Genes and the Environment

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Genes and the Environment

• Viruses and Cancer.
• Environmental Exposure and Cancer.

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Carcinogenesis
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What Causes Cancer?

• Carcinogens 
• Age 
• genetic make up 
• immune system 
• diet 
• day-to-day environment 
• Viruses

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Age
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Diet
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Factors in Carcinogenesis
Chromosomes/DNA
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In addition to chemicals and radiation, a few
viruses also can trigger the development of
cancer. In general, viruses are small infectious
agents that cannot reproduce on their own, but
instead enter into living cells and cause the
infected cell to produce more copies of the
virus. Like cells, viruses store their genetic
instructions in large molecules called nucleic
acids. In the case of cancer viruses, some of the
viral genetic information carried in these
nucleic acids is inserted into the chromosomes of
the infected cell, and this causes the cell to
become malignant.
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Viruses

• Viruses contribute to development of some
  cancers.  Typically, the virus can cause genetic
  changes in cells that make them more likely to
  become transformed. These cancers and viruses
  are linked
• Cervical cancer and the genital wart virus, HPV
• Primary liver cancer and the Hepatitis B virus 
• T cell leukaemia in adults and the Human T cell
  leukaemia virus

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HTLV-1

• naturally infects CD4 T lymphocytes and can be
  transmitted between close contacts through blood
  transfer or from mother to infant through cells
  in breast milk.
• In most cases the infection is harmless. However,
  as many as 1 in 20 infected individuals
  eventually develop a type of adult T cell
  leukaemia in which every tumour cell carries a
  clonally integrated HTLV1 provirus.
• HTLV1 differs from the standard 'chronically
  oncogenic' and 'acutely oncogenic' retroviruses
  in its mechanism of action
• it appears to drive cell growth through
  expression of a particular viral protein, Tax, in
  latently-infected cells

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HTLV retrovirus and adult T-cell leukaemia
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Mode of action

• Tax can transactivate expression of a number of
  key cellular genes that enhance cell growth.
• The best examples are the genes encoding
• interleukin 2 (a T cell growth factor) and
• the interleukin 2 receptor (a molecule that
  allows cells to respond to the growth factor).
• As a consequence, the infected cells not only
  make their own growth signals, but also respond
  to them

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Mode of action

• HTLV1 induces a weak growth transformation of T
  cells in the laboratory but, in the body, is
  probably never sufficiently strong to induce T
  cell leukaemia on its own.
• BUT, a virally infected cell in which growth
  controls have even partly broken down, is more
  susceptible to further genetic accidents.
• During persistent infection a gradual build-up of
  HTLV1-positive T cells which have accumulated
  additional genetic changes may occur.
• Eventually this can lead to selection and
  outgrowth of a fully malignant, HTLV1-positive
  clone
• At this stage malignant cell growth can occur in
  the absence of tax gene expression.

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Epstein Barr Virus

• Epstein-Barr virus (EBV), also called Human
  herpesvirus 4 (HHV-4), is a virus of the herpes
  family (which includes Herpes simplex virus and
  Cytomegalovirus),
• one of the most common viruses in humans.
• Most people become infected with EBV,
• often asymptomatic
• but commonly causes infectious mononucleosis.
• It is named after Michael Epstein and Yvonne
  Barr, who together with Bert Achong discovered
  the virus in 1964.

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• On infecting the B-lymphocyte, the linear virus
  genome circularises and the virus subsequently
  persists within the cell as an episome.
• The virus can execute several distinct programmes
  of virally-encoded gene expression
• broadly categorised as being lytic cycle or
  latent cycle.
• The lytic cycle or productive infection results
  in staged expression of a host of viral proteins
  with the ultimate objective of producing
  infectious virions. Formally, this phase of
  infection does not inevitably lead to lysis of
  the host cell as EBV virions are produced by
  budding from the infected cell.
• The latent cycle programmes are those that do not
  result in production of virions.

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EBV-associated malignancies

• The strongest evidence linking EBV and cancer
  formation is found in Burkitt's lymphoma and
  Nasopharyngeal carcinoma

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Burkitts Lymphoma

• a type of Non-Hodgkin's lymphoma
• most common in equatorial Africa
• co-existent with the presence of malaria.
• Malaria infection causes reduced immune
  surveillance of EBV immortalised B cells, so
  allowing their proliferation. This proliferation
  increases the chance of a mutation to occur.
  Repeated mutations can lead to the B cells
  escaping the body's cell-cycle control, allowing
  the cells to proliferate unchecked, resulting in
  the formation of Burkitt's lymphoma. Burkitt's
  lymphoma commonly affects the jaw bone, forming a
  huge tumour mass. It responds quickly to
  chemotherapy treatment, namely cyclophosphamide,
  but recurrence is common.
• Other B cell lymphomas arise in immunocompromised
  patients such as those with AIDS or who have
  undergone organ transplantation with associated
  immunosuppression.
• Smooth muscle tumours are also associated with
  the virus.

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Nasopharyngeal carcinoma

• found in the upper respiratory tract, most
  commonly in the nasopharynx, and is linked to the
  EBV virus.
• It is found predominantly in Southern China and
  Africa, due to both genetic and environmental
  factors. It is much more common in people of
  Chinese ancestry (genetic), but is also linked to
  the Chinese diet of a high amount of smoked fish,
  which contain nitrosamines, well known
  carcinogens (environmental).

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Kaposi's sarcoma

• form of skin cancer that can involve internal
  organs. It most often is found in patients with
  acquired immunodeficiency syndrome (AIDS), and
  can be fatal.

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K.S.

• Kaposi's sarcoma (KS) was once a very rare form
  of cancer, primarily affecting elderly men of
  Mediterranean and eastern European background
  (tumours on lower legs), until the 1980s, when it
  began to appear among AIDS patients.
• AIDS-related KS, emerged as one of the first
  illnesses observed among those with AIDS. Unlike
  classic KS, AIDS-related KS tumours generally
  appear on the upper body, including the head,
  neck, and back. The tumours also can appear on
  the soft palate and gum areas of the mouth, and
  in more advanced cases, they can be found in the
  stomach and intestines, the lymph nodes, and the
  lungs.

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Kaposi Sarcoma and HHV8

• Studies in 2000 showed that HHV-8 was the culprit
  behind KS.
• It does not work alone.
• In combination with a patient's altered response
  to cytokines (regulatory proteins produced by the
  immune system) and the HIV-1 transactivating
  protein Tat which promotes the growth of
  endothelial cells, HHV-8 can then encode
  interleukin 6 viral proteins, specific cytokines
  that stimulate cell growth in the skin.
• This becomes KS.

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HHV-8

• HHV-8 destroys the immune system further by
  directing a cell to remove the major
  histocompatibility complex (MHC-1) proteins that
  protect it from invasion.
• These proteins are then transferred to the
  interior of the cell and are destroyed.
• This leaves the cell unguarded and vulnerable to
  invaders which would normally be targeted for
  attack by the immune system.

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The natural history of HPV infection and cervical
cancer
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Cervical Screening
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Genomic Map of HPV
(von Knebel Doberitz/European Journal of Cancer
2002 38 2229-2242).
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The interacting worlds of HPV and p16
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The Hepatitis B Virus (HBV)

• HBV is a mostly double-stranded DNA virus in the
  Hepadnaviridae family.
• HBV causes hepatitis in human.
• The HBV genome has four genes pol, env, pre-core
  and X that respectively encode the viral
  DNA-polymerase, envelope protein, pre-core
  protein (which is processed to viral capsid) and
  protein X.
• The function of protein X is not clear but it may
  be involved in the activation of host cell genes
  and the development of cancer.

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Consequences of HBV Infection

• HBV causes acute and chronic hepatitis.
• The chances of becoming chronically infected
  depends upon age.
• About 90 of infected neonates and 50 of
  infected young children will become chronically
  infected.
• In contrast, only about 5 to 10 of
  immunocompetent adults infected with HBV develop
  chronic hepatitis B.

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Hepatocellular carcinoma

• cancer that arises from hepatocytes, the major
  cell type of the liver.
• Hepatocellular carcinoma is one of the major
  cancer killers.
• It affects patients with chronic liver disease
  who have established cirrhosis, and currently is
  the most frequent cause of death in these
  patients.
• The main risk factors for its development are
  hepatitis B and C virus infection, alcoholism and
  aflatoxin intake.

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• integrated HBV can generate chromosomal
  instability
• it has been suggested that viral DNA sequences
  encompassing the encapsidation signal may exhibit
  intrinsic recombinogenic activity via binding to
  a putative recombinogenic cellular protein

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Genes and the Environment Thyroid Disease
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• The thyroid gland is a small gland located at the
  front and sides of the neck.
• It is made up of right and left lobes connected
  by a bridge called the isthmus and weighs between
  20 and 30 grams.
• It is slightly heavier in females and becomes
  enlarged during menstruation and pregnancy.

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• The thyroid gland produces three hormones called
  thyroxine (T4), tri-iodothronine (T3) and
  calcitonin. These hormones help to regulate the
  body's metabolism.

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• Iodine is essential for the production of the
  thyroid hormones.
• Good sources of iodine include fish such as cod,
  sea bass and haddock.
• Dairy products and plants grown in soil that is
  rich in iodine are also good sources.

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• The thyroid participates in these processes by
  producing thyroid hormones, principally thyroxine
  (T4) and triiodothyronine (T3).
• These hormones regulate the rate of metabolism
  and affect the growth and rate of function of
  many other systems in the body.
• Iodine is an essential component of both T3 and
  T4.
• The thyroid also produces the hormone calcitonin,
  which plays a role in calcium homeostasis.

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Physiology

• The primary function of the thyroid is production
  of the hormones thyroxine (T4), triiodothyronine
  (T3), and calcitonin.
• Up to 80 of the T4 is converted to T3 by
  peripheral organs such as the liver, kidney and
  spleen.
• T3 is about ten times more active than T4.

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• The function of the thyroid gland is to take
  iodine, found in many foods, and convert it into
  thyroid hormones thyroxine (T4) and
  triiodothyronine (T3).
• Thyroid cells are the only cells in the body
  which can absorb iodine.
• These cells combine iodine and the amino acid
  tyrosine to make T3 and T4.

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• T3 and T4 are then released into the blood stream
  and are transported throughout the body where
  they control metabolism (conversion of oxygen and
  calories to energy).
• Every cell in the body depends upon thyroid
  hormones for regulation of their metabolism.
• The normal thyroid gland produces about 80 T4
  and about 20 T3, however, T3 possesses about
  four times the hormone "strength" as T4.

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• The thyroid gland is under the control of the
  pituitary gland, a small gland at the base of the
  brain.
• When the level of thyroid hormones (T3 T4)
  drops too low, the pituitary gland produces
  Thyroid Stimulating Hormone (TSH) which
  stimulates the thyroid gland to produce more
  hormones.
• Under the influence of TSH, the thyroid will
  manufacture and secrete T3 and T4 thereby raising
  their blood levels.

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• The thyroid gland is invested by a thin capsule
  of connective tissue, which projects into its
  substance and imperfectly divides it into masses
  of irregular form and size.
• Cut surface reveals a number of closed vesicles,
  containing a yellow fluid, and separated from
  each other by intermediate connective tissue

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Significance of iodine

• In areas of the world where iodine (essential for
  the production of thyroxine, which contains four
  iodine atoms) is lacking in the diet, the thyroid
  gland can be considerably enlarged, resulting in
  the swollen necks of endemic goitre.

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Significance of iodine

• Because of the thyroid's selective uptake and
  concentration of what is a fairly rare element,
  it is sensitive to the effects of various
  radioactive isotopes of iodine produced by
  nuclear fission.
• In the event of large accidental releases of such
  material into the environment, the uptake of
  radioactive iodine isotopes by the thyroid can,
  in theory, be blocked by saturating the uptake
  mechanism with a large surplus of non-radioactive
  iodine, taken in the form of potassium iodide
  tablets.

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Significance of iodine

• While biological researchers making compounds
  labelled with iodine isotopes do this, in the
  wider world such preventive measures are usually
  not stockpiled before an accident, nor are they
  distributed adequately afterward.
• One consequence of the Chernobyl disaster was an
  increase in thyroid cancers in children in the
  years following the accident.

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Chernobyl

• On April 26, 1986, the fourth reactor of the
  Chernobyl Nuclear Power Plant, exploded at 0123
  AM local time. All permanent residents of
  Chernobyl and Zone of alienation were evacuated
  because radiation levels in the area had become
  unsafe.

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Chernobyl
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Chernobyl
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Chernobyl
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Identifying molecular mechanisms that reveal
underlying pathobiology in Thyroid Neoplasia
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Background

• Thyroid Cancer
• most frequently occurring endocrine malignancy,
• sub-divided into a number of diagnostic
  /morphological categories.
• Papillary thyroid carcinoma (PTC)
• Most common thyroid malignancy
• Irelandlt100 cases/yr, U.S.24,000 cases/yr
• Incidence on the rise - global estimate 0.5
  million new cases this year
• Fastest growing cancer in women worldwide

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Pathological Pathways
ret/BRAF
Follicular Epithelial Cell
RAS
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Molecular markers in PTC

• ret/PTC
• To date 15 chimeric mRNAs involving 10 different
  genes have been described
• Ret/PTC-1 and ret/PTC-3 are the most common
  types, accounting for 90.
• Morphological variants are likely to reflect
  variations in tumour biology which have yet to be
  fully defined.

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ret/PTC-1 expression and associated thryoiditis.
Number of cases
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E-cadherin expression and radiation dose
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ret/PTC-3 activation

• commonly seen in children exposed to ionizing
  radiation.
• ? Radiation signature
• Literature demonstrates correlation with
  solid/follicular variant morphology, poorer
  prognosis and aggressive tumor behavior.
• Correlation between tumor morphology and specific
  ret rearrangements

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Ret/PTC-3 activation

• commonly seen in children exposed to ionising
  radiation.
• Literature demonstrates correlation with
  solid/follicular variant morphology, poorer
  prognosis and aggressive tumour behavior.
• ? prevalence of the common ret chimeric
  transcripts (ret/PTC-1 and ret/PTC-3) in a group
  of sporadic PTC.
• Correlation between tumour morphology and
  specific ret rearrangements

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Ret/PTC-3 analysis in sporadic cases

• Ret/PTC-1 and ret/PTC-3 transcripts were detected
  in 43 and 18 of PTCs respectively.
• Ret/PTC-3 correlated strongly with the
  solid/follicular variant 55 and was not
  detected in conventional PTC.
• Ret/PTC chimeric transcripts in an Irish Cohort
  of Sporadic Papillary Thyroid Carcinoma.
• SP Finn, P Smyth, O Leary JJ, Sweeney EC, Sheils
  O.
• J Clin Endocrinol Metab 2003 Feb
  88(2)938-41.PMID 12574236 PubMed - in process
  
• Real time analysis of beta and gamma catenin mRNA
  expression in ret/PTC-1 activated and
  non-activated thyroid tissues. Smyth P, Finn S,
  OLeary J, Sheils O.
• Diagn Mol Pathol 2003 Mar12(1)44-9 PMID
  12605035 PubMed - in process

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Conclusion of ret/PTC 3 analysis in sporadic cases

• Significant prevalence of ret/PTC transcripts in
  this sporadic group of PTC.
• Higher incidence than previously reported in
  areas not affected by ionising radiation
• ? More sensitive technique
• ? Effect of exposure to ionising radiation..

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Ret activation and morphology

• In the setting of radiation induced PTC it is
  apparent that specific ret/PTC rearrangements are
  associated with specific tumour morphology
• ret/PTC-1 associated with classic morphology
• ?low dose/long latency
• ret/PTC-3 associated with solid/follicular
  morphology and adverse prognosis.
• ?higher dose/short latency

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BRAF

• Raf kinases
• Serine/threonine kinases
• Function in Ras/Raf/MEK/ERK pathway
• 3 isoforms A-Braf, B-Raf, C-Raf/Raf-1
• BRAF implicated in many cancers

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Temporal trends?
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Microarray analysis
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Effect of BRAF mutation on transcriptome

• Normal thyroid cell line (left)
• PTC Cell Lines with BRAF mutation (right)

• BRAF (mut) transfection experiment
• Left cluster wt braf-
• normal cell line
• Right Cluster (mut) braf-
• Normal cell line transfected with braf (mut).

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Effect of ret/PTC-1 activation on transcriptome

• ret/PTC-1 transfection experiment
• Left cluster wt ret
• normal cell line
• Right Cluster ret/PTC-1-
• Normal cell line transfected with ret/PTC-1

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Genes differentially expressed (upregulated) in
PTC vs benign
Name Panther Function
Galectin 3 Signalling molecule, cell adhesion molecule
Calcium Calmodulin-dependant protein kinase 1 Non-receptor serine/threonine protein kinase
TGFB1 Angiogenesis
Syndecan 3 Cell adhesion, proliferation, differentiation
TGFb1 Receptor protein serine/threonine kinase signalling. Cell cycle control
Calgizzarin (S100A11) Tumour suppressor, calmodulin related protein
CD44 Cell adhesion, inhibition of apoptosis
TIMP1 Metalloprotease and its inhibitor
MMP14 Metalloprotease and its inhibitor
Data derived from array analysis of surgically
resected lesions
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Genes differentially expressed (downregulated) in
PTC vs benign
Name Panther Function
TIMP4 Metalloprotease Inhibitor
RAB23 Small GTPase Member RAS oncogene family
FGFR2 Tyrosine Protein Kinase receptor, MAPKKK cascade, JAK-STAT cascade
Metallothionein Homeostasis activities
Syndecan 2 Cytoskeletal protein, defence and immunity protein, extracellular matrix glycoprotein
MAPK4
TSHr G-protein receptor
Lectin, mannose-binding1 (LMAN1) Immunity and Defence
Data derived from array analysis of surgically
resected lesions
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Archival ffpe Tissues
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Archival FFPE Tissues