Transcript Molecular-Biology-of-Tumours

Molecular Biology
of Tumours 2
Dr Orla Sheils
Introduction
• All features and properties of
individual cells, and ultimately
the organisms composed of
them are a function of the
genes expressed by those
cells.
Background
• Genes of human somatic cells
reside in 46 chromosomes.
– 22 pairs of autosomes and 2 sex
chromosomes.
• Genes consist of stretches of
DNA encoding the synthetic
function for amino acid chains
representing the polypeptide
product.
Introduction
• Carcinogenesis is a multi step
process that leads to
uncontrolled growth of cells
with unchecked potential for
proliferation.
• Cancer is caused by the
activation or amplification of
oncogenes, or by the
deactivation or loss of genes
that protect against cancer,
such as tumour suppressor
genes and DNA repair genes.
Molecular Pathology
Lymphomas
Introduction
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Diverse group of tumours.
Form a spectrum of disease
Clonal malignancies
Characteristic aspects are
related to molecular biology of
normal B-cells
Classification of lymphomas
• Historically lacked precision
and accuracy
• Potentially compromising
diagnosis and therapy
• Characterisation of key
oncogenic events increased
understanding
• Combination of molecular and
conventional diagnostic tools =
optimal
Classification of lymphomas
• 25 different lymphoma
classification schemes in past
80 years
• Initial schemes exclusively
morphological
– Small vs large cell
– Nodular vs diffuse
• These criteria remain
germaine to contemporary
classification –but as
components
Introduction
• Last two decades of 20th
century
– First real insights into
fundamental biological
mechanisms involved in
development of human cancer.
• Discoveries are based on
rapid developments in field of
molecular biology.
Antigen Receptor gene rearrangements
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Most general diagnostic marker in
lymphoma.
Relevant to all lymphocytic
cancers.
Antigen receptor genes encode
polypeptide units that make up
immunoglobulins and T cell
receptors.
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–
These are structurally and
functionally homologous
glycoproteins.
Mediate recognition of antigens and
account for the specificity of normal
immune response.
B-cells and Immunoglobulin
Gene Rearrangement.
 B-cells normally produce
antibodies
 heterodimeric proteins with
2 identical heavy chains,
and 2 identical light chains
Immunoglobulins
 Ig proteins must be capable of
reacting with an infinite
number of potential foreign
antigens.
 need to minimise the amount
of info. required at germline
level.
• recombination
• somatic mutation
Immunoglobulins
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The three immunoglobulin gene
loci IgH, Igk and Igl are located on
chromosomes 14q32, 2p12 and
22q11.
All three have the same basic
structure.
Gene segments - variable(V),
diversity(D),
joining(J)
Segments brought together by
genetic recombination.
Antigen Receptor Structure
• Gathered into three main sets:
–V
–J
–C
variable
joining
constant
• IgH gene and b and d T-cell
receptor genes also have D
(diversity) segment.
Antigen Receptor Structure
• Collectively V,D and J
segments contain the
repertoire of sequence
information for the synthesis of
all possible variable regions
within expressed antigen
receptor subunits.
Errors in rearrangement are
Lymphomagenic
 aberrant joinings can occur
between immunoglobulin gene
segments and some other
cellular gene, that confer a
survival on the cell.
 Burkitt’s Lymphoma - c-myc
 Follicular Lymphoma - bcl-2
Burkitt’s Lymphoma
• C-myc oncogene (8q24)
rearranges into one of the Ig
gene loci.
• Gene becomes aberrantly
expressed as a result of the
rearrangement
– Continuous proliferative stimulus
to the cell.
Follicular Lymphoma
• Most common indolent form of
lymphoma in Western
Hemisphere
Follicular Lymphoma
• bcl-2 gene 18q21 is aberrantly
translocated into Ig gene locus,
leading to inappropriate expression.
• bcl-2 provides a growth advantage
by extending the lifespan of the cell
through its anti-apoptotic function.
• Geographical variation in frequency
of t14:18 (lower in Asian than North
America)
– ?environmental effect rather than
genetic
Follicular Lymphoma and bcl-2
 about 50% non-Hodgkins lymphoma
 generally low grade
 90% have reciprocal translocation
between chromosomes 14q32 and
18q21.
 Juxtaposition of bcl-2 oncogene to an
Ig joining gene.
 over-expression of bcl-2 gene
product due to juxtaposed Ig and
enhancer sequences.
 inhibits apoptosis
Follicular Lymphoma and bcl-2
• Chromosome breakpoints
are clustered
– Facilitates PCR based
detection using genomic
junctional sequences.
• bcl-2 protein localises to
membrane systems of
nuclei, mitochondria and ER
– Provides a survival advantage
by inhibiting programmed cell
death.
Mantle Cell Lymphoma and bcl-1
 Lymphocytic lymphoma of
intermediate differentiation.
 Characterised by
t(11;14)(q13;q32)
 Bcl-1 encodes cyclin-D
 cell cycle regulator involved in
progression through G1 phase
of cell cycle.
Mantle Cell Lymphoma and bcl-1
• Can operate with other oncogenes
to transform cells in-vitro.
• Several minor breakpoints have
been identified on 11q13.
• Using a panel of probes it is
possible to detect bcl-1
rearrangements in >70% of MCL.
• Other genetic markers:
– Deletions of INK4a/ARF locus (p16) =>
p53 and Rb
– Disablement of p27
Diffuse Large Cell Lymphoma bcl-2 and bcl-6
 Heterogeneous group of
lymphomas.
 Generally have an aggressive
clinical course.
 Most frequent abnormalities:
 t(14;18)(q32;21)
 t(3q27)
 ~1/4 have t14:18 and bcl-2
rearrangement.
Diffuse Large Cell Lymphoma bcl-2 and bcl-6
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Molecular cloning of breakpoints have
identified the gene bcl-6 - translocated
into IgH gene (22q11).
Translocations may also involve other
non-immunoglobulin genes on other
chromosomes.
Normal function of bcl-6 – unknown.
Translocation results in deregulation and
over-expression of bcl-6
~5-10% have c-myc involvement +/- EBV
As these tumours share a common
molecular pathogenesis with BL ? variant
of BL
Small non-cleaved cell lymphoma,
Burkitt’s Type and c-myc.
 BL is highly aggressive
 association with EBV
 Association with unique
chromosomal translocation
involving c-myct(8;14)(q24;q32) and Ig gene locus.
 ~85% have t(8;14)(q24;q32)
 Remainder have t(2;8)(p12;q24) or
t(8;22)(q24;q11) involving Igk or Igl
gene loci.
 Tumour Suppressor genes – p53
T cell receptors
• Expressed by T lymphocytes
• Membrane bound
heterodimers
 ab or gd chains
• Variable region – differs widely
between receptors produced
by different lymphocytes.
T-Cell Lymphoma
 Majority involve 14q11
 analogy between Ig genes and
B-cell malignancy and TCR
and T-cell Lymphoma
 Trisomy 3,5,7,19
T-Cell Lymphoma
 TCR genes organised similarly
and rearranged during
maturation
 t(10;14)(q24;q11)
 c-myc - TCR ad elements
c-myc Fusion Transcript
 t(2;8)(q34;q24)
 increased stability of c-myc
Chromosome translocations
• Common in lymphoma
• Oncogenes that are modified
in structure or expression due
to the translocation process
are implicated in malignant
behaviour of cells harbouring
them.
• Good markers for malignancy
• Constant through course of
disease.
Viral genomes
• EBV
– (Epstein-Barr Virus)
• HTLV-1
– (human t-cell leukaemia/lymphoma virus –1)
• HHV8
– (human Herpesvirus type 8)
EBV
• Maintained in nuclei of latently
infected cells as extracellular
DNA circles / episomes
• Episomes express:
– 5 genes for nuclear antigens
(EBNAs).
– Latent membrane protein
(LMP1) – promotes cellular
proliferation.
HTLV-1
• 8.6 kb
• 5 genes
• Oncogenic mechanism not well
understood
• After infection RNA genome is
reverse transcribed to ds DNA
/provirus
– Inserts at random points into host
genome.
– Expresses viral proteins for life of cell.
HTLV-1
• Tax protein interacts with
cellular transcription factors
e.g. IL2 and IL2r
– Stimulates proliferation in
infected cells.
HHV8
• Discovered by analysis of KS
biopsies.
• Body cavity based lymphoma
• Castleman’s Disease
• ?? Multiple Myeloma
Molecular
Pathology Thyroid
Neoplasia
Papillary Thyroid Cancer
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
– Ireland<100 cases/yr, U.S.~20,000
cases/yr
– Incidence on the rise - global estimate
0.5 million new cases this year
– Fastest growing cancer in women
worldwide
Introduction
• In relation to thyroid
carcinoma, particularly PTC,
ionizing radiation is the most
notorious initiator of
carcinogenesis.
• In general, radiation is more
likely to induce DNA strand
breaks rather than point
mutations.
Introduction
• When DNA strand breaks occur,
especially double stranded breaks, the
ability to fully repair the sequence is
limited and the consequences are
chromosomal rearrangements such as
inversions, translocations, gains and
deletions.
• If these consequences are non-lethal
for the cell and cannot be otherwise
reversed, they may lead to malignant
transformation via altered gene
expression, formation of chimeric
genes, or loss of tumour suppressor
gene function.
Pathological Pathways
Papillary Thyroid Carcinoma
Follicular Epithelial Cell
Follicular Carcinoma
Papillary Thyroid Cancer
Normal thyroid
PTC Variants:
•Follicular Variant PTC
•Tall Cell Variant PTC
•Solid PTC
•Solid Trabecular PTC
PTC
Existing markers of 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.
ret/PTC-1 expression
• ret/PTC-1
– Inflammatory response
• Associated with altered expression of CAM
• ? Response to oxidative stress
• Classical variant PTC
ret/PTC-1 expression and
associated thryoiditis.
20
18
16
14
12
10
8
6
4
2
0
Hashimoto
thyroiditis ret
pos
Hashimoto
thyroiditis ret
neg
Lymphocytic
thyroiditis ret
pos
Lymphocytic
thyroiditis ret
neg
Graves disease
ret pos
Graves disease
ret neg
•Risk factors for autoimmune diseases are genetically linked to the
presence of specific Class I or Class II HLA alleles.
•Viruses implicated in triggering HT –Coxsackie virus
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
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
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
BRAF mutation detection in ffpe archival PTC
9
8
7
6
5
4
3
2
1
0
ret/PTC
-0
2
-9
9
20
00
-9
6
19
97
-9
3
19
94
-9
0
Year
19
91
-8
7
19
88
19
85
19
82
-8
4
T1799A mutation
Hierarchical clustering – thyroid resections
BRAF mut vs BRAF wt PTC
•Hierarchical clustering of 1139
genes after t-test (p<0.05).
•The column dendrogram clearly
shows BRAF mut cases clustering to
the right with BRAF wt cases in a
distinct left-sided cluster.
•Red denotes genes with relative
increased expression and green
denotes genes with relative
decreased expression.
TSHr expression
TSHr Expression
TSHr index
• The synthetic
function and growth
of follicular cells in
the normal thyroid
are predominantly
regulated by TSH
through the medium
of the TSHr
30
25
20
15
10
5
0
1=FTC/well diff
2=FTC/poorly diff
3=PTC/well diff
4=PTC/poorly diff
5=FA/norm
6=ATC
7=MTC
0
1
2
3
4
Tumour Type
5
6
7
Cell Adhesion Molecules
(CAM)
• Cadherins are considered
one of the most important
Cell Adhesion Molecules,
and are key to the
formation of intercellular
cell junctions.
• Variable down-regulation
of E-cadherin among PTC
occurs with a gradual
reduction from normal to
well-differentiated
carcinomas to its absence
in anaplastic lesions.
Galectin-3
• The gene lgalS3
codes for galectin-3
(GAL3), a bgalactosil-binding
lectin involved in
regulating cell-cell
and cell-matrix
interactions.
• GAL3 expression to
be of value in
discriminating
between benign and
malignant thyroid
nodules
0.4
1.6
0.35
1.4
0.3
1.2
0.25
1
LGALS3 TQ
0.2
0.8
0.15
0.6
0.1
0.4
0.05
0.2
0
0
8505C
B-CPAP
HTH74
Cell Line
TPC-1
LGALS3 ARR
Expression profile from normal
through well to un-differentiated PTC
Marker
Thyroglobulin
E-Cadherin
Ki-67
p53
Cyclin D-1
p27
Normal
Well
Differentiated
Poorly
Differentiated
Undifferentiated