Cancer cytogeneticsCancer cytogenetics

5th year

seminar

RNDr Z.Polívková

Cancers = heterogenous diseases – initiation

and progression are promoted by

aberrant function of genes, that regulate DNA repair, genome stability, cell proliferation, cell death, cell adhesion, angiogenesis, invasion and metastasis – so called „ cancer genes“

Cancer is caused by stepwise accumulation of

numerous genetic and epigenetic changes

Driving force of tumorigenesis - genomic instability

= increased tendency to alteration in genome

Major mechanisms involved in maintaining the genomic stability1. Fidelity of DNA replication (S- phase)2. Accurate segregation of chromosomes (mitosis)3. Precise repair of DNA damage (throughout cell cycle))4. Cell cycle check point

4.Cell cycle check points:- G1/S check points- G2/M check points- Intra S check points- Spindle check points -Post mitotic check points

1. Fidelity of DNA replication:- DNA polymerase- Mismatch repair- Replication licensing- Maturation of Okazaki fragments- Restart of stalle replication fork- Telomere maintenance- Preservation of epigenetic signatures

2.Segregation of chromosomes in mitosis:- Chrom. condensation- Sister chromatid cohesion- Kinetochor assembly and attachment- Centrosome duplication and separation- Spindle formation-Chromatid segregation- Cytokinesis

3. DNA repair- DNA damage signalling- DNA repair pathways: Excision repair: NER, BER Repair of double strand breaks: HR, NHEJ

According Shen, 2011

Overview of the major mechanisms to maintain genomic stability during the cell cycle

Deregulation of responsible genes leads to genomic instability

Interindividual variability in cancer expression due to:

• differences in the amount of DNA damage

• capacity to repair DNA damage

Both influenced by genetic predisposition and by environmental

factors, including life-style.

Individual response to exogenous and endogenous genotoxins

due to genetic polymorphisms:

• of xenobiotic-metabolizing enzymes

• of genes of DNA repair or genes of folate metabolism

= „low penetrant genes“

Approx. 350 genes is connected with tumors

„Cancer genes“: • protooncogenes• tumor suppressor genes

DNA repair genes - mutator genes

Gene functions can be influenced by: • gene polymorphisms

• alteration of copy number (amplifications, deletions,

duplications, changes in chromosomal number)

• changes of gene structure, chromosome structure

(translocations, inversions etc.)

• gene mutations (substitution, deletion, insertion in coding

sequences or splicing sites)

• epigenetic modifications (imprinting, DNA methylation and

histone modification – histone acetylation/deacetylation,

methylation or phosphorylation)

Activation of oncogenes (change of protooncogene to oncogene) through:

• mutation

• structural rearrangement (reciprocal translocation, inversion)

• amplification (double minutes or HSR=homogenously staining regions)

• epigenetic changes

• virus insertion

Inactivation of tumor suppressor genes through:

• mutation

• deletion

• epigenetic modification

• mitotic recombination

v

Prokarcinogene

Metabolic.activationIst phase enzymes

Ultimative carcinogen

Normal cell

Iniciated cell

Preneoplasticcells

Tumor cells

DetoxicationIInd phase enzymes

Iniciation1-2 days

Promotion10 years

Progression 1 year

Iniciation/promotion theory of tumor origin

CHA and tumors

1. Specific CHA in tumors - CHA is primary event in tumor origin

rearrangement in neighborhood of protooncogenes:

- abnormal activity of product

- abnormal gene expression

rearrangement only in tumor cells (chronic myelogenous leukemia, Burkitt

lymphoma)

- deletion of tumor suppressor genes

in tumor cells or constitutional aberrations (heterozygosity) (e.g. retinoblastoma)

2. Heritable syndromes with increased chromosome breakage

defect of reparation or replication high risk of malignancies

Chromosomal study in tumors:Role: - diagnosis and subclassification of haematologic malignancies

- rational selection of therapy, targeted therapies

- prognostic informations

- monitoring of treatment effect, residual leukemia ..

- study of mechanism of carcinogenesis

CHA in tumors:balanced without loss or gain of material :

translocations, inversions

unbalanced: with loss of material: deletions, monosomies

with gain of material: duplications, trisomies, polyploidies,

amplifications

Primary changes connected with initiation of malignant process

Secondary changes – connected with progression of disease, with

genome instability

Chromosomal aberrations as primary changes connected

with initiation of malignancy

Translocations – 2 types of translocations

1. Translocations leading to fused genes (genes with function in cell

division regulation or differentiation)

Ph1 chromosome in chronic myelogenous leukemia (CML)

= reciprocal translocation 46,XX or XY,t(9;22)(q34;q11)

protooncogen abl transfered from 9q to 22q near the gene bcr

fused gene bcr/abl abnormal product = chimeric protein with

constitutively active tyrosin kinase activity –

breaks in introns of genes Ph1 in CML good prognosis during blastic crisis another chromosome changes In ALL (acute lymphoblastic leukemia) other site of break in bcr gene Ph1 in ALL = bad prognosis

Cme.medscape.com

Wysis 1996/97

Fused gene brc/abl

Other examples of fused genes:

ALL t(1;19) good prognosis

der(19)t(1;19) bad prognosis

t(12;21) good prognosis

acute promyelocyt.leu (M3) t(15;17) good prognosis

acute myelocytic leu (M2) t(8;21) good prognosis

ALL and AML t(4;11) bad prognosis

2. Translocation of protooncogenes to position, where they are

abnormally stimulated to transcription

Burkitt lymphoma (BL) – B lymphocytes

t(8;14)(q24;q32) also in other lymphomas

protooncogen myc transfered from 8q to 14q – next to promotor of

broken gene for heavy chain of immunoglobulin abnormal

stimulation of gene activity abnormal amount of normal product

t(8;22) or t (2;8) – next to strong promotor of genes for Ig light chains

T-lympho malignancies - breaks near genes for T-cells receptors

Restricted to cells in which genome undergoes somatic rearrangement (e.g.VDJ

recombination of Ig genes) as a part of process of maturation to effector cells

(B,T lymphocytes)

ncbi.nlm.nih.gov

Translocation produces premalignant clone – probably other genetic

changes (mutations, epigenetic changes..) are necessary for full

malignancy

Translocations (balanced) are relatively frequent cause of

malignancies

Most of translocations or inversions were detected in haematologic

malignancies,

In solid tumors translocations are less frequent (and rearrangements are

more complex)

Fused genes encoding: • transcription factors necessary for haematopoetic differentiation –

chimeric product of fused gene increases aberrant transcription or represses transcription of genes involved in

differentiation

e.g. product of fused gene PML/RARα = t(15/17) = chimeric receptor activates

histon deacetylase complex* → transcription repression of genes for myeloid differentiation

→ accumulation of immature myeloid cells in acute promyelocytic leukemia

• tyrosine kinases (regulators of proliferation) – fused gene product = chimeric protein – constitutive activity – uncontrolled cellular proliferation

•Histon acetylation removes positive charge of histones – it decreases interaction with negatively charged DNA in open (active) chromatinHiston deacetylation restore positive charge leading to tight interaction DNA with histones and condensation of chromatin to inactive state - inaccesible to transcription factors

CLINICAL UTILITY OF TRANSLOCATIONS:

Targeted therapy:

e.g. first successfully targeted therapy = Imatinib (Gleevec) = tyrosin kinase

inhibitor - good response to treatment in patients with

bcr/abl fusion (t 9/22)

acute lymphoblastic leukemiae (ALL), acute myeloid leukemia (AML)

-second-generation bcr/abl inhibitors = dasatinib, nilotinib

(in resistancy to Imatinib)

All-trans retinoid acid (ATRA) effective in patients with fusion PML/RARα

(t15/17) in acute promeylocytic leukemia (APL)

ATRA reverse transcriptional repression by disrupting interaction of

PMR/RARα protein with histon deacetylase complex that promotes

transcriptional repression

Origin of fused genes and other chromosomal rearrangements:

Critical lesions = DSB (double strand breaks) - caused by exogenous

(radiation, chemicals) and endogenous factors (reactive oxygen species..)

DSB also consequences of normal cell processes as V(D)J recombination of

B cells and T cell receptor genes, class switching, meiotic recombination …

missrepair of double strand breaks – aberrant recombination

Specific fusion – influenced by position of chromosomes in interphase and by

the presence of sequentional homology in the sites of breaks

role of „ fragile sites“ (=sites of genome instability)

„ Fragile sites“ and tumors

„Fragile sites“ (FS)

• sites of genome instability on chromosomes

• late replicating

• nonrandom loci – disposed to breaks and exchanges

• manifested as gap or break under condition of replicative stress (i.e.inhibition of DNA synthesis by aphidicoline, 5.azacytidine, BUdR)

FS – common - rare in < 5% of population (connected with expansion of triplet repeats, e.g.FRAXA)

In sites of common FS tumor supressor genes and protooncogenes

are located

Common FS = target site of mutagenes/carcinogenes action, site of

integration of oncogene viruses

52% of all translocations in tumors have sites of breaks in FS (Burrow et al. 2009)

CHA as primary event in initiation of malignancy:

Deletions of tumor suppressor genesRetinoblastoma (Rb) – eye cancer of children

heritable type (familiar or „de novo“ origin) - AD (with reduced

penetrance)

sporadic type – nonheritable – usually afects only one eye

• heritable Rb – 1st step - germinal mutation or deletion in all cells of body

= heterozygote (constitutional abnormality)

2nd step: somatic mutation in one cell of retina = loss of

heterozygosity (LOH)

loss of heterozygosity by somatic recombination

del(13)(q141-142)

• sporadic Rb – both mutations somatic in one cell of retina

Heterozygosity for mutation or deletion = predisposition to tumor

Imprinting of tumor suppressor gene = only single functional copy

Retinoblastoma

HeritableRB/rb or RB/-Mutation or deletion

Sporadic

heterozygote

Mutation of second allele in one somatic cell = loss of heterozygosity

Mutation of both alleles consecutively in one somatic cell

→

→ →

Loss of function: mutation, deletion, loss of whole chromosome, mitotic recombination

Knudson´s two-hit hypothesis of loss of function of tumor suppressor gene

heterozygosity

replicationmitotic recombination

chromatid segregationIn mitosis

Heterozygosity in daughter cells

Loss of heterozygosity

Loss of heterozygosity by mitotic recombination

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Interstitial deletion 11p

Wilms tumor = nephroblastoma

WT1 locus on 11p13 mutation or deletion

Wilms tu = isolated or a part of

syndrome - WAGR association

(Wilms, aniridia, urogenital anomaly,

mental retardation)

Deletions of tumor suppressor genes –esp. in epithelial tumors

Gain of material

Amplification of oncogenes:

• „double minutes“ = amplified circular oncogenes (extrachromosomal)

• HSR (homogenously stainin regions) = amplification and recombination of oncogenes

tandemly to chromosome

• or insertion of amplified sequences to different sites on chromosomes

Amplification especially in solid tumors:

e.g.: N-myc in neuroblastoma, cyclin D1 in many tumors (carcinoma of oesophagus), cyclin D2 in ovarian and testicular cancers

Amplification (gene overexpression) often includes more

than one gene – probably contribute to tumor phenotype

Targeted therapy: Herceptin (trastuzumab)=monoclonal

antibody targeted to Her2/ERBB2 oncogene (= tyrosin-

kinase receptor) in women with breast cancer and

amplification of this oncogene

Amplification of some „cancer genes“ is associated with

therapeutic resistance (e.g. amplification DHFR gene connected

with resistance to methotrexate, amplification of bcr/abl gene

in CML patients resistant to imantinib/Gleevec, amplification of

gene for androgenic receptor in prostate cancers resistant to

endocrine therapy)

Imprinting and tumors

Tumors: - inhereted or induced mutations of protooncogenes, tumor supressor genes

- epigenetic changes = changes in methylation (imprinting) of these genes

Imprinted protooncogenes – error in imprinting → activation

of imprinted allele (biallelic expression) = oncogenes

Imprinted tumor supressor genes – loss of function of

one allele only (active allele) = loss of gene function

only 1 step needed for loss of function = increased sensitivity to tumors

Polymorfism of imprinting of some genes in population

e.g.tumor supressor genes WT1(11p13)

IGF2R(6q26)=receptor for IGF2 (used for intracellular degradation of IGF2)

usually biallelic expression, but in some peoples monoallelic (=imprinted)

imprinting of these genes = predisposition to tumors

Methylation = reversible process – possibility of therapy of

tumors caused by aberrant methylation??

Chromosomal changes as a consequences of malignant

process, genomic instability

→ deregulation of genes resposible for segregation of chromosomes or cytokinesis – changes in chromosome number

Gains of material: intragene duplications, duplications of genes,

groups of genes, chromosomal parts or whole chromosomes

Duplication, trisomy : e.g.+8 in blastic crisis in CML, ANLL, MDS

Hyperdiploidiy, polyploidy – hyperdiploidy in ALL = good prognosis

but hypodiploidy = bad prognosis

Loss of genetic material: deletions inside genes, deletions of whole genes, groups of genes, chromosomal parts or whole chromosomes - deletions of tu su genes, loss of noncoding genes (e.g.micro RNA –role in posttranscriptional regulation of gene expression)

Loss of genetic material = loss of tumor suppressor genes,

loss of micro RNA encoding genes (miRNA - role in

posttranscriptional regulation of gene expression)

Gain of genetic material = gain of (proto)oncogenes

Oncogene Her-2/neu amplification in breast cancer cells– FISH method

Trisomy and tetrasomy in cells of breast tumor

Chromosome loss in cells of breast tumor

New methods of chromosomal study in tumors:

FISH, comparative genomic hybridization and variants of

these methods (mFISH, m-band, array CGH,..)

Array CGH: comparison of tested and normal DNA, both stained

by different fluorochromes – mixture of both is applied to a slide

with thousands of spots of reference DNA sequences to hybridize

- gains and losses of genetic material are detected by computor

as spots of different colours

Specific aberrations = markers for prediction of

disease outcome or response to treatmennt

Identification of genes for therapy or prevention

!! Complex, multiple changes = bad prognosis,

bad response to treatment !!

Karyotype of breast cancer cell

Chromosome instability syndromes

Common features:

• AR inheritance

• increased sensitivity to UV light (sun light)

• hyper - or hypopigmentation

• small stature

• defects of immunity

• increased sensitivity to radiation, chemical mutagens (breaks, chromosome exchanges, sister chromatid exchanges)

• increased spontaneous level of chromosome aberrations or increased level of CHA after induction by mutagenes

• increased risk of malignancy !!!

error in DNA reparation or replication

Fanconi anemia (FA) panmyelopathy with bone marrow failure leading to pancytopenia

skeletal anomalies (thumb, radius), growth retardation

hyperpigmentation

microcephaly, defect of thumb and radius - in 50% of patients

CHA: breaks and chromatid exchanges (multiradials, komplex changes)

heterogenic: several genes (7genes FANCA-G)

- defect in DNA repair activation

Bloom syndrome (BS) low birth weight, stunted growth

sun sensitivity of the skin

immunodefect (B-lymphocytes)

facial butterfly-like lesions with telangiectasia

most families of Ashkenazi Jewish origin

CHA: breaks, exchanges between homologs, increased level of sister chromatid

xchanges (SCEs)

defect of replication, DSB reparation (DNA helicase-gene BLM)

Ataxia teleangiectasia (AT - Louis-Bar syndrome) progressive cerebellar ataxia, growth retardation

sensitivity to radiation

oculocutaneous teleangiectasia

immune deficiency (cell immunity)

„café au lait“ spots on skin

CHA: rearrangement of chromosomes Nos 7, 14 or 2,22

= sites of T-cell receptors genes and Ig heavy chains genes

defect of DNA repair (ATM gene – protein kinase regulates TP53, signal recognition)

Xeroderma pigmentosum (XP) erythema after UV irradiation of the skin - atrophy,

teleangiectasias

sensitivity to UV and ionizing radiation

skin cancer

CHA: spontaneous level not increased, increased respons to induction of aberrations (UV)

defect of DNA repair (excision, postreplication repair, repair of DNA strand breaks ) 7types: genes XPA-G + XPV

Nijmegen breakage sygrowth retardation, mental retardation

microcephaly, atypic facies

immunodefect

CHA: rearrangement of chromosomes Nos 7, 14

Rearrangement→failure to produce fully functional immunogluobulins

and T-cell receptors → immunodeficiency

Error in reparation of DNA double strand breaks

gene NBS1 (nibrin)

Syndromes connected with premature aging

Werner sy

Cataracts, subcutaneous calcification, changes of skin, premature hair greying,

premature arteriosclerosis

defect of exonuclease and helicase activity, gene WRN

Cockayne sy

Stunted growth, mental retardation, deafness, premature senility

defect of DNA excision repair (NER)

• Willman CH.L. and Hromas R.A.: Genomic alterations and chromosomal aberrations in Human Cancer - Google

• Fröling S.and Dohner H.: Chromosomal abnormalities in Cancer,. N Engl J Med 2008, 359, 722-34

• Mitelman Database of Chromosome Aberrations and Gene Fusions in Cancer (2013). Mitelman F, Johansson B and Mertens F (Eds.),

http://cgap.nci.nih.gov/Chromosomes/Mitelman

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• Thompson &Thompson: Genetics in medicine, 7th ed.

Chapter 16: Cancer genetics and genomics: Oncogenes, Tumor- suppressor genes (including Retinoblastoma,Caretaker genes in autosomal recessive chromosome instability syndromes, Cytogenetic changes in cancer, Gene amplification)

Chapter 6: Principles of clinical cytogenetics:Mendelian disorders with cytogenetic effects, Cytogenetic analysis in cancer

+ informations from presentation