Kurdistan Region Government Ministry of Higher Education and Scientific Research University of Sulaimani College of Medicine Department of Pathology and Forensic Pathology
Fibroblastic ketone production fuels mitochondrial biogenesis in breast cancer cells/ an immunohistochemical study in Sulaimani city - Iraq A THESIS
SUBMITTED TO THE COUNCIL OF THE College OF MEDICINE SULAIMANI UNIVERSITY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN PATHOLOGY
By
Rawand Tajuldeen Sahib M.B. Ch.B.
Supervised by
Assistant Prof. Dr. Hadeel Adnan
F.I.B.M.S. (Path) M.B. Ch.B.
2017AD
2717K
1438 H
} وما أُوتِيتُم ِ ّمن ال ِع ْل ِم ِإالَّ ق ِليلا { صدق هللا العظيم سورة اإلسراء/آية ٨٥
II
Certification I certify that this thesis entitled “Fibroblastic ketone production fuels mitochondrial biogenesis in breast cancer cells/ an immunohistochemical study in Sulaimani city/ Iraqˮ was prepared under my supervision at the Department of Pathology and Forensic Pathology, College of Medicine, Sulaimani University, as a partial fulfillment of the requirements for the degree of M.Sc. in Pathology.
Signature
Supervisor Assistant Prof. Dr.Hadeel Adnan Yasseen F.I.B.M.S. (Path) Department of Pathology and Forensic Pathology College of Medicine Sulaimani University Date:
May, 2017
In view of the available recommendations, I forward this thesis for debate by the examining committee
Signature Assistant Prof. Dr. Sana Dlawar Jalal F.R.C.Path., F.I.B.M.S. Path. Chairman-Department of Pathology and Forensic Pathology College of Medicine Sulaimani University III
We, the examining committee, certify that after reading this thesis entitled “Fibroblastic ketone production fuels mitochondrial biogenesis in breast cancer cells/ an immunohistochemical study in Sulaimani city/ Iraq” in its contents and what is connected with it, have examined the student “Rawand Tajuldeen Sahib” and in our opinion, it meets the basic requirements toward the Degree of Master of Science in Pathology.
Assistant Professor
Assistant Professor
Dr. Nabil AM. Salmo
Dr. Hassanain Hafidh Khudheir
MSc. (path)
F.I.B.M.S. (Path)
Chairman
Member
Lecturer
Assistant Professor
Dr. Karokh Hassan Salih
Dr. Hadeel Adnan Yasseen
F.I.B.M.S. (Path)
F.I.B.M.S. (Path)
Member
Member and supervisor
Date: Approved by the dean of college of medicine Dr. Kosar Mohammad Ali Assistant Professor of Medicine University of Sulaimani
Acknowledgement I would like to express my sincere thanks and gratitude to my teacher and supervisor Assistant Prof. Dr. Hadeel Adnan Yasseen for her support and help, encouragement and great advice throughout this work. I'd also like to thank Assistant Prof. Dr. Nabil. AM. Salmo for his help in the research and IHC technique. A special thanks to Dr. Hemin Abdulwahab Hassan for his help and support in the research process. Special thanks to all pathologists and the technical staff working at the Pathology Lab of Shorish Hospital for their assistance in the collection of samples.
IV
Dedication
To My beloved Father, Mother, Sister and Brothers
V
Abstract Background Breast cancer is the second leading cause of cancer death in women. Thousands of researches are done in breast cancer regarding risk factors, genetics, response to treatment and prognosis, but cancer cell interaction with its surrounding microenvironment has become one of the most interesting topics. Aims of the study to detect HMGCS2 and ACAT1 mitochondrial enzymes; responsible for ketone bodies production and utilization respectively in cancer associated fibroblasts and cancer epithelial cells, to understand Warburg and reverse Warburg effects and indirectly the influence of ketogenic diet on breast cancer patients. Materials and methods this prospective cross-sectional study was carried out in pathology research lab in college of medicine Sulaimani University. Forty cases of invasive ductal carcinoma (N.O.S) type were collected from Department of Surgical Pathology-Shorsh Hospital and Sulaimani Teaching Hospital in Iraq. Information
from
previous
histopathology
reports
were
gathered,
Immunohistochemistry stain was made for all cases with two mitochondrial enzymes named ACAT1 and HMGCS2. The intensity and percentage of staining were scored in cancer epithelial cells and cancer associated fibroblasts. Correlations between the two antibodies and with all collected clinicopathological parameters were done. Cancer epithelial cells and cancer associated fibroblasts correlation to ketone body production and utilization were analyzed by using the Chi square test and Z test for proportion. Results no significant correlations were found between the two antibodies and all clinicopathological parameters apart from low grade tumors. ACAT1 expression was positive in 87% and 67% in cancer epithelial cells and cancer associated VI
fibroblasts respectively while HMGCS2 expression was positive in 97% and 95% in cancer epithelial cells and cancer associated fibroblasts respectively. The results were highly consistent with reverse Warburg effect. Warburg effect also occurs at the same time. Conclusions this study highly supports the
reverse Warburg effect, although
Warburg effect does occur in breast cancer and indirectly concludes that ketogenic diet may have limited beneficial effect in controlling breast cancer growth since both cancer epithelial cells and cancer associated fibroblasts do express the mitochondrial enzymes needed for ketone body utilization and production..
VII
List of Contents Subject Acknowledgment Dedication Abstract List of Contents List of Figures List of Tables List of abbreviations Chapter One: Introduction 1.1 Introduction 1.2 Aims of the study Chapter Two: Review of Literature 2.1 Breast cancer 2.1.1 Epidemiology 2.1.2 Risk factors 2.1.3 Histological classification of breast cancer 2.1.4 Staging and Grading of breast cancer 2.1.5 Prognosis of breast cancer 2.2 Tumor micro environment 2.3 Cellular respiration 2.3.1 Glycolysis 2.3.2 Aerobic respiration 2.3.3 Krebs cycle 2.3.4 Anaerobic respiration 2.3.5 Metabolism in fasting and starvation 2.4 Ketone bodies 2.4.1 Ketone body production
Page IV V VI VIII X XII XIV 1 3 4 4 5 8 13 17 20 23 23 24 25 26 26 27 28
2.4.2 Ketone body utilization
29
2.4.3 Role of HMGCS2 and ACAT1 in ketone body metabolism
30
2.5 Warburg effect
30 32 33 34
2.6 Reverse Warburg effect 2.7 Mechanism of metabolic changes in cancer cell 2.8 Role of ketone bodies in tumor Chapter Three: materials and methods 3.1 Case selection 3.2 Sample preparation
35 35 VIII
3.3 Equipments and materials 3.4 Preparation of reagents 3.5 Immunestaining procedure 3.6 Immunohistochemical scoring 3.7 Statistical Evaluation Chapter Four: Results 4.1 Age distribution 4.2 Clinicopathological findings 4.2.1 Tumor grade 4.2.2 Lymph node status 4.2.3 Molecular Subtypes 4.3 Immunohistochemistry results 4.3.1 ACAT1 expression in cancer epithelial cells and cancer associated fibroblasts. 4.3.2 HMGCS2 expression in cancer epithelial cells and cancer associated fibroblasts 4.4 Correlations between staining intensity and percent in ACAT1 and HMGCS2 4.5 Correlation ACAT1 and HMGCS2 with clinicopathological parameters 4.5.1 ACAT1 correlation 4.5.2 HMGCS2 correlation 4.5.3 Correlation of ACAT1 and HMGCS2 in both cancer epithelial cells and cancer associated fibroblasts Chapter Five: Discussion 5.1 Clinicopathological findings 5.1.1 Age distribution 5.1.2 Tumor grade 5.1.3 Molecular subtypes 5.2 Warburg and reverse Warburg effect 5.2.1 ACAT1 expression in cancer epithelial cells and cancer associated fibroblasts 5.2.2 HMGCS2 expression in cancer epithelial cells and cancer associated fibroblasts Chapter Six: Conclusions and Recommendations 5.1 Conclusions 5.2 Recommendations Chapter Seven: References Appendix A
IX
36 37 38 38 39 40 40 40 41 42 42 42 46 49 51 51 54 56 60 60 60 61 62 63 64 66 66 67 87
List of Figures Figure
Subject
No.
Page
2.1
Glycolysis
24
2.2
Aerobic respiration
25
2.3
Citric acid cycle
25
2.4
Anaerobic respiration
26
2.5
Acetyl-CoA metabolicm in fasting and starvation
27
2.6
Ketone body production
28
2.7
Ketone body utilization
29
2.8
Aerobic glycolysis in cancer cells
31
4.1
Age distribution in 40 patients
40
4.2
Tumor grade
41
4.3
Lymph node status
41
4.4
Molecular subtypes
42
4.5
4.6
4.7
4.8
4.9
4.10
ACAT1 positivity a) in cancer epithelial cells b) in associated fibroblasts. Kidney tissue used as positive control for ACAT1 showing cytoplasmic staining in renal tubules X400 Moderate staining intensity of ACAT1 in cancer epithelial cells and strong in CAFs (arrow) (oil X1000) Strong staining intensity of ACAT1 in both cancer epithelial cells and CAFs (X400) Strong staining intensity of ACAT1 in cancer epithelial cells and moderate in CAFs (arrow) (X400) Moderate staining intensity of ACAT1 in both cancer epithelial cells and CAFs (X400) X
43
43
44
44
45
45
4.11
HMGCS2 positivity a) in cancer epithelial cells b) in cancer associated fibroblasts
46
Hepatocytes as positive control for HMGCS2 showing 4.12
4.13
4.14
4.15
4.16
strong cytoplasmic staining X400.
Strong staining intensity of HMGCS2 in CAFs (arrows) and low in cancer epithelial cells (oil X100) Strong staining intensity of HMGCS2 in CAFs and moderate in cancer epithelial cells. (X400) Strong staining intensity of HMGCS2 in cancer epithelial cells and weak in CAFs (X400) Moderate staining intensity of HMGCS2 in both cancer epithelial cells and CAFs (X400)
XI
47
47
48
48
49
List of Tables Table
Subject
No. 2.1
Page
American Joint Committee on Cancer (AJCC) of breast cancer
15
2.2
Nottingham grading system
16
3.1
materials and equipments
36
3.2
General reagents and solutions
37
4.1 4.2 4.3
4.4
4.5 4.6 4.7
4.8
4.9 4.10
Correlation
between
ACAT1
epithelial
intensity
and
percentage Correlation between ACAT1 stromal intensity and percentage Correlation between HMGCS2 epithelial intensity and percentage Correlation between HMGCS2 epithelial intensity and percentage Correlation of ACAT1 in cancer epithelial intensity and other parameters Correlation of ACAT1 stromal intensity and other parameters Correlation of HMGCS2 epithelial intensity and other parameters Correlation of HMGCS2 cancer associated fibroblast intensity and other parameters Correlation of HMGCS2 and ACAT1 in cancer epithelial cells Table 4.10 Correlation of ACAT1 and HMGCS2 in CAFs. XII
49 50 50
51
52 53 54
55
56 57
4.11
4.12
4.13
4.14
Correlation of HMGCS2 positivity in CAFs and ACAT1 in cancer epithelial cells. Correlation of ACAT1 in CAFs and HMGCS2 in cancer epithelial cells Correlation of ACAT1 positivity in both epithelial and stromal fibroblasts Correlation of HMGCS2 positivity in both cancer epithelial cells and cancer associated fibroblasts
XIII
57
58
58
59
List of Abbreviations ACAT1 AJCC ATP BRCA1 BRCA2 CAFs DAB DCIS ER PR HER2 HMGCS2 Ki-67 KD TCA TME NADH NAD+ NOS PBS 17q21 17q12.3
Acetyl-Coenzyme A acetyltransferase 1 American Joint Committee on Cancer Adenosine Tri-Phosphate BReast CAncer susceptibility gene 1 BReast CAncer susceptibility gene 2 Cancer Associated Fibroblasts 3,3' diaminobenzidine Ductal Carcinoma In Situ Estrogen Receptor Progesterone Receptor Human Epidermal Growth Factor receptor hydroxymethylglutaryl CoA synthase 2 Proliferation index Ketogenic Diet tricarboxylic acid Tumor Micro Enviroment Nicotinamide Adenine Dinucleotide (reduced) Nicotinamide Adenine Dinucleotide (oxidized) Not Otherwise Specified Phosphate Buffer Saline Chromosome 17 long arm, region 2, band 1 Chromosome 17 long arm, region 1, band 2, sub band 3.
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Chapter One
Introduction
Introduction 1.1: Introduction Breast cancer is the second leading cause of cancer death in women, exceeded only by lung cancer, and each year nearly 1.7 million new cases diagnosed worldwide. This represents about 12% of all new cancer cases and 25% of all cancers in women (Breast cancer statistics 2012). Thousands of researches are done in breast cancer, regarding risk factors, genetics, types, response to treatment, and prognosis, but the relationships to the surrounding microenvironment has now become a great topic in cancer research in general which is called (tumor microenvironment). Researches try to find out how cancer cells get energy and what role the surrounding microenvironment play in supplying energy to the cancer cells. Nowadays tumor microenvironment has become a dominant theme in upcoming and new researches, which study the way tumor cells get the energy that fuels their growth, and try to find out what role, if any, tumor microenronment has to play in local invasion and metastastic potential of tumor. For this purpose we remind the reader that tumor mass is composed of tumor cells, cancer associated fibroblasts, endothelial cells, and extracellular matrix. In this research we have concentrated on ketone bodies, which are high-energy mitochondrial fuels that burn more efficiently than other mitochondrial fuels, e.g. more efficient fuel than glucose (Prince et al 2013; Henderson 2008). It is important to have an idea about how ketone bodies are formed. In human beings and most other mammals, acetyl-CoA is formed in the liver during oxidation of fatty acids and this may enter the citric acid cycle (Krebs cycle) or it 1
Chapter One
may
be
Introduction
converted
to
the
ketone
bodies, “acetoacetate,
D-β-
hydroxybutyrate, and acetone” for export to other tissues. Then these ketones can be converted back to Acetyl-CoA, and used via oxidative mitochondrial metabolism to generate high amounts of ATP in Krebs cycle (Fukao et al 2004),
and provide the energy required by different tissues, most importantly, they can be utilized under conditions of hypoxia, when oxygen is scarce (Zou et al 2002; Suzuki et al 2002). Potentially, in case of tumor mass this would allow a tumor to grow even in the absence of vascular supply. Thus, ketone body fuels may be important in tumor cell growth (before the establishment of a vascular supply) or tumor spread, and ketone rich tissues are targets for metastasis of breast cancer especially liver (Martinez-Outschoorn. et al. 2011b; Martinez-Outschoorn. et al. 2012; Stogia et al 2012). Understanding Warburg effect and reverse Warburg effect is the milestone of tumor microenvironment and tumor metabolism, The Warburg Effect which was adopted by Otto Warburg in 1924, demonstrates that tumor cells change their metabolism in order to fuel their own growth (Vander et al 2009; Warburg et al 1924). There are debates on this finding, as recent researches demonstrate that when cancer progresses, increasing numbers of the stromal cells eat themselves to provide recycled nutrients to tumor cells and this is named “The Reverse Warburg Effect” (Pavlides et al 2009).
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Introduction
1.2: Aims of the study: 1- To show that the cancer associated fibroblasts play a role in ketone bodies production through highlighting the upregulated HMGCS2 enzyme which is required for ketone bodies production. 2- To demonstrate that cancer epithelial cells can utilize ketone bodies as a source of energy by highlighting the upregulated ACAT 1enzyme which is needed for ketone bodies utilization. 3- Understand Warburg and revere Warburg effect. 4- Role of ketogenic diet in breast cancer patient.
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Chapter Two
Review Of Literature
Review of Literature 2.1 Breast cancer 2.1.1 Epidemiology Breast cancer is the most common cancer in women worldwide, according to recent available data on breast cancer nearly 1.7 million new cases diagnosed in 2012 (second most common cancer overall). This represents about 12% of all new cancer cases and 25% of all cancers in women (Breast cancer statistics 2012). In Iraq, breast cancer ranks first among cancers diagnosed in women, a study performed for the 23,792 registered cases from 2000 to 2009, and the study showed that incidence rate increased from 26.6 per 100,000 in 2000 to 31.5 per 100,000 in 2009 for all age groups (Al-Hashimi and Wang 2014). In USA about 1 in 8 women (12%) will develop invasive breast cancer over the course of her lifetime. In 2015, an estimated 231,840 new cases of invasive breast cancer are diagnosed in women in the United State, along with 60,290 new cases of noninvasive (in situ) breast cancer and about 2,350 new cases of invasive breast cancer were estimated to be diagnosed in men in 2015 (Breasr cancer statistics 2015). A man‟s lifetime risk of breast cancer is about 1 in 1,000. (DeSantis et al. 2015).
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2.1.2 Risk factors 1. Age and gender Although breast cancer is occurring in both sexes and at any age, it is 100 times more common in female than male and nearly two third of the cases occur after age of 55. The average age at diagnosis is 61 for white women, 56 for Hispanic and 46 for African American women (Hopkins Medicine Health Library 2017). 2. Family and personal history of breast cancer Women with first degree relatives (mother, sister, and daughter) with breast cancer are at higher risk about (2-3 times) than general of population, although majority of breast cancer cases have negative family histories. A history of breast cancer in one breast increases the development of subsequent breast cancer in other breast, and this will allow early diagnosis of breast cancer (Schacht et al. 2014; Heisey and Carroll 2016). 3. Genetic Two classes of inherited susceptibility genes are considered as etiological factor for breast cancer development, which are BRCA1 and BRCA2 which are located on the chromosome 17q21 and chromosome 13q12.3 respectively. The risk of developing breast cancer with mutation in these two genes is 3% by age of 30, 19% by age 40, 51% by age 50, 54% by age 60, 85% by age 70 (Donegan and Spratt 2002). Genetic predisposition causes only 5-10% of all breast cancers (Tan et al. 2008)
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4. Reproductive life and menstrual history The higher incidence of breast cancer is observed in nulliparous women. In addition, it has been shown that early parity is associated with a pronounced decrease in risk of breast cancer, and additional live births confer greater risk reduction (Lambe et al. 1996), younger age at menarche and older age at menopause increase breast cancer risk (Chang-Claude et al., 2007). 5. Alcohol and smoking Alcohol even in moderate amount increases the risk of breast cancer, although the mechanism of action is unknown at the present time, it is seen more frequently in women with ER-positive/PR-positive breast cancer. Alcohol consumption increases the risk of breast cancer in men (Chen et al. 2014; Boyle and Boffeta 2009; Guenel et al. 2004). There is consistent evidence for a low to moderate increase in the risk of breast cancer in both passive and active smokers (Macacu et al. 2015; Xue et al. 2011). 6. Obesity In relation to menopausal state, central obesity is an important risk factor for postmenopausal breast cancer as compared to lean women. Research data has shown that Obesity in premenopausal women may be protective against breast cancer (Morimoto et al. 2002; Van Den Brandt et al. 2000).
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7. Radiations Exposure to ionizing radiation, especially during breast development, is associated with increased risk of breast carcinoma (Goss and Sierra 1998). 8. Hormones Studies showed that hormone replacement increases the risk of breast cancer particularly in combined progesterone-estrogen replacement (Schairer et al. 2000; Ross et al. 2000), and oral contraceptives have been showing no increased risk, or at most a very low increase among young long-term users (Hunter et al. 2010; White et al. 1994). 9. Benign breast diseases Proliferative lesions without atypia are associated with a 1.5 to 2-fold increase in risk of getting invasive breast cancer in life time, while proliferative diseases with atypia has shown (4-5 fold) increased risk of getting breast cancer (Schnitt 2003). 10. Physical activity Many studies support that regular aerobic exercise lowers the of breast cancer although the picture is not clear; exercise also helps in control of side effects associated with breast cancer therapy (Kim et al. 2013). 11. Diet High fiber diet is believed to be protective against breast cancer, there are several debates about relation of fat to breast cancer; high-fat, low-carbohydrate ketogenic diets (KD) enhances mitochondrial oxidative metabolism while limiting glucose consumption, this could represent a safe, easy, and effective approach to 7
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Review Of Literature
selectively affect metabolic stress in cancer cells versus normal cells. KD has become popular in recent decades for their demonstrated positive effects on weight loss (Bueno et al. 2013). KD induces a ketosis which is not a pathological, regarded as physiological condition occurring on a daily basis. Hans Krebs was the first to use the term “physiological ketosis” (Krebs 1966); this physiological condition, i.e., ketosis, can be reached through fasting or through a drastically reduced carbohydrate diet (below 20 g per day). Blood levels during a normal diet, K.D. Ketone body levels will be 7-8 mmol/L, and pH level 7.4. (Paoli et al. 2012). 12. Association Association between breast carcinoma and meningioma has been repeatedly noted in women, but this is not seen in men. Some studies have suggested this association. Two of these studies reported significant associations, with relative risk estimates mostly between 1.5 and 2.0, while the other studies reported relative risk (Rao et al 2009; Custer et al. 2002).
2.1.3 Histological classification of breast cancer To classify breast cancer, two key determinations are important: whether the tumor is confined to basement membrane or is invading the stroma, and morphological pattern of the tumor (Sin and Kreipe 2013):
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A- Non- invasive cancers; - Ductal carcinoma in situ - Lobular carcinoma in situ B- Invasive breast cancer; - Invasive ductal carcinoma - Invasive lobular carcinoma - Medullary carcinoma - Mucinous (colloid) carcinoma - Tubular carcinoma - Invasive papillary carcinoma - Metaplastic carcinoma - Inflammatory carcinoma
A- Non-Invasive breast carcinoma Include the type of tumors that are confined to the basement membrane, the myoepithelial cells are preserved and morphological patterns include; ductal carcinoma in situ, and lobular carcinoma in situ. 9
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Ductal carcinoma in situ (DCIS): Include cellular proliferations in large ducts and terminal duct lobular unit, with many distinct patterns; solid, micro papillary, cribriform, clinging, and cystic hypersecretory and comedo (Sakorafas and Tsiotou 2000).
Lobular carcinoma in situ (LCIS): The lobules filled with relatively uniform cells, small to medium sized, commonly identified incidentally in breast biopsies, and also associated with 70% multicentricity and 30-40% bilaterality (Hanby and Hughes 2008).
B- Invasive breast carcinoma Include tumors that invade basement membrane and invade the surrounding stroma.
Invasive ductal carcinoma not otherwise specified (NOS): This type of tumor does not fulfill the definition of any other categories of breast cancer, the size and shape are highly variable, it accounts for most common breast cancer according to WHO classification, and for 65% of all breast cancers. Grossly the tumor appears firm, irregular with crab-like outline, and gray to whitish in color, and microscopically there are sheets of well defined nests, cords, or individual cells; may show marked tubule formation, or sometimes the tubules are barely detected, and may not be seen at all cases (Berg and Hutter 1995).
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Invasive lobular carcinoma: Invasive lobular carcinoma (ILC) composes of non-cohesive cells individually dispersed or arranged in single-file linear pattern in fibrous stroma and characterized by small, round cells that are bland in appearance and have scanty cytoplasm. Infiltration typically does not destroy anatomic structures or incite a substantial connective tissue response. Lobular carcinoma often fails to form distinct masses that can easily be diagnosed by palpation or mammography (Rakha and Ellis 2010; Dicostanzo et al. 1990).
Medullary carcinoma: Medullary carcinoma account for 5-7% of all breast carcinomas. It usually appears in patients under 50 years of age and is said to be particularly common in Japanese women. Microscopically, the borders are always of the „pushing‟ type. The pattern of growth is diffuse, with minimal or no glandular differentiation or intraductal growth and absence of mucin secretion (Rosai and Ackerman 2011, p. 1701).
Mucinous (colloid) carcinoma: Mucinous carcinoma, also known as mucoid, colloid, or gelatinous carcinoma, Mucinous carcinoma of the breast is a rare entity with a favorable prognosis due to low incidence of lymph node metastases, accounts for 1-7% of breast cancers. Grossly, it is well circumscribed, and soft to palpation due to high mucin content. Microscopically consist of small clusters of tumor cells „floating in lakes of mucin (Dumitru et al. 2015; Ha et al. 2013; Anderson et al. 2004).
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Tubular carcinoma: The age of the patient is usually around 50 years, and accounts for less than 5% of breast cancers. Grossly the tumor is not well circumscribed with hard consistency, microscopically it simulates a benign condition because of the well-differentiated growth of the glands, necrosis or mitoses are absent, with little pleomorphism. The tumor carries good prognosis and lymph node metastasis usually is uncommon (Cabral et al. 2003).
Invasive papillary carcinoma: Invasive papillary carcinomas and invasive micropapillary carcinomas are rare, representing 1% or fewer of all invasive cancers. Usually this pattern occurs in old aged groups around 63-67 years (Pal et al. 2010).
Inflammatory carcinoma: Inflammatory breast cancer (IBC) is a rare and aggressive form of invasive breast cancer accounting for 2.5% of all breast cancer cases. It is characterized by rapid progression, local and distant metastases, younger age of onset, and lower overall survival compared with other breast cancers. IBC is a lethal disease with less than a 5% survival rate beyond 5 years when treated with surgery or radiation therapy (Robertson 2010).
Paget disease of the nipple: Is a rare manifestation of breast cancer, and almost always associated with an underlying invasive or non-invasive breast cancer, it accounts for 4% of all breast cancer cases. Paget disease is the name given to a crusted lesion of the nipple 12
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caused by breast carcinoma, as described by Sir James Paget in 1874 (Karakas 2011).
2.1.4 Staging and Grading of breast cancer TNM system for breast cancer classification is now used internationally; this system was adopted by American Joint Committee on Cancer (AJCC), breast cancer staging according to TNM 7th edition (2009), T represents the size of primary tumor, N for regional lymph node, and M for distant metastasis. (Edge and Compton, 2010) TX: Primary tumor cannot be assessed. T0: No evidence of primary tumor. Tis: Carcinoma in situ. Tis (DCIS): Ductal carcinoma in situ. Tis (LCIS): Lobular carcinoma in situ. Tis (Paget‟s): Paget‟s disease of the nipple NOT associated with invasive carcinoma and/or carcinoma in situ (DCIS and/or LCIS) in the underlying breast parenchyma. T: Tumor ≤ 20 mm in greatest dimension. T2: Tumor > 20 mm but ≤ 50 mm in greatest dimension. T3: Tumor > 50 mm in greatest dimension.
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T4: Tumor of any size with direct extension to the chest wall and/or to the skin (ulceration or skin nodules) Note: Invasion of the dermis alone does not qualify as T4. NX: Regional lymph nodes cannot be assessed (for example, previously removed). N0: No regional lymph node metastases. N1: Metastases to movable ipsilateral level I, II axillary lymph node(s). N2: Metastases in ipsilateral level I, II axillary lymph nodes that are clinically fixed or matted; or in clinically detected ipsilateral internal mammary nodes in the absence of clinically evident axillary lymph node metastases. N3: Metastases in ipsilateral infraclavicular (level III axillary) lymph node(s) with or without level I, II axillary lymph node involvement; or in clinically detected* ipsilateral internal mammary lymph node(s) with clinically evident level I, II axillary lymph node metastases; or metastases in ipsilateral supraclavicular lymph node(s) with or without axillary or internal mammary lymph node involvement. M0: No clinical or radiographic evidence of distant metastases. M1: Distant detectable metastases as determined by classic clinical and radiographic means and/or histologically proven larger than 0.2 mmNotes.
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Table 2.1: American Joint Committee on Cancer (AJCC 7th edition 2009) of breast cancer 0
Tis
N0
M0
IA
T1
N0
M0
T0
N1mi
M0
T1
N1mi
M0
T0
N1
M0
T1
N1
M0
T2
N0
M0
T2
N1
M0
T3
N0
M0
T0
N2
M0
T1
N2
M0
T2
N2
M0
T3
N1
M0
T3
N2
M0
T4
N0
M0
T4
N1
M0
T4
N2
M0
IIIC
Any T
N3
M0
IV
Any T
Any N
M1
IB
IIA
IIB
IIIA
IIIB
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Breast cancer grading; Nottingham grading system (the Elston-Ellis modification of Scarff-BloomRichardson grading system) (Elston and Ellis 1991) is used for grading of breast cancer, three tumor characteristics are depended upon, which are; tubule formation, nuclear pleomorphism, and mitotic count per 10 high power microscopic fields. Table 2.2: Nottingham grading system Feature
Score
Tubule and gland formation 1 Majority of tumor (>75) 2 Moderate degree (10-75%) 3 Little or none (<10%) Nuclear pleomorphism 1 Small, regular, uniform cells 2 Moderate increase in size and variability 3 Marked variation Mitotic count/10 HPF 1 0-9 2 10-19 3 >20
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The grade is detected after summation of all three scores of tumor characteristics as follows; Grade 1 (low grade): scores of 3, 4, or 5 Grade 2 (intermediate grade): scores of 6 or 7 Grade 3 (high grade): scores of 8 or 9
2.1.5 Prognosis of breast cancer
Age; Younger age at breast cancer diagnosis confers a bad prognosis when compared to older women (Andres et al. 2009).
Early diagnosis; The relative 5-, 8-, and 10-year survival rates for asymptomatic breast carcinomas detected in a large screening project were 88%, 83%, and 79%, respectively (Rosai and Ackerman 2011, p 1719).
Invasive carcinoma versus in situ disease; The great majority of women with adequately treated DICS are cured. In contrast, at least half of invasive carcinomas have invade locally or distantly at the time of diagnosis (Robbins et al. 2010; p: 1089).
Distant metastases; Once distant metastases are present, cure is unlikely, although long-term remissions and palliation can be achieved, especially in women with hormonally responsive tumors (Guth et al. 2009; Kang 2006). Lymph node metastases; biopsy is necessary for accurate assessment of lymph nodes. With no nodal involvement, the 10-year disease-free survival rate is close to 70% to 80%; the rate falls with the number of lymph nodes that are involved by the tumor (Wu et al. 2013).
Tumor size; The risk of axillary lymph node metastases increases with the size of the primary tumor, but both are independent prognostic factors. Women with 17
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node-negative carcinomas <1 cm in size have a 10-year survival rate of over 90%, whereas survival drops to 77% for cancers >2 cm. When tumor size is greater than 5 cm in diameter it has 100% chance of lymph node metastasis (Tan et al. 2005; Chu et al. 1999).
Locally advanced disease; Carcinomas invading into skin or skeletal muscle are associated with poor outcome and usually recur in the next 5 years, and may be difficult to treat surgically (Iqbal et al. 2010).
Histologic subtype; the 30-year survival rates of women with special types of invasive carcinomas (tubular, mucinous, medullary, lobular, and papillary) are
associated with a somewhat better prognosis than ductal carcinomas not otherwise specified. A major exception is inflammatory carcinoma, which has a poor prognosis; the 3-year survival rate is only 3% to 10% (Robertson et al. 2010).
Histologic grade; Survival for patients with well-differentiated grade 1 carcinomas (approximately 20% of the total) is 70% at 24 years. In contrast, most deaths for poorly differentiated grade 3 carcinomas (approximately 46% of the total) occur in the first 10 years. (Kumar et al. 2014; P:1089)
Estrogen and progesterone receptors; Current assays use immunohistochemistry to detect nuclear hormone receptors, a finding that is correlated with a better outcome and is an important predictor of response to hormonal therapy, ER and PR positive status and low grade tumors (Siadati et al. 2015; Srijaipracharaoen et al. 2010).
HER2/neu; HER2/neu overexpression is associated with poorer survival, but its main importance is as a predictor of response to agents that target this transmembrane protein (e.g., trastuzumab or lapatinib). The relationship between
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HER-2/neu and high grade tumor and lymph node involvement was significant (Siadati et al. 2015; Srijaipracharaoen et al. 2010).
Lymphovascular invasion; Lymphatics are major route of entry for tumor cells to axillary lymph nodes, lymphovascular invasion is associated with worse outcome in invasive breast cancers ( Gurleyik et al. 2007).
DNA content; The amount of DNA per tumor cell can be determined by flowcytometric analysis or by image analysis of tissue sections. Aneuploid tumors are those with abnormal DNA indices and have a slightly worse prognosis (Robbins et al. 2010; p: 1090).
Proliferative rate; Tumor cell proliferation can be assessed by mitotic counts, and by immunohistochemical study of cellular proteins produced during the cell cycle (e.g., Ki-67), Ki-67 is widely applied in routine clinical work. High level of Ki-67 associated with poor prognosis. In breast cancer, ki-67 considered low if the rate is less than 10%, 10-20% regarded as intermediate or borderline, and high if more than 20% (Inwald et al. 2013).
Screening program; Early screening program have a good effect in prognosis of the breast cancer cases. According to National Health Services in United Kingdom, the likelihood of getting breast cancer increases with age. All women who are aged 50-70 and registered with a general practitional are automatically invited for breast cancer screening every three years, although peoples with breast cancer symptoms, such as breast lump, nipple discharge, nipple retraction, are directly admitted to the screening program (Breast cancer statistics 2012).
Molecular subtypes of breast cancer; This is a surrogate classification categorizing breast cancer by the ER/PR/HER2 and tumor grade, that is simple, 19
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easy to interpret, accessible, reproducible, and readily available for clinicians in all breast cancer reports without additional tests. 1. Luminal A is ER+/PR+/HER2−, ER+/PR−/HER2−, ER−/PR+/HER2−, and low tumor grade. 2. Luminal
B/HER2−
is
ER+/PR+/HER2−,
ER+/PR−/HER2−,
and
ER−/PR+/HER2− and has high tumor grade. 3. Luminal B/HER2+ is ER+/PR+/HER2+, ER+/PR−/HER2+, ER−/PR+/HER2+, and regardless of tumor grade. 4. Triple negative is ER−/PR−/HER2−. 5. HER2 overexpressing is ER−/PR−/HER2+. In surrogate classification the tumor prognosis is somewhat different for example; the luminal B/HER2− had poorer survival and a higher risk of mortality than the luminal B/HER2+ subtypes. The luminal B/HER2− category consists solely of tumor grades 3, whereas the luminal B/HER2+ category contains cases of all tumor grades, suggesting that tumor grade, in this instance, may be a more important predictor of mortality than HER2-positivity (Parise and Caggiano 2014; Goldhirsch et al 2011).
2.2 Tumor micro environment Tumors are highly complex tissues composed of cancer cells and surrounding stroma, which is built up by different types of mesenchymal cells and an extracellular matrix (ECM) (Egeblad et al 2010). Once the tumor is formed, regardless of the mechanism, it doesn‟t only modify the stroma drastically but also initiates an inflammatory reaction and complex immune response (Rakoff-Nahoum 2006).
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The malignant features of cancer cells cannot be manifested without an important interplay between cancer cells and their local environment. The tumor mass composed of leukocytes, endothelial cells (both blood and lymphatic), and other stromal components like cancer-associated fibroblastic cells actively play a role in cancer progression. The ability to change these surroundings is an important property by which tumor cells are able to acquire some of the hallmark functions necessary for tumor growth and metastatic dissemination (Shiga et al. 2015). A better understanding of how the tumor environment (TME) affects cancer progression should provide new targets for the isolation and destruction of cancer cells via interference with the complex crosstalk established between cancer cells, host cells, and their surrounding extracellular matrix (Aboussekhra 2011). Tumor mass is composite of many different cellular and non-cellular constituents that surround the malignant cancer cells harboring activating mutations in oncogenes or loss of tumor suppressors that drive tumor growth (Egeblad et al 2010). As mentioned TME plays a significant role in disease progression, but the precise function of each constituent remains unknown. A variety of infiltrating immune cells, cancer-associated fibroblasts, and angiogenic endothelial cells play expanding and critical functions in sustaining cell proliferation, evading growth suppressors, promoting survival, activating invasion and metastasis, and reprogramming energy metabolism. Some constituents of the TME are also involved in restraining tumor growth and metastasis (Mao et al. 2013). The TME has also been implicated in determining location of metastatic disease, and impacting the outcome of therapy. While the stromal cells are not malignant 21
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per se, their role in supporting cancer growth is so vital to the survival of the tumor that they have become an attractive target for chemotherapeutic agents. various signaling mediators are derived from tumor itself, that promote tumor malignancy through local and systemic signals, Some of those signals are known to act on the bone-marrow and stimulate the release of bone-marrow derived cells (BMCs), preparing the pre-metastatic niche for spreading cancer cells and support their survival and outgrowth in the new environment (Augsten 2014). Thus in the clinical setting the targeting of the TME to encapsulate or destroy cancer cells in their local environment has become mandatory. (Sounni and Noel 2012). Cancer associated fibroblasts (CAFs) in tumor microenvironment A specialized group of fibroblasts called cancer associated fibroblasts, CAFs, is believed to actively participate in the growth and invasion of the tumor cells by providing a unique tumor microenvironment (Xing et al. 2010). Because of the close relationship between the cancer cells and CAFs, it is increasingly clear that the development of cancer cannot be dissociated from its local microenvironment. The origin of CAFs, and the criteria to distinguish CAFs from normal fibroblasts is not well established (Palvides et al. 2012). Tumor cells, regardless of their site of origin, behave like parenchymal cells in normal tissues and proliferate by interacting with the stroma (Dvorak 2015). Fibroblasts are a major cell type within the stroma and contribute to tissue remodeling in development and tissue homeostasis, this is achieved by providing structural scaffolding and growth regulatory mediators.
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For example after tissue injury, fibroblasts exhibit an activated and contractile phenotype with enhanced expression of α-smooth muscle actin (α-SMA) (Klingberg 2013). Another role of CAFs is abundantly expressing growth factors such as hepatocyte growth factor (HGF) (Koliaraki et al. 2012), epiregulin (Neufert et al. 2013), and heparin-binding epidermal growth factor-like growth factor (HB-EGF) (Sasaki et al. 2014) CAFs, is a major cellular component of cancer-associated inflammation, they can mediate tumor-enhancing inflammation by expressing a proinflammatory gene signature in an NF-κB-dependent manner (Erez et al. 2010).
2.3 Cellular respiration Cellular respiration is the process that cells use to harvest the energy in organic compounds, particularly glucose, which occurs in two stages: Stage 1 Glucose is converted to pyruvate, producing a small amount of ATP and NADH. This process is called glycolysis. Stage 2 when oxygen is present, pyruvate and NADH are used to make a large amount of ATP. This process is called aerobic respiration.
2.3.1 Glycolysis Glycolysis is a series of enzyme-catalyzed reactions that breaks down one sixcarbon molecule of glucose to two three carbon pyruvate ions, Glycolysis uses two ATP molecules but produces four ATP molecules. The net result of glycolysis is the production of two pyruvate molecules, two ATPs, and two NADH/H+s. 23
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Figure 2.1 Glycolysis (cell respiration 2017)
2.3.2 Aerobic respiration Normal cells, under normal conditions, undergo aerobic respiration, which is a metabolic pathway that requires oxygen. Aerobic respiration produces most of the ATP made by cells; pyruvate produced during glycolysis, in the presence of oxygen, then enters mitochondria produces one carbon dioxide molecule, one NADH molecule, and one two-carbon acetyl group, the acetyl group is attached to a molecule called coenzyme A (CoA), forming a compound called acetyl-CoA which then enters Krebs cycle (Ferrier and Harvey, 2014; p: 194).
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Figure 2.2 Aerobic respiration (Moriah Beck, Carbohydrate metabolism 2017).
2.3.3 Krebs cycle (Citric acid cycle) Citric acid cycle is the final common oxidative pathway that oxidizes acetyl CoA to CO2, the process is explained in the Fig. (2.3) (Nelson and Cox 2013; p:648).
Figure 2.3 Citric acid cycle (Nelson and Cox 2013; p: 649) 25
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2.3.4 Anaerobic respiration When there is no enough oxygen for aerobic respiration to occur, anaerobic respiration will be an alternative way, which occurs in two stages; first glucose will break down into two molecules of pyruvate and construct ATP, then in last stage produce lactic acid. Under anaerobic conditions, electrons are not transferred from NADH and NAD+, they are recycled in another way, electrons carried by NADH are transferred to pyruvate produced during glycolysis, and this will be available source for ATP that must continue. The reduction of pyruvate to lactate will provide that source (Nelson and Cox 2013; p: 563).
Figure 2.4 Anaerobic respiration (Moriah Beck, Carbohydrate metabolism 2017).
2.3.5 Metabolism in Fasting and Starvation •During the first few days of starvation, protein is used. •Lipid catabolism is mobilized, and acetyl-CoA molecules derived from breakdown of lipids accumulate. 26
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•Acetyl-CoA begins to be removed by a new series of metabolic reactions that transform it into ketone bodies (Nelson and Cox 2013; p : 686).
Figure 2.5 Acetyl CoA in fasting and starvation (Acetyl CoA Crossroads, 2017).
2.4 Ketone bodies Ketone
bodies
are
three
water-soluble
compounds
(acetoacetate, D-β-
hydroxybutyrate, and acetone). Acetoacetate regarded as primary ketone body, Dβ-hydroxybutyrate and acetone are secondary ketone bodies. They are formed exclusively in liver mitochondria. Ketone bodies transported by the blood stream to the extrahepatic tissues, where they are oxidized via the citric acid cycle to provide energy required by tissues for e.g. heart, brain, and skeletal muscles. Acetone is produced in lesser amount and excreted from the body. Citric acid cycle has a major role in utilization of ketone bodies (Nelson and Cox 2013; p: 686).
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2.4.1 Ketone body production In the liver acetyl CoA is formed from oxidation of fatty acids, then two molecules of acetyl CoA unite to form acetoacetyl CoA. There one more acetyl CoA is added to form HMG CoA, then in the presence of 3-hydroxyl 3-methyl glutaryl CoA (HMG-CoA) synthase (present in the mitochondria) form HMG-CoA, which is cleaved to form acetoacetate (primary ketone body) and acetyl CoA. Acetoacetate may directly enter blood stream or by reduction form D-3Hydroxybutyrate. Acetoacetate may also undergo spontaneous decarboxylation to form acetone (Ferrier and Harvey, 2014; p: 194).
Figure 2.6 Ketone body production (Ferrier and Harvey, 2014; p: 194).
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2.4.2 Ketone body Utilization The ketone bodies are formed in the liver; but they are used as fuel by extrahepatic tissues for e.g. heart muscle and renal cortex prefers ketone bodies. Tissues like skeletal muscle and brain can also utilize the ketone bodies as alternate sources of energy, when glucose is not available .In extra hepatic tissues, D- βhydroxybutyrate is oxidized to acetoacetate by D- β- hydroxybutyrate dehydrogenase. The acetoacetate is converted to acetoacetyl-CoA by 3-ketoacylCoA transferase. The acetoacetyl-CoA is then cleaved by thiolase to yield to two acetyl-CoAs, which enter the Krebs cycle. Thus the ketone bodies are used as fuels in all tissues, except the liver. The liver cannot utilize ketone bodies because it lacks the mitochondrial enzyme succinyl CoA:3-ketoacyl CoA transferase required for activation of acetoacetate to acetoacetyl CoA (Nelson and Cox 2013; p:686).
Figure 2.7 Ketone body utilization (Nelson and Cox 2013; p: 687). 29
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2.4.3 Role of HMGCS2 and ACAT1enzymes during ketone body metabolism HMGCS2 In the liver the formation of acetoacetyl CoA occurs by reversal of the thiolase reaction of fatty acid oxidation, this is achieved by mitochondrial HMG CoA synthase (HMGCS2), which combines a third molecule of acetyl CoA with acetoacetyl CoA to produce HMG CoA, furthermore HMG CoA is cleaved to produce acetoacetate and acetyl CoA. So HMGCS2 plays a direct role in synthesis of the primary ketone body; acetoacetate (Cotter 2013).
ACAT1 (also known as thiolase) The ACAT1 gene provides instructions for making an enzyme that is found in the energy-producing centers within cells (mitochondria), this enzyme is also involved in processing ketones. The ACAT1 enzyme carries out the last step in ketone breakdown (ketolysis) during the processing of fats. The enzyme converts a molecule called acetoacetyl-CoA into two molecules of acetyl-CoA, which can be used to produce energy in citric acid cycle (Martinez-Outschoorn. et al. 2012a).
2.5 Warburg Effect Developed by German researcher Otto Warburg in 1924 (for which he won a Nobel prize), says that tumor cells change their metabolism in order to fuel their own growth, The paradox is that cancer cells rely on glycolysis even if oxygen is available. This phenomenon is called aerobic glycolysis or the Warburg effect. 30
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Otto Warburg discovered that most cancer cells rely only on the first part of the energy process: glycolysis. During anaerobic respiration, cells break down glucose into pyruvate and construct ATP, but produce lactic acid instead of carbon dioxide. Aerobic respiration produces far more ATP molecules, 32 per molecule of glucose, than anaerobic respiration, which produces a mere two. So cancer cells need more glucose to satisfy their enormous appetite. Compared to normal cells which can, from a single molecule of glucose, produce 36 to 38 servings of ATP, cancer cells will need 19 molecules of glucose to produce an equivalent quantity. Although cancer cells produce far less ATP per molecule of glucose, they produce it much faster. Cancer cells produce ATP almost a hundred times faster than normal cells (Li et al. 2016; Phan et al. 2014). Some medical imaging techniques can help us locate tumours when they reach a certain size. Radio-active glucose is injected in patients. The Positron Emission Tomography (PET) scan tool is sensitive to radio-active material. Since cancer cell will consume 18 to 19 times more glucose than normal cells, they will accumulate more radio-active material.
Figure 2.8 Aerobic glycolysis in cancer cells (Samarasinghe 2014). 31
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2.6 Reverse Warburg Effect The reverse Warburg effect in human breast cancers was first proposed by Dr. Michael P. Lisanti and colleagues in 2009. According to this model, aerobic glycolysis (Warburg Effect) actually takes place in tumor associated fibroblasts, and not in cancer cells (Martinez-Outschoorn et al. 2010; Palvlides et al. 2010a, 2009; Bonuccelli et al. 2010) The researchers termed this new idea “The Reverse Warburg Effect”, to distinguish it from the conventional Warburg Effect, which was originally thought to take place in epithelial cancer cells.
A logical prediction of this hypothesis is that tumors must consist of two distinct metabolic compartments: one that provides the fuel (the host tumor stroma), and the other that burns the energy (the epithelial cancer cells) (Martinez-Outschoorn. et al. 2011, 2011c) Relative to the tumor stroma and normal adjacent epithelial cells, epithelial cancer cells have dramatically amplified their capacity to undergo oxidative mitochondrial activity (Sotgia et al. 2012; Whitaker-Menesez et al. 2011). It‟s clear that the tumor microenvironment plays a critical role not only in early oncogenesis, but also in tumor progression as well as in the response of cancer cells to therapy. In the last decade, great efforts have indeed been dedicated to the characterization of the molecular and cellular interactions between cancer cells and their stroma. This intense wave of research led to the elucidation of multiple mechanisms whereby stromal cells interact with their malignant neighbors, for instance, cancer-associated fibroblasts can secrete pro-tumorigenic cytokines and promote the establishment of an immunosuppressive microenvironment. 32
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Cancer cells and their stroma also are connected at the level of bioenergic metabolism in this case the stromal cells are facing oxidative stress and undergo a glycosis based metabolism (Pavlides et al. 2009). The net result is liberation of high level of lactic acid and ketone bodies which then used as fuel by cancer cells for oxidative phosphorylation.
2.7 Mechanism of metabolic changes in cancer cells The role of mitochondrial function in cancer pathogenesis remains a controversial topic, it is generally agreed upon that cancer cells undergo metabolic reprogramming (Fiaschi et al. 2012) Cancer cells accumulate defects in the mitochondrial genome, leading to deficient mitochondrial respiration and ATP generation (Chatterjee, A et al. 2011). It appears that acquired mutations in mitochondrial DNA fall into two classes. A first category includes severe mutations that inhibit oxidative phosphorylation, increase the production of reactive oxygen species (ROS) and promote tumor cell proliferation. Another category of milder mutations could permit tumors to adapt to new microenvironments, especially when tumors progress and metastasize (Branden et al. 2006). Genetic changes in epithelial cancer cells (conveyed by either oncogenic mutations or by loss of tumor suppressor function) induce the production of hydrogen peroxide by cancer cells. Hydrogen peroxide released from cancer cells then functions to fertilize their surrounding microenvironment via the induction of oxidative stress in tumor stromal cells, especially cancer-associated fibroblasts (Lisanti et al. 2011).
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Cancer-associated fibroblasts, which suffer from oxidative stress, then undergo a variety of catabolic processes to produce mitochondrial fuels in order to feed cancer cells. These catabolic cellular programs include autophagy, mitophagy and aerobic glycolysis. Cellular catabolism in the tumor microenvironment induces stromal mitochondrial dysfunction, driving the production of high-energy mitochondrial fuels, such as L-lactate and ketone bodies (Pavlides et al. 2012).
2.8 Role of ketone bodies in tumor Ketone body utilization may be important in tumor before the establishment of a vascular supply or after the tumor has outgrown its blood supply. Studies showed that ketones promote mitochondrial “health” in cancer cells, effectively shutting off their apoptotic machinery, causes continuous proliferation. As such, ketone body utilization could have important implications for both cancer prevention, as well as the effective treatment of advanced metastatic disease.In treatment of breast cancer cells with ketones was coupled with genome-wide transcriptional profiling, to generate a prognostic gene signature. This ketone-induced gene signature was specifically associated with increased tumor recurrence, metastasis and poor clinical outcome in human breast cancer patients (Martinez-Outschoorn et al. 2011a).
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Materials and Methods 3.1: Case selection: This prospective cross-sectional study was carried out in the period from March 2016 to December 2016, whereby paraffin-embedded tissue blocks of forty female patients with invasive ductal carcinoma (NOS) of the breast were collected from the Department of Surgical Pathology-Shorsh Hospital and Sulaimani teaching Hospital in Iraq. Information from previous histopathology reports were collected including age of the patient, estrogen receptor (ER) positivity, progesterone receptor (PR) positivity, HER2, Ki-67 index, Tumor size (T), lymph node status (N), and grade. Categorization of cases to molecular subtypes was done into five groups according to (ER), (PR), (Her2/neu) positivity, and (Ki-67) index (Parise and Caggiano 2014). Two cases of normal liver and renal tissues were used as positive control for the ACAT1 and HMGCS2 respectively. The IHC technique was done in research lab of Pathology department in the College of Medicine in Sulaimani University.
3.2: Sample preparation Additional four sections were obtained from each paraffin embedded tissue blocks and stained as below: One section was mounted on ordinary glass slides for hematoxylin and eosin stain to select the proper tissue area. Two sections were mounted on positive charge slides for IHC stain for the two different antibodies; ACAT1 and HMGCS2 (appendix A).
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One section was mounted on positive charge slides from each block as negative control.
3.3 Equipments and materials: All used materials and equipments are listed in Table 3.1. Table 3.1: materials and equipments Equipments and materials
Manufacturing company/Country
Oven
B.T Memmert (Germany)
Glass staining jars
Hangzhou Liandong (China)
Pressure cooker
Geepas (China)
Humidity chamber
Acelya (Turkey)
Graduated cylinder
Boeco (Germaney)
Positively
charged
microscopic Cancerdiagnostics.com (U.S.A.)
slides (Superfrost/plus) Cover slips
Sail Brand (China)
Micro pipettes
Slamed (Germany)
Pap pen
Ihcworld
Light microscope
Olympus (Japan)
Reagents and solutions brought from Santa Cruz Biotechnology Inc., (U.S.A.), Include; I. Specific reagents a. Primary polyclonal antibody ACAT1 (SC-161307) and primary monoclonal antibody HMGCS2 (SC-367092). b. ImmunoCruz™ ABC detection kit which contains the following reagents: 36
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ImmunoCruz™ ABC Staining Systems include 1.0 ml normal blocking serum, 250 µg biotinylated secondary antibody, 0.5 ml each avidin and biotinylated horseradish peroxidase (AB reagents), 1.0 ml 50x peroxidase substrate, 1.0 ml 50x DAB chromogen and 3.0 ml 10x substrate buffer. The detection kit also includes mixing bottles for the preparation of reagent working solutions. II. General reagents and solutions: All are listed in table 3.2. Table 3.2: General reagents and solutions General reagents and solutions
Manufacturing company/Country
Absolute ethanol
Scharlau (European union)
Antigen retrieval solution (citrate ABCAM (U.K.) buffer pH 6) (20x) Hematoxylin (Ready-to-use)
Scharlau (European union)
Mounting medium (DPX)
Marienfelb (Germaney)
Phosphate buffer saline (PBS)
ChemCruz (U.S.A.)
Xylene
Scharlau (European union)
DAB chromogen
DAKO (U.S.A.)
Substrate buffer
DAKO (U.S.A.)
Antibody diluent
ABCAM (U.K.)
3.4 Preparation of reagents: The preparation reagents were conducted according to company protocol. 1. Phosphate buffer saline (PBS) was prepared by dilution 10x concentrated PBS in distilled water.
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2. Primary antibodies, ACAT1 and HMGCS2 were diluted at 1:100 with antibody diluent by the addition of 1µl of primary antibody solution to 99µl of antibody diluent. 3. Chromogen Solution prepared in the substrate bottle by the addition of one drop of DAB chromogen to 1 ml of substrate buffer.
3.5 Immunostaining procedure ImmunoCruz™ mouse ABC Staining System was used with few changes as mentioned in details in appendix A.
3.6 Immunohistochemical scoring The intensity and percentage of staining were scored in cancer epithelial cells and CAFs as follow: The intensity was scored as - 0 = negative or no stain - 1 = weak - 2 = moderate - 3 = strong i.e. Equal intensity to positive control tissue. The percentage of stained cells was performed at 400x total magnification per 10 fields as: - sporadic i.e.< 10% - focal i.e. ≥ 11% and < 50% - diffuse i.e ≥ 50% Positivity cut off was established with staining intensity of 2 or 3 combined with focal or diffuse pattern. Weak intensity and/or sporadic staining were regarded as negative (De Melo Maia et al 2012). 38
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Correlations between the different variables were done include: - Correlations between the staining intensity and percent of the two antibodies with ER, PR, Her2/neu, Ki-67, tumor size (T), lymph node metastasis (N), grading, and with molecular subtypes separately. - Correlations between staining intensity and percent of the two antibodies in cancer epithelial cells and stromal fibroblasts.
3.7: Statistical Evaluation Data were analyzed using SPSS version 21, Chi-square test, and Z test for proportion used to find relation between variables, P.values <0.05, <0.01, <0.001 are regarded as significant, highly significant, and very highly significant respectively.
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Results 4.1 Age distribution A total of 40 cases of invasive ductal carcinoma (NOS) were studied. The age of the patients ranged from 31 to 88 years, (mean ± S.D. = 47.83 ± 12.04), 50% of cases were in the range of 41-50 years, 22.5 % were ≤ age of forty, as shown in Figure 4.1.
60.00% 50%
50.00% 40.00% 30.00%
22.50% 20.00% 12.50% 7.50%
10.00%
5%
2.50%
0.00%
31-40 years
41-50 years
51-60 years
61-70 years
71-80 years
81-90 years
Figure 4.1 Age distribution in 40 patients.
4.2 Clinicopathological findings:
4.2.1 Tumor grade In the present study 3 cases (8%) were grade I, 9 cases (23%) were grade II, and 28 cases (69%) were grade III, Figure 4.2
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8% 23% Grade I Grade II
69%
Grade III
Figure 4.2 Tumor grade.
4.2.2 Lymph node status Eleven cases (28%) were N0, 14 cases (34%) were N1, 4 cases (10%) were N2, and 11 cases (28%) were N3. Figure 4.3
N0
28%
28%
N1
N2
10% 34%
N3
Figure 4.3 Lymph node status
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4.2.3 Molecular subtypes In this study; 14 cases (34%) were luminal A, 9 cases (23%) were luminal B HER2 negative, 8 cases (20%) were luminal B HER2 positive, 5 cases (13%) were triple negative and 4 cases (10%) were HER2 overexpressing, Figure 4.4
Luminal A
10% 13%
Luminal B Her2 negative
34%
Luminal B Her2 positive
20%
Triple negative
23% Her2 over expressing
Figure 4.4 Molecular subtypes
4.3 Immunohistochemistry results: 4.3.1 ACAT1 expression in cancer epithelial cells and cancer associated fibroblasts. Regarding ACAT1 expression, 35 (87%) cases show positivity in the cancer epithelial cells, while 27(67%) cases were positive in CAFs, Figure 4.5, a and b. Kidney tissue was used as positive control, in which the stain is shown in cytoplasm of renal tubules, Figure 4.6. Variable staining intensities were shown in both cancer epithelial and CAFs, Figures 4.7-10. 42
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13% 33% 67%
87%
ACAT1+ve
ACAT1+ve
ACAT1-ve
a.
ACAT1-ve
b.
Figure 4.5 ACAT1 positivity a) in cancer epithelial cells b) in cancer associated fibroblasts.
Figure 4.6 Kidney tissue used as positive control for ACAT1 showing cytoplasmic staining in renal tubules X400.
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Figure 1.7 Moderate staining intensity of ACAT1 in cancer epithelial cells and strong in CAFs (arrow) (oil X1000)
Figure 4.8 Strong staining intensity of ACAT1 in both cancer epithelial cells and CAFs (X400) 44
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Figure 2.9 Strong staining intensity of ACAT1 in cancer epithelial cells and moderate in CAFs (arrow) (X400)
Figure 3.10 Moderate staining intensity of ACAT1 in both cancer epithelial cells and CAFs (X400)
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4.3.2 HMGCS2 expression in cancer epithelial cells and cancer associated fibroblasts Thirty nine cases (97%) were positive in the cancer epithelial cells, while 38 (95%) cases were positive in CAFs, Figure 4.11 a and b. Liver tissue was used as positive control showing strong cytoplasmic positivity of hepatocytes, Figure 4.12. Variable staining intensities were detected in both cancer epithelial cells and cancer associated fibroblasts, Figures 4.13-16
3%
5%
95%
97%
HMGCS2+ve a.
HMGCS2-ve
HMGCS2+ve
HMGCS2-ve
b.
Figure 4.11 HMGCS2 positivity in a) cancer epithelial cells b) CAFs
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Figure 4.12 Hepatocytes as positive control for HMGCS2 showing strong cytoplasmic staining X400.
Figure 4.13 Strong staining intensity of HMGCS2 in CAFs (arrows) and low in cancer epithelial cells (oil X100)
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Figure 4.14 Strong staining intensity of HMGCS2 in CAFs and moderate in cancer epithelial cells. (X400)
Figure 4.15 Strong staining intensity of HMGCS2 in cancer epithelial cells and weak in CAFs (X400)
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Figure 4.16 Moderate staining intensity of HMGCS2 in both cancer epithelial cells and CAFs (X400)
4.4 Correlations between staining intensity and percent in ACAT1 and HMGCS2. There is statistically very highly significance between ACAT1 epithelial intensity and percent, Table 4.1 Table 4.1 Correlation between ACAT1 epithelial intensity and percentage ACAT1 cancer epithelial cells intensity ACAT1 epithelial
cancer cells
percent
low
in
intermediate
P-
High
value No.
%
No.
%
No.
%
No stain
0
0.0
0
0.0
0
0.0
sporadic 1-10%
5
100.0 0
0.0
0
0.0
focal 11-50%
0
Diffuse more than 0
0.0 0.0
<0.001
8
80.0 2
20.0 <0.001
4
16.0 21
84.0 <0.001
50% 49
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There is statistical significance to very high significance between ACAT1 CAFs intensity and percent, Table 4.2 Table 4.2 Correlation between ACAT1 stromal intensity and percentage cancer ACAT1 cancer associated fibroblasts intensity
ACAT1 associated
low
fibroblasts
intermediate
P-
High
value
in
percent
No.
%
No stain
2
sporadic 1-10% focal 11-50%
No.
%
No.
%
100.0 0
0.0
0
0.0
0.11
11
100.0 0
0.0
0
0.0
<0.001
0
0.0
6
75.0
2
25.0
0.02
Diffuse more than 0
0.0
5
26.3
14
73.7
<0.001
50%
There is statistical significance to very high significance correlation between HMGCS2 epithelial intensity and percent, Table 4.3 Table 4.3 Correlation between HMGCS2 epithelial intensity and percentage HMGCS2 epithelial
cancer cells
percent
HMGCS2 cancer epithelial cells intensity low
in
intermediate
P-
High
value No.
%
No.
%
No.
%
no stain
0
0.0
0
0.0
0
0.0
sporadic 1-10%
1
100.0 0
0.0
0
0.0
<0.001
focal 11-50%
0
0.0
6
85.7
1
14.3
0.02
Diffuse more than 0
0.0
10
31.3
22
68.8
0.005
50%
50
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There is statistically very highly significance between HMGCS2 CAFs intensity and percent, Table 4.4 Table 4.4 Correlation between HMGCS2 epithelial intensity and percentage HMGCS2 HMGCS2
cancer
associated
fibroblasts
cancer intensity
associated
low
Pintermediate
value
High
fibroblasts in percent No.
%
No.
%
No.
%
no stain
0
0.0
0
0.0
0
0.0
sporadic 1-10%
2
100.0 0
0.0
0
0.0
<0.001
focal 11-50%
0
0.0
11
78.6
3
21.4
<0.001
Diffuse more than 0
0.0
3
12.5
21
87.5
<0.001
50% The previous tables has shown that intensity and percentage can be a substitute to each other, so here we will compare intensity of staining with other clinicopathological parameters of breast cancer in next few tables to avoid repetition.
4.5 Correlation of ACAT1 and HMGCS2 with clinicopathological parameters
4.5.1 ACAT1 correlation. Correlation analyses between ACAT1 epithelial intensity and other parameters was done, and the result shows no statistical significance with any of them Table 4.5
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Table 4.5 Correlation of ACAT1 in cancer epithelial intensity and other parameters ACAT1 epithelial intensity low
Parameters
No.
%
No.
%
No.
%
3
21.4
3
21.4
8
57.1
0.38
0
0.0.
3
33.3
6
66.7
0.43
2
25.0
1
12.5
5
62.5
0.17
0
0.0
3
60.0
2
40.0
0.25
0
0.0
2
50.0
2
50.0
0.55
T1
1
14.3
2
28.6
4
57.1
0.98
T2
4
16.0
8
32.0
13
52.0
0.57
T3
0
0.0
2
25.0
6
75.0
0.39
T4
0
0.0
0
0.0
0
0.0
N0
1
9.1
6
54.5
4
36.4
0.35
N1
2
14.3
4
28.6
8
57.1
0.33
N2
1
25.0
0
0.0
3
75.0
0.41
N3
1
9.1
2
18.2
8
72.7
0.32
low grade
0
0.0
1
33.3
2
66.7
0.52
Intermediate
1
11.1
1
11.1
7
77.8
0.5
high grade
4
14.3
10
35,7
14
50.0
0.48
luminal B HER2 negative molecular
luminal
subtypes
HER2positive
B
triple negative HER2
over-
expressing
N
Grade
P-
High
value luminal A
T
intermediate
52
Chapter Four
Results
Also correlation between ACAT1 staining intensity of CAF with other parameters was done and was statistically not significant Table 4.6 Table 4.6 Correlation of ACAT1 CAFs intensity and other parameters ACAT1 CAFs intensity low
Parameters
intermediate high
value No. % luminal A
No. %
21.4
4
28.6 0.22
0
0.0
4
44.4
5
55.6 0.06
4
50.0 2
25.0
2
25.0 0.46
1
20.0 1
20.0
3
60.0 0.61
1
25.0 1
25.0
2
50.0 0.90
T1
3
45.5 1
18.2
3
36.4 0.65
T2
10
40.0 6
24.0
9
36.0 0.42
T3
0
0.0
4
50.0
4
50.0 0.06
N0
5
45.5 2
18.2
4
36.4 0.56
N1
6
42.9 2
14.3
6
42.9 0.51
N2
2
50.0 1
25.0
1
25.0 0.49
N3
0
0.0
54.5
5
45.5 0.61
low grade
2
66.7 0
0.0
1
33.3 0.67
intermediate
3
33.3 3
33.3
3
33.3 0.71
high grade
8
28.6 8
28.6
12
42.9 0.65
B
molecular
luminal
subtypes
HER2positive
HER2
B
triple negative HER2
over-
expressing
Grade
%
50.0 3
negative
N
No.
7
luminal
T
P-
4.5.2 HMGCS2 correlation.
53
6
Chapter Four
Results
Correlation of HMGCS2 cancer epithelial intensity with other parameters show no statistical significance. Table 4.7 Table 4.7 Correlation of HMGCS2 epithelial intensity and other parameters HMGCS2 epithelial intensity Low
Parameters
%
No.
%
No.
%
1
7.1
6
42.9
7
55.6
0.34
0
0.0
4
44.4
5
55.6
0.83
0
0.0
4
50.0
4
50.0
0.74
0
0.0
2
40.0
3
60.0
0.92
0
0.0
0
0.0
4
100.0
0.19
T1
0
0.0
2
28.6
5
71.4
0.67
T2
1
4.0
10
40.0
14
56.0
0.73
T3
0
0.0
4
50.0
4
50.0
0.74
N0
0
0.0
5
45.5
6
54.5
0.47
N1
0
0.0
5
35.7
9
64.3
0.41
N2
1
25.0
0
0.0
3
75.0
0.39
N3
0
0.0
6
54.5
5
45.5
0.51
low grade
0
0.0
0
0.0
3
100.0
0.30
intermediate
1
11.1
2
22.2
6
66.7
0.10
high grade
0
0.0
14
50.0
14
50.0
0.06
negative molecular
luminal
subtypes
HER2positive
B
Triple negative HER2
over-
expressing
Grade
P-
No.
luminal B HER2
N
high
value
luminal A
T
intermediate
54
Chapter Four
Results
The statistical analysis of cancer associated fibroblast HMGCS2 intensity with other parameters was not significant except for low grade tumors, Table 4.8 Table 4.8 Correlation of HMGCS2 cancer associated fibroblast intensity and other parameters HMGCS2 CAFs intensity Parameters
low
intermediate
P-
high
value No.
%
No.
%
No.
%
1
7.1
7
50.0
6
42.9
0.26
0
0.0
4
44.4
5
55.6
0.11
1
12.5
1
12.5
6
75.0
0.22
0
0.0
2
40.0
3
60.0
0.84
0
0.0
0
0.0
4
100.0
0.22
T1
0
0.0
3
42.9
4
57.1
0.74
T2
2
8.0
8
32.0
15
60.0
0.50
T3
0
0.0
3
37.5
5
62.5
0.76
T4
0
0.0
0
0.0
0
0.0
N0
0
0.0
4
36.4
7
63.6
0.27
N1
1
7.1
6
42.9
7
52.0
0.11
N2
1
25.0
0
0.0
3
75.0
0.31
N3
0
0.0
4
36.4
7
63.6
0.25
low grade
0
0.0
3
100.0
0
0.0
0.04
intermediate
1
11.1
2
22.2
6
66.7
0.67
high grade
1
3.6
9
32.1
18
64.3
0.64
luminal A luminal B HER2 negative molecular
luminal
B
subtypes
HER2positive triple negative HER2
over-
expressing
T
N
Grade
55
Chapter Four
Results
4.5.3 Correlation of ACAT1 and HMGCS2 in both cancer epithelial cells and cancer associated fibroblasts
Studying Warburg effect in a single compartment of cancer epithelial cells; the correlation between ketone body production and utilization was analyzed using HMGCS2 and ACAT1 enzymes respectively. Figures 4.5a and 4.11a show that the cancer epithelial cells in 39/40 cases express HMGCS2 enzyme and in 35/40 cases express ACAT1 respectively, and table 4.9 shows that this correlation was not significant. Table 4.9 Correlation of HMGCS2 and ACAT1 in cancer epithelial cells
ACAT1 cancer epithelial cells
HMGCS2 cancer epithelial cells Positive
Negative
No. (%)
No. (%)
Positive
35 (88%)
0 (0%)
Negative
4 (10%)
1 (2.5%)
Pvalue
0.09
The same previous correlation in the other compartment of CAFs was also studied as shown in Table 4.10 and shows a statistically high significance between ketone body production and utilization in CAFs.
56
Chapter Four
Results
Table 4.10 Correlation of ACAT1 and HMGCS2 in CAFs. HMGCS2 cancer associated fibroblasts ACAT1 CAFs
Positive
Negative
No. (%)
No. (%)
Positive
27 (67%)
0 (0%)
Negative
11 (28%)
2 (5%)
Pvalue
0.003
Investigating the reverse Warburg effect through studying the correlations of ketone production and utilization between the two compartments of cancer epithelial cells and CAFs; first ketone production by CAFs and utilization by cancer epithelial cells was done and shows no statistical significant difference between the two compartments. Figure 4.5b shows in 38/40 cases the CAFs express HMGCS2 that produces ketone bodies and figure 4.5a 35/40 cases the cancer epithellial cells can utilize these ketone bodies using ACAT1 enzyme, and correlation between them were not significant (Table 4.11).
Table 4.11 Correlation of HMGCS2 positivity in CAFs and ACAT1 in cancer epithelial cells. ACAT1 cancer epithelial cells HMGCS2 CAFs
Positive
Negative
No. (%)
No. (%)
Positive
34 (85%)
4 (10%)
Negative
1 (2.5%)
1 (2.5%)
57
Pvalue
0.23
Chapter Four
Results
Then the opposite correlation between the two compartments was done and shows a statistically high signifcant difference. Table 4.12 Table 4.12 Correlation of ACAT1 in CAFs and HMGCS2 in cancer epithelial cells HMGCS2 cancer epithelial cells ACAT1 CAFs
Positive
negative
No. (%)
No. (%)
Positive
27 (67%)
0 (0%)
Negative
12 (30.5%)
1 (2.5%)
Pvalue
0.002
For the sake of interest the difference significance was studied between the two compartments in their production and utilization of ketone bodies. Figure 4.5 highlights ketone body utilization in both compartments using ACAT1. Out of 40 cases 35 cases of cancer epithelial cells express ACAT compared to 27 of CAFs, Table 4.13 shows there is a statistically significant difference. Figures 4.7-10. Show that both compartments can utilize the ketone bodies as a source of energy.
Table 4.13 Correlation of ACAT1 positivity in both epithelial and stromal fibroblasts
ACAT1 cells
cancer
epithelial
P-
ACAT1 CAFs
value
Positive
negative
No. (%)
No. (%)
Positive
26 (65%)
9 (22.5%)
Negative
1 (2.5%)
4 (10%) 58
0.03
Chapter Four
Results
Table 4.14 highlights the correlation of ketone body production in the two compartments using HMGCS2; statistically there was no significant difference between the two. Expression of HMGCS2 was seen in 39/40 cases in cancer epithelial cells compared to 38/40 CAFs (Figure 4.11). Figures 4.13-16 show that both compartments can efficiently produce the ketone bodies as a source of energy. Table 4.14 Correlation of HMGCS2 positivity in both cancer epithelial cells and cancer associated fibroblasts
HMGCS2 cancer epithelial cells
P-
HMGCS2 CAFs
value
Positive
negative
No. (%)
No. (%)
Positive
38 (95%)
1 (2.5%)
Negative
0 (0%)
1 (2.5%)
59
0.55
Chapter Five
Discussion
Discussion Breast cancer is the most common invasive cancer in female worldwide, accounting for 14.6% of all new cancer diagnosis in USA, including both males and females. Breast cancer regarded as commonest cancer in Asia, although it is lower as compared to United States. It is lower than Asian women who are long resident in USA (Wu et al 2014; Ziegler et al 1993). Screening program and patient education about breast cancer are saving lives by detecting breast cancer in earlier and more treatable stages and increase life expectancy.
5.1: Clinicopathological findings 5.1.1: Age distribution In 40 cases, fifty percent of cases were ranging 41-50 of age, this is consistent with Alwan (2010) who studied the demographic characteristics and clinicopathological presentation of patients in Iraq in 721 patients, and found one third of breast cancer cases were among 40 to 49. In Sulaimani 60% of patients with breast cancer were less than 50 years of age, Majid et al (2009) wich is comparable with our result.
5.1.2: Tumor grade In this study majority of the cases were grade II and grade III, 23% and 69% respectively, which is comparable with a study done by Rakha et al. (2008) who studied the Prognostic significance of Nottingham histologic grade in invasive breast carcinoma in 2219 cases and found that 35.6% and 45.6% grade II, and grade III respectively.
06
Chapter Five
Discussion
In this study we found a significant correlation between expression of HMGCS2 (a mitochondrial enzyme involved in ketone body production) in CAFs and low tumor grade. This may be related to small number of the sample that was studied in our research, and bigger sample study may show significant correlation with other tumor grades.
5.1.3 Molecular subtypes This study shows that 34% were luminal A, 23% luminal B HER2 negative, 20% luminal B HER2 positive, 13% triple negative and 10% HER2 over expressing, this result was comparable with many other studies as Brouckaert et al. (2012) who studied prognosis and survival of breast cancer patients in relation to molecular subtypes for 4318 patients and they had quite comparable distributions of breast cancer subtypes; 42 % for Luminal A, 27 % for Luminal B/HER2 negative, 14 % for Luminal B/HER2 positive, 11 % for triple negative, 7 % for HER2 over expressing. also are comparable to Minicozzi et al. (2013) who studied the role of molecular subtypes in breast cancer in 3381 patients and their result shows 56 % were Luminal A, 22 % Luminal B/HER2 negative, 7 % Luminal B/HER2 positive, 10 % triple negative and 4 % HER2 over expressing. While Hennigs et al. (2016) in their large cohort study on Prognosis of breast cancer molecular subtypes in routine clinical care for 4102 patients found that luminal A type is most frequent type 44.7 %, 31.8 % Luminal B/HER2 negative, 6.2 % Luminal B/HER2 positive, 12.3 % Triple negative, and 5.0 % HER2 over expressing. Wang et al. (2016) worked on Breast Cancer Molecular Subtypes and Ki67 role for the prediction of efficacy and prognosis of neoadjuvant chemotherapy in a Chinese population for 240 patients, they revealed that 25.4% of their cases were luminal A, 52.9% luminal B type, 12.6% HER2 over expressing, and 8.8%
06
Chapter Five
Discussion
were triple negative, the slight difference is due to sample numbers and they also put luminal B as one category. In general, in all studies luminal A type constitutes the majority of breast cancer cases and the other types come in sequence with the HER2 overexpressing being the least which is consistent with our finding, except the last study which triple negative was minority.
5.2 Warburg and reverse Warburg effect During his research on the energy metabolism of tumors, Otto Warburg found that cancer epithelial cells prefer to produce ATP by glycolysis; this finding is termed the Warburg effect. His hypothesis is that cancer epithelial cells undergo increased glycolysis even in the presence of oxygen, a process also known as aerobic glycolysis. (Warburg et al. 1924) Otto Warburg hypothesis was first described assuming that the tumor is a single compartment made of cancer epithelial cells. This research highly supports the Warburg hypothesis that cancer epithelial cells can produce and utilize significantly the ketone bodies through expression of both mitochondrial enzymes in 97% and 87% respectively. In other words, the cancer cells can feed themselves by glycolysis. There is much evidence that the Warburg effect has many questionable points. Researchers suggested that cancer epithelial cells might induce the Warburg effect in neighboring stromal fibroblasts. Based on a mass of research, another hypothesis has emergerged whhich is the reverse Warburg effect in which glycolysis occurs in mesenchymal stromal cells under influence of neighboring cancer cells. From this aerobic glycolysis, the cancer cells will get the fuel metabolites like pyruvate and lactate that enter mitochondria for TCA cycle (Balliet et al. 2011; Palvides et al. 2009). 06
Chapter Five
Discussion
In this research comparable percents of both compartments; CAFs and cancer epithelial cells, 95% and 87% respectively, were found to express the mitochondrial enzymes suggesting that CAFs produce the ketone bodies while cancer epithelial cells utilize them which is highly consistent with reverse Warburg effect and potentiate the role of microenvironment in supporting tumor growth and nutrition. This is consistent with many new researches as Vincent et al (2008). who demonstrated that human skin keloid fibroblasts display similar bioenergetic changes as cancer cells in generating ATP mainly from glycolysis. This finding led to a hypothesis that cancer epithelial cells can induce Warburg effect in surrounding stromal tissue (reverse Warburg effect). Sotgia et al. (2012a; 2011) suggest that stromal cells that lack cav-1 will undergo aerobic glycolysis and feed cancer cells and if co-cultured with cancer cells promotes Cav-1 down-regulation in adjacent fibroblasts which enhance aerobic glycolysis, but when cultured under homotypic conditions, cancer cells have a very low mitochondrial mass (the conventional Warburg effect). In other words, cancer epithelial cells can feed themselves but much less efficiently than if cultured and supported by fibroblasts, which is highly consistent with this research in which both Warburg and reverse Warburg effects do occur in cancer(the tumor sample).
5.2.1 ACAT1 expression in cancer epithelial cells and cancer associated fibroblasts Apart from Warburg and reverse Warburg effects, this research is also concerned about the utilization of ketone bodies by the two compartments. Both show positivity to ACAT1 enzyme as 87% and 67% in cancer epithelial cells and CAFs respectively with strong correlation between them, i.e. when cancer epithelial cells utilize ketone bodies the CAFs also can utilize them, which was little clarified by other authors. Many previous literatures concentrate on the 06
Chapter Five
Discussion
idea that fibroblasts produce the ketone fuel for cancer epithelial cells, but in this research it is proven that the CAFs also feed on ketones. ACAT1 positivity in cancer epithelial cells was demonstrated by Martinez-Outschoorn et al. (2012b) who used Western blot analysis with isoform-specific antibodies and IHC stained paraffin embedded breast cancer tissue to confirm the overexpression of ACAT1 antibody in cancer epithelial cells. Our finding is consistent with Martinez-Outschoorn. et al. (2011b) who Showed that fibroblasts and cancer cells shared parallel glucose uptake and they suggest that tumor cells and stromal fibroblasts in co-culture were metabolically coupled. ACAT1 enzyme in human fibroblasts was used by Li et al. (1999) for location of human ACAT1 coding region, this is highly consistent with this research that fibroblasts can utilize ketone bodies.
5.2.2 HMGCS2 expression in cancer epithelial cells and cancer associated fibroblasts Many studies comment only on HMGCS2 positivity in CAFs as Bonuccelli et al. (2010) who demonstrate the expressing enzymes need for ketone body production in fibroblast associated tumor microenvironment by using xenograft model for explanation of the role of 3-hydroxy-butyrate in increasing tumor size without significant increase in angiogenesis and they used a list of genes that are transcriptionally upregulated in human breast cancer epithelial cells, relative to the adjacent stromal cells. In the current study the cancer epithelial cells and CAFs show high positivity for HMGCS2 in 97% and 95% respectively, which means that both compartments contain this enzyme needed for ketone body synthesis. This was also shown by Martinez-Outschoorn et al. (2012a) in their microscopical figures that clearly demonstrate the positivity in both compartments by using IHC stained paraffin embedded human breast cancer 06
Chapter Five
Discussion
tissue and Western blot technique, but they didn’t comment on epithelial HMGCS2 positivity. The idea of K.D in patients with malignancy is based on the assumption that malignant cells cannot consume ketone bodies so the malignant cells will be starved and die. Through this research we can assume that K.D. have no role in controlling in breast cancer growth since both compartments can efficiently produce and consume the ketone bodies i.e. fuel themselves in addition to the undeniable interaction between both. This may have some therapeutic impact on management of breast cancer.
06
Chapter Six
Conclusions and Recommendations
6.1 Conclusions 1. The studied mitochondrial enzymes may have no correlation with clinicopathological parameters. 2. It was evident that breast cancer epithelial cells can produce the ketone bodies and utilize them at the same time, which supports Warburg effect. 3. The CAFs can produce the ketone bodies that fuels the cancer epithelial cells, which supports the reverse Warburg effect. 4. Each compartment can efficiently consume and produce ketone bodies i.e. can fuel its own cell. 5. Ketogenic diet may be of no benefit in controlling breast cancer growth.
6.2 Recommendations 1- We recommend that a study to be held on genes that are transcriptionally upregulated in human breast cancer epithelial cells to clarify the role of tumor microenvironment in potentiation of tumor growth. 2- an experimental study to be carried out to evaluate the efficacy of ketogenic diet in breast cancer patient. 3- A bigger sample has to be studied to get more precise correlations. 4- More mitochondrial enzymes in relation to ketone body production and utilization have to be studied.
66
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Appendix A 1. Baking overnight at 56˚C. 2. Deparaffinization in oven by raising temperature to 65˚C for 30-minutes. 3. Clearing in two changes of xylene in oven at 65˚C, 10 minutes each. 4. Rehydration by serial dipping of the slides in descending concentration of ethanol: two changes of absolute ethanol at room temperature, 10 minutes each, two changes of 90% and 70% ethanol at room temperature, 10 minutes each. Then washing in running tap water at room temperature for 5 minutes. 5. Antigen retrieval: in pressure cooker for 6 minutes. 6. Cooling: slides inside antigen retrieval solution were left to cool to room temperature. 7. Washing in three changes PBS buffer, 2 minutes each. 8. The slides were placed in a glass jar containing PBS buffer over night for the work completed on next day. Note: The subsequent steps must be in humid chamber to prevent dryness. 9. Encircling tissue sections with pap pen. 10. Peroxidase block: two to three drops on each tissue section for 30 minutes at room temperature, then washed in four changes PBS buffer, 2 minutes each.
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11. Serum block: two to three drops on each tissue section, for 30 minutes at room temperature then slides were tapped on filter paper but with no wash in PBS. 12. Primary antibody: few drops of Anti HMGCS2 or anti ACAT1 antibody were applied to cover the tissue sections and then incubated for 60 minutes. The slides were washed in four changes of PBS buffer, 2 minutes each. 13.Biotinylated secondary antibody: two to three drops were applied to cover the tissue sections and incubated for 30 minutes at room temperature. The slides were washed in four changes of PBS buffer, 2 minutes each. 14. Avidin-biotinylated horseradish peroxidase: two to three drops of AB reagent for 30 minutes then washing in four changes of PBS buffer, 2 minutes each. 15. DAB chromogen: two to three drops each tissue section for 3-10 minutes until brown color appears. The slides were rinsed with tap water for 10 minutes. 16. Counterstain with Hematoxylin stain for 5 seconds, and then rinsed with running tap water till water become clear. 17. Dehydration of tissue sections was done by serial dipping of slides in ascending concentration of ethanol alcohol 70%, 90%, for 5 minutes each then two changes of 100% ethanol at room temperature, 10minutes each. The slides were placed in oven at 65oC for 10 minutes. 17. Clearing: The slides were put in a glass staining jar of xylene for 5 minutes.
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18. Mounting with permanent mounting medium, then covering with over slips and left to dry.
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اىخالصت الخلفية سشطبُ اىزذي ٕ٘ ربًّ أمزشاّ٘اع اىسشطبُ اىَسببت ىي٘فٍبث فً اىْسبء .األف اىبح٘د أصشٌج فٍَب ٌخؼيق بؼ٘اٍو اىخط٘سة ,اىَ٘سربث ,االسخضببت ىيؼالس ٗىنِ حبقى ػالقت اىخالٌب اىسشطبٍّت ببىبٍئت اىَضٖشٌت ٍِ امزش اىَبضٍغ اربسة. اهذاف الذراسة; ٕزٓ اىذساسٔ حٖذف اىى ابشاص اّضٌٍَِ ٍِ اّضٌَبث بٍ٘ث اىطبقت اىَخؼيقت ببّخبس ٗاسخٖالك اىنٍخّ٘بث ٍِ قبو اىخالٌب اىسشطبٍّت ٗاىخالٌب اىيٍفٍت اىَضبٗسة فً سبٍو ح٘ضٍح حأرٍش ٗاسبشؽ ٗحأرٍش ٗاسبشؽ اىَؼبمس ٗببىخبىً حخٍَِ فؼبىٍت اىْظبً اىغزائً اىَ٘ىذ ىينٍخّ٘بث ىَشضى سشطبُ اىزذي. المواد والطرق اُ ٕزٓ اىذساست ًٕ ٍِ ّ٘ع اىذساسبث اىخقذٍٍٔ حٍذ اخذث اىَْبدس ٍِ اسبؼٍِ حبىٔ ٍشضٍت ىسشطبُ اىزذي االّب٘بً اىغبصي ػيى شنو مخو ببسافٍٍْٔ ّسٍضٍٔ ٍغ اىخقبسٌش ٍِ ٍحف٘ظبث اسشٍف قسٌ االّسضت اىَشضٍت ٗاىضشاحٍتٍ -خخبش ٍسخشفى ش٘سش ٗاىَسخشفى اىخؼيًٍَ حٍذ حٌ ححضٍش ٍضَ٘ػخٍِ ٍِ اىسالٌذاث ٗصبغٖب ببسخخذاً حقٍْت اىنٍٍَبٍْبػٍٔ اىْسضٍت ٗرىل ىخحذٌذ حؼبٍشاالّضٌٍَِ .HMGCS2 ٗ ACAT1حٌ قشاءة شذة ّٗسبت اىخالٌب اىَصب٘غت ٗ ٍقبسّخٖب ٍغ مبفت اىَؼيٍ٘بث اىسشٌشٌت اىَشضٍت ٗرٌ ححيٍو اىؼالقت بٍِ ٕزٌِ االّضٌٍَِ فً مو ٍِ اىخالٌب اىسشطبٍّت ٗاىخالٌب اىَخؼيقت اىيٍفٍت . النتائج ىٌ حنِ ْٕبك ػالقت احصبئٍت بٍِ مال اىْضٌٍَِ ٗاىؼ٘اٍو اىسشٌشٌت اىَشضٍٔ ػذا ٍغ حبالث اىخَبٌض اىَْخفض .حؼبٍش ACAT1مبُ ٍ٘صبب فً %78 ٗ %98فً مو ٍِ اىخالٌب اىسشطبٍّت
ٗاىخالٌب اىَخؼيقت اىيٍفٍت بٍَْب مبُ حؼبٍش %:8 ٗ %:9 HMGCS2فً مو ٍِ اىخالٌب اىسشطبٍّت ٗاىخالٌب اىَخؼيقت اىيٍفٍتٗ .مبّج اىْخبئش حخطببق ٍغ حبرٍش ٗاسبشؽ اىَؼبمس. األستنتاج: ىقذ اسخْخضج ٕزٓ اىذساست اُ حأرٍش ٗاسبشؽ اىَؼبمس مبُ ٗاضحب فً سشطبُ اىزذي االّب٘بً ببىشغٌ ٍِ أُ حأرٍش ٗاسبشؽ مبُ ٌحذد اٌضب ٗىنِ ىٌ ٌنِ رٗ دالىت احصبئٍت ٗطبىَب اُ اىخالٌب اىسشطبٍّت ٗاىَخؼيقت اىيٍفٍت مالَٕب حح٘ي االّضٌَبث اىَخخصصت فً اسخٖالك اىنٍخّ٘بث ٗاّخبصٖب فٍَنِ االسخذاله بأُ اىْظبً اىغزائً اىَ٘ىذ ىينٍخّ٘بث ىَشضى سشطبُ اىزذي سٍنُ٘ رٗ فبئذة ٍحذٗدة.
کورتکراوە دیوی پشتووە ؛ شێرپَّجّی يّيک دٔٔەو بأتریٍ ْۆکاری يردَّ بّ شێرپَّجّ نّ ژَاٌ دا ،بّ ّْزارەْا تٕێژیُّٔە ئَّجاو درأەنّسّر شێرپَّجّی يّيک نّ بارەی ْۆکارە يّترسی دارەکاٌ، بۆيأەییٔ ،ەاڵو دأَّە بۆ چارەسّر نّگّڵ پێشبیُی بۆ چارەَٕسی َّخۆشییّکّ ،بّاڵو کارنێکی َێٕاٌ خاَّی شێرپَّجّ ٔ دۀرٔبّری خاَّکّ بٕٔەتّ یّکێک نّ گرَگتریٍ بابّتّکاٌ. موبوست لو توێژینووەکو؛ بۆ دەرخستُی ئَّسیًّکاَی ْ-٣ایذرۆکسی-٣يّثیهگهٕتریم-کۆئّی سیُثّیس )HMGCS2( ٢نّگّڵ کۆئَّسیى ئّی ئّسیتیم تراَسفیرەیس ،)ACAT1( ١کّ نّ َێٕ يایتۆکۆَذریا ب َٕیاٌ ّْیّ ،بّکاردێٍ بۆ بّرّْو ْێُاٌ ٔ بّکار ْێُاَی تَّی کیتۆٌ بّدٔای یّکذا ،نّ ّْریّک خاَّی شێرپَّجّ ٔ خاَّی درٔستکّری ریشاڵی دۀرٔبّری بۆ رَٔکردَّٔەی ّْر یّک نّ دیاردەکاَی ٔاربیگ ٔ پێچّٔاَّی ٔاربیگٔ ،ە بّ شێٕازێکی َاراستّخۆ کاریگّی ژەيی کیتۆٌ بّرّْيٓێٍ نّسّر شێرپَّجّی يّيک. ماددە و ڕێگای کارکردن؛ ئّو تٕێژیُّٔە پرۆسپێکتیڤّ ئَّجاو درأە نّ سّر سايپڵّکاَی بّشی پاثۆنۆجی َّخۆشخاَّی شۆڕش ٔ َّخۆشخاَّی فێرکاری سهێًاَیَّ ٤٤ ،خۆشی جۆری شیربّ َجّ ي يّ يكي رِ كذاكٕترأ ( )NOSکۆ کرأەتّٔە ،زاَیاری کۆکرأەتّٔە بّ سٕد ٔەرگرتٍ نّ ڕاپۆرتّکاَی پێشٕی َّخۆشّکّّْ ،ئٕ سايپڵّکاٌ رەَگکرأٌ نّگّل ّْردٔٔ ئَّسیًی (َ )ACAT1()HMGCS2أ يایتۆکۆَذریا .خّستی ٔ ڕێژەی سّدی رەَگ ٔەرگرتُّکّ تۆيارکرأە نّ ّْردٔٔ خاَّی شێرپَّجّ ٔ خاَّی درٔستکّری ریشاڵی دۀرٔبّی .پّیٕەَذی َێٕاٌ ّْردٔٔ ئَّسیًّکّ نّگّڵ ئّٔ پێٕەرە سّرجێیّ َّخۆشییّکاٌ ،کّ نّ ڕاپۆرتی پێشٕی َّخۆشّکّ ٔەرگیرأە ،ئَّجاو درأۀ .ە ّْرٔەْا پّیٕەَذی َێٕاٌ ّْردٔٔ ئَّسیًّکّ نّگّڵ خاَّی شێرپَّجّ ٔ خاَّی درٔستکّری ریشاڵی دۀرربّری. ئونجام؛ْیچ پّیٕەَذییّک َّبیُرا نّ َێٕاٌ ّْردٔٔ ئَّسیًّکّٔ کارا سّرجێیّ َّخۆشییّکاٌ جگّ نّ حانّتّکاَی جۆری شێرپَّجّی ًَرە َسو .ئَّسیًی ACAT1بّ پۆزەتیڤ بیُرا نّ ٦٨٪ ٔ٧٨٪نّ ّْر یّک خاَّکاَی شێرپَّجّٔ درٔستکّری ڕیشاڵی بّدٔای یّکذا نّ کاتیکذا ٥٥٪ HMGCS2 ٔ ٥٨٪بیُرا نّ ّْر یّک خاَّکاَی شێرپَّجّٔ درٔستکّری ڕیشاڵی بّدٔای یّکذا نّ کاتیکذا ،ئّو ئَّجايّ ْأڕایّ نّگّڵ ّْردٔٔ دیاردەی ٔاربیگ ٔ پێچّٔاَّی ٔاربیگ.
ده ره خامو كان؛ ئَّجايی ئّو تٕێژیُّٔەیّ دەریذەخات کّ دیاردەی پێچّٔاَّی ٔاربیگ نّ شێرپَّجّی يّيک دا رٔٔدەدات سّرەڕای ئّٔەی کّ دیارەی ٔاربیگ نّّْياٌ کاتذا رٔٔ دەدات ٔە بّ شێٕەیّکی َاڕاستّخۆ رَٔٔی دەکاتّٔە کّ خۆراکّ کیتۆٌ بّرّْيٓێُّکاٌ کاریگّرێکی سُٕرداریاٌ ّْیّ نّسّر شێرپَّجّی يّيک بّ ْۆی بَٕٔی ّْردٔٔ ئَّسیى نّ خاَّی شێرپَّجّ ٔ خاَّی درٔستکّری ڕیشاڵی دەرٔرٔبّری.
حكومت أقليم كزدستان وسارة التعليم العالي والبحث العلمي جامعت السليماويت كليت الطب فزع الباثولوجي والطب العذلي
اوتاج الكيتون مه الخاليا الليفيت يغذي العملياث الحيويت لبيوث الطاقت في سزطان الثذي /دراست كيميامىاعيت وسجيت في مذيىت السليماويت -العزاق بحث هقدم الى كليت الطب /هيأة العلىم الطبيت جاهعت السليوانيت كجزء هن هتطلباث نيل شهادة ال ماج س ت ير في علن االهراض هن قبل
رووذ تاج الذيه صاحب بكالىريىس في الطب والجراحت العاهت باشراف
د .هذيل عذوان ياسيه استاذ هساعد -فرع الباثىلىجي F.I.B.M.S.(Path), M.B.Ch.B.
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حکومەتی هەرێمی کوردستان وەزارەتی خوێىدوی بااڵو توێژیىەوەی زاوستی زاوکۆی سلێماوی کۆلیژی پسیشکی بەشی وەخۆشی زاوی و پسیشکی دادوەری
بەرهەم هێىاوی کیتۆن لە الیەن خاوەی دروستکەری ڕیشاڵی وبەکارهێىاوی وەك سەچاوەی ووزە بۆ زیىدەکردارەکاوی مایتۆکۆودریای خاوەی شێرپەوجەی مەمك/توێژیىەوەیەکی ئیمیووۆهیستۆکیمستریە لە شاری سلێماوی-عێراق نامەیەکە پێشکەشی کۆلێژی پسیشکی زانکۆی سلێمانی کراوە وەك بەشێك لە پێىیستیەکانی بەدەستهێنانی بروانامەی ماستەر لە نەخۆشی زانی دا لە الیەن ڕەوەود تاج الدیه صاحب بەکالۆرێۆش لە پسیشکی و وەشتەرگەری گشتی بە سەر پەرشتی
د .هەدیل عەدوان یاسیه پ.ی .لە بەشی وەخۆشی زاوی F.I.B.M.S.(Path), M.B.Ch.B.
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