Medical Biology I

Genome stability and instability – DNA damage,

mutations, DNA repair and defects in DNA
repair

Ondřej Slabý
Department of Biology, Faculty of Medicine MU
 Various factors (internal, external) cause different types of DNA
damage and induce various mechanisms of DNA repair

Causes:

Type
of damage:

DNA repair:
 Types of DNA damage and their causes

There are two principal types of DNA damage causes:

• Internal factors – reactive oxygen species (ROS) and other endogenous molecules capable for
interaction with DNA originating as a products of the cellular metabolism.
DNA replication errors.

• External factors
Physical factors– UV a ionizing radiation (thymin dimers, single- and double-strand breaks in DNA)
Chemical mutagens (chemical modification of bases- deamination, alkylation, oxidation, and
methylation, disruption of the phosphodiester bonds in DNA, formation of DNA cross-links.
Biological mutagens – insertion of the viral DNA
 DNA lesion Number of repaired in 24 hours
Hydrolysis
Depurination 18 000
Depyrimidination 600
Cytosine deamination 100
5-Methylcytosine deamination 10
Oxidation
8-oxo G 1500
Ring-saturated pyrimidines (thymine 2000
glycol, cytosine hydrates)
Lipid peroxidation productes (M1G, 1000
etheno-A, etheno-C)
Nonenzymatic methylation by S-adenosylmethionine
7-Methylguanine 6000
3-Methyladenine 1200
Nonenzymatic methylation by nitrosated polyamines and peptides
O6-Methylguanine 20-100

Endogenous DNA lesions created and repaired in 24 hours in a normal mammalian cell - errors as a
result of normal chemical reactions taking place in the cell, so the table does not include external
influences from both chemical and physical mutagens.
 Types of DNA damage – depurination, deamination
During the time needed to read this sentence, 1012 purines (A and G) will be lost from DNA in all cells of the human body
by a spontaneous reaction called depurination - the formation of gaps in the nucleotide sequence, phosphodiester binding
is usually unaffected.
Deamination - spontaneous loss of the amino group of cytosine.

AP site (apurine/apyrimidine→
depurination/depyrimidination) results from
various chemical modifications of bases
leading to breaking the N-glycosidic bond
(between sugar and base) – removal of the
base (mainly depurination – removal of
adenine or guanine)

Uracil pairs with A

In the 2nd round replication
CG – AT

Depurination and deamination are the most common chemical reactions that cause serious DNA damage.
Depurination releases guanine and adenine.
Deamination most often converts cytosine to uracil.
 Types of DNA damage – depurination, deamination
How chemical modifications induce mutations…

Chemical modification of nucleotides causes mutations if the errors are not eliminated in time.

A - deamination of cytosine leads to base substitution for another pairing of uracil with adenine.
B - depurination leads to loss of nucleotide pair - the replication apparatus skips the missing base.
Types of DNA damage – deamination, methylation

A special DNA glycosylase recognizes deaminated

methylated C nucleotides and mismatched T's
with G, which it then removes.

Deamination of nucleotides in DNA.

The added oxygen is marked in red in each reaction. Spontaneous deamination
of A and G is recognized as unnatural and base is removed immediately, the
figure also shows the deamination C to U. T does not have an amine group, so it
is not subject to deamination .
 Types of DNA damage – errors made during DNA replication

Chyby
DNA DNA polymerázy
polymerase při replikaci.
errors during replication.
ItProjeví se naatúrovni
manifests jedné
the level z dceřiných
of one buněk. cells
of the daughter
 Types of DNA damage – oxidative DNA damage
- single-strand breaks in DNA, oxidation of bases

Free oxygen radicals (reactive oxygen species, ROS) are intermediates of

electron transport during oxidative phosphorylation in mitochondria and
metabolism in peroxisomes (superoxide, hydrogen peroxide, especially
hydroxyl radical).
They oxidize bases, disrupt the sugar phosphate backbone and cause single-
chain breaks
8-Oxo-7,8-dihydroxyguanine (8-oxoG) is the result of
modification of guanine by oxygen radicals under
conditions of oxidative stress.

Hydroxyl radical attack

sites on the DNA molecule.
Oxo-guanine is formed
from guanine by the action
of free oxygen radicals,
which is coupled with
adenine → CG / TA
substitution Formation of the "C to A" mutation through 8-oxoG-
adenine base mismatch. Oxidative stress is a direct
path to mutations.
 Types of DNA damage – ionizing and UV radiation
Ionizing radiation (X-ray, gamma, cosmic): causes DNA breaks
non-ionizing radiation (UV): specific absorption at a wavelength of 280 - 315 nm (UVB-B), formation of thymine dimers
the degree of DNA damage corresponds to the dose of radiation absorbed

Formation of a covalent bond between two adjacent thymes

- replication fork stop - increase the probability of a shift in DNA replication (addition or deletion of bases)
leading to a shift of the reading frame during translation

80% of UVB induced mutations are pyrimidine

dimers.
This type of DNA lesion is repaired by nucleotide
excision repair (NER). Errors in this system cause
hereditary disease xeroderma pigmentosum,
Damage to DNA by ultraviolet radiation - connection of two linked extreme photosensitivity and higher
adjacent thymin bases by covalent bonds - thymine dimers. cancer predisposition.
 Types of DNA damage - chemical mutagens- base analogs, substances modifying bases
Base analogs are compounds with a structure similar to nucleic acid bases. They can have a mutagenic
effect because they integrate into the DNA strand during replication and induce pairing changes

Chemical mutagens:
- base analogs
- substances modifying bases (alkylating agents,
deaminating or oxidizing agents)

Alkylating agents
Yperite, mustard gas
Di-(2-chlorethyl)sulfide
Chemical warfare in WWI

Mutagenic effect of 5-bromouracil - bidirectional transition

5-bromouracil (BU) in its less common enol form present at
the time of incorporation into DNA, induction of G: C -> A: T
transition.
When BU is incorporated in its more frequent keto form and
Pairing between 5-bromouracil and transitions to the enol form during replication, induction of the
adenine or guanine A: T -> G: C transition.
Types of DNA damage - chemical mutagens- formation of DNA adducts
Interaction of a chemical with DNA and formation of a bond within one strand or between
two strands
- Leads to blockage of DNA metabolic activity (transcription, replication)
- Use for example in chemotherapy (cis-platinum)

Cisplatin activation and DNA damage - exchange of

one or both chloride ions for water (A). Cisplatin can
form covalent bonds with DNA, thereby damaging it
- DNA adducts and interstrand cross-links (B).
Types of DNA damage - DNA breaks (disruption of phosphodiester bond in DNA strand)

DNA breaks are caused mainly by ionizing radiation and oxidative DNA damage (damage of sugar-phosphate backbone
by free radicals).
Ionizing radiation can induce DNA damage directly (energy transfer) or indirectly through radiolysis of the water and
reactive oxygen species (ROS).
Several phosphodiester bonds close to each other are often damaged, which leads to the formation of a double-strand
break (DSB). DSB are also formed by replication of damaged or incorrectly repaired DNA.

Direct and indirect effects of

ionizing radiation on the DNA.
photon

Indirect
effect
Water radiolysis

photon
Consequent reactions

DIrect
effect
Extent of DNA damage - an overview of the most common DNA
damage in one cell in 1 day (approximately)

• Base loss – tens of thousands

• Cytosine deamination – thousands

• Base alkylation – tens of thousands

• Pyrimidine dimerization – tens of thousands

• DNA breaks – hundreds of thousands

Up to 500 000 damages per cell per day in total

L. Krejčí, Biology Lecture, LF MU

Genotoxicity test - Ames test

Ames Mutagenicity Test - The medium in each Petri dish

contains traces of histidine and a known number of the
Salmonella typhimurium test strain with a frameshift
mutation in the histidin operon. The control dish on the
left is used to estimate the frequency of spontaneous
revertants. The experimental dish on the right shows the
frequency of reversion induced by a potential mutagen.
 Various factors (internal, external) cause different types of DNA
damage and induce various mechanisms of DNA repair

Causes:

Type
of damage:

DNA repair:
 Type of mutations and necessity of genome stability

If the DNA damage is not repaired and the cell with the damaged DNA is not removed, a mutation occurs.
Mutations are associated with the development of cancer and many other diseases.
On the other hand, they play an important role in adapting to changes in the environment and thus evolution.

• Gene - nucleotide changes at the level of individual genes

• Chromosomal - structural and numerical changes at the chromosomal level (rearrangements, aneuploidy)
• Genomic - changes in the number of chromosome sets (polyploidy)

- The stability of genetic information is key to the proper functioning of all organisms
from bacteria to humans
- Various reactive chemicals and physical influences can lead to chemical modification of
DNA leading to a change in the coding sequence of genes - any structural change in DNA
can lead to disruption of cell function and disruption of cell division.

- Every day, each cell is affected by many such effects that have the potential to
cause changes in the structure of DNA

Therefore, cells are equipped with a number of mechanisms by which

they can prevent the negative consequences of DNA damage (DNA
repair mechanisms, cell cycle checkpoints, apoptosis).

L. Krejčí, Biology Lecture, LF MU

Balance between the DNA damage and repair

L. Krejčí, Biology Lecture, LF MU

DNA repair pathways
1. Direct reversal (photoreactiovation, dealkylation)
2. Mismatch repair (MMR)
3. Excision repair
Base excision repair (BER)
Nucleotide excision repair (NER)
4. Single-strand breaks repair
5. Double-strand breaks repair
Homologous recombination
Non-homologous end joining (NHEJ)
6. Tolerance repair mechanim (lesion bypass)
 Direct reversal (photoreactiovation, dealkylation)

Light-dependent repair or photoreactivation.

Only in plants, lower animals and marsupials
- the specific light-activated enzyme photolyase directly recognizes the thymine
dimer (binding in the dark) and, upon absorption of visible light (especially the blue
region of the spectrum), cleaves the covalent bond between adjacent thymes,
cytosines, or between cytosine and thymine
- Increasing the number of mutations in bacteria - growing in the dark
- Not present in humans

Dealkylation along the way O6 -

methylguanine DNA methyltransferase
(MGMT)

Cleavage of thymine dimers

by light-activated photolyase.
Therapy of glioblastoma with alkylating agent temozolomide
O6-metylguanin-DNA metyltransferase (MGMT) in prediction of thrapy response to TMZ

Methylated
No expression
« turned off »

gene MGMT
Promotor region expressed

Non-methylated Hegi et al., NEJM 2005

GBM with methylated promoter

• There is no DNA reparation protein to fix DNA methylations
• DNA chemical modifications induced by alkylating agent temozolomide are not fixed
• Good therapeutic response
• Longer survival of patients with glioblastoma
 Mismatch repair (MMR)

- ensures the correction of errors caused during DNA replication - inclusion of the wrong
nucleotide.
- the MMR pathway is highly evolutionarily conserved. It was first described in more detail in
yeast and the identified genes were named MutS, MutHa MutL. Their homologues also occur in
humans (e.g. MSH2, MutS homolog 2).
- In humans, MMRs occur simultaneously with replication to ensure that a newly emerging DNA
strand with a mismatched nucleotide is repaired.

Mismatch

Excision,
DNA synthesis,
ligation

Insertion/
deletion loop
 Postreplicative mismatch repair (MMR) and Lynch syndrome

Congenital defects in the genes of the MMR

system are the basis of a hereditary tumor
syndrome called hereditary non-polyposis
colorectal cancer (HNPCC, Lynch's syndrome),
which accounts for 2–5% of all cases of colorectal
cancer.

A characteristic feature of the impaired function of

the MMR system is the so-called microsatellite
instability (MIN) - tandem repeats of one to six
nucleotides, the length of which remains stable in
healthy individuals.
In people with HNPCC, microsatellites are unstable
and their length varies - used in diagnosis.
Excision repair – base excision repair (BER)

Based on excision of the wrong section of DNA and its replacement by the correct section.
Base excision repair is based on excision of only one base or nucleotide.

- DNA glycosylase enzymes - recognize a specific type of modified base and catalyze its
hydrolithic removal.
- At least six types of glycosylases
- They test each base separately by turning it out of the chain
- AP endonucleases - detect blank space after base removal by glycosylase (AP site)
- It cleaves the phosphodiester bond and corrects the gap

Repair of minor damage - oxidative damage, deamination and alkylation of the base, AP
site (abasic site - site where the base was removed).

Despite the key role of BER in maintaining genome stability, hereditary syndromes or
diseases caused by mutations in individual genes of the BER system have not been
described. This may be partly due to the relative redundancy of DNA glycosylases, partly
due to the fact that mutations in essential genes of the BER system lead to embryonic
lethality or are yet to be discovered.
Excision repair – nucleotide excision repair (NER)
Cutting out a larger section of several nucleotides.
Repair of DNA adducts, dimers after UV irradiation, after treatment with some cytostatics and helical
disruptors in general

Nucleotide excision repair

in bacteria - the multienzymatic complex recognizes lesions (eg pyrimidine dimers)
- cleaving of the strand on both sides of the lesion
- DNA helicase in the complex removes part of the strand including the
dimer (approximately 12 nucleotides)

in humans - upon recognition of the damaged strand, the DNA helicase locally
unwinds the DNA duplex and the excision nuclease cleaves a region of
approximately 30 nucleotides, including the dimer.

The NER repair mechanism recognizes many different types of damage.

Excision repair – nucleotide excision repair (NER)

- In addition to chemical base changes, NER activation also distorts the normal
structure of the double helix caused by UV radiation
- Complex system, two subsystems:
- Global genome repair (GGR) - a slow continuous repair of the entire
genome. Significant role of seven proteins called XPA-XPG (discovered
in the patients with Xeroderma pigmentosum).
- Repair initiated by XPC protein binding.
- Transcription coupled repair (TCR) - repair of defects on the template
DNA strand
- repair activated when RNA polymerase II stops at the site of
injury - CSB protein (Cockayne syndrome B) binds - binding of
transcription factor TFIIH (among other subunits XPB, XPD) and
XPG protein - local unfolding of DNA - XPA protein and RPA
stabilize DNA, attract nucleases ERCC1 and XPF - defect excision,
followed by DNA synthesis, ligation.
 Diseases associated with defective NER

Occurrence in the population of about 1-4 / million for each of them.

All are autosomal recessive - both alleles of a given gene must be mutated in order for the
relevant DNA repair mechanism to be knocked out of function.

Xeroderma pigmentosum - 7 different genes (XPA-XPG) Disorders of DNA damage repair after
UV radiation
- Skin damage, frequent skin tumors, neurological abnormalities, visual
disturbances

Cockayn syndrome - several genes (CSA, CSB)

- Nervous system development and growth disorders, sensitivity to light, visual
disturbances, premature aging; there is no increased risk of developing tumors.

Trichothiodystrophy - several different genes (TTDA, XPB)

- Fine and brittle hair, mental and growth retardation, receding loin, hardened skin,
sensitivity to light; there is no increased risk of tumor development

- NER is associated with transcription - very important in embryonic development to ensure

that transcription and thus cell differentiation will proceed smoothly and without errors -
therefore some disorders of NER mechanisms lead to developmental defects

L. Krejčí, Biology Lecture, LF MU

Single-strand breaks DNA repair

Single-strand breaks can be formed indirectly (indirect SSB) as an

intermediate of base excision repair (BER), or directly, for example, after
ROS-based damage (direct SSB)

Key repair regulators are poly (ADP-ribose) polymerases 1 and 2 (PARP-1

and PARP-2), with PARP-2 being responsible for only 10-15% of total DNA
damage-stimulated PARP activity.
PARP-1 is a highly conserved enzyme which, after binding to damaged DNA,
activates covalent automodification, catalyzing the transfer of ADP-riboses
from NAD (nicotinamide adenine dinucleotide) to its own molecule, but also
to H1 histones, whose modification loosens the chromatin structure and
allows the damaged site to access to other proteins necessary for DNA
repair.

The figure shows the process of repairing indirect breaks (a-g) and direct
breaks (b-h). Under certain circumstances, polymerase β may be involved in
the repair of both long and short paths (patch). Direct single-strand breaks
(sugar-phosphate backbone damage) also occur during BER, PARP-1 and
PARP-2 are bound, and gaps after cleavage of the damage-site can be filled
with either Polβ or Polδ / ϵ.
 Double-strand breaks DNA repair

DNA repair triggered by ATM kinases (DSB response) and ATR (SSB
response at the site of replication arrest)
- phosphorylation of other target molecules:
- histone H2AX - chromatin release
- CHK2 kinases, CHK1 – checkpoint kinases, further
phosphorylation (eg p53) and signaling leading to cell cycle arrest /
senescence / apoptosis.

Mutations in ATM
- ataxia telangiectasia disease - DSB recognition disorder
- neurodegenerative diseases, increased risk of acute lymphoblastic
leukemia, lymphoma, radiosensitivity

Mutations in CHK2 - Li-Fraumeni syndrome, familial breast cancer

and some sporadic tumors
 Double-strand breaks DNA repair

Double-strand breaks can be repaired by homologous recombination

- according to the sister chromatid as a template (therefore only applicable to dividing
cells, in the G2 phase of the cell cycle).

DSBs are recognized by the MRN complex (MRE11 + RAD50 + NBS1), which is a
substrate of ATM kinase - 5´-3´ exonuclease activity - the formation of single-chain
overlaps covered with RPA protein.
Unclear involvement of BRCA1 / 2 proteins, binding site for RAD51, stimulation of the
formation of the nucleoprotein complex RAD51, which subsequently searches for its
homologous sequence (sister chromatid) as a template for complementation.
During synthesis and ligation, a Holliday junction is formed, which is finally disrupted
by BLM helicase and topoisomerase 3α.

Non-dividing cells usually repair non-homologous fractures by means of non-

homologous end joining (NHEJ), which is, however, mostly erroneous.
The process began with very rapid recognition of the DSB by the heterodimer Ku70-Ku80
and binding of the DNA-PK kinase. The ends are ligated by XRCC4 complex with DNA
ligase IV.
Deficiency of the NHEJ system does not necessarily lead to an increased risk of
developing tumors.

DNA
synthesis/ligation

Two Holliday junctions

 Diseases associated with defects in double-strand break repair

Predisposition to breast and ovarian tumors

• Mutations in the BRCA1 or BRCA2 gene (= breast cancer type 1 or type 2
susceptibility protein), RAD51C, RAD51

Ataxia telangiectasia
• Occurrence in the population of about 10-20 / million
• Autosomal recessive, mutations in the ATM gene (ataxia telangiectasia mutated)
• Neurodegenerative diseases with various symptoms manifesting from childhood

Other syndromes (rare) associated with mutated genes for proteins involved in DSB
repair and recombination:
• Bloom's syndrome, Werner's syndrome, Seckel's syndrome, Nijmegen
breakage syndrome

In general, disorders in DSB repair can lead to chromosomal structural

aberrations, excessive cell death, fertility disorders, immune disorders
(mutated Artemis protein gene found in combined immunodeficiency
syndrome), mutations in the ku70 and Ku80 protein genes in mice cause
premature aging, etc.

L. Krejčí, Biology Lecture, LF MU

 Tolerance repair mechanism (translesion synthesis)

When DNA damage is an obstacle to replication, it is more advantageous

for the cell in a given situation to bridge the damage and replicate the
DNA and complete the division of the cell with the damage (even at the
cost of creating a cell with a mutation in DNA).

Bridging of damage during replication is enabled by special DNA

polymerases called translesion DNA-polymerases (types of DNA
polymerases that are able to synthesize over a damaged DNA region,
where common types of polymerases would stop)
- but they are error-prone (introduces mutations into DNA )

L. Krejčí, Biology Lecture, LF MU

Inherited human disease caused by defects in DNA repair
gfdgdfgdgfgdfgdgdfgf
DNA repair mechanisms and introduction of the errors

Most DNA repair mechanisms enable DNA repair without introducing other errors
(„error-free“ repairs) according to the complementary DNA strand or a sister
chromatid. These repair mechanisms are frequently preferred by cells.

- e.g., direct reversal, excision repairs (NER, BER), mismatch repair (MMR),
homologous recombination (HR).

But in some cases, the cell does not contain the sister chromatid or non-mutated
complementary DNA strand usable as a template for the DNA repair; or the speed
of the DNA repair is more important than being error-free. In that case the cell uses
DNA repair mechanisms which are prone to introduction of new errors in DNA
(„error-prone“ repairs).

- such as non-homologous end joining (NHEJ), translesion synthesis (TLS)

 Cancer is caused by an accumulation of genomic alterations

All cancers arise as a result of changes in the DNA of cancer

cells, though not all of these changes present in a cancer
would have been involved in its development. These are so-
called ‘passenger mutations
A ‘driver mutation’ is causally implicated in cancer; it has
conferred a growth advantage on the cancer cell and as
such is implicated in oncogenesis

The tumor cell genome typically consists of

10 000 passenger mutations
5-10 driver (biologically relevant ) mutations
1-2 “actionable” (clinically-relevant) mutations

Stratton, M.R. (2013) EMBO Mol Med. 5(2):169-72.

Patient case 3: Secondary glioblastoma

Patient presentation, initial diagnosis and treatment

Jan 2014
Patient profile

• Male, 14 years old

• Diagnosis: bifocal invasive colon adenocarcinoma
(G2, pT2, pN1a, pM0, IIIA)
• Mutations: KRAS
• Treatment: hemicolectomy + FOLFOX
1. CCR OS 58 months

…but a very strange tumour for a child...

Case courtesy of Jaroslav Sterba, University Hospital Brno, Czech

Republic.

37
 Patient case 3: Secondary glioblastoma

Medical background

Germinal exome sequencing identified biallelic CMMR-D: Constitutional mismatch repair-deficiency

• Both parents: healthy carriers syndrome (CMMR-D)
• Both sons: composed heterozygotes • Syndrome related to changes in
MLH1, MSH2, MSH6, PMS2 or EPCAM
genes
Gene Variant • Frequent and early occurence of colon
PMS2 c.2521delT/p.W841fs cancer, haematological malignancies and
c.2T>A/p.M1K brain tumors (malignant gliomas, high
MET c.2975C>T/p.T992I grade glioma)
c.1792C>T/p.H598Y • Typically TMB-high
NTRK1 c.1820G>T/p.G607V

MYC c.1213G>T/p.A405S

CMMR-D: constitutional mismatch repair deficiency syndrome;

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TMB: tumour mutational burden.
CZ/ONCO/0917/0113
 Patient case 3: Secondary glioblastoma

Diagnostic work-up on proven CMMR-D

Jan 2014 Mar 2017

Secondary malignancy glioblastoma diagnosed

Diagnostic MRI Histopathology

PNET component
Based on
proven
bCMMR-D
follow-up,
paradigm
changed to
whole body
MRI

Glial component

CMMR-D: constitutional mismatch repair deficiency syndrome;

MRI: magnetic resonance imaging;
PNET: primitive neuroectodermal tumour.
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 Patient case 3: Secondary glioblastoma

Treatment decision and early progression

Jan 2014 Mar 2017 April 2017

MRI scans revealed

early progression
• Subtotal tumour
resection with early
postoperative
progression

• Radiotherapy +
concomitant
alkylating
chemotherapeutic agent
/metronomic schedule/

MRI: magnetic resonance imaging.

40
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 Patient case 3: Secondary glioblastoma

Genomic profiling performed

Germline exome analysis Somatic exome analysis

• DNA purified from peripheral blood • DNA purified from FFPE sample

• Whole dataset (exome) evaluated • Whole dataset (exome) evaluated +

• Variants were filtered on the FoundationOne® gene

set (315 genes)

Exome analysis methods: TruSeq® Exome kit, NextSeq 500 (Illumina®)

FFPE: formalin-fixed paraffin-embedded.

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 Patient case 3: Secondary glioblastoma

Somatic mutations in glioblastoma sample

Gene Variant Gene Variant

PIK3CA c.1360G>T/p.D454Y BRCA2 c.52C>T/p.R18C
c.2422C>T/p.R808W c.5292dupA/p.S1764fs
c.2746C>T/p.R916C c.6952C>T/p.R2318X
PIK3R1 c.37G>A/p.G13R DNMT3A c.2162C>T/p.A721V
c.418C>T/p.R140W c.1679G>A/p.R560H
PDGFRA c.863A>G/p.Y288C c.1492G>A/p.V498I
c.1715A>C/p.Y572S c.233C>T/ p.S78F
c.3265C>A/p.L1089M TP53 c.448C>T/p.R150W
PDGFRB c.2765G>A/p.R922H
RET c.2437C>T/p.R813W
KDR c.3352C>T/p.R1118X
PTEN c.180G>T/p.K60N
c.2830C>T/p.R944X c.922C>T/p.R308C

• MSI-high
• TMB-high (384.23 / MB)

MSI: microsatellite instability; TMB: tumour mutational burden.

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Tumor mutational burden (TMB) is predictive biomarker of response to
immunotherapy
TMB IS THE NUMBER OF SOMATIC MUTATIONS IN TUMOR GENOME EXPRESSED RELATED TO 1 MB OF DNA (mut/Mb)

The origin of somatic mutations in tumor DNA is multifactorial

-includes disorders in DNA repair systems, carcinogens, disorders in the
activity of DNA polymerases.

Some of these mutations lead to the formation of surface neoantigens

that induce an anti-tumor immune response.

Mutation load can be determined by whole genome or whole exome

sequencing (WES) possibly using sequencing panels focused on tumor-
relevant genes (in the order of hundreds of genes).

The determination of the mutation charge is influenced by a number of

experimental and bioinformatics factors (width and definition of the
sequenced area, methodology of filtering variants).

The mutation charge correlates with the response on immunotherapy

checkpoint inhibitors.

Braun DA et al. Clin Cancer Res 2016

TMB correlates with the number of neoantigens -> TMB correlates with
immunotherapy response
High TMB is thus associated with a higher rate of activation
immune responses.

The immune response checkpoint at the PD-L1 / PD1 level is a

critical factor in the extent of the immune response.

Sharabi et al. Oncologist 2017

TMB differs in various tumors

Chung-Han Lee et al, Trends in Immunology 2018

 Patient case 3: Secondary glioblastoma

Molecularly-guided therapy
Prior immunotherapy 3 months 6 months 18 months

Based on high TMB: immune

checkpoint inhibitor (anti
PD-1) + autologous dendritic
cell vaccine* producing IL-12
+ valproic acid +
metronomic low dose
alkylating agent

46

* EudraCT number 2014-003388-39.

TMB: tumour mutational burden.
.
CZ/ONCO/0917/0113

CZ/ONCO/0917/0113
 Patient case 3: Secondary glioblastoma

Patient follow-up

Prior immunotherapy Recent follow-up (week 73)

• Gradual response on FLT-PET MRI

• Decreasing tumour size and PET avidity
• Able to attend school (outpatient treatment)
• Karnofsky 90

FLT-PET: fluoro-L-thymidine-positron emission tomography;

MRI: magnetic resonance imaging;
PET: positron emission tomography.
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 Cíle přednášky – Nestabilita genomu

Types of DNA damage. Spontaneous mutations in DNA - replication errors. Induced mutations
in DNA - physical and chemical mutagens. Principles of action of mutagens on DNA. Barriers
protecting genome stability. DNA damage sensors (ATM and ATR). Tumour suppressor p53.
DNA repair mechanisms. Base mismatch repair and microsatellite instability. Nucleotide
excision repair. Base excision repair. Repair of double-stranded DNA breaks. Homologous
recombination. Non-homologous ends joining. Chromosomal instability and aneuploidy.
Diseases associated with DNA repair disorders (xeroderma pigmentosum, Fanconi anaemia,
cancer). DNA repair as a target of anticancer treatment.

Figures adapted from (if not stated otherwise):

ALBERTS, Bruce. Základy buněčné biologie : úvod do molekulární biologie buňky. Translated by Arnošt Kotyk. 2. vyd.
Ústí nad Labem: Espero Publishing, 2004. xxvi, 630. ISBN 8090290620

D. Peter Snustad Michael J. Simmons. Genetika. Brno, 2017. ISBN 9788021086135

Thank you for your attention –
any questions?