الفهرس 
Incidence and mortality of breast cancer worldwide
A- Myeloid-derived suppressor cells and cancerOnly 14 pages are availabe for public view
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Abstract
Breast cancer (B.C.) is the most commonly diagnosed cancer among women worldwide. In addition, considered as the second most common cancer with nearly 1.7 million new cases in 2012; this represents about 12% of all new cancer cases and 25% of all cancers in women. In Egypt, according to statistics on all cancer (2014), B.C. is the most common cancer that leading to death among woman’s accounting 38.8% of all cancer among woman’s. In 2018, according to (WHO), B.C. was approximately 15% of all cancer deaths among women. There are different types of treatment for B.C. including surgery, radiation doses, chemicals doses, anti-hormones, and target antibodies. Despite of the reasonable success of these therapies, recurrence still occurs. The mechanisms underlying this relapse are still obscure. One possible mechanism that has been suggested and tested is the suppression of the immune cells by several immunoregulatory (checkpoints) molecules, which are expressed on immune cells, as well as cancer cells. The immune system has evolved an array of regulatory mechanisms to protect against tissue damage from autoimmunity or during active response to pathogen. Immune checkpoint pathways such as those mediated by PD-1 receptors are critical aspects of normal physiologic function the hallmark of these pathways is generation a negative feedback loop that preserves self-tolerance and prevents excessive tissue damage in the setting of immune response. The pathways regulated by PD-1 molecules have somewhat distinct modes of action on the immune cells and is induced upon effector T cell activation and highly expressed on Tregs. The cognate ligands for PD-1 include PDL1. These are constitutively expressed on the antigen presenting cells and are induced in peripheral tissues during inflammatory responses or on the surface of cancer cells. Aims from this study Our study aimed to analyze the phenotypic and the functions of immune T cells in the peripheral blood of breast cancer patients. Further to analyze the expression of checkpoint molecules (PD-1, PDL-1) on immune cells, also analyze the expression of PDL-1 on circulatory tumor cells and cancer stem cells in the peripheral blood of breast cancer patients and correlated the results with clinical status of the patients compared to healthy volunteers’. (2) Materials and methods This study was approved by Ethics Committee from Faculty of Medicine Tanta University, and executed in Centre of Excellence in Cancer Research (CECR), Tanta University Educational Hospital, Egypt in project with title ”Correlating immunologic and clinical performance between genomic, transcriptomic and proteomic profiling of circulating tumor cells versus primary tumor cells” with code Number (3012/01/15 ). The study included breast cancer patients (n=28). All patients were females with mean ages 47 ± 10.48 years and range (31 – 68) years. Five ml of (PB) were collected on (EDTA) from patients before surgery. The stages and grades of B.C. were diagnosed histopathologicaly from biopsies that were collected after surgery. Six patients had stage II (21.4%), nine patients had stage III (32.1%) and thirteen patients had Stage IV (46.5%). Twenty-two (n=22) of patients had not chemotherapy and Six (n=6) of patients (21.4%) had chemotherapy. These different cases of patients compared with healthy volunteers (n=10) with mean age 31.75±6.02 years and range (24-37) years. 1-Firstly, Complete blood pictures (CBC) were measured within 1hours after drawn to calculated percentages of haemoglobin (Hb), RBC and (WBCs) and its differential. 2-Secondly, Processing whole blood samples occurred through BD Bioscience protocol: 100 μ L of whole blood were mixed with aliquots monoclonal antibodies that were divided two categories. Category I: CD279 (PD1), CD4, CD25, CD127, CD8. Category II: CD274 (PDL-1), CD44, CD24, EpCAM, CD133. The samples were incubated in dark condition for 30 minutes then incubated for 15 minutes in dark conditions after addition 2ml lysing solution (1X) to lyses (RBCs) then spin down the samples at 1500 r/m in the centrifuge for five minutes. The supernatant discarded, the pellets washed with 2 ml phosphate buffer saline (PBS). The supernatant discarded, and the pellets were suspended in (PBS). The samples were analysed by (BD FACS Canto II) software and FlowJo software version Vx.0.7. The percentages of phenotypic subsets CD4+ cells, Treg (CD4+CD25+), and CD8+ were measured in different stages (II, III, and IV) of B.C. woman’s and compared with healthy individuals. Gates were taken around total leucocytes, myeloids, monocytes and lymphocytes to estimates percentages of PD-1 and PDL-1 expression on these populations. The study compared between two groups with late stages (III-IV) before and post chemotherapy treatment. (3) Results and discussion Our data showed red blood cells (RBCs) count, Hb, and its parameters slightly decreased in stages II, III, and IV compared to healthy women. In addition, Platelets count decreased at stages (II, III, and IV), respectively, compared to controls. These decrease due to cancer progression and forming metastasis in bone marrow and lung. Total leucocytes count of B.C. patients significantly increased in stage II compared to other stages (III and IV) and healthy women as result of increasing in neutrophils. Lymphocytes significantly decreased with cancer progression due to migrating lymphocytes to large numbers of lymph nodes especially in stage IV. Our data showed percentages of CD4 significantly decreased in (PB) of B.C. patients from stage II to stage III, and the least percentage was in stage IV, compared to healthy women. PD-1 expression on immune cells with immunophenotypes CD4+ significantly increased from stage II to stage III, and the highest expressions were in stage IV of B.C. patients, compared to healthy women. The percentages of CD8+ in (PB) of B.C. patients significantly decreased from stage II to stage III and the lowest percentages were in stage IV, compared to percentage in (PB) of healthy women: Conversely, the percentages of PD-1 expression on CD8 significantly increased from stage II to stage III, and the lowest expressions of PD-1were in stage IV. Treg (CD4+CD25+) significantly increased with cancer progression from stage II to stage III, and the highest percentages of Treg were in stage IV, compared to healthy women. This clarify the suppression effect of Treg. Our data showed the important role of CD127 expression on Treg and proved that this marker had low expression on Treg in healthy women and this low expression significantly decreased with B.C. progression so it is possible to differentiate between Treg in normal and adaptive condition as B.C. development. Treg had expression of PD-1 that significant increased with B.C. progression compared to healthy women, these clarify the important role of PD-1 expression on increasing suppression effect of Treg on other immune cells as CD4 and CD8. Such these cells mediated immune system, activate, and protect immune system from pathogen and cancer. Our data showed the conjugation between PD-1 and PDL-1 on populations of total WBCs, lymphocytes, monocytes, and granulocytes has important role in B.C. progression. Our data showed PDL-1 expression on circulatory tumor cells and cancer stem cells significantly increased in (PB) of B.C. post chemotherapy treatment compared to before chemotherapy. Our data suggested, however, chemotherapy (4) eradicate cancer cells of B.C. patients and gave chance to immune cells to refresh temporally but gave chance also to PDL-1 expression to conjugate with new PD-1 expression on immune cells and re-suppression and recurrence. Therefore, it is important to breast cancer patients with late stages to blocked PDL-1 by immunotherapy or take it with combination chemotherapy to prevent B.C. recurrence. Conclusion Detection of T regulatory cells levels in peripheral blood of breast cancer patients and expression of checkpoint molecule PD-1 (CD279) on immune T cells CD4, CD8 and Treg help us to understand the immunomodulatory nature of Treg and PD-1 in immunotherapy. The study help us to understand the nature of CTCs that responsible of a recurrence and metastasis of breast cancer through precision these cells. The expression of PDL-1 on CTCs has main role in behaviour these cells; furthermore, the patients need seriously to anti PDL-1 as immunotherapy with considerable specificity of cells that target to this type of treatment.