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Chromosomes and tumors RNDr Z.Polívková Lecture No 523 course: Development of cells and tissues 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“ 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, on boundary of exons and introns) • epigenetic modifications (imprinting, DNA methylation and histone modification – histone acethylation/deacethylation, 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 CHA and tumors 1. Specific CHA in tumors - CHA is primary event in tumor origin rearrangement in neighborhood of protooncogenes: consequences: - abnormal activity of product - abnormal gene expression rearrangement only in tumor cells (e.g.chronic myelogenous leukemia, Burkitt lymphoma) - deletion of tumor suppressor genes in tumor cells or present as 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 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) =abnormal chrom. 22 from 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 stable tyrosinkinase 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, Ph1 in ALL= bad prognosis Cme.medscape.com Fused gene brc/abl Wysis 1996/97 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) - present also in other lymphomas protooncogen myc transfered from 8q to 14q – near genes for heavy chains of immunoglobulins abnormal stimulation of gene activity abnormal amount of normal product similar translocations: t(8;22) or t (2;8) – light chains of Ig (on chrom.2 and 22) 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 of 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 or represses transcription of genes involved in differentiation • tyrosine kinases (regulators of proliferation) – fused gene product = chimeric protein - uncontrolled cellular proliferation CLINICAL UTILITY OF TRANSLOCATIONS: Targeted therapy: ex. imatinib = tyrosin kinase inhibitor - good response to treatment in patients with bcr/abl fusion (t 9/22) „ 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, ex.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 oncogenne 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 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 Heterozygosity for mutation or deletion = predisposition to tumor Imprinting of tumor suppressor gene = only single functional copy Retinoblastoma Mutation of second allele in one somatic cell = loss of heterozygosity heterozygote Heritable RB/rb or RB/Mutation or deletion Sporadic Loss of function: mutation, deletion, loss of whole chromosome, mitotic recombination → → → Mutation of both alleles consecutively in one somatic cell Heterozygosity in daughter cells replication mitotic recombination chromatid segregation In mitosis + heterozygosity Loss of heterozygosity by mitotic recombination + Loss of heterozygosity 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) Gain of material Amplification of oncogenes: „double minutes“ = amplified circular oncogenes (extrachromosomal) HSR (homogenously stainin regions) = amplification and recombination of oncogenes to chromosome tandemly or to different sites Amplification present 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 Targeted therapy: Herceptin =monoclonal antibody against ERBB2 oncogene (=Her2/Neu= tyrosin-kinase receptor) in women with breast cancer and amplification of oncogene Chromosomal changes as a consequences of carcinogenic process: caused by deregulation of genes resposible for segregation of chromosomes or cytokinesis = consequences of chromosomal instability Gains of genetic 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 posttransctriptional regulation of gene expression) - Loss of genetic material = loss of tumor suppressor genes - Gain of genetic material = gain of (proto)oncogenes Oncogene Her-2/neu amplification in breast cancer – 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 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 as spots of different colours by computor !! 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 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) 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) http://dl1.cuni.cz/course/view.php?id=324 presentation http://dl1.cuni.cz/course/view.php?id=324 supplementary text to cytogenetics Thompson &Thompson: Genetics in medicine, 7th ed. Chapter 16: Cancer genetics and genomics: Oncogenes, Tumorsuppressor 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