1. Cancer cytogenetics 5 th year seminar RNDr Z.Polívková

2. 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“

3. Cancer is caused by stepwise accumulation of numerous genetic and epigenetic changes Driving force of tumorigenesis - genomic instability = increased tendency to alteration in genome

4. Major mechanisms involved in maintaining the genomic stability 1. 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

5. 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“

6. Approx. 350 genes is connected with tumors „Cancer genes“: protooncogenes tumor suppressor genes DNA repair genes - mutator genes

7. 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)

8. 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

9. v Prokarcinogene Metabolic.activation I st phase enzymes Ultimative carcinogen Normal cell Iniciated cell Preneoplastic cells Tumor cells Detoxication II nd phase enzymes Iniciation 1-2 days Promotion  10 years Progression  1 year Iniciation/promotion theory of tumor origin

10. 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

11. 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

12. 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

13. Cme.medscape.com

14. Wysis 1996/97 Fused gene brc/abl

15. 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

16. 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)

17. ncbi.nlm.nih.gov

18. 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)

19. 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) chromatin Histon deacetylation restore positive charge leading to tight interaction DNA with histones and condensation of chromatin to inactive state - inaccesible to transcription factors

20. 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

21. 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)

22. „ 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)

23. CHA as primary event in initiation of malignancy: Deletions of tumor suppressor genes Retinoblastoma (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

24. Heterozygosity for mutation or deletion = predisposition to tumor Imprinting of tumor suppressor gene = only single functional copy

25. Retinoblastoma Heritable RB/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

26. heterozygosity replication mitotic recombination chromatid segregation In mitosis Heterozygosity in daughter cells Loss of heterozygosity Loss of heterozygosity by mitotic recombination + +

27. 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

28. 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

29. 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)

30. 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

31. 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??

32. 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)

33. 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

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

35. Trisomy and tetrasomy in cells of breast tumor

36. Chromosome loss in cells of breast tumor

37. 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

38. 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 !!

39. Karyotype of breast cancer cell

40. 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

41. 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)

42. 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

43. Nijmegen breakage sy growth 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)

44. 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)

45. 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 --- 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