1. Chromosomal abnormalities and tumors Seminar No 524, course: Cells and tissues development

2. Key words: chromosomal aberrations, translocation, amplification,double minutes, HSR (homogenously staining regions), genotoxicity, DSB (double strand breaks), fused gene, LOH („loss of heterozygosity“), LOI („loss of imprinting“), chronic myelogenous leukemia (CML), Burkitt lymphoma (BL), retinoblastoma (RB), Wilms tumour (WT), syndromes with increase chromosome breakage = chromosomal instability syndromes: Bloom syndrome (BS), Fanconi anemia (FA), Ataxia teleangiectasia (AT), Xeroderma pigmentosum (XP), Nijmegen breakage syndrome (NBS), syndromes of prematury ageing: Werner syndrome, Cockayne syndrome

3. Structural chromosomal aberrations (CHA) = result of unrepaired or misrepaired DNA damage = early biological effect of genotoxicity Late biological effect of genotoxicity = tumors Primary event leading to CHA origin are double-strand breaks (DSB)

4. Primary changes connected with initiation of malignant process: Rearrangement changing position 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)

5. Primary changes connected with initiation of malignant process: 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 is transfered from 9q to 22q near the gene bcr  fused gene bcr/abl  abnormal product=chimeric protein with increased tyrosinkinase activity Ph1 in CML good prognosis during blastic crisis - other chromosome changes In B-ALL (acute lymphoblastic leukemia) other site of break in bcr Ph1 in ALL = bad prognosis

6. Wysis katalog 1996/97 Fused gene brc/abl detected by FISH method in interphase cell t 9/22

7. 2. Translocation of protooncogenes to the 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 – near genes for heavy chains of immunoglobulins  abnormal stimulation of gene activity  abnormal amount of normal product Other translocations: t(8;22) or t (2;8) – to neighbourhood of light chains of immunoglobulins T-lympho malignancies - breaks near genes for T-cells receptors

8. Translocation produces premalignant clone – probably other genetic changes (mutations, epigenetic changes..) are necessary for full malignancy This changes (translocations, inversions involving protooncogenes) are present only in malignant or premalignant cells, it is not constitutional change (present in all cells) !!!

9. Primary events: 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 familiar Rb – 1st step - germinal mutation or deletion in all cells of body = heterozygote (constitutional abnormality) 2nd step : mutation in one cell of retina = loss of heterozygosity (LOH) del(13)(q141-142) sporadic Rb – both mutations in one cell of retina heterozygosity for mutation or deletion = predisposition to tumor

10. Retinoblastoma heritable sporadic heterozygote Mutation of second allele in one somatic cell = loss of heterozygosity Mutation of both alleles consecutively in one somatic cell → → →

11. Interstitial deletion 11p Wilms tumor = nephroblastoma WT1 locus on 11p13 mutation or deletion isolated or a part of syndrome WAGR association (Wilms, aniridia, urogenital anomaly, mental retardation)

12. Deletion of one allele of tumor suppressor gene can be constitutional aberration, present in alll cells of body - heterozygote

13. Gain of material Amplification of oncogenes: „double minutes“ = amplified circular oncogenes (in solid tumors) HSR (homogenously stainin regions) = amplification and recombination of oncogenes into chromosome tandemly or into different sites Amplification especially in solid tumors Targeted therapy: Herceptin =monoclonal antibody against ERBB2 oncogene (=Her2/Neu= tyrosin-kinase receptor) in women with breast cancer and amplification of oncogene

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

15. Tetraploid nucleus with amplification of Her2/neu

16. Trisomy and tetrasomy in cells of breast tumor

17. Chromosome loss in cells of breast tumor

18. Secondary changes – consequences of malignancy (genome instability in tumor cell): Losses or gains of whole chromosomes, structural chromosomal rearrangements

19. UroVysion Bladder Cancer FISH Probe Panel CEP 3 (spektrum red) CEP 7 (spektrum green) CEP 17 (spektrum aqua) LSI p16 (spektrum gold) = 9p21- tumor suppressor gene (p16)

20. Chromosomal changes in bladder cancer cells – FISH method (a) normal cell (c) Homozygous 9p21 loss (b) Trisomy 7

21. Karyotype of breast cancer cells from tissue culture – G-bands

23. Chromosomal changes after radiotherapy, chemotherapy: Breaks and rearrangements – detected in peripheral lymphocytes Aberrations after irradiation - chromosome: dicentrics, tricentrics, chromosome breaks (on both chromatids), ring chromosomes Aberrations after chemicals: – chromatid: chromatid breaks, chromatid exchanges

24. Dicentric chromosome + difragment Aberrations after irradiation

25. Dicentric chromosome Aberrations after irradiation

26. Ring chromosomes and difragments Aberrations after irradiation

27. Aberrations after bleomycine (BLM) in vitro BLM was added for the last 5 hours of cultivation

28. Chromatid breaks

29. Chromatid break

30. Multiple breaks

31. Chromatid exchange

32. and breaks

33. Chromatid exchange and breaks

34. Irradiated mouse cells in tissue culture

35. Questions: Explain why translocations in somatic cells sometimes lead to cancer? What is Ph 1 chromosome? Can you describe cytogenetic manifestation of oncogene amplification? Which chromosomal aberrations are primary events in malignancy? Explain mechanisms of activation of protooncogene to oncogene. Explain mechanisms of inactivation of alleles of tumor supressor gene. Describe origin of heritable and sporadic retinoblastoma. State expamples of malignancy connected with deletion of tumor suppressor gene. Describe chromosomal abnormalities secondary to malignant process. Describe translocation leading to origin of fused gene. Describe translocation leading to abnormally increased synthesis of product.

36. What is the cause of Li Fraumeni syndrome? Describe chromosomal aberrations followed in peripheral lymphocytes of patients after chemotherapy, radiotherapy. What is the role of viruses in tumor origin? Describe difference between oncogene of DNA tumor viruses and RNA tumor viruses.