ hybridization is a technique advanced for identifying particular nucleic acid DNA and RNA sequences for metaphase spreads, morphologically well-preserved tissue parts, and cell preparations by hybridizing the DNA probe’s complementary strand to the target sequence. In the past few decades, cytogenetic cancer diagnosis and treatment methodologies have progressed enormously. The discovery of a landmark link between cancer and chromosome aberration was first discovered in 1960 by Peter No well and David Hungerford, see [19]. In addition, several previous studies using ISH techniques have shown ISH to be an efficient tool for identifying and concentrating CMV-DNA and HPV in the infected tissues. In the present study, CMV DNA-ISH was distinguished in 25 percent (25 out of 100 cases), and HPV was detected in 45 percent (45 out of 100 cases). Unexpectedly, Shakir and Al-Alwany [20] observed that 34.3 percent (24 out of 70) of breast cancers show positive signals, HPV-DNA ISH identification of 18/70 cases of breast cancer (25.7 %) revealed positive red signals. No positive signals for the HCMV and HPV ISH tests were registered in the control group. Breastfeeding is the primary way HCMV is transmitted during the first year of lifespan in nation-state where record women are HIV-positive and breastfeeding (Stagno and Cloud, 1994 [21]). The HPV method of transmission identified in breast tumors remains unknown. The reported frequency of HPV infection in breast tumors has seen a significant change worldwide, reaching from 0 to 86 percent [22]. Demographic features and inherited history may lead to global geographic differences in the prevalence of HPV in breast tumor tissues [23].

In this study, high oncogenic risk genotypes of HPV, such as HPV 16, 1 8, and 31, were found in breast cancer cases. The common HPV forms distinguished are from the high oncogenic community (HPV 16 + 18) despite significant variations in worldwide HPV recognition rates [24]. The relationship of high oncogenic-risk forms of HPV in invasive breast carcinomas has been reported to be higher, indicating a potential interaction with pathology and disease score [25]. Although it is well known that the main causative agent for cervical cancer is high-risk HPVs, the role of the virus in breast cancer is more controversial. This debate could be affected by technological limitations, multiple primer sets and/or detection probes, HPV epidemiology in different geographical regions, different sexual behavioral trends, different incidences of anogenital HPV infection, and probably different population genetics. This makes it much more difficult to diagnose HPV in the breast and can, therefore result in the lack of HPV in breast cancers identified by some investigators [26, 27, 28]. Any research, on the other hand, did not provide signs to support the role of HPV in breast cancer. Wrede et al. [28] and Gopalkrishna et al. [29] performed experiments to determine the involvement of HPV types 16 and 18 in breast tumors. However, nobody of the samples revealed any relationship. A study has been conducted on the presence of HPV DNA in breast tissue in Korean women and the relationship between HPV and breast cancer development, however, no visible associations have been found [30]. During the last period, multiple independent trainings in various areas have recorded no substantial association between HPV and the prevalence and development of breast tumors [31]. The role of HPV in tumors such as uterine cervical cancer has been commonly recognized.

Virons have been reported to be shed from desquamating keratinocytes (goal cells from HPV infections), and high-danger HPVs can be transferred by near-human non-sexual communication [32]. We therefore speculate that HPVs may be transferred by finger from the female perineum to the breast, which may happen during a shower or a bath. It has been well recognized that hormonal influences (estrogen and its derivatives) are identified to be precipitation in breast tumors [33, 34].

The results of this study showed a connection between HCMV and high-oncogenic genotypes of HPV in breast cancer patients..

HCMV: Human Cytomegalovirus
HPV: Human Papilloma Virus
ISH: In Situ Hybridization (ISH) technique

This research paper received no external funding.

The authors declare no conflicts of interest.

All authors contributed equally to this paper. They have all read and approved the final version.

Informed consent was obtained from all participates in the study as needed.

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