Transitional Cell Carcinoma Of The Bladder Biology Essay

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The bladder functions to store and voluntary discharge urine. It can store between 400-500ml of urine at low pressure.(1) The bladder needs to have specialised cells that enhance it purpose. The bladder consists of transitional epithelium in the mucosa that is designed to stretch once the bladder is full. (2) There are three more layers underneath the epithelium- (1) lamina propria, ( 2) muscularais propria and (3) adventia. (Figure 1)

Transitional epithelium is an impermeable layer of cells that line's the whole of the urinary tract from the renal papillae to halfway along the urethra in women and to the navicular fossa in men. (3)

Figure 1-Illustration shows the functional subunit of the bladder. Note that the diagram doesn't show the adventia layer. (1)

Transitional cell carcinoma can occur anywhere along the urinary tract. However they are most likely to arise in the bladder because the transitional epithelium is continuously in contact with urine carcinogens. Transitional cell carcinoma has the highest incidence among bladder tumours- accounting for more than 130, 000 deaths annually across the world and was responsible for 4,295 deaths in England and Wales in 2005.(4) The incidence of bladder cancer in men is 2.5 times greater than that in women. (5) There is a decreased chance of tumour growth in the rest of the urinary tract since the urine just passes along the tract.

The main presenting symptom in bladder cancer is painless haematuria. Haematuria can be microscopic or macroscopic. Microscopic haematuria doesn't discolour the urine and is only detected using a Dipstick analysis which causes a positive reaction for blood on urine-reagent strip. (6) Macroscopic haematuria does cause discolouration of the urine. It's visible to the human eye. Patients often have mixed symptoms-some have multiple episodes of haematuria while others may only complain of one episode. Patients will often be distressed to see blood in their urine and seek medical help. The risk of bladder cancer in patients who are 50 years of age or older that present with painless haematuria is 35% and for those under 50, there's still a 10% chance they have bladder cancer. (6) In addition, patients can also complain of dysuria, increased frequency and reoccurring urinary tract infections. In patients with a progressively developed tumour, anaemia and a suprapubic mass is palpable. (3)

Initially heamaturia will be investigated further via urine analysis and Intravenous Pyelogram (IVP). (7) The urine analysis confirms the presence of red blood cells in the urine under the microscope. In addition, the presence of sterile pyuria can be suggestive of urothelial tumour. (6) IVP involves injecting iodinated contrast material into the veins and a series of X-ray images are taken to assess how efficiently the body copes with the fluid injected. Iodine is radio-opaque and shows up where urine is. This allows imaging of the whole urinary tract and therefore to exclude other possible diagnoses like renal cell carcinoma and upper urinary tract tumour. (8) However a plain abdominal film and ultrasound are used by some urologists which are known to be very sensitive at detecting bladder tumours but it's not as useful in detection of the upper urinary tract rumours (9).

Furthermore any patients presenting with macroscopic haematuria, must always undergo a rigid or flexible cystoscopy. (10) This is the gold standard test for detecting bladder cancer. The high incidence of bladder cancer compared to the upper urinary tract tumours, which accounts for less than 5% of the tumours makes it a very likely diagnosis. Urine cytology has a high specificity and low sensitivity as it's related to tumour grade. It's more sensitive for high-grade carcinomas because that causes more desquamation of the malignant cells. (11) During cytology, biopsies must be taken for histological assessment, which is essential for diagnosis from which treatment options can be considered.

a)

b)

Figure 2: a) Intravenous urography showing tumour (arrow) on the left side of the bladder. B)Illustrates a cytoscopic view of the bladder (9).

The staging of bladder cancer is done via the Tumour, Node, and Metastasis (TNM) classification system. (12) As well as being staged the cancers are graded depending on their stage of differentiation. Biopsies of the bladder must include a specimen of the muscularis layer to determine whether or not the tumour has invaded the underlying muscle.

TNM Classification of the Bladder Cancer (12)

T-Primary Tumour

TX

Primary Tumour cannot be assessed

T0

No evidence of Primary tumour

Ta

Non-invasive papillary carcinoma

Tis

Carcinoma in situ

T1

Invasion of subepithelial connective tissue

T2

T2a

T2b

Invasion of muscle

Tumour invades inner half of superficial muscle

Tumour invades outer half of deep muscle

T3

T3a

T3b

Invasion of preivesicle Tissue

Microscopically

Macroscopically (extravesical mass)

T4

Tumour invades other structures like prostate or uterus..

N-Regional Lymph Nodes

NX

Regional lymph nodes cannot be assessed

N0

No lymph node metastases

N1

Metastasis in a single lymph node (2cm in greatest dimension)

N2

Metastasis greater than 2cm in dimension but not more than 5cm

N3

Metastasis in a lymph node more than 5cm in greatest dimension

M-Distant Metastasis

MX

Distant metastasis cannot be assessed

M0

No distant metastasis

M1

Distant Metastasis

A non-invasive Bladder cancer involves the Ta and T1 staging and invasive Bladder Cancers refer to the T2, T3 and T4 stages. Invasive Bladder Cancers are those that have invaded the muscle leading to an increased risk of metastases since the tumour can invade the lymphatic plexus. Additionally there is an increased blood supply in the muscularis layer. This allows for haematogenous spread of the tumour once the blood vessel are invaded.

They can also be graded histopathologically which involves three stages (12):

G1-Well differentiated

G2-Moderately differentiated

G3-Poorly differentiated or undifferentiated

It is important to monitor the change from transitional cell carcinoma to either Carcinoma in Situ (CIS) or invasive bladder cancer. Carcinoma in Situ is a non-invasive form of aggressive superficial cancer staged at T1 and graded as G3. CIS can be identified by cytology without a histopathalogical analysis of the tumour because of its unique characteristics. This form of transitional cell carcinoma involve's appearance of anaplastic cells in the mucosa urothelium. With CIS, there are often malignant cells in the urine which can be useful in diagnosing cancer initially.

Bladder Cancer is associated with many risk factors. Genetic mutations, urinary carcinogens and environmental exposures are the most common causes of bladder cancer. Smoking is the most prevalent environmental risk factor but others including occupational exposure, arsenic in drinking water and radiation. There are over 60 carcinogens in tobacco smoke including aromatic amines causing an estimation of 65% of bladder cancer in men and 35% of bladder cancers in women.(13) Furthermore, smokers are four times more likely to develop bladder cancer than non-smokers. Molecular aetiology of bladder cancer is just as important. There is evidence of cigarette smoking causing an increase in mutagenic DNA in bladder tumours and deletions of chromosome 9 and 17 is seen in 60% of bladder cancers. (10) In addition, mutations in specific genes have also been implicated in bladder cancer. Tumour suppressor genes and oncogenes play a vital role in growth and development of the cancer. These risk factors are associated with transitional cell carcinoma occurring anywhere along the urinary tract. However the percentage of cancer development is higher in the bladder because the transitional epithelium is in continuous exposure to carcinogens.

I have decided to focus particularly on aetiology of Transitional cell cancer since it's the most common type of bladder cancer and affects many thousands of people worldwide. Additionally, upon initial diagnosis, 30% of the tumours are advanced invasive tumours patients with a T4 stage live between 1 year and more whereas patients with a T2 stage tumour can live up to five years or more. (10) Furthermore, in majority of patients a single causative factor can never be identified and therefore it's suspected that endogenous carcinogens are the reason for the development of the cancer. It is of particular interest to me to find examples of such carcinogens and understand how they cause the mutations in the DNA that leads to over proliferation and a change in cell types of the bladder mucosa. I believe that by understanding such aetiological factors will help in prevention of bladder cancer.

Discussion

There are variable factors contributing to transitional cell carcinoma of the bladder. In I will focus on the process of carcinogenesis and explain what actually causes the cells to proliferate uncontrollably. I have decided to focus on two main factors. These are:

Environmental factors

There are various carcinogens in the environment that increase the risk of bladder formation but I will be specially talking about Smoking, Occupational hazard and Radiation.

Genetic factors

There are several genes where mutation in any of them can lead to cancer generation.

Several changes occur in the urothelium and other layers of the bladder during carcinogenesis but to understand the differences we need to appreciate the normal physiology and anatomy of the bladder. The urothelium consists of three to seven transitional cell layers that rest on a basement membrane. These cells are actively proliferating cells where as the cells of the lumina are large, umbrella-like cells that form tight junctions with one another. The urothelium is covered by sulphated polysaccharides that function as a permeability barrier, preventing the entry of proteins, bacteria and ions. The lumina propia consists of loose connective tissue and occasional muscle fibres. Then the muscularis layer is composed of detrusor muscle with its fibres running in all directions. (1) The bladder is a highly adaptable organ that responds to insults by cell proliferation and tissue reorganisation. However, many factors cause uncontrolled proliferation of the transitional cells that ultimately lead to transitional cell carcinoma by the process of carcinogenesis.

Carcinogenesis

Carcinogenesis involves alteration in genes that function to control cell growth. Endogenous and exogenous carcinogens cause direct or indirect DNA damage. The process of carcinogenesis is thought to be a two part process involving initiation and promotion. Initiation is a fast irreversible stage of cell transformation. Promotion involves cell proliferation and is reversible.(14) Initiation requires direct contact with DNA, resulting in chromosomal mutation, gene amplification and duplications as well as deletions. However promotion involves continuous exposure to a single or multiple carcinogens. Alternations caused by carcinogens affect various cell properties from insensitivity to anti-growth signals, unlimited replication potential, maintained angiogenesis and ability of invasion and metastasis. (15)

Figure 3: a) Illustration showing the use of Microdissection to separate normal cells from tumour cells and how they can be amplified using polymerase chain reaction. b) The amplified DNA can be sequenced to show a point mutation in the tumour cell.(16)

The two major pathways involved are activation of oncogenes and inactivation of tumour suppressor genes. Chromosomal deletions are seen in many tumours and led to the discovery of Tumour Suppressor Genes (TSGs) of which p53 and RB are most investigated. (17) Loss of heterozygosity (LOH) analysis has been broadly used to identify the location of TSGs. (14) The heterozygosity of other body cells is not affected which allows analysis of the many tumour suppressor sites in bladder cancer. Translocations can also be associated with carcinogenesis, in which genes may be relocated on a different chromosome that will enhance the expression of oncogenes. Moreover a point mutation (figure 2) causing a single amino acid change in the oncogenes can lead to increased growth of the cancer. Additionally RNA messengers can be up or down-regulated which can lead to over production or under production of important growth regulating proteins or influence transcriptional factors. These changes in the DNA can be caused by environmental factors which become causative factors for transitional cell carcinoma of the bladder.

Environmental Factors

Several factors in the environment can increase the risk of transitional cell carcinoma. Some have been identified for several years while others are relatively new. The research is ongoing and many more could possibly be identified.

Table 1: Summary of environmental risk factors for the development of bladder cancer (13)

Environmental Factor

Comment

Smoking

50% of cases

Aromatic amines

Aniline dyes and other industrial chemicals

Ionizing radiation

Pelvic radiotherapy

Radio-contaminated areas

Arsenic

Contamination of water

Dietary nitrites/nitrates

Fertilizers and pesticides

Chloride

Chlorinated water

Weak correlation

Chlorinated hydrocarbons

Dry-cleaning solvents

Contamination of soil

Polycyclic aromatic hydrocarbons

Aluminium production, coal gasification, coke production, tar and tar-related products

Alkylating agents

Cyclophosphamide

Carcinogenic metabolite (acrolein)

Coal miners

Coal

Schistosoma haematobium

Schistosomiasis

Endemic in Africa, Asia and South America

Squamous-cell carcinoma

Occupational Exposure

The aetiological factors for bladder cancer were first investigated and highlighted by Ludwid Rehn, a German surgeon. On 20th April 1895 he gave a lecture titled "Bladder Cancers among fuchsine workers."(18) He was the first to recognise the link between chemical dye factory workers and bladder cancer development. These men were in constant contact with aniline and he proved the link scientifically.

He studied the factory workers and found similar symptoms in all the men: haematuresis, dysuria, exhaustion, dizziness and classified the cancer as "occupational cancer."(18). From his investigation Rehn summarised three main points(sourced from an article on Ledwig Rehn): 1) The gases produced in fuchsine production lead to urinary problems, 2) Constant contact with fuchsine could possibly lead to bladder tumours over time due to irritation. 3) These occur due to inhalation of aniline fumes.(18) He further highlighted how bladder tumours are the most common because once urine settles in the bladder for storage, the chemicals are stimulated leading to cancer formation.

However his research took several years to cause a change. It took decades before alinine cancer was recognised by the government and classified as an occupational cancer. This meant that workers were entitled to compensation from the government if their past history revealed occupational exposure. The first epidemiological study then occurred in 1954. The results of this study illustrated how exposures to certain occupation carcinogens like aromatic amines (used in rubber and dye industries), polycyclic aromatic hydrocarbons (used in aluminium and coal industries) can cause an increased risk of developing bladder cancer. Rehn's research promoted the research in bladder cancer which is still ongoing.

A study by Bernard C.K. Choi et al (1994) investigated whether urinary mutagens are a cause of environmental exposure.(19) A case control study (from 1983-1990) was conducted with 37 patients (19 bladder cancer cases and 18 controls) involving a questionnaire asking about their occupational history, exposure to toxic materials and smoking habits. Urine samples were collected at both their home and their workplace for analysis. The results highlighted that bladder cancer patients had more previous occupation experience in places known as high risk jobs like chemical dye industry, spray painting and metal machinery. In general 17/19 case patients had worked in a high risk job where as only 14/18 of the control reported to working in these occupations. The bladder cancer patients were linked with history of high-risk jobs, current exposure to hazardous materials at their job and had a positive Ames Test (a test to determine whether a chemical is a mutagen). However, only current exposure to hazardous materials was statistically significant. The Ames test had a positive predictive value of 72% and negative predicting value of 59%. The study highlights how occupational exposure was then investigated and considered to be a major risk factor for bladder cancer.

Thereafter many occupational carcinogens have been identified. These include benzidine, aniline dyes, chlorinated aliphatic hydrocarbons, paints, hair dyes and 4-aminobiphenyl. Occupations that most expose workers to these carcinogens are painters, truck drivers, dry cleaners, metal workers and rope makers. (13) These substances are known as carcinogenic due to their ability to cause nuclear damage. Genotoxic carcinogens that are DNA reactive interact and modify DNA and epigenetic carcinogens act directly on the cell. They cause immunological or hormonal effects which lead to abnormal cell proliferation and gene expression. (9)

Presently, occupational exposure is responsible for around 20% of bladder cancer cases. (19) Along with factory carcinogens that increase the risk of bladder cancer in the workers- the increased use of fertilizers and pesticides in the farming industry has lead to contamination of soil and water with nitrates. Since nitrates are a known carcinogen already, it leads to an increasing risk of bladder carcinoma in the general population. Additionally, the increased levels of arsenic and chloride in drinking water have been correlated with increased number of bladder cancer cases.

Radiation

Radiation is used to treat a variety of pelvic cancers in both men and women. However the use of radiation as a form of treatment has serious side effects including radiation-induced carcinomas. Pelvic tumours usually invade surrounding tissues and so it's not always possible to irradiate the pelvic tumour without harming normal tissue.(1) As a result the urinary bladder is often incidentally irradiated.

A study conducted by Kaldor et al (1995) investigated bladder cancer in women who were being treated for ovarian cancer.(20) 63 cases of bladder cancer were identified and 188 controls were selected for comparison. Each participant was asked about his or her treatment regime for ovarian cancer. The results illustrated an increased risk of bladder cancer in patients treated with radiotherapy alone or chemotherapy alone and in those who were treated with both chemotherapy and radiotherapy compared to those treated by surgery alone. Chemotherapy was split into 2 groups depending on whether or not they had cyclophosphamide.

The results demonstrated an approximately 4-fold increase of bladder cancer in those who did receive cyclophosphamide. The risk doubled if they also received radiotherapy as part of their treatment. The patients who received other chemotherapy drugs along with radiation had an increased risk of bladder cancer than those who only received other chemotherapy agents alone. Furthermore the risk continued to increase even after ten years of treatment. The study highlights how radiation and chemotherapy can increase the risk of bladder cancer and are also important environmental causes of bladder cancer. Ionizing radiation is thought to activate oxidative stress causing DNA damage, increase in DNA repair genes and epigenetic alterations such as DNA methylation. (21)

Radiation causes pathological changes including bladder contracture, mucosal ulceration and a decrease in bladder volume.(21) The symptoms can present within four years or more serious complications can arise even after ten years from when radiation was first given. The formation of bladder carcinoma is dependant upon the radiation dose and volume of bladder affected. Tolerance to radiation by the bladder is limited by high dose rates. Parez et al undertook an investigation to quantify the dose rate and ratio of doses that lead to damages of the bladder and rectum urothelium when treating patients with radiation for cervical cancer. (50) In relation to the bladder they found that doses above 80 Gy correlated to incidence of morbidity at 5%. The incidence of bladder problems was 6.9% when a high dose (above 80Gy) was used compared to 2.9% when low doses were used. There seems to be a pattern on grading of the cancer depending on the dose rate. Those that receive high doses generally tend to have a poorer prognosis since the grading of the tumor is higher in these patients.

Table 2: highlights how those given higher dose of radiation to treat prostate cancer usually had a diagnosis of Grade 3 tumour compared to those given lower doses of radiation. (51)

Radiation causes many changes to the bladder urothelium. After three months of irradiation, the urothelium of a mouse showed several changes. There was nuclear irregularity and cellular oedema. After six to twelve months, the proliferative activity of the urothelium enhanced and the cells became undifferentiated. (1) There is evidence of loss of tight junctions and the polysaccharide layer which reveals the isotonic cells of the bladder mucosa to the hypertonic urine. The tissues underneath are at a higher risk of damage when this layer is not present and correlate to clinical symptoms of urine urgency and increased frequency.(1) Even though radiation is clearly a causative factor for bladder urothelial carcinoma, it can also be used as a mean to follow up patients that had previously received radiotherapy as part of their treatment. The Bladder Antigen test (BTA) is particularly sensitive to radiation and can be used as a marker for detecting reoccurrence of transitional cell carcinoma in patients who had previously received radiotherapy. A study by Crane et al (1999) used a sample of 18 patients that were receiving external beam radiotherapy (ERBT). The results demonstrated that 10 out of the 18 patients had a positive BTA test. The study illustrates that radiation can cause positive results in the BTA test and therefore it should be used to follow up patients who had previously been given radiotherapy. (23)

Smoking

In addition to occupational exposure, smoking is considered as one of the highest risk factor for transitional cell carcinoma of the bladder. The cases of bladder cancer attributed to smoking include those who smoke and those who smoke passively. Smokers are twice more likely to develop transitional cell carcinoma. (24) Smoking was initially considered as a risk factor in the late 20th century. The 1964 Surgeon General's report analysed a series of case controlled and cohort studies. The results of the cohort study showed a mean relative risk of bladder cancer morality in smokers compared to non-smokers was 1.9.(52) These studies were the first in their series to show an association between bladder cancer and smoking. The results suggested the association between the two increased with frequency and depth of smoking.

In the following years the trend continued with smokers developing or dying from bladder cancer- two or three times more than non-smokers in both men and women.

Another factor was highlighted when results illustrated that the risk of developing bladder cancer was higher in persistent smokers than in those who had quit.(52) It was then concluded that cigarettes are a bladder carcinogenic factor.

A case controlled study in Northern Italy conducted by Barbara D'Avanzo et al (1990) investigated the strength of the association between smoking and bladder cancer.(25) The study included 337 cases of histologically diagnosed invasive bladder cancer and 392 controls from the same hospital with acute non-cancerous and non-urological conditions. Trained interviewers questioned the participants on sociodemographic factors, personal and lifestyle questions, alcohol consumption, medical history, drug abuse and occupation history. Smoking related questions included smoking status, average amount smoked per day, and three most used cigarette brands.

The results confirmed the association between bladder cancer development and smoking. The risk ratio was 1.8 for ex smokers and 3.3 for current smokers compared to non-smokers. The risk ratio rose with increase in quantity of cigarettes. The risk for light smokers(less than 10 cigarettes a day) was 2.6 compared with 3.6 for heavy smokers (more than 20 cigarettes a day). Moreover the same trend existed for duration of smoking period. The risk of bladder cancer initiation increased with the increase in duration of smoking. The risk ratio was consistent among men and women. The results of this study suggest a causal relationship between bladder cancer and smoking.

Cigarettes are known to contain more than 60 carcinogens which ultimately cause transitional cell carcinoma of the bladder.(24). These include 4-amino-biphenyl, acrolein, and oxygen free radicals. Arylamines are recognised as the primary carcinogen inducing bladder cancer. These exogenous substances induce DNA damage and alter genes involved in cellular growth. Many amines in the cigarette smoke react with nitrates in the diet to produce nitrosamines that in turn produce unstable electrophilic intermediates which are direct mutagens to DNA.(14) Tobacco carcinogens create smoke-related DNA adducts and cause base changes in specific genes.

With bladder carcinoma, mutations in the p53 gene have been directly linked with exposure to cigarette carcinogens. The p53 gene functions as a transcriptional factor and is a known tumour suppressor gene. An investigation by Charles H et al analysed distinct pattern of p53 mutation and its relationship to tobacco usage.(26) Eighty Bladder cancer samples were taken from patients diagnosed with grade 3 transitional cell carcinoma of the bladder. Smoking history was acquired through interviews of the patient and spouse and through medical records. The eighty samples were screened for p53 mutations by Single-strand conformation Polymorphism (SSCP) analysis which was than compared in smokers and non-smokers.

In non-smokers, mutations were found in 33% of the tumours along with a single base-pair change that altered the amino acid sequence in the protein. In current smokers 40%of the samples had p53 mutations and ten had a single base change that altered the amino acid sequence in the protein. Five of the samples had multiple base changes in the p53 gene, and one sample had a single base deletion that resulted in a frameshift. Restriction fragment analysis illustrated three of the current smoker's samples were reduced to homozygosity for chromosome 17q where the p53 gene exists. It was found that in bladder cancer there seemed to be an increased frequency of base changes involving a G:C to a C:G change.(26) The results of the study propose that carcinogens in cigarettes cause enhanced DNA damage in the urothelial cells of the bladder.

Passive Smoking

Those classified as non-smokers could still be at risk of bladder carcinoma due to exposure to carcinogens in the cigarette smoke. Therefore it's important to consider smoking as a risk factor for bladder carcinoma in among those who do not smoke. Many epidemiological studies have investigated the effects of smoking on non-smokers but the results have been contradictory to one another.

Bladder cancer in non-smokers was studied by Geoffrey C et al in 1986 involving 76 male and 76 females diagnosed with bladder cancer and 238 male and 354 females as control that reported to have never smoked before. Many risk factors were investigated involving occupation, smoking by smoke, exposure to smoke at home or at work and many others. However the results showed no association between bladder cancer and smoke exposure. This was the case for both spouse smoking and smoke exposure at work and home. (Bladder cancer in non smokers)

However other studies have showed significant DNA damage with exposure to tobacco smoke which increases the risk of bladder carcinoma. A study conducted by Collier AC et al in 2005 investigated 49 non smokers from non smoking household but worked in bars across Las Vegas.(27) The participants DNA was analysed and so were their cotinine levels that verified them as non-smokers and quantified their environmental tobacco smoke (ETS) exposure. The results illustrated a general increase in DNA damage with increasing cotinine-a dose dependant relationship. In addition a more significant increase in DNA damage was observed with increasing cotinine level in men than females. ETS is a genotoxic substance and a pro-oxidant stressor and hence a carcinogen.(diff in DNA damage) Therefore exposure induces damage which increases risk of uncontrolled cell growth and initiation of urothelial tumour.

Many other epidemiological studies have been conducted but they too have inconsistent results and analysis of several studies would not be reliable since exposure to smoke was measured differently in each of the study. (28)

Genetic Factors

The specific characteristics of bladder cancer suggest alterations in certain genes and their pathways which contribute to the cancer. In addition some genes initiate the tumour while others are involved in its progression. By understanding such pathways and changes, we can enhance our knowledge and predict possible molecular markers that will be essential for the diagnosis but also for reoccurrences of bladder carcinoma.

There are generally four levels of molecular pathways involved in the carcinogenesis of transitional cell carcinoma (Fig 4) (translating molecular bio):

Chromosome level alternations

Gene level alterations ( including mutation, amplification and deletions)

Expression level alterations

Protein level alterations-up or down regulations of proteins

Of these levels, I will focus on the chromosome and gene level alterations.

Figure 4:- Carcinogenesis of the epithelium can due to functional or structural alterations in the chromosome or the gene change the cell properties. (15)

Chromosomal Abnormalities

Ananlysis of the genome has identified abnormalities associated with chromosomes 1, 3, 5, 7, 9, 11, 17, X and Y. (29)

Chromosome 9

More than half of transitional cell carcinoma of variable grades and stages show deletions of chromosome 9. More than 67% of bladder carcinoma examined by LOH showed homozygous deletions on chromosome 9 and the common location of the deletion suggest that a gene important for initiation of carcinoma exists here.(30) Many methods have been used to examine the chromosome including LOH, cytogenetics, comparative genomic hybridization (CGH) and array CGH have all, showed deletions in both arms of the chromosome in urothelial carcinoma of the bladder. (41) Interestingly it has been noted that the surrounding tissue around the tumour also contains chromosome 9 abnormalities. It has been suggested that abnormality of this chromosome predisposes affected urothelium to more genetic alterations and therefore create a pathway for tumorigenesis. In addition it's also possible that the deleted regions contain the locations of tumour suppressor genes. (41) A known TSG cyclin-dependant kinase inhibitor 2A (CDKA2A) is located at 9p21 on chromosome 9.(genetic initiation) Deletion in this region inactivates CDKN2A and this encodes the INK4A and ARF53 Pathways. These pathways initiate cell cycle arrest through RB and p53 signalling pathways. (41) Loss of these pathways can make an individual be at a higher risk of carcinogen-induced carcinoma.

The second deletion of chromosome 9 that's been investigated in detail is at the location of 9q34 where tuberous sclerosis 1 (TSC1) resides. (31) This gene is linked to multi-organ hamartoma conditions. A recent study conducted in 2003 established that at least 12% of transitional cell carcinoma showed TCS1 mutations. (53) This indicates that deletions or mutations in the gene may play an important part in carcinogenesis.

Trisomy 7

Many studies have documented trisomy 7 (figure 5) in early stages of bladder cancer which is often even seen as the only chromosomal change.(32) In a study conducted by Matsuyama et al found 60% of the tumours expressed trisomy 7.(33) They suggested that genetic instability could be the cause of the increased copy number which is advantageous for the tumour. In addition they noticed trisomy 7 in the normal mucosa surrounding the tissue, suggesting the possible initiation role of trisomy 7. The epidermal growth factor receptor (EGF) gene is located on chromosome 7 and trisomy could lead to an increased number of EGF receptors on the tumour cells, which enhance its growth. (34)

Figure 5:- Karyotype illustrating trisomy 7 in a patient with transitional cell carcinoma.(34)

Chromosome 11

Many studies have also highlighted mutations on chromosome 11 which are particularly involved in the progression of the tumour. Around 40% of high-grade transitional cell carcinoma showed mutations on chromosome 11.(ref to 60 on genetic initiation and cytogenesis) Mostly the changes involve deletions of the short arm or other structural alterations in 11q. These changes correlate with tetroploidization and are seen in higher stages of tumour progression.(35)

Gene Level Alterations

Oncogenes

These genes are responsible for the normal differentiation and growth of cells. (5) Tumour growth is enhanced if these genes are mutated or over-expressed. Oncogenes dominate the malignancy phenotype of transitional cell carcinoma which could be done in two ways. There could be an increase in the gene product or an in increase of an altered protein product. In most cases there is an over expression of the normal product which can be achieved through chromosomal translocations or gene amplification. (31)

In transitional cell carcinoma, H-RAS from the RAS family represents one of the transformed oncogenes. Ras genes are involved in transducing intracellular messages via the RAS-MEK-ERK pathway. (54) The mutation of RAS into an oncogenic H-RAS is reported to be via mutation in the enzyme coded by RAS protein or via internal splicing.(36) Its been reported that 30% of urothilial malignancies show a substitution of glycine to valine at codon 12 of the H-RAS gene.(36) This leads to loss of GTPase function and therefore the protein in continuously active in the GTP-bound state and is constantly sending messages for cell growth and thus causing uncontrolled proliferation of the mucosa of the bladder. (54)

In addition 14% of bladder tumours showed amplification of ERBB2- a gene encoding a transmembrane receptor. This is particularly a case with high grade TCC. (35) The over-expression of the gene without gene amplification may also occur, suggesting more than one pathway for the alteration of the gene. Analysis of invasive TCC by CGH has shown increased amplification at 17q21-the location where ERBB2 resides.(37) The results of many studies show a correlation between over expression of ERBB2 and high tumour grade, suggesting a possible involvement of ERBB2 in tumour progression.

FGFR3, another oncogene, belongs to the fibroblast growth factor receptor family of which there are four types that are related to tyrosine kinase receptor genes. Mutations in FGFR3 are seen in more than 70% of superficial low grade tumours.(15) Its located on chromosome 4 at the locus 4p16.3. There is an extracellular component to the protein consisting of 3 domains and a single transmembrane division along with cytoplasmic tyrosine kinase domains. (15) The protein interacts with fibroblast growth factors in the extracellular region and the interaction promotes a signalling cascade that affects cell growth, migration and differentiation.

Activation of FGFR3 initiates several tyrosine pathways including the RAS pathway. This is known to induce mutagenesis through over proliferation of epithelial cells. Consequently, FGFR3 are RAS mutations found mutually exclusive to one another.(38)

FGFR3 is a very frequent mutated oncogene in many studies with mutations mostly found on exon 5, 7 and 10.(39) Around 50-80% of all mutation in FGFR3 is found in exon 7.(15) These mutations are corresponding with low recurrence rate in superficial TCC with 74% mutations found in Ta stage TCC.(40) Since FGFR3 activated a series of tyrosine kinase pathways, mutation in FGFR3 is a key event in low grade tumours.

Figure 5:-An illustration of the pathways activated during FGFR3 and RAS gene activation. (41)

Tumour Suppressor Genes

Tumour Suppressor Genes (TSG) have a negative regulatory function in cells. These act as supervisors of the cell cycle and terminate the cell cycle if a defected genome or mitotic spindle is detected. Loss of tumour suppressor genes, lead to mutagenesis with unregulated growth and differentiation of cell. (16) These genes have been known to play a role in transitional cell carcinoma and the two main pathways are named p53 and pRB pathways. (16) It was previously thought that both alleles of the gene must be inactivated or mutated in order to change the cell's phenotype but recent research has shown that inactivation of just one allele is sufficient to change the phenotype of a cell.

The p53 TSG Gene

The p53 gene is located on chromosome 17 at the locus of 17p13.1.(42) Cytogenic studies have consistently shown aberrations of chromosome 17. This gene can be inactivated through loss of both or one copy or by mutated copy acting in the hemizygous state. One of the properties of the mutated p53 is that it has a much longer half life than the normal p53 gene.(17) In addition, one of the downstream targets of p53 called p21 is downregulated in most of the transitional carcinoma that involve p53 mutations.(43) Normally, p21 binds and inhibits cyclin-dependant kinase 2(CDK2). This functions to drive the cell into the next stage of cell cycle. The inhibition of CDK2 by p21 allows time for DNA repair which is lost with a mutated p53 gene.(17)

Many studies have illustrated a link between p53 mutations and tumour grade and stage. A study by Olumi et al examined 43 transitional cell carcinomas of the bladder. The results showed that some chromosomes are grade-independent while others like chromosome 17 where mutated in the higher grades three TCC. (30) Its been observed that deletions of 17q are more common in invasive and CIS tumours rather than superficial tumours.(43) In superficial TCC, the status of p53 mutation has been noted to be a predictor marker for recurrence, progression and survival of the patients.(44)

There is contradictory evidence as to how p53 mutated tumours respond to chemotherapy and radiotherapy. Hinata et al (2003) investigated p53 status and radiation response in 5 tumours. They found that in cells with a normal p53 gene, the ionizing radiation induced p53-dependant cell apoptosis more readily than those with a mutated p53.(45) However other studies have shown that p53 mutations are more responsive to DNA-damaging reagents like chemotherapy drugs like cisplatin and have a longer survival with adjuvant chemotherapy than those with a normal p53. (51)

In additional to structural mutations, the p53 gene can be inactivated by over expression of an oncoprotein MDM2. (51) MDM2 downregulates p53 by forming an auto regulatory loop with the p53 pathway.(46) In 30% of invasive high grade TCC, mutations of MDM2 were demonstrated.(46) This illustrates another method of p53 mutation through pathways that is independent of mutations in the p53 gene.

Figure 7:-Figure illustrates the mutation in p53 and FGFR3 gene in relation to tumour grade. The results are based on a collection of studies. It can be clearly seen that FGFR3 mutations are associated with low grade tumours while p53 mutations is linked to high grade tumours.(15)

The RB Pathway

Retinnoblastoma (RB) gene has several roles including cell differentiation and development, cell cycle regulation and apoptosis. Many of these processes are involved in urothelium carcinogenesis. (51) Alteration in the RB gene is common in invasive TCC where often either over expression or hyper phosphorylated version of the gene is found. (47) This lead's to loss of inhibition of transcriptional factors and increase cell proliferation. It's also been noted that in tumour cell with Rb gene deficiency, there is an up regulation of MDM2. This compromise cell cycle progression and increases genetic instability in cells.(47)

The RB pathway is also associated with another TSG called INK4 A/ARF. This is found on chromosome 9 at the loci of 9p21 along with INK4B All these are negative regulators of cell cycle. In most cases homozygous deletions of both alleles was found indicating that this is the common way for mutations to occur in TCC.(31) Inactivation of these genes could cause genetic instability and expose the cell to carcinogenesis.

More than 50% of high grade invasive transitional cell carcinomas show alteration in both p53 and RB genes.(51) p53 is involved at the G1/S stage of cell cycle where as Rb is involved in G2/M stage of the cell cycle and therefore its indicated that both these stages need to be instable for progression of TCC into higher grades.

Conclusion

The importance of knowing the aetiological factors for transitional cell carcinoma of the bladder is vital. TCC is a huge burden on the health system with at least 12 million new cases occurring worldwide annually.(48) TCC is much more common in developed countries-with 5.4 million cases out of the 12000 occurring in developed countries.(48) The burden of TCC is likely to rise in developing countries due to increased risk of exposure to risk and because of the increase in ageing population.

However, much research has gone into the causes of transitional cell carcinoma in the bladder. Many factors increase the risk of TCC of the bladder. Some are environmental like smoking, radiation, occupational exposure and other variables like drugs and dietary components. Endogenous carcinogens like those in the urine can also increase the risk. In addition genetic instability leads to mutagenesis. These findings can be used practically to reduce the rate of transitional cell carcinoma.

Majority of the TCC can be prevented by smoking cessation and reducing risk of occupational exposure. Further action should be taken to protect people working in hazardous jobs or for those that are in contact with harmful compounds. In addition, government's should focus on primary prevention of smoking within the population. Moreover, the detection of TCC due to occupational hazard needs to be improved. This will increase the chances of finding a tumour in its early stage and therefore offer the patient a better prognosis. Many occupational cancers are currently missed due to a lack of appropriate patient assessment which can be due to a number of reasons. Occupational cancers do not differ anatomically, pathologically or clinically from non-occupational cancers.(49)

Many doctors do not ask for a detailed work history from their patients, which would give doctors details about risk factors for that patient. In addition those that do ask may only ask about current or previous jobs but occupational carcinogens have a long latency period so the exposure may have been very early in the working life of the patient. Also many strong occupational carcinogens are capable of inducing cancer in anyone that was in that environment, even if they weren't in direct contact with the carcinogen. Additionally many environmental carcinogens may be involved-for example occupational exposure like amines and smoking. Therefore the doctor may focus on the smoking alone and miss the occupational exposure to carcinogens. There is definitely a need for improvement in detecting occupation cancer. One of the simplest solutions to this is taking more detailed medical histories which give an insight into the patient's life from very early on so that risk factors can be highlighted for a particular patient.(49)

Moreover, genetic evaluation allows marking of genes most involved in the different types of TCC. Further analysis will allow prognostic factors to become available which will allow physicians to stratify patients into the right treatment groups much quicker. In addition, findings of prognostic markers will allow for new treatments that will target the cancer at a molecular level and therefore lead to a better prognosis.

The methods for diagnosing and following transitional cell cancer need to be improved. The current methods of cancer detection (urine cytology and cystoscopy) are not 100% efficient in the detection of transitional cell carcinomas.(5) Cytology is valuable for specificity but lacks sensitivity and often low-grade tumours are missed. Cystoscopy is currently the routine test used for following development of the cancer.

In the meantime, the urologist's judgement and oncologists review are necessary for determining the treatment options that will maximise the patient's survival as well as preserve their quality of life.

Word Count: 7004

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