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Lecture 6 – Brain Tumours and Cancer Stem Cells
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What are brain tumours?
o Malignant transformation of cells of the CNS (rarely metastases from other organs)
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o What are the cells of the CNS?
 Gliomas – astrocytomas, oligodendrocytomas, and neuronal tumours (e
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medulloblastoma)
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What’s the prognosis?
o Vary variable – some very treatable, some almost 100% fatal
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 Among the deadliest of cancers
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 Mean survival – 9-12 months
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o Chemo – most don’t cross blood brain barrier
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o What do we need:
 Drug delivery;
effective chemo;
surgery not an option
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 Most in 5-10 year olds
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Medulloblastoma – problems:
o Like other brain tumours of children, treatments very
damaging so need biological therapies
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Medulloblastoma – a case study:
o Aetiology identified from rare inherited disorders
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o Animal models used to dissect aetiology
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o Drug targets defined and developed
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o Gorlin’s Syndrome – non-melanoma skin cancer (BCC) and brain tumours
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o Autosomal dominant, 1/500,000
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o 14 families, 10 had APC mutations
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 The other 4 families developed GBM (had MMR mutations)
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 Result – nuclear β-catenin
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 Nuclear β-catenin reflects level of activating mutations at
around 18%
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 Binding of WNT activates its receptor, which then blocks the
phosphorylation-activated (by glycogen synthase kinase-3-β (GSK3β) in a
complex with APC and axin) degradation of β-catenin (β-Cat)
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Gorlin’s Syndrome – Shh signalling:
o Gorlin’s Syndrome (nevoid basal cell carcinoma syndrome)
skin cancer
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o Most get basal cell carcinoma (BCC) early adulthood
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 Medulloblastoma in about 3% in early childhood
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PTCH and the Shh pathway – importance in MB’s:
o Shh is a key factor in the proliferation of granule
neuron precursors of the cerebellum during
normal development
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o Smo causes release of Gli to nucleus to act as
transcriptional activator
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o SUFU mutated in 12%, all exhibit LOH in
tumours
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o Mutations in PTCH or Smo or any downstream
component could activate the pathway,
increased Gli function resulting in increased gene expression and proliferation
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 SMO represses the cleavage of GLI (the cleaved form has a transcriptionalrepressor role in the nucleus), resulting in its release from a complex
including fused (FU) and costal (COS)
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 So, there is a great deal of similarity between these two pathways
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 SUFU can also directly repress the transcriptional activity of intact GLI
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Mitogens feed into R point transition:

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Germ line models:
o PTCH +/- heterozygote mice – only 3-7% develop tumours
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g
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 PTCH1+/- p53-/- mice all spontaneously develop tumours as early as two
weeks
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PTCH – tumour suppressor:
o But… p53 mutations rare in these tumours in humans
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o Humans both PTCH alleles affected in about 50% of PTCH mutation cases
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o Therefore differs from classical tumour suppressor model, but also differs from
simple haploinsufficiency since a second event is needed
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o Subtypes of medulloblastoma have distinct developmental origins:
 SHH subtype:
 Arise in very young children
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 Poor prognosis
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 WNT subtype:
 Arise in older children
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 Highly curable
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Subtypes are anatomically distinct at diagnosis:

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Subtypes are anatomically distinct in mouse models:
o WNT mouse model:
 Blbp-cre/floxed β-catenin (Ctnnb1) mice (VZ
cells)
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 Target mutated β-catenin to brainstem, disrupted
migration and increased proliferation in dorsal
brain tumours
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 15% of mice develop classic
medulloblastomas after 290
days
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o Can identify WNT and SHH subgroup patients and treat
accordingly
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o Note lack of relationship between MB and normal
cerebellum
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o Gli pathway strongly implicated in
desmoplastic type
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o By matching gene expression profile
of human tumours to animal tumours with specific molecular
trigger, can classify by cause
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 What’s gone wrong – global analyses
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o Medulloblastoma – many tumours have activated shh pathway, so block shh
receptor
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 Decrease in tumour cell proliferation
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 Treatment prolonged tumour free survival
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So far smo inhibitors are used regardless of profiling to see where mutations are – SUFU vs
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Glial tumours:
o Grade I – benign
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o Grade III – death in a few years
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o II – convert to III and IV after several years
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Age-based genomic and epigenomic features of biological
glioblastoma subgroups:
o Approaching personalised approaches
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 However, the other becomes a “transit-amplifying cell”
(sometimes termed a “progenitor cell”; gray), which is
committed to enter a differentiation pathway but does not
yet participate in end-stage differentiation
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o Because a single stem cell can spawn dozens if not hundreds of post-mitotic
descendants, this cell does not need to proliferate frequently
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The cancer stem cell:
o Many cancers contain proliferating cells and differentiated cells & areas of cell death
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o However, there may be different types of dividing cells recapitulating the ideas of
stem cells and TA cells
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 5,000 minority (1%) cells could produce cancer
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Breast cancer stem cell:
o The computer determines how the cells will be sorted before
the drop forms at the end of the stream
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o This charged drop is then deflected left or right by charged
electrodes and into waiting sample tubes
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o New tumours contained same mix of cells
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 The intensity of staining is plotted logarithmically on each
axis
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 In this experiment, a 12% subpopulation of cells that
expressed low CD24 and high CD44 antigen staining (green
box) was separated from cells (T1-P) that showed high CD24
expression and high CD44 expression (blue bracket)
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o But some features would be expected to protect them from becoming cancerous:
 Stem cells often more distant from sources of damage (gut/lung lumen,
surface of skin) and protected (e
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mucins)
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 Strand retention provide protection against genotoxic damage
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Are cancer stem cells derived from normal stem cells?
o Stem cells:
 For:
 Immortal – express telomerase
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 Against:
 Slow dividing/quiescent; Protected from mutations
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 Against:
 Not immortal; Many soon lost from body
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 TA cells have acquired immortality and these are target of the initiating
mutations
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 Some tumour types frequently exhibit such mutations, general in
cancer derived from tissues with low level of self-renewal, i
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low in
stem cells
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o One cell amid a large cell population
sustains an initiating mutation (red
sector, top) that confers on it a
proliferative and/or survival
advantage over the other cells
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o When this clone expands to a large enough size (e
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, 106 cells), a second
mutation—one that strikes with a frequency of ~10–6 per cell generation—may
occur (green sector), resulting in a doubly mutated cell that has even greater
proliferative and/or survival advantage
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o This results once again in a large descendant population, in which a third mutation
(blue sector) occurs, and so forth
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o Importantly, this scheme does not take into account the fact that clonal successions
may require greatly different time intervals to reach completion
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Cancer stem cells and the evolution of cancers:
o The existence of cancer stem cells has
important implications for how tumour
progression and clonal succession occur
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e
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o Moreover, the cancer stem cells represent a
minority of the neoplastic cells in tumour
masses, while the transit-amplifying cells
represent the majority of neoplastic cells
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o Conversely, mutations that strike the genomes of transit-amplifying cells cannot be
transmitted further, because these cells have only limited replicative ability
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Cancer stem cells and the evolution of cancers?
o As tumour progression proceeds, the genomes of
tumour cells often become increasingly unstable
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o Consequently, the tumour mass develops an
increasing number of distinct sectors, each
dominated by a genetically distinct sub-clone
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o Tumours are made up of genetically distinct subclones, several of
which might be able to function as CSCs/TICs
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Thus, the simple linear model of clonal succession (above) should be
replaced, at least in latestage tumours, by dynamic changes
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Does the existence of cancer stem cells affect how we think about
therapy?
o Stem cells are often slow dividing or near quiescent
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Does the existence of cancer stem cells affect how we think about
therapy?
o Drug pumps
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Why are cancer stem cells important?
o It is the cancer stem cell that can seed a new tumour
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o If cancer stem cells escape from therapies designed to damage DNA, it is likely that
the worst and even more mutagenised cells will seed the new tumour
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o Is it the/a stem cell that escapes therapy?
Key questions:
o What is the relationship to normal cells?
 Many early molecular studies of cancers weakened by inappropriate
assumptions of this relationship
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o What special issues do CSCs present for therapy?
Brain cancer stem cell:

Do tumours arise from endogenous stem cells?
Malignant astrocytomas originate from neural stem/progenitor cell;s:
o Previously – GFAP driven loss of
P53/PTEN/NF1 100% high grade
astrocytomas
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o Here – Nestin promoter driving ER
linked cre – so not in mature
astrocytes
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o Used with floxed P53/NF1/PTEN or just floxed
P53/NF1
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5 or 4 weeks old animals – all got
high grade-astrocytomas
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o Some are clearly related to other types of
precursor (Shh-medulloblastoma)
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CD133+ cells exhibit strong evidence of being tumour
stem cells:
o The CD133+ cells enriched by irradiation, formed
neurospheres (rarely seen from CD133- cells) and
expressed markers of neural/cancer stem cells
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 CD133- cells were not
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o The CD133+ cells increased expression of key DNA-repair machinery genes in
response to radiation more than CD133- cells
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 Shielded by mucus – shields stem cells from the actions of toxins
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o Self-renewing cells of various types are the direct targets of the mutagenesis that
leads, sooner or later, to the formation of tumors
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 Still, in many tissues, committed progenitors, which normally have only a
limited ability to renew themselves, may represent more plausible targets
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 Inactivating mutations of p53 and Rb; increased incidence of glioma is LiFraumeni
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 2
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 2x1013 platelets
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 Goal is to produce a bunch
of various cell types
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o Replace 1% each day
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5x1011 red blood cells are
produced daily
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 Leukaemia (literally “white blood”)
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o Symptoms (other cells ‘crowded out’)
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 Abnormal bruising/bleeding
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 Increased risk of infection
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 Anemia, shortness of breath and fatigue
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o Either lymphocytic (B and T-cells) or myeloid
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 Acute Myelogenous Leukaemia – AML
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 Chronic Myelogenous Leukaemia – CML
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 Kids – almost entirely acute – 80% ALL and 20% AML
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Oncogenes and leukaemias:
o 65% of acute leukaemias involve chromosomal
translocations
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 Transcription factors or receptors often
affected by translocation
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o Results in either:
 The inappropriate expression of a gene
in the wrong cell
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 Expression of a novel gene
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 Causes big increase in expression of oncogene
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o It is the result of a reciprocal translocation between chromosome 9 and
chromosome 22, which is specifically designated t(9;22)(q34;q11)
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 While the chromosomal translocation generating the two altered
chromosomes (9q+,22q–) is reciprocal (for example, involving a loss and a
gain by both), the sizes of the exchanged chromosomal arms are unequal,
leading to the greatly truncated Chromosome 22 (for example, 22q–)
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o The relatively minor change to the tumor cell
karyotype that is created by the CML
translocation is apparent in this SKY analysis, in
which chromosome-specific probes are used,
together with fluorescent dyes and computergenerated coloring, to visualize the entire
chromosomal complement of CML cells
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 Reciprocally, one of the two Chromosomes 22 has acquired a white region
(characteristic of Chromosome 9) at the end of its long arm
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 Breakpoint cluster region (bcr)
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 Acts as a constitutively activated tyrosine kinase
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 A serine/threonine kinase and GTPase
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 Tyrosine kinase activity not activated – now has aberrant activity –
phosphorylates wrong targets
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 Two other alternative breakpoint sites in the bcr
gene are involved in bcr–abl translocations (not
shown here) arising in certain other hematopoietic
malignancies
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 Transcription factors including Jun, Myc and NF-ϰβ
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 Most important phenotype is tyrosine kinase signaling
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o Translocations might be entirely random –
only reason you see them in leukaemia is
because they generate a product which gives
that cell a selective advantage
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o More than 95% of cases of chronic
myelogenous leukemia (CML) exhibit the
Philadelphia chromosome, which results from
a reciprocal translocation between
Chromosomes 9 and 22
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 The net result is a truncated Chromosome 22 (i
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, 22q–), often termed the
Philadelphia chromosome (Ph), and a fusion of the 5ʹ portion of the ABL
gene with a 3ʹ-proximal portion of the BCR gene, which normally resides at
22q11
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 Each of these BCR-ABL fusion genes encodes a multidomain (and thus
multifunctional) protein
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o Or – translocations are non-random, and some cause clonal expansion
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Why do leukaemias exhibit specific translocations?
o Answer 1:
 Only some translocations capable of giving a B or Tcell at a given developmental stage a selective
advantage and so promoting clonal expansion
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 Double strand breaks (DSBs) and incorrect repair
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g
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o 2 – Some genes just have fragile regions – open due to transcription?
 Associated with proteins that make them fragile, e
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topoisomerase
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g
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Other pathways to leukaemia – somatic
hypermutation:
o Only in B-cell lineage (about 50% of
adult leukaemias, 80% of kids)
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o B-cells express Activation-induced-cytidine-deaminase (AID), which semi-randomly
converts ‘C’s to ‘U’s
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o So when genes moved into an Ig locus they may be subject to the same increased
mutation rate
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o The resulting
uridine:guanosine
mismatch can be repaired – but
mistakes happen
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 Requires ongoing transcription
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o One model predicts that a collection of transacting factors bound to immunoglobulin
enhancers form a specific structural platform
that recruits AID to the immunoglobulin locus
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What is cell of origin and is translocation the 1st event?
o The translocation generally appears to be an
early, or the earliest, event
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e
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Leukaemia accounts for about ¼ of cancers in children, 4/5 of these ALL
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o TEL-AML1 fusion is the most common genetic alteration in childhood ALL
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What is the cell of origin and is the translocation the 1st event?
o TEL gene from chromosome 12 to the AML1 region of
chromosome 21
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o Red = AML; green = Tel1
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o Twins share blood
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o Therefore very early event
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Is the translocation the 1st event, and why so young?
o 1% of newborn babies carry cells with this translocation in blood, ‘pre-leukaemic
clones’
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o However, the occurrence of this first step towards this particular cancer in the blood
cells at birth, could provide one explanation for the early age of onset of this cancer
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 The process seems irrevocable and sometimes, rapid
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o Remember retinoblastoma, patients who carry one mutation, also get cancer as
children
Preleukaemic clones could provide reservoir for relapse:
o AML (adults) often have dominant negative DNMT3a mutation plus other gene
mutations
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o Hence this mutation is early and arises in the HSC
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o Preleukaemic clones have some growth advantage and persist after therapy
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Evolution of leukaemia:

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The patient:
o Treatment:
 Chemotherapy:
 Actively dividing cells
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 Radiotherapy:
 Used if leukaemia has spread to the CNS
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o Must replace the stem cells to restore heamatopoietic system to
patient
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 Gleevec/imatinib mesylate/glivec
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o Clinical trials in 1998 demonstrated remission in all 31 treated CML
patients
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Treatment problems:
o 90% of patients respond positively
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o But, 60% of patients who have already progressed to blast crisis respond, but
subsequently relapse
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o Relapsing patients:
 29/32 patients have new mutations in the BCR-ABL gene prevent Gleevec
binding to catalytic cleft
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o Treatment reduces BCR-abl
transcripts
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o But – the prognosis of CML appears now extremely positive
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 For patients with Gleevec intolerance or progression, new tyrosine kinase
inhibitors are providing excellent and durable responses
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o Sequenced transcriptome – 20 missense mutations specific to relapse – hetero so
dominant negative?
o 2 mutations in NT5C2 a 5’ nucleotidase
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o Mutations caused increase in the enzymes activity
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 Deep sequencing is going through the genome multiple times
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o More resistant to purine analogues used as part of chemo for most ALL cases
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 Deep sequencing of primary tumours found this mutation in some:
 Suggests that relapse after therapy had selected for these clones
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Children’s Cancers
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Children get different solid tumours to adults
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o Frequency of cancer in children reduces as age increases (up to a point)
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 Infants – brain, liver
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o Brain tumours
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 Children – glial and neuronal –
predominantly cerebellum
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 Proliferation – developing tissues provide a huge resource for tumourigenic
transformation
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 Children’s tissues more proliferative because they are growing
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 Many more cells in a growing body contain stem cell features than
normal cells
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 Growth factors, etc
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 Together these make the developing individual an ideal starting point for
cancer
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 Maybe CSTs don’t need all of the hallmarks of cancer
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Summary:
o The number of necessary oncogenic events is a function of tissue ontogeny
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Children get different solid tumours to adults:
o Some cancers restricted to children (cancers of development)
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 Neuroblastoma (PNS)
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Wilm’s tumour – connecting tumourigenesis and organ development in the kidney:
o Wilm’s tumour, or nephroblastoma, is a common childhood tumour that is
intimately linked to early kidney development and is often associated with
persistent embryonic renal tissue and other kidney abnormalities
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o Tumour in newborn child is massive – been growing for a while
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 Doesn’t carcinogenic model
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o Over 10 million sufferers under 15 years old in USA
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 Almost all under 10 years, 40% under 1 year
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o Origins – neural crest
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o Neural crest migrates throughout
the body
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o Most NBs are spontaneous somatic
events – ALK mutations/amplifications in 10-14%
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 Near diploid means damage must be within chromosomes
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Neuroblastoma – a developmental disorder:
o Most common genetic lesion found to date – amplification of MYCN
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 Or homogenously staining regions (HSRs) – intrachromosomal
amplification
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o Mechanism unclear
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o MYCN associates with Max to drive transcriptional
cell cycle
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o Survival of infants with metastatic neuroblastoma
based on MYCN status:
 A Kaplan-Meier survival curve of infants
less than 1 year of age with metastatic
neuroblastoma who were treated in a recent study
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Myc is an important signalling molecule
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o Has the highest rate of spontaneous
regression amongst human cancers –
IV
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o Genome can look relatively normal
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o Sequenced genomes of 240 cases:
 Very few non-silent exon mutations – 14/12 per tumour
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 Found several rare germ line mutations
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o Age of incidence defines the cancer type
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o Explanation for the unusual distribution of brain tumours
in kids (cerebellum), prolif of other regions earlier,
therefore tumours earlier, therefore kill patient before or
around birth

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Are children’s cancers less abnormal than adult cancers?
o So: children’s cancers may be quite different because of
the time they arise and may have a much closer
relationship to their progenitor cell in the normal
child/foetus
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Teratocarcinoma – a cancer of a normal cell?
o Study of Rickert – of 8 foetal teratocarcinomas, none had
karyotypes differing from their host
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 Are children’s cancers actually more ‘normal’ than adults and therefore
more susceptible to correction by environment or therapy
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 Some tumours regress
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 Lack mutations in p53
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 Cancers exhibit less genetic damage than adult cancers
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o Mutation of P53 allows DNA damage to lead to inheritance of additional
mutations/chromosomal damage in daughter cells and therefore predisposes to
cancer initiation and progress
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o So, lack of P53 mutations is because carcinogenesis is unlikely to play a major role,
but rather epigenetics or mismatch repair errors drive children’s cancers
Is the underlying mechanism of children’s cancers different from adults?
o Lack of geographical variation
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Replication error (mismatch repair):
 Error rate = 1 mutation in any single gene each 107 cell divisions
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e
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 1010 cell divisions in developing cerebellum
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 If any of only 20 genes can play a role: any 3 gene in the same cell
1/4,000,000
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 Therefore the likelihood of a tumour initiating mutation is high
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Summary:

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Children’s cancers may be less abnormal than adults because:
 Replication error drives their formation
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Therefore more closely related to their normal progenitor than would be the case
for an adult cancer
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o What about inherited predisposition – not classical tumour suppressors generally
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Summary:
o Children’s tumour cells are very closely related to normal progenitors, they may
have less genetic damage than adult cancers
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 Fewer mutations needed
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 More prone to mutation through replication error
o If replication error is the cause, these are unavoidable
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o If children’s cancers are closer to normal cells, they may be capable of normal
growth/differentiation, so there is more chance that therapies can be developed to
make them behave normally
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o Therapy – Side effects even more pronounced than adults
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 E
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Differentiation – RA for NB
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 Driver mutations:
 Tumour initiation and progression
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o Growth advantage might be activating mutation in
oncogene or loss of TS gene
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 Usually more than one per cancer
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 Can provide information about mutational processes
...

 Candidate genes, or
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 Everything!
Example of candidate gene study success:
o First aims of cancer genome project – sequence candidate cancer genes in
all forms of cancer
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o Activating mutations in BRAF in 50-70% human melanomas:
 BRAF mutated in other cancers
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o 10 years from finding mutation to clinical trials
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o Ras pathway includes good drug targets
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o Could target exons (exome)
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o Whole genome:
 Will find 10s or 100s or 1,000s of
mutations
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o Improvements in the rate of DNA sequencing over
the past 30 years and on:
 Million-fold improvement in the rate of
sequence generation
...

 The median number of nonsynonymous
mutations per tumor in a variety of tumor
types
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o Mutations may be acquired while the cell lineage is phenotypically normal, reflecting
both the intrinsic mutations acquired during normal cell division and the effects of
exogenous mutagens
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o Passenger mutations do not have any effect on the cancer cell, but driver mutations
will cause a clonal expansion
...


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Key issues:
o Distinguishing driver mutations from passenger mutations
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o Genetic heterogeneity in tumour:
 Tumour evolution
...

Early sequencing studies looked at a single tumour:
o Used COLO-829 cell line
...

 Also a matched lymphblastoid line (COLO-829BL)
...

 Subtracting variants in COLO-829BL from COLO829 gave a catalogue of somatic mutations in the
tumour
...

 292 coding changes (105 silent)
...

Key points from this paper:
o Most somatic base substitutions were C>T transitions,
with a high frequency of CC>TT changes
...

 UV causes formation of pyrimidine dimers
...

o Substitution patterns differ in other tumour types
...

o Lines show where 2 pieces of DNA are not joined
together where they aren’t normally joined together
...

 Part of catalogue of somatic mutations in the small-cell lung cancer cell line
NCI-H2171
...

 Arrows indicate examples of the
various types of somatic mutation
present in this cancer genome
...

 The Y axis is a log scale
...

o Many adult solid tumours follow a multi-step model
...

 May take decades for cancer to fully develop, so increased time for more
replication errors (evolutionary clock model)
...

o Leukaemia/children’s don’t seem to follow this multistep model
...

 The major signaling pathways that drive tumorigenesis are shown at the
transitions between each tumor stage
...

 Patient age indicates the time intervals during which the driver genes are
usually mutated
...

 Genome very stable
...

 Average number somatic mutations 167
...
25/case resulted in amino acid
change (compare with 187 in melanoma genome)
...

o Loss of RB sufficient for cancer formation?
 Fits with early onset of disease and Knudson’s 2-hit hypothesis
...

 P53, RB, MYC, etc
...

o Driver genes show more mutations than random expectation
...

 Slow growing, pituitary brain tumours
...
18 per 100,000
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 Papillary form (mostly adults)
...

 CTNNB1 gene (encodes β-catenin) mutated in 11/12 adamantinomous
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o Targeted sequencing
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 BRAF mutations in 36/39 papillary
tumours
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Clinical relevance of findings:
o Immunostaining:
 Difference in β-catenin localisation between
tumour types
...

 Diagnostic tool
...

 Wnt pathway for adamantinomous tumours
...

 Vemurafenib already trialled for
melanoma
...

Spectrum of mutations
differs across cancers:
o The end point is a
result of the
different mutational processes that the tumour has been subjected to as it develops
...

 Protect against retrovirus and retrotransposons propagation
...

 APOBEC3B overexpressed in many cancers, associated with APOBEC
mutation
signature
...

o Shows the variation in
the contribution of
different signatures to a particular cancer
...

o Signature 11 represents chemotherapy (TMZ) signature
...

o Order in horizontal axis represents position in human genome, vertical axis shows
the distance of each
mutation from the
previous mutation
...

Genome rearrangements are very common in cancer
cells:
o Chromosome gains and losses
...

 Previously recognised in leukaemia,
since found to be prevalent in solid tumours
...

 Recently described in prostate cancer genomes
...

 Elevated levels in bone cancers, pediatric medulloblastoma and
neuroblastoma
...

Chromothripsis – possible mechanism:
o Single catastrophic event
...

 These vary between tumours
...

o Most likely mechanism
...

 Probably occurs when chromosomes
condense for mitosis as this can occur
prematurely in micronuclei
...

 May disrupt several TS genes
...

o May be associated with more aggressive tumours
...

 In neuroblastoma, survival rates worse for tumours
that have chromothripsis
...

 Also seen in AML
...

 Different pathways, different driver genes
...

o Heterogeneity within cancer types
...

 Same pathway mutated, but at different points (e
...
RAS/BRAF in
melanoma)
...

 Can be important for therapy
...

 Tumour evolution
...

 Relatively homogenous tumour
...

o Branched evolutionary pattern (B and C):
 Multiple distinct clones exist and co-evolve
...

 Potential for multiple subclonal driver
events
...

 Seen in many cancer types
...


-

The cancer genome:
o Approximately 90% of the known somatically mutated cancer genes are dominantly
acting, that is, mutation of just one allele is sufficient to contribute to cancer
development
...

 Ten per cent act in a recessive manner, requiring mutation of both alleles,
and the mutations usually result in abrogation of protein function (these are
sometimes known as tumour suppressor genes)
...

 In each dominantly acting cancer gene, however, the repertoire of cancercausing somatic mutations is usually more constrained, both with respect to
the type of mutation and its location in the gene
...

 Most, however, are activated through genomic rearrangement
...


Epigenetics and Cancer
-

Smokers have different mutations:

o

o

o

Heat map:
 G>T; C>A – transversions
...

G>T; C>A transversions dominated smoker-only group
...

In contrast, G>A; C>T transition dominated the never-smoker group
...

Epigenetics and cancer:
o Heritable changes in gene expression caused by mechanisms other than changes in
the underlying DNA sequence
...


-

-

-

-

Discoveries from cancer genome characterisation:
o Epigenetics DNA methylation – DNMT3A
...

o Chromatin histone methyltransferases – MLL, MLL2, MLL3, EZH2, NSD1, and NSD3
...

o Chromatin histone acetyltransferases – CREBP, and EP300
...

o Chromatin other – CHD1, CHD2, and CHD4
...

o Epigenetic alterations are also inherited in a semi-permanent manner, and might
also change with cancer progression
...

 Changes to DNA methylation
...

o Could be reversible!
Histones – modifications:

Histone code offers massive complexity:
o A nucleosome contains 146bp of DNA wrapped twice
around an octamer composed of two copies of each
histone protein – H2A, H2B, H3, and H4
...



There are also several variant histones
...

Where genomics and epigenetics meet – mutation of histone modifiers:
o Medulloblastoma:
 Inactivating mutations of the histone-lysine N-methyltransferase genes
MLL2 or MLL3 were identified in 16% of MB patients
...

 For example, 32% of DLBCL and 89% of FL cases had somatic
mutations in MLL2,which encodes a histone methyltransferase
...

o Bladder:
 Genetic aberrations of the chromatin remodeling genes (UTX,
MLL-MLL3, CREBBP-EP300, NCOR1, ARID1A and CHD6) in 59%
...
e
...

Where genomics and epigenetics meet – mutation of histones
...
3 and chromatin remodeling genes in
paediatric glioblastoma
...

 21 had mutations in the H3
...

o ATRX and DAXX are components of a complex that affects recruitment
of H3
...

o H3
...

 Also found mutations in H3
...

DNA methylation changes:
o Almost every tumour type analysed shows global DNA hypomethylation
...

o Many tumours exhibit a CpG island methylator phenotype (CIMP)
...

DNMT 3A and 3B: de-novo methyltransferase
...

Can affect:
 Genomic Stability (mobile elements), (especially lung cancer)
...

 Gene expression (mainly tissue specific genes)
...

 Dnmt1 mutants get sarcomas and gut cancer
...

 Dnmt3b mutant cells develop aneuploidy and chromosomal breaks
...
e
...

 Tumour suppressor genes
...

 Some (RASSF1A) almost always inactivated this way
...

 Greater than 50% GC
...

 Observed-to-expected CpG ratio = ((Num of CpG/(Num of C × Num
of G)) × Total number of nucleotides in the sequence)
...

 DNMTs (DNA-methyltransferases)
...

 Polycomb etc
...

What causes a methylator phenotypes?
o Epigenetics DNA methylation – DNMT3A
...

o Metabolism – IDH1 and IDH2
...

o AML (adults) often have dominant negative DNMT3A mutation
...

What causes a methylator phenotypes – mutation of demethylators
...

o Mutation of IDH1 or IDH2 actually changes
activity so leads to decreased α-KG increased
synthesis of 2-HG which inhibits activity of
TET family of DNA hydroxylases
...

o Mutations events are in changes of the
machinery that takes methyl group off –
demethylation machinery broken
...


-

What causes a methylator phenotype – inhibition of demethylation
...

 TET mutations – LOF
...

o Mutation exclusion indicates a common pathway
...

o Different classes of cancer exhibit different
signature ‘methylomes’
...

Antitumour effects of a combined 5-Aza-2’ Deoxycytidine
and valproic acid treatment on Rhabdomyosarcoma and
Medulloblastoma in Ptch mutant mice
...

 Preventative, but ineffective on advanced tumours
...

o Female breast cancer – 48,000
cases per year in UK, 12,000
deaths
...

Most cancers – survival over 2-4 years more likely to survive long term
...

o Need good new alternatives:
 For unresponsive
cancer
...

Screening programmes:
o The earlier a cancer is
diagnosed and treated, the
better the chances of cure
...

o Efficacy/accuracy and potential
for successful treatment must
outweigh cost and/or risk
...

o But, many would not progress to a lifethreatening cancer during the lifetime of the
women – not aggressive or death from another cause
...

o However, in 2010 it was found that for every 56
cases diagnosed, 5 lives saved and 2 cases overdiagnosed
...

o Pap smear (after George Papanicolaou,
introduced it in the 1930s)
...

o Developed countries, mortality now 70% lower
than 30 years ago
...

o But long-term survival only gone from 46%
to about 66%
...
e
...

Screening programmes – bowel cancer:
o 2002, rolled out in several countries
...

o Tests:
 Foecal occult blood test (FOBT)
...

o So why not adopt colonoscopy as the method of initial screening?
o Budget for screening the UK population using FOBT is £20-30 million per year
...

o Virtual colonoscopy (CT colonography) – computerised tomography (CAT scan)
series of X-rays – 3D picture of colon and rectum
...


-

-

-

How therapies work – radiotherapy:
o X-rays or radioisotopes
...

o To damage cells to such an extent that they cannot survive
...

o Often fractionated or brachytherapy used
...

How therapies work – chemotherapy:
o Developed in WWII
...

o Non-specific DNA alkylating agent leading to
cross-linking
...

o Most other new chemotherapeutics are natural products of bacteria or plants
...

o Advantages:
 Very toxic to target cells so high efficiency of cell death
...

 Radiation can be highly targeted
...

 Toxicity is not specific to cancer cells, treatments limited by toxicity
...

o So for all cancers it would be better if we could understand them to find a more
specific way to kill them
...

 Tumour suppressors are poor targets (need to restore gene function)
...

 Biochemical inhibition
...

o Synthetic lethality approaches
...

 Most drugs fail during
development/trials
...

 Molecules that affect the target (hits) must then be evaluated for chemical
properties and potency before those that are worth pursuing (leads) can be
identified, synthesized, evaluated and modified for drug qualities (lead
optimization)
...

 All of these processes must occur before the first dose of any new molecule
can be tested in humans
...

o Following initial clinical testing for safety in
humans (PHASE I), the molecule enters the
most crucial phase, PHASE IIA, during which
the desired clinical effect (that is, efficacy) is
addressed in a relatively small, but still
expensive, clinical trial
...

 With costs as high as this, only molecules for which there is good evidence
of efficacy and a reasonable biological rationale for its mechanism of action
are selected for full development
...

 By selecting out patients in Phase-II and -III clinical trials who would be
predicted to be non-responders (red figures in parts b and c), subsequent
clinical trials can be made smaller, faster and less expensive
...
g
...

o Optimised – recommended, but with restrictions (perhaps only a subset of patients)
...

 Used only as part of a well-designed clinical trial
...

 Lacks evidence to support clinical effectiveness
...

o NICE guidance supports the use of quality-adjusted life years (QALY) as the primary
outcome
...

 Used to estimate an incremental cost-effectiveness ratio (ICER)
...

 Since 2008, for end-of-life cancer drugs the threshold has been increased
above £30,000
...

 Considers the national costing implication
...


-

-

-

NICE – case study 1:
o Vemurafenib for treating locally advanced metastatic BRAF V600 mutation-positive
malignant melanoma
...

 Mutation testing provided
free by Roche
...

 Patient access agreement
...

NICE – case study 2:
o Ipilimumab (antibody therapy for malignant melanoma)
...

 Patient access scheme
...

 Approved
...

P53 and cancer therapy:
o Obvious target for therapy
development (remember cells may
contain mutant p53)
...


o

-

Two main approaches
...

 Rather than restore wt p53 expression, use approach that relies on
p53 deficiency
...

 Approved for treatment of head and neck cancer
...

ONYX-015 action:
o The virus lacks the EIB gene, this normally
binds to p53 and causes its degradation
...


-

-

-

-

-

Head and neck cancer:
o A type of squamous cell carcinoma
...

 Associated with alcohol and tobacco use
...

o Treated with surgery and/or radiotherapy
...

 High level of morbidity and palliative surgery difficult
...

o Tumours with p53 mutations can be targeted with ONYX-015 (Advexin/Gendicine)
...

 Some response, but limited efficacy
...

 Restoring p53 activity in tumours where p53 itself is not mutated
...
g
...

 Aim is to block action of mutant or overexpressed protein, restore
p53 expression – apoptosis
...

o Nutlins promising approach (cell and animal model studies)
...

 Nutlin-3 in clinical trials
...

 What happens in normal cells?
 Development of resistance
...

 Herceptin; BRAF inhibitors; Gleevac
...

 Parp-1 inhibitors
...

 Target not expressed or mutated in tumour
...

 Vemurafenib (only suitable for BRAF V600E mutations)
...

 Down regulation of target
...

 Use of alternative pathway
...


-

-

-

Acquired resistance to kinase inhibitors:
o ABL gene (mutated in CML, drug
imatinib)
...

 Mutations in ABL (block drug
interaction, alter protein
conformation)
...

o BRAF (mutated in melanoma, colon cancer, drug vemurafenib)
...

 NRAS mutation – pathway activation by alt mech
...

 MEK mutation – activation downstream effector
...

 Alternative splicing of BRAF
...

 Kinase inhibitors are effective clinical therapies in subsets of cancers, but
resistance inevitably emerges
...
e
...

 All of these lead to reactivation of the critical signaling pathway for the
specific kinase and clinically to cancer
progression
...

 Different pathways, different driver genes
...

o Heterogeneity within cancer types
...

 Same pathway mutated, but at different points
(e
...
RAS/BRAF in melanoma)
...

 Can be important for therapy
...

 Tumour evolution; Resistance to therapy
...

o Cancer cells do not mutate their genes to become resistant
...

 These may be already present in the tumour
...

o Illustrated by studies on clear cell renal carcinomas (ccRCC) and glioma
...

 Affects 300,000 people worldwide every year
...

o Sporadic and heritable forms
...

 VHL mutated or hypermethylated in >90% of sporadic ccRCC
...

 VHL mutation is a very early event
...

o Used to study genomic architecture and evolution of tumours
...

Identifying tumour heterogeneity in ccRCC:

-

Patterns of mutations in one tumour:

-

o Dark boxes represent non-synonymous mutations
...

Different tumours follow different (but related) evolutionary:

o

-

-

Branch and trunk lengths are proportional to the number of nonsynonymous
mutations acquired on the corresponding branch or trunk
...

 Driver mutations defining parallel evolution events are highlighted by color
...

Sequencing study of recurrent tumours in gliomas:

Therapy-driven tumour recurrence in glioma:
o Looked at recurrent tumours in patients treated with
temozolomide (TMZ)
...

 Causes C>T/G>A transitions at CpC and CpT
dinucleotides
...

o 6/10 recurrent tumours hypermutated
...

 These tumours show signature of TMZ-induced mutagenesis
...

 Cancer cells express abnormal/selective target
...

 Typically use monoclonal antibodies (mAbs)
...

FcγR effector mechanisms:

-

From animal Abs to near human by protein engineering
...

 Humira is now (2012) the world’s top selling
product ($9 billion pa)
...
g
...

o Direct tumour cell killing can be elicited by receptor agonist activity, such as an
antibody binding to a tumour cell surface receptor and activating it, leading to
apoptosis (represented by the mitochondrion)
...

o An antibody binding to an enzyme can lead to neutralization, signalling abrogation
and cell death, and conjugated antibodies can
be used to deliver a payload (such as a drug,
toxin, small interfering RNA or radioisotope) to
a tumour cell
...

o Anti-CTLA4 – cytotoxic T lymphocyte-associated antigen
4
...

 Anti-CTLA4 – metastatic melanoma
...

o Immune-mediated tumour cell killing can be carried out
by the induction of phagocytosis; complement activation; antibody-dependent
cellular cytotoxicity (ADCC); genetically modified T cells being targeted to the
tumour by single-chain variable fragment (scFv); T cells being activated by antibodymediated cross-presentation of antigen to dendritic cells; and inhibition of T cell
inhibitory receptors, such as cytotoxic T lymphocyte-associated antigen 4 (CTLA4)
...

o Vascular and stromal cell ablation can be
induced by vasculature receptor antagonism
or ligand trapping (not shown); stromal cell
inhibition; delivery of a toxin to stromal cells;
and delivery of a toxin to the vasculature
...

 Increases patients immune
response – clinical trials underway
(pancreatic cancer) – so far not
elicited a very impressive response
...

Priming the immune response against cancer – dendritic cell therapy:
o FDA, May 2010 – first therapeutic cancer vaccine, sipuleucel-T (Provenge)
...

 APCs cultured with prostatic acid phosphatase (PAP)
...


-

-

-

-

-

-

Homeopathy costs NHS £4,000,000/year:
o Survey of more than 1,000 clinical staff – 55 per cent want to see an immediate ban
on funding homoeopathy and herbal medicine
...

 Easy for viruses, e
...
HPV in cervical cancer
...

 Green tea, curcumin
...

o In 1828, Johann Buchner, professor of
pharmacy at the University of Munich, isolated
a tiny amount of bitter tasting yellow, needlelike crystals, which he called salicin
...

o 27% less likely than controls to die from
cancer
...

o Reduced the risk of metastasis in patients without initial metastasis by 55%
(colorectal cancer by 74%)
...

o COX-2 inhibition, since many colorectal cancers aberrantly express COX-2
...

Cancer free babies born:
o First used for the severe, inherited cancer predisposition Li Fraumeni Syndrome,
resulting in the birth in the USA in 2001 of a child that had been selected to be free
of the mutant gene that was carried by its parents
...

 USA – BRCA1, APC, PTCH, P53, RB1, VHL,
many others
...


-

Antibody therapy of cancer:
o Antibody-based therapy for cancer has become established over the past 15 years
and is now one of the most successful and important strategies for treating patients
with haematological malignancies and solid tumours
...

o The killing of tumour cells using monoclonal antibodies (mAbs) can result from direct
action of the antibody (through receptor blockade, for example), immune-mediated
cell killing mechanisms, payload delivery, and specific effects of an antibody on the
tumour vasculature and stroma
...

o Serological, genomic, proteomic and bioinformatic databases have also been used to
identify antigens and receptors that are overexpressed in tumour cell populations or
that are linked to gene mutations identified as driving cancer cell proliferation,
including EGFRvIII, MET, CTLA4 and fibroblast activation protein (FAP)
...

o A major objective for the clinical evaluation of mAbs has been determining the
toxicity and therapeutic efficacy of the antibody alone or as a delivery system for
radioisotopes or other toxic agents
...

o Twelve antibodies have received approval from the US Food and Drug
Administration for the treatment of various solid tumours and haematological
malignancies, and a large number of additional therapeutic antibodies are currently
being tested in early stage and late-stage clinical trials

---