Potential gene network for the health effect of exposure to pcb fs on human diffuse large cell lymphoma

THAI NGUYEN UNIVERSITY UNIVERSITY OF AGRICULTURE AND FORESTRY NATIONAL TSING HUA UNIVERSITY THAVISACK MIVONGSACK TITLE: POTENTIAL GENE-NETWORK FOR THE HEALTH EFFECT OF EXPOSURE TO PCB/

THAI NGUYEN UNIVERSITY

UNIVERSITY OF AGRICULTURE AND FORESTRY NATIONAL TSING HUA UNIVERSITY

THAVISACK MIVONGSACK

TITLE: POTENTIAL GENE-NETWORK FOR THE HEALTH EFFECT

OF EXPOSURE TO PCB/FS ON HUMAN DIFFUSE LARGE CELL

LYMPHOMA BACHELOR THESIS

Study Mode:Full-time

Major: Environmental science and management

Faculty:International Programs Office

Batch: 2013-2017

Thai Nguyen, 21/12/2017

DOCUMENTATION PAGE WITH ABSTRACT

Thai Nguyen University of Agriculture and Forestry

Degree Program Bachelor of Environmental Science and Management

Thesis Title Potential gene-network for the health effect of

exposure to pcb/fs on human diffuse large cell lymphoma

Supervisor (s) Prof Chun-Yu Chuang,

Associate Prof DRTran Van Dien Abstract:

The thesis describes the Lymphoma is the most top cancers in the worldwide, and the incidence rises strikingly since the last half of 20thcentury Lymphoma is a cancer affecting the immune system; the major risk factor is associated with exposure to occupational or environmental chemicals Polychlorinated biphenyls (PCBS) are a class of organic chemicals, known as congeners that have been used in a variety of commercial products PCBs were used in caulking, electronics, fluorescent light ballasts and other building materials from the 1950s to the late 1970s Buildings built

or renovated during that time may contain PCBs in caulking and other materials PCBare very stable mixtures that are resistant to extreme temperature and pressure PCBS were used widely in electrical equipment like capacitors and transformers They also were used in hydraulic fluids, heat transfer fluids, lubricants, and plasticizers

PCBs have been released into the environment through spills, leaks from electrical and other equipment, and improper disposal and storage It is estimated that more than half

of the PCBS produced have been released into the environment Once in the environment, PCBS can be transported long distances and they bind strongly to soil and sediment so they tend to be persistent in the environment They have been found

in air, water,soil, and sediments throughoutthe world.PCBs can enter the body through inhalation, ingestion, and dermal routes of exposure They are readily absorbed but are slowly metabolized and excreted In particular, PCBs initially distribute to the liver and muscle tissues, but eventually accumulate in lipid-rich tissues This leads to greater concentrations of PCBS in adipose tissue, breast milk, the liver, and skin The data analysis was subsequently performed using Network Analyst, a standard web browser for network analysis and interactive exploration

Keywords TCDD, Furans, DBLCL, bioinformatics, GEO, Array

Express

Date of submission October,2017

Supervisor’s signature

ACKNOWLEDGEMENT

First of all, we know that knowledge is just only can be proved by our works, and internship is one of the best opportunity for a student whose can do their first project before they find their jobs to enroll in the future Besides that, we are not only improving ourselves by knowledge in company environment, institute or laboratory but also making more friends whose are having many experiences in environment, and

it will help us in the near future From my perspective, this internship is absolutely needed, helpful and important

Because of that, and be assigned by the International Programs Office and also the allowed of Department of Biomedical Engineering and Environmental Sciences (National TsingHua University, Taiwan) To well done this thesis, I want to express profound gratitude to Advanced Education Program, the school administrators, the staffs in Department of Biomedical Engineering and Environmental Sciences, the

staffs of YC laboratory, and particularly my supervisor,Associate Prof DRTran Van Dien and Prof Chun-Yu Chuang whose were always supporting me every single time

I got troubles I would like to send both of supervisors a warmly thanks for the supporting me, and for their sacrifice for education, as same as environmental issues in Taiwan and Vietnam as all countries in the world

Finally, I would like to say that I had tried my best to finish this thesis in the best way, I guess However, to be honest, I partly believe that my thesis still have some problems because of the limitation of knowledge and reality experiences, especially in our environmental circumstances these days It is totally happy if I can

get feedbacks and comments from you, my Teachers, Professors, and Supervisors, to finish my thesis in a fantastic way, to get the best results

Sincerely,

Thai Nguyen October, 2017

THAVISACK MIVONGSACK

TABLE OF CONTENTS

DOCUMENTATION PAGE WITH ABSTRACT i

ACKNOWLEDGEMENT iii

TABLE OF CONTENTS v

LIST OF FIGURES vii

ABBRIVIATION ix

PART I: INTRODUCTION 1

1.1 Rationale 1

1.2 Objectives 3

PART II:LITERATURE REVIEW 4

2.1 Polychlorinated biphenyls (PCBs) 4

2.1.1 Polychlorinated Biphenyls (PCBs) Toxicity 5

2.1.2 The industrial production of PCB 6

2.1.3 PCBs and Environment 7

2.1.4 Health Effects of PCBs 9

2.2 Lymphoma 12

2.3 Biological pathway 13

2.3.1 Diffuse large B cell lymphoma (DLBCL) 15

2.4 Gene-network components 17

2.4.1 Gene-network database:Array Express and (GEO) 18

2.4.2 Statistical analysis 19

3.2 Gene network analysis and Cytoscape for gene-network analysis 24

PART IV: RESULTS AND DISCUSSIONS 29

4.1 Genetic datasets 29

4.1.1 Differentially expressed genes 32

4.1.2 Gene-network construction of DLBCL and PCBs 35

4.2 Discussion 38

PART V: CONCLUSION AND RECOMMENDATION 41

5.1 Conclusion 41

5.2 Recommendation 42

REFERENCES 43

LIST OF FIGURES

Figure 2.1: Diagram of Polychlorinated Biphenyls (PCBs)(Shoemaker, 2005) 6

Figure2.2: Polychlorinated biphenyls – levels in foods 11

Figure 2.3: Diffuse large Bcell lymphoma 16

Figure 3.1: The flowchart of methodology 24

Figure 3.2: Cytoscape home page(Shannon P, 2003) 26

Figure 4.1: Diagram of Gene-network construction of DLBCL & PBCs Sources: (Rosenwald,2002) 37

Figure 4.2: The potential regulatory pathway of NHL progression in response to PCB exposure Source: (Miller, 2001) 38

LIST OF TABLES

Table 4.1: Genetics Datasets of DLBCL 30 Table 4.2: Datasets on Array Express used for PCB analysis 31 Table 4.3: Differentially expressed genes, including –up and down – regulate genes in Diffuse Large B cell lymphoma compared to normal cells 33 Table 4.4: Differentially expressed genes, including up-and down – regulated genes activated by PCB compared to control group 35

ABBRIVIATION

ABC Activated B cell

AML Acute myeloid leukemia

ALL Acute lymphoblastic leukemia

B-Cells B-lymphocytes

B-NHL Non Hodgkin lymphoma

DLBCL Diffuse large B cell lymphoma

DMSO Dimethyl sulfoxide

DEGS Differentially expressed genes analysis

FL Follicular lymphoma

FDR False discovery rate

GEO (NCBI) Gene expression omnibus National center for

IARC International agency for research on cancer

MIAME Minimum information about microarray

experiment MAGE-ML Microarray and Gene Expression Markup

Language NPL N-acetylneuraminate pyruvate lyase

NHL Non Hodgkin lymphoma

PCB Polychlorinated biphenyls

SNPs Single Nocleotide Polymorphisms

T-Cells T-lymphocytes

PART I: INTRODUCTION 1.1 Rationale

Polychlorinated biphenyls, in short PCBs, are a group of man-made chemicals.PCBs have been used in many different products, including electrical equipment, surface coatings, inks, adhesives, flame-retardants, and paints PCBs may

be released into the environment, for instance when waste that contains PCBs is incinerated or stored in landfills(Morris, 2017).PCBs are probable human carcinogens and can also cause non-cancer health effects, such as reduced ability to fight infections, low birth weights, and learning problems The risks and hazards associated with PCBs in the Hudson River are a function of the toxicity of PCBs and an individual's exposure(Hudson, 2016).The most commonly observed health effects in people exposed to large amounts of PCBs are skin conditions such as acne and rashes Studies in exposed workers have shown changes in blood and urine that may indicate liver damage PCB exposures in the general population are not likely to result in skin and liver effects Most of the studies of health effects of PCBs in the general population examined children of mothers who were exposed to PCBs(Buford Hwy

NE, 2014)

Polychlorinated biphenyls (PCBs) were powerful carcinogens A carcinogen is any substance or agent that tends to produce a cancer Polychlorinated biphenyls are found in things such as asbestos and radon However, we are also forced to consume it

in our foods There are exposed to many substances at any given time This includes those we encounter at work, school, or home, in the food we eat and in the air we breathe(Jeni, 2011)

But it’s almost impossible anyone could know precisely what they’ve been exposed to PCBs are toxic They can provoke serious health effects that go beyond cancer They can also play a role in hormone disruption, reduced ability to reproduce, skin toxicity, immune system disorders

Lymphoma is the most common blood cancer that develop in lymphatic system The two main categories of lymphoma are Hodgkin lymphoma and non-Hodgkin lymphoma (NHL)(Ferlay, 2013) Lymphoma occurs when cells of the immune system called lymphocytes, a type of white blood cell, grow and multiply uncontrollably Cancerous lymphocytes can travel to many parts of the body, including the lymph nodes, spleen, bone marrow, blood, or other organs, and form a mass called a tumor The body has two main types of lymphocytes that can develop into lymphomas: B-lymphocytes (B-cells) and T-lymphocytes (T-cells)

Diffuse large cell lymphoma (DLBCL or DLBL) is a cancer of B cells, a type

of white blood cell responsible for producing antibodies It is the most common type

of non-Hodgkin lymphoma among adults (Clinical, 1997), with an annual incidence of 7–8 cases per 100,000 people per year in the USA and the UK (Morton et al., 1992-2001) This cancer occurs primarily in older individuals, with a median age of diagnosis at approximately 70 years of age (Smith et al., 2011), though it can also occur in children and young adults in rare cases(Smith at el., 2010) DLBCL is an aggressive tumor which can arise in virtually any part of the body (Kumar et al., 2009),and the first sign of this illness is typically the observation of a rapidly growing mass, sometimes associated with B symptoms: fever, weight loss, and night sweats (Freeman et al, 2012)

1.2 Objectives

• The aims of the study was tosummarize the health implications associated with exposure topolychlorinatedbiphenyls (PCBs)

• To identify the potential pathway exposure to dioxin corresponding to DLBCL

• To express the gene number that exposure to dioxin can induce DLBCL and PCBs

• To evaluate diffuse large B-cell lymphoma (DLBCL) and construct regulatory networks of genes and miRNAs to gradually reveal the underlying mechanisms of DLBCL development

PART II:LITERATURE REVIEW 2.1 Polychlorinated biphenyls (PCBs)

Polychlorinated biphenyls (PCBs) is a group of organic compounds used in the manufacture of plastics, as lubricants, and dielectric fluids in transformers, in protective coating for wood, metal and concrete, and in adhesives, wire coating and so forth (New York, 2003) It can cause a number of different harmfuleffects They are highly toxic to aquatic life and persist in the environment for long periods of time They can accumulate in food chains and may produce harmful sideeffects at high concentrations There are no known natural sources of PCBs in the environment PCBs are either oilyliquids or solids and are colorless to light yellow Some PCBs are volatile and may exist as avapor in air They have no known smell or taste(Buford Hwy NE, 2014) PCBs enter the environment as mixturescontaining a variety of individual chlorinated biphenyl components, known as congeners, as wellas impurities Because the health effects of environmental mixtures of PCBs are difficult toevaluate, most of the information in thistoxicological profile is about seven types of PCBmixtures that were commercially produced These seven kinds of PCB mixtures include 35% ofall the PCBs commercially produced and 98% of PCBs sold in the United States since 1970.Some commercial PCB mixtures are known in the United States by their industrial trade name,Aroclor For example, the name Aroclor 1254 means that the mixture contains approximately54% chlorine by weight, as indicated by the second two digits in the name Because they do not burn easily and are good insulating materials, PCBs were used widely as coolants and lubricantsin transformers, capacitors, and other electrical equipment The manufacture of PCBs stopped inthe

United States in August 1977 because there was evidence that PCBs build up in theenvironment and may cause harmful effects Consumer products that may contain PCBs includeold fluorescent lighting fixtures, electrical devices or appliances containing PCB capacitors madebefore PCB use was stopped, old microscope oil, and old hydraulic oil

2.1.1 Polychlorinated Biphenyls (PCBs) Toxicity

Since PCBs are found throughout the environment, it is likely that everyone has been exposed to them PCBs can enter the body by eating or drinking contaminated food, through the air we breathe, or by skin contact PCBs are easily absorbed by the body and are stored in fatty tissue PCBs are not eliminated well, so they can accumulate in the body(Gray et al.,2003; Thrasher and Crawley, 2009)

Most people are exposed to PCBs by eating contaminated fish, meat, and dairy products Catfish, buffalo fish, and carp usually have the highest PCB levels Plants take up only small amounts of PCBs from the soil, so amounts in grazing animals and dairy products are generally lower than in fish Dust contaminated with very small levels of PCBs may be found on the outer surfaces of fruits and vegetables PCBs do not dissolve well, so exposure to them from water is usually not a concern Some private wells may use old submersible pumps that contain PCB oil If the pump seal fails, PCBs can leak into the well and contaminate the drinking water older fluorescent lights found in schools, offices and homes may still contain transformers or ballasts that contain PCBs If the ballasts fail, PCBs can leak out and contaminate exposed surfaces and the air PCB levels measured in the air after a light ballast failure can be

significant The workplace also may be a source of PCB exposure(Crago et al., 2003,

Etzel, 2006)

Figure 2.1: Diagram of Polychlorinated Biphenyls (PCBs) (Shoemaker, 2005)

PCBs are chemicals formed by attaching one or more chlorine atoms (at the Xs

in Figure 1 above) to a pair of connected benzene rings.Depending on the number and position of chlorine atoms attached to the biphenyl ring structure, 209 different PCB congeners can be formed PCB congeners can be divided into the coplanar, the mono-ortho-substituted PCBs, and other non-dioxin-like PCBs The significance of this designation is that coplanar and some of the mono-ortho-substituted PCBs have

dioxin-like toxicological effects(Shoemaker, 2005)

2.1.2 The industrial production of PCB

PCBs were used for many different industrial purposes, including the afore mentioned electrical industry and: Hydraulic fluids; casting wax; carbonless carbon paper; compressors; heat transfer systems; plasticizers; pigments; adhesives; liquid cooled electric motors; fluorescent light ballasts There is little wonder that this group

of chemicals was so readily accepted

One of the qualities that make PCBs so desirable is one of the characteristics that make PCBs so hazardous to the environment The high thermal and chemical resistance of PCBs means that they do not readily break down when exposed to heat

orchemical treatment This is a very desirable trait for lubricants However, since PCBs do not break down they remain in the environment and continue to build up as more are introduced into the environment Worms consume organic matter contaminated with PCBs, and small fishes eat the worms Small fish are then eaten by larger fish, and perhaps the large fish are eaten by an eagle Worms may only live for a short time, but eagles live for a long time They continue to eat lots of large fish that ate lots of small fish that ate lots of contaminated worms Over a lifetime, the PCB concentration in the fatty tissue of the eagle would continue to increase as it ate more and more contaminated fish This accumulation of a chemical in animals at the top of the food chain is known as bioaccumulation If the chemical happens to be toxic, the consequences become obvious

2.1.3 PCBs and Environment

Before 1977, PCBs entered the air, water, and soil during their manufacture and use in theUnited States Wastes that contained PCBs were generated at that time, and these wastes wereoften placed in landfills PCBs also entered the environment from accidental spills and leaksduring the transport of the chemicals, or from leaks or fires in transformers, capacitors, or otherproducts containing PCBs Today, PCBs can still be released into the environment from poorlymaintained hazardous waste sites that contain PCBs; illegal or improper dumping of PCBwastes, such as old transformer fluids; leaks or releases from electrical transformers containingPCBs; and disposal of PCB-containing consumer products into municipal or other landfills notdesigned to handle hazardous waste PCBs may be released into the environment by the burningof some wastes in municipal and industrial incinerators.Once in the environment, PCBs

do not readily break down and therefore may remain for verylong periods of time They can easily cycle between air, water, and soil For example, PCBs canenter the air

by evaporation from both soil and water In air, PCBs can be carried long distancesand have been found in snow and sea water in areas far away from where they were released intothe environment, such as in the arctic As a consequence, PCBs are found all over the world Ingeneral, the lighter the type of PCBs, the further they may be transported from the source ofcontamination PCBs are present as solid particles or as

a vapor in the atmosphere They willeventually return to land and water by settling as dust or in rain and snow In water, PCBs maybe transported by currents, attach to bottom sediment or particles in the water, and evaporate intoair Heavy kinds of PCBs are more likely to settle into sediments while lighter PCBs are morelikely to evaporate

to air Sediments that contain PCBs can also release the PCBs into thesurrounding water PCBs stick strongly to soil and will not usually be carried deep into the soilwith rainwater They do not readily break down in soil and may stay in the soil for months oryears; generally, the more chlorine atoms that the PCBs contain, the more slowly they breakdown.Evaporation appears to be an important way by which the lighter PCBs leave soil As agas, PCBs can accumulate in the leaves and above-ground parts

of plants and food crops.PCBs are taken up into the bodies of small organisms and fish

in water They are also taken upby other animals that eat these aquatic animals as food PCBs especially accumulate in fish andmarine mammals (such as seals and whales) reaching levels that may be many thousands oftimes higher than in water PCB levels are highest in animals high up in the food chain

Environmental contamination from PCBs has been caused by accidental releases, careless disposal practices, and leaks from industrial facilities or chemical waste-water disposal sites PCBs degrade very slowly, are cycled and transported within the ecosystem, and bioaccumulate as they move up the food chain

Once released into the environment, PCBs adsorb strongly to soil and sediment

As a result, these compounds tend to persist in the environment, with half-lives for most congeners ranging from months to years PCBs leach from soil slowly, particularly the more highly chlorinated congeners, and translocate to plants via soil insignificantly Cycling of PCBs through the environment involves volatilization from land and water surfaces into the atmosphere, with subsequent removal from the atmosphere by wet or dry deposition, then revolatilization In the general population, inhalation of these airborne PCBs is one route of exposure, in addition to the food source of exposure to PCBs

Once released into the environment, PCBs may undergo all of the volatilization from land and water surfaces into the atmospheres, biotransformation into more complex undefined mixtures, fast degradation within the ecosystem and strong adsorption to soil and sediment

2.1.4 Health Effects of PCBs

A growing number of studies have found serious health effects from exposure

to PCBs PCBs are likely to cause cancer and are classified as a “probable carcinogen”

by the federal Environmental Protection Agency and the International Agency for Research on Cancer In addition, a study found children’s risk of developing acute lymphocytic leukemia, increased by two-fold when PCBs were detected in the dust of

a room in which the child spent a significant amount of time(Ward M H, 2009) People with potentially high exposures to PCBs include: Recreational and subsistence fishers who typically consume larger quantities of locally caught fish than the general population, Children with in utero and lactational exposure to PCBs from mothers who eat large quantities of contaminated fish during pregnancy and while nursing,Certain farmers and their families who consume PCB-contaminated food via their own farm-raised beef and dairy cattle, andpeople living near incinerators, other PCB-disposal facilities, or NPL hazardous waste sites where PCBs have been detected (Buford Hwy

NE A , 2016)

PCBs can enter human cells and tissues when contaminated air is breathed in, when contaminated food enters the digestive system, or through contact with the skin Tests on laboratory animals show that PCBs are readily absorbed through the digestive tract when swallowed, and to a lesser extent through the skin The main PCB elimination routes are through the faeces, urine, and breast milk Once in the gastrointestinal tract, ingested PCBs diffuse across cell membranes and enter blood vessels and the lymphatic system PCBs, especially those that contain a greater number

of chlorine atoms, are readily soluble in fats and thus tend to accumulate in fat-rich tissues such as the liver, brain and skin.In mothers, PCBs have also been found to pass into the placenta, umbilical cord blood, and breast milk PCBs can undergo different transformations in the body and then either be stored in certain tissues or excreted Transformations that lead to accumulation,PCBs can easily be transformed into

persistent metabolites that are not readily excreted and that can accumulate in

specifictissues and body fluids The biological half-life of these persistent metabolites depends on the structure of the original PCB

Transformations that lead to excretion, PCBs can leave the blood and enter tissues very rapidly and be transformed into water-soluble substances These can combine with glutathione and glucuronic acid, that are naturally present in the body, forming a substance that is then excreted in urine and feces The speed of this process depends on the number and position of chlorine atoms of the original PCB It is, for instance, slower when there are more chlorine atoms on both phenyl rings and faster if there are two carbon atoms without any attached chlorine atoms next to each other

In a case where both children and their mothers were exposed to PCBs through their diet, levels of PCBs in blood dropped more rapidly in the children This may be partly explained by the children’s growth, as their increasing tissue mass may lower the PCB concentration in blood due to dilution, rather than elimination

Figure2.2: Polychlorinated biphenyls – levels in foods

Source: USDA Nutrient Database for Standard Reference, Release 16 (2003)

This graph shows the absolute amount of polychlorinated biphenyls in various foods It is taken from a FDA market basket study

2.2 Lymphoma

Lymphoma is a type of cancer that begins in infection-fighting cells of the immune system, called lymphocytes The two main types of lymphoma are Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL) It isonly possible to determine the difference between HL and NHL under a microscope In HL, a particular cell called the Reed-Sternberg (RS) cell is found NHL is much more common than HL about 90% of lymphoma is NHL According to the America Cancer Society, the 5 year survival rate for HL is 85%, with a 10 year survival rate of 81% There are many different forms of NHL, and prognoses vary, but HL tends to be easier to treat Nearly 40% of NHL diagnosed cases are at stage iv, which are more likely to spread and not

as predictable in terms of their progression

NHL incidence rate has increased markedly in recent year International Agency for Research on Cancer (IARC) declared the incidence rate and the mortality rate of NHL were 9.8 and 3.5 per 100,000 populations during 1990 to 2010 in forty countries of Europe In Taiwan, from 2000 to 2012, the standardized cancer incidence rate of NHL was from 4.03 per 100,000 populations rising to 7.23 NHL is a diverse group of cancers originating in the cells of immune system, including B-lymphocytes, t-lymphocytes and natural killer cells Most types of NHL from B-lymphocytes, called B-cell non-Hodgkin lymphoma (B-NHL) Types of NHL vary significantly in their severity, from indolent (slow-growing) to vary aggressive (fast-growing) types Based

on a WHO classification system, more than 30 different subtypes have been

identified.The most common histologic subtype of NHL is diffuse large B-cell lymphoma (DLBCL), which is an aggressive, fast-growing lymphoma Follicular lymphoma (FL) is the second most common, which is an indolent, slow-growing lymphoma Several epidemiologic studies explored that the risk factors of NHL include human immunodeficiency virus (HIV) infection, other viral and bacterial infections, and increased use of immunosuppressive drugs, changes in diet and lifestyle, and exposure to various chemicals Although the descriptive epidemiology of NHL has been well characterized using population-based cancer registry data, the etiology of NHL is less well understood

2.3 Biological pathway

A biological pathway is a series of actions among molecules in a cell that leads

to a certain product or a change in the cell Such a pathway can trigger the assembly of new molecules, such as a fat or protein Pathways can also turn genes on and off, or spur a cell to move

For your body to develop properly and stay healthy, many things must work together at many different levels - from organs to cells to genes From both inside and outside the body, cells are constantly receiving chemical cues prompted by such things

as injury, infection, stress or even the presence or lack of food To react and adjust to these cues, cells send and receive signals through biological pathways The molecules that make up biological pathways interact with signals, as well as with each other, to carry out their designated tasks

Biological pathways can act over short or long distances For example, some cells send signals to nearby cells to repair localized damage, such as a scratch on a

knee Other cells produce substances, such as hormones, that travel through the blood

to distant target cells These biological pathways control a person's response to the world For example, some pathways subtly affect how the body processes drugs, while others play a major role in how a fertilized egg develops into a baby Other pathways maintain balance while a person is walking, control how and when the pupil in the eye opens or closes in response to light, and affect the skin's reaction to changing temperature Biological pathways do not always work properly When something goes wrong in a pathway, the result can be a disease such as cancer or diabetes

Researchers have discovered many important biological pathways through laboratory studies of cultured cells, bacteria, fruit flies, mice and other organisms Many of the pathways identified in these model systems are the same as, or are similar

to, counterparts in humans Still, many biological pathways remain to be discovered It will take years of research to identify and understand the complex connections among all the molecules in all biological pathways, as well as to understand how these pathways work together

Finding out what pathway is involved in a disease - and identifying which step

of the pathway is affected in each patient may lead to more personalized strategies for diagnosing, treating and preventing disease Researchers currently are using information about biological pathways to develop new and more effective drugs It likely will take some time before we routinely see specifically designed drugs that are based on information about biological pathways However, doctors are already beginning to use pathway information to choose and combine existing drugs more effectively(Español, August 27, 2015)

2.3.1 Diffuse large B cell lymphoma (DLBCL)

The lymphoma organs include the bone marrow, thymus, spleen, and lymph nodes These help to fight infection throughout the body The organs of the lymphatic systems are connected by a network of lymphatic and blood vessels Lymphatic fluid contains white blood cells called lymphocytes

There are two primary types of lymphocytes: B cells and T cells Diffuse large

B cell lymphoma (DLBCL) is a cancer of B lymphocytes Almost all lymphocytes begin growing in the bone marrow or lymph nodes T cells leave the bone marrow before they are completely matured, and finish maturing in the thymus gland B cells instead continue to develop and mature in the bone marrow and lymph nodes In DLBCL, the abnormal B cell lymphocytes are larger than normal, and they have stopped responding to signals that usually limit the growth and reproduction of cells

This subtype of lymphoma is called diffuse large B cell because of the way the malignant large B cells are distributed within the lymph nodes when examined with a microscope

There are different varieties of DLBCL that can be identified by performing advanced tests on the lymph node specimen Particular varieties of DLBCL may be more likely to respond to certain treatments Terms that may be used to describe the varieties of DLBCL include the following: Germinal center B cell type (GCB), Activated B cell type (ABC), double hit lymphoma

DLBCL can either develop as a transformation from a less aggressive form of lymphoma or as a first occurrence of lymphoma

The body has two main types of lymphocytes that can develop into lymphomas:

B lymphocytes (B cells) and T lymphocytes (T cells) Be-cell lymphomas are much more common than T-cell lymphomas and account for approximately 85 percent of all NHLs Diffuse large Be-cell lymphoma (DLBCL) is the most common form of NHL, accounting for about 30 percent of newly diagnosed cases of NHL in the United States DLBCL occurs in both men and women, although it is slightly more common

in men Although DLBCL can occur in childhood, its incidence generally increases with age, and roughly half of patients are over the age of 60(Amengua, (2015) )

DLBCL is an aggressive (fast-growing) lymphoma that can arise in lymph

nodes or outside of the lymphatic system, in the gastrointestinal tract, testes, thyroid, skin, breast, bone, or brain Often, the first sign of DLBCL is a painless, rapid swelling

in the neck, underarms, or groin that is caused by enlarged lymph nodes For some patients, the swelling may be painful Other symptoms may include night sweats, fever, and unexplained weight loss Patients may notice fatigue, loss of appetite, shortness of breath, or pain (Amengua, 2015)

Figure 2.3: Diffuse large Bcell lymphoma

(Micrographor Field stainof a diffuse large B cell lymphoma)

Source: (Oncogene,2004)

2.3.2 Diffuse large B cell Lymphoma risk factors

The age, gender, and ethnicity affect a person's likelihood of developing DLBCL Although DLBCL has been found in people of all age groups, it is found most commonly in people who are middle-aged or elderly The average age at the time

of diagnosis is 64 years Men are slightly more likely to develop DLBCL than women

In the United States, white people are more likely to develop this type of lymphoma than are Asians or Blacks

DLBCL is not an inherited disease Siblings and children of patients with DLBCL do not have a substantially increased risk of developing DLBCL The majority of patients have no family history, but approximately 9 percent of patients have a first degree relative (eg parent or sibling) with lymphoma or chronic lymphocytic leukemia

2.4 Gene-network components

DNA microarray techniques have been applied to predict DLBCL, treatment success and explain disease heterogeneity five clinical features (age, tumor stage, serum lactate, dehydrogenate concentrations, performance status, number of extra nodal disease sites) (Gohlmanm and Talloen, 2009) In fact, this techniques is most widely used to profile gene expression of an organism on a whole genome scale, and available for spawning a series of microarray-based expression studies of DLBCL in order to the refine prognosis referring to molecular-level information (Segal, 2005) Besides, DNA microarray was also carried out to analyze the changes of human B-cell gene expression induced by dioxins (Kovalovaet al., 2017)

In this study, the gene expression profiling representing DLBCL and dioxins (TCDD and furans) created by DNA microarray techniques were conducted for further

analytical steps The datasets of genes expression are collected by two main kinds of database: Gene Expression Omnibus (GEO) and Array express databases that will be discussed more detailed in the following part

2.4.1 Gene-network database:Array Express and (GEO)

ArrayExpress is a public database for high throughput functional genomics data ArrayExpress consists of two parts—the ArrayExpress Repository, which is a MIAME supportive public archive of microarray data, and the ArrayExpress Data Warehouse, which is a database of gene expression profiles selected from the repository and consistently re-annotated Archived experiments can be queried by experiment attributes, such as keywords, species, array platform, authors, journals or accession numbers Gene expression profiles can be queried by gene names and properties, such as Gene Ontology terms and gene expression profiles can be visualized ArrayExpress is a rapidly growing database, currently it contains data from

>50 000 hybridizations and >1 500 000 individual expression profiles ArrayExpress supports community standards, including MIAME, MAGE-ML and more recently the proposal for a spreadsheet based data exchange format: MAGE-TAB Availability: www.ebi.ac.uk/arrayexpress

The Gene Expression Omnibus (GEO) repository at the National Center for Biotechnology Information (NCBI) archives and freely disseminates microarray and other forms of high-throughput data generated by the scientific community The database has minimum information about a microarray experiment (MIAME)-compliant infrastructure that captures fully annotated raw and processed data Several data deposit options and formats are supported, including web forms, spreadsheets,

XML and Simple Omnibus Format in Text (SOFT) In addition to data storage, a collection of user-friendly web-based interfaces and applications are available to help users effectively explore, visualize and download the thousands of experiments and tens of millions of gene expression patterns stored in GEO This paper provides a summary of the GEO database structure and user facilities, and describes recent enhancements to database design, performance, submission format options, data query and retrieval utilities(Bethesda, 2011) GEO is accessible at http://www.ncbi.nlm.nih.gov/geo/

2.4.2 Statistical analysis

The Affymetrix Gene Chip system is one of the most widely adapted microarray platforms However, due to the overwhelming amount of information available, many Affymetrix users tend to stick to the default analysis settings and may end up drawing sub-optimal conclusions Written by a molecular biologist and a biostatistician with a combined decade of experience in practical expression profiling experiments and data analyses Gene Expression Studies Using.Affymetrix Microarrays tears down the omnipresent language barriers among molecular biology, bioinformatics, and biostatistics by explaining the entire process of a gene expression study from conception to conclusion (Chapman&Hall, 2009)

Every multiple testing correction procedure uses some error rate to measure the occurrence of incorrect conclusion The focus is generally on errors due to false positive conclusions (the non-differentially expressed genes called significant) whilekeeping the number of false negatives (the differentially genes not called