Molecular studies of breast cancer in Iran: A Systematic Review

Background: The present study aimed to review the existing cellular and molecular research systematically carried out in the eld of breast cancer in Iran. This study was part of a big project, which evaluated a variety of studies conducted on breast cancer in Iran. Methods: All molecular articles about breast cancer in Iran which were published from January 2005 to October 21, 2015 were included. English and Persian databases consisted of Web of Science, PubMed, Scopus, Scientic Information Database (SID) and IranMedex. Finally of 3331 abstracts, a total of 379 articles, including 219 English and 160 Persian papers related to molecular studies were included in this study. Results: After initial evaluations, 240 studies were included. Molecular studies conducted in Iran were divided into four main groups based on their focus. They evaluated breast cancer susceptibility genes, Prognosis and clinicopathologic features of the patients, compounds with therapeutic properties, and diagnosis. Moreover, ethical statements in manuscripts were investigated. Conclusions: Result of this review showed that most of the researches in Iran categorize to 4 main subjects. The majority of the studies in Iran were conducted sporadically and with small sample sizes. Stronger studies would require the development of more biological banks and execution of multicenter comprehensive projects dened with orientation to main problems of Breast Cancer patients in the country. Ki67, E-cadherin, and BRCA1 were found to have signicant relationships with the stage of the disease. Twelve studies identied signicant associations between tumor grade and 22 genes/proteins including Bcl-2, E-cadherin, FGFR1, ADAM9, IKBKB, PRDM14, MTDH, MYC, HER2, CCND1, TOP2A, CDC6, CD10, BRCA 1,hTERT,p53, PSA, Telomerase, Hsp70, HER2, MHC I and ICAM-I (9–20). and nanoparticle forms of doxorubicin (32–39). Of these, four were conducted on human samples and 20 on rats or mice (in-vivo). The studies mainly involved cells and cell lines (particularly MCF7). Most studies (n = 42) used MTT assay to determine the effects of various compounds on specic genes, mainly cell proliferation and apoptosis pathway genes. (qRT-PCR) (MLPA) inter- and of assessment of HER2/neu immunostaining in invasive (220). blue (MB) (EIS) and cyclic (CV) (221). Other study developed a High-Performance Liquid Chromatography (HPLC) method for assessment of ErbB1 and ErbB2 genes amplication (222). Salimi et al. evaluate the gene amplication using dual-colour primed in situ labelling (PRINS) in comparison with uorescence in situ hybridization (FISH) (223). Conjugation of monoclonal antibodies to SPIO informed as a useful method for the detection of tumor cells in the other study (224).

Totally 379 articles included in a molecular subgroup. Full texts of the selected papers were found and, on some occasion, an e-mail was sent to corresponding authors to get the necessary information. Three reviewers used a checklist to appraise the full texts of selected articles critically. The reviewers discussed and resolved any cases of disagreement.

Data extraction
After reviewing all selected articles, the necessary information was extracted and entered into the study characteristics sheet. Because of the wide variation in the methodology and presented results of the studies chosen, an Excel sheet was used for data extraction. The rst part of the datasheet contained general information about the rst author, number of authors, title, study design, year of publication, year of the study (midpoint), city and place of the study, study population, sample type, total sample size, and ethnicity, sex, and age of the participants. The second and third parts of data consisted of the method of research and main results, respectively.
Three persons extracted all articles. The manager of the research team organised the three extracted forms in one sheet. The main objective of this study was to clarify the distribution of breast cancer researches in Iran. Therefore, no studies were excluded due to low quality. Duplicate and unrelated articles were excluded from the study. For better quality assessment of the articles, all their limitations were highlighted by entering NA (not assigned) in relevant columns of the results tables.

Data analysis
In the third step, the extracted data were analyzed. Due to the variety in study elds, the results were categorized according to study designs. The reviewed articles were categorized into ve groups, including susceptibility, treatment with the different type of compounds, Prognosis, survival and clinicopathologic features, and diagnosis. However, 17 studies could not be included in any of the mentioned groups. They were, hence, not inserted in the tables but described in the text.
This research has been approved in Breast Cancer Research Center Ethical Committee with ethical code number "IR.ACECR.IBCRC.REC.1394.54"

Results
A total of 379 articles, including 219 English and 160 Persian papers, were found eligible. After initial evaluations, 139 studies were excluded from the systematic review due to irrelevance (n = 37), unavailability of full text (n = 14), duplicates (n = 54), irrelevance of publication date (n = 12), inaccessibility (n = 5), and poster (n = 3), review studies (n = 12), or note articles (n = 2). Ultimately, 240 studies were included. (Fig. 1) Duplicates found during this phase were articles published in both Persian and English languages. The reviewed articles were categorized into ve groups according to their main themes. The results of the analysis of the papers in each group were as follows: Susceptibility Table 1 summarises the studies in Iran which examined genes related to breast cancer susceptibility. Based on these studies, susceptibility genes were divided into three groups, including high, intermediate, and low penetrance genes. Accordingly, from a total of 62 studies, 21 examined high-penetrance genes, four studied intermediate-penetrance genes, and 38 investigated low-penetrance genes. Moreover, 47 studies were conducted on blood samples, nine on tissue samples, three on blood and tissue samples, and three on cell lines.  Thirteen studies focused on BRCA 1/2 genes. The total sample size in these studies was 1413 cases and controls and ve families. While six studies determined the sequencing of all the exons of these two genes, the others examined their speci c areas. P53, from the high-penetrance group of genes, was also observed in six studies. Few (mainly one) studies assessed other genes. The largest sample size (1457 participants) belonged to a study on the CAT gene (8).
The majority of studies were conducted in Tehran (n = 18), Shiraz (n = 11), and Tabriz (n = 8). None of the studies investigated the survival rate of mutation carriers.

Prognosis, survival and clinicopathologic features
Another group of studies investigated the relationship of gene or miRNA expressions, mutations, and epigenetic changes with clinicopathological characteristics and invasiveness of the disease ( Table 2  B, MMP-9, MMP-3 and p53 and metastasis was signi cant in these studies (19,(26)(27)(28)(29)(30) Of the two studies evaluating the relationship between gene expression and response to chemotherapy; one found the relationship between MRP1and metastasis to be signi cant (31).

Treatment
The third group of studies focused on the therapeutic effects of different compounds, including herbal extracts, medicinal compounds, nanoparticles, and bacteria, on breast cancer ( Table 3). Some of these studies (n = 104) assessed the effects of various compounds on the expression of speci c genes involved in apoptosis or cell proliferation. The most studied compounds are drug conjugate to Trastuzumab and nanoparticle forms of doxorubicin (32)(33)(34)(35)(36)(37)(38)(39). Of these, four were conducted on human samples and 20 on rats or mice (in-vivo). The studies mainly involved cells and cell lines (particularly MCF7). Most studies (n = 42) used MTT assay to determine the effects of various compounds on speci c genes, mainly cell proliferation and apoptosis pathway genes.

Diagnosis
The fourth group included 9 studies with applications in breast cancer diagnosis. The majority of these studies compared various molecular methods such as differential PCR (216) The fth group comprised 18 studies that could not be assigned to any of the abovementioned groups. They examined various issues including functional mechanisms of genes (225)(226)(227)(228)(229)(230)(231) animal models of the disease (232)(233)(234)(235), familial breast cancer assessment (236)(237)(238), tumor environment simulation (239) and informatics studies (240,241). One study optimised a method to prepare liposomes containing HER2/Neu-derived peptide as a vaccine delivery system for breast cancer (242).

Ethical considerations
Out of the 240 selected studies, 174 studies were conducted on human or animal samples. However, only 53.45% of these papers (n = 53) had ethical approval, and the rest did not address ethical issues (the other 71 studies did not consider ethical issues, either).

Discussion
Cellular and molecular studies conducted on breast cancer in Iran were divided into ve groups: I) focusing on breast cancer susceptibility genes, II) genes affecting prognosis and survival, III) genes affecting therapeutic effects of different compounds on different grades of breast cancer cell lines, IV) studying about genes affecting breast cancer diagnosis, and V) studies which are focus on functional mechanisms of genes and establishment of animal models of breast cancer, respectively.
Studies in the rst group investigated either germline or somatic mutations. Most of the researches were about BRCA1/2 gene. Most of the investigations on other susceptible genes did not consider family history. The critical problem in these studies was patient sampling di culties and collecting clinical data and follow up variables. It seems that further studies on family pedigrees and germline mutations are required to con rm the role of mutations in susceptibility to cancer. Moreover, establishment and strengthening the cancer biobanks are very important which has been neglected these years (243)  Numerous studies in the world have examined the effects of epigenetic changes on cancer. However, the number of such studies is very limited in Iran. Among all of the studied HIC 1 promoter hypermethylation was correlated with nodal involvement and tumor size. Since most epigenetic changes occur due to environmental and non-genetic factors (22,67,164), studying the subject in sporadic patients can help clarify the role of the environment in the incidence of cancer. Moreover, these changes may affect the invasiveness of cancer or its response to medications (244,245).
Hence, it is essential to conduct further studies to clarify susceptibility genes and their relationship with prognosis. Such studies will lead to the discovery of new genes or gene panels and facilitate decision-making during the treatment of patients with breast cancer. Conducting these studies in populations from different geographical locations is highly essential. However, most studies in Iran have been sporadic and with limited sample sizes. The majority of these studies have not assessed the patient survival rate or different subgroups of breast cancer separately. Performing reliable studies in this eld would require access to not only the patients' clinical data but also biological banks and systems for regular collection and storage of biological samples. Limited reports of such banks are available in the country (e.g. BCRC-BB) (243). Moreover, since limited research resources are available, more extensive studies cannot be easily performed.
Another problem is that the majority of studies are researcher-oriented. Determining research priorities in speci c elds by relevant institutions, e.g. the Ministry of Health or various universities, and aggregating and integrating the results of multi-center studies would probably yield stronger and more valid results. However, sporadic studies in single centers will not help the expansion of the boundaries of knowledge.
The majority of studies on the effects of herbal and medicinal compounds were at cell level or on mice. Considering the high therapeutic potentials of these compounds, the great interest of pharmaceutical companies in the screening and discovery of new medications based on the geographical area, vegetation cover, and microorganism populations in Iran, more serious studies in this eld may be essential.
This review also assessed the ethical considerations in the selected articles. The absence of ethical approval, despite its necessity, indicates two points. Firstly, ethical approvals were not mandatory for biological studies before 2015 in Iran, and many researchers did not consider it as an essential issue in non-clinical studies. Now, the Ministry of Health has obliged researchers to obtain approval from an ethics committee. Secondly, and more importantly, it is evident that journals publishing such papers had no strict rules about the absence of ethical approval. In these cases, most of the researcher might have had ethical approval but did not provide the data due to journal policies. Considering the current policies in ethical aspects of researches in Iran, It is expected that this issue is modi ed in future studies.

Availability of data and materials
Not applicable.

Competing interests
The authors declare that they have no con ict of interest.

Funding
This study was a part of a comprehensive project to systematically review the different aspects of breast cancer in Iran. A grant from Roche Company funded the leading research.
Authors' contributions RE managed the group and was responsible for the literature search, manuscript categorization, data extraction, writing manuscript. SHD and ZES participated in the literature search, data extraction, and preparation of tables and rechecking the data. TO was responsible for data extraction and rechecking the data. SH and KM managed the team in developing the idea in all steps of research including search, data extraction, preparing tables, writing the manuscript. All authors have participated in providing the rst draft of the manuscript and they have approved the nal version.