Genetic analyses in tumor diseases l Developopmnet of tumor genetics (cytogenetics) l David Hanseman (1890) Theodor Boveri (1914) l l l l l 46,XXam Tjio and Levan (1956) Mit5c Nowell a Hungerford (1960) Rowley (1973) Boveri The first theories on the relationship between mitotic disorders and tumorigenesis Discovery of Ph chromozomu …the findings suggest a causal relationship between the chromosome abnormality observed and chronic granulocytic leukemia… A minute chromosome in human granulocytic leukemia. Science 132, 1960, 1497. PC Nowell, DA Hungerford, University of Pennsylvania in Philadelphia 1960 The origin of the Philadelphia chromosome (Ph) and the BCR - ABL fusion gene after translocation Ph chromozome - 95 % patients with CML Chronic myeloid leukaemia (CML) - 25% of all adult leukaemias with an incidence of 1-2 cases per 100,000 population Definition of BCR-ABL fusion gene - NCI Dictionary of Cancer Terms - NCI resistance to apoptosis influence on the cell cycle t(9;22) negative effects can be trated! 1998 - Clinical trilas of STI 571 agens(Glivec – Novartis) - inhibitor of TKs Tyrosine kinase inhibitors - imatinib, dasatinib and nilotinib - a model for modern non-cytostatic treatment of malignant diseases !!Personalized medicine !! Figure 1 from STI571: targeting BCR-ABL as therapy for CML. | Semantic Scholar Database of chromosomal aberrations in cancer Prof. Felix Mitelman mitartwl 398px-Felix_Mitelman l The role of cytogenetic testing in oncology nan integral part of oncohaematological examinations (initial examinations) as well as examinations of some solid tumours ncytogenetic and molecular genetic methods used in oncology are part of the diagnosis and treatment of many malignancies in the sense of: nclarifying the diagnosis, determining the prognosis nstratification of patients, determination of treatment strategy ndifferential diagnosis ntreatment monitoring (prediction of sensitivity to treatment) nmonitoring residual disease after transplantation nprediction of the likely course of the disease nlocalization of proto-oncogenes and tumor suppressor genes n nthe importance of cytogenetic and molecular genetic testing in hematological malignancies is also emphasized in the classification of myeloid certain cancers, where separate entities with specific genetic changes are distinguished n Techniques used for detection of CHAs in tumor diseases lStandard G- banding l lFluorescence in situ hybridization (FISH) - especially I-FISH - specific probes for tumor investigation - deletion, translocation, inversion....!!! l- always set a cut off for a given probe ! l lAdditional methods lMulticolor FISH (M-FISH, SKY) l lMulticolor banding (M-BAND - complex rearrangements lMicrochip technology (array-CGH) G-banding in tumor diseases lbasic examination method - tumor karyotype lwe use cells cultured in vitro, mainly from bone marrow, less from peripheral blood or other types of tissues lcultured in culture medium at 37°C without stimulation lsuccess rate of cytogenetic examination of BM around 80% l150 - 200 bands (G-banding) lbut...worse chromosome morphology, few mitoses l lExamination of karyotype in tumors is still of great importance- we evaluate individual mitoses, we can also detect small clones.... l l Clonal CHAs in tumors lTumor cell populations are composed of heterogeneous cells....important !!!! lMost changes are clonal, but we often find cells with normal karyotypes lThe karyotype of a tumor evolves gradually ....we find several clones - selection advantage !!! l lCLONE l= the same chromosome is missing in 3 mitoses l= 2 mitoses have the same supernumerary chromosome or the same structural aberration l Clonal heterogeneity is hidden in the mix – Wellcome Sanger Institute https://www.sanger.ac.uk/wp-content/uploads/140529-clonal_0.png Genome destabilization and the multistep process of tumor formation lTumor formation is the result of mutations affecting genes: l l1) cell aging (telomerase) l2) cellular proto-oncogenes – le.g. MYC, ERBB, MYB, INT1, RAS, NEU, ABL (activation by insertional mutagenesis, point mutation, translocation, gene amplification) behave in a dominant manner l l3) tumour suppressor genes TP53, Rb1 l mutation, loss of gene or whole chromosome, loss of heterozygosty (LOH), recessive character l l1% of tumours - gametic mutations l99% of tumours - somatic mutations Chromosomal aberrations in tumours - basic classification lPrimary basis for tumorigenesis, a single change, triggers a multistep process of carcinogenesis - e.g. t(9;22) lSecondary secondary occur during the course of the disease, image of the stage of the disease, expression of tumour progression, sometimes a prognostic factor for disease progression lSpecific regularly in a certain type of tumour, the same chromosomes represent a specific tumour marker, genes involved in the tumour process have been identified at the breakpoints - influence on prognosis !!! lRandom they occur randomly, affecting different chromosomes Chromosomal changes in tumours - classification according to mechanism –loss of genetic material –(deletion, monosomy) – –multiplication of genetic material –duplication, amplification, trisomy, polyploidy) – –translocation without loss of material –(translocation, inversion, insertion) l Numerical CHAs in tumor diseases laneuploidy x polyploidy - common in tumours ! l lhyperdiploidy (more than 46 chromosomes) - often better prognosis (ALL, multiple myeloma) ...more active tumor suppressor genes? l lhypodiploidy - less than 46 chromosomes - worse prognosis l lMethods of investigation - classical cytogenetics, I-FISH, flow cytometry, array-CGH (not ploidy) Demonstration of hyperdiploid karyotype in a patient with myeloma - 63 chromosomes Hypotriploid karyotype from patient with extramedular myeloma relapse at the time of diagnosis detected by G-banding. ISCN: 63,X,-X,-X,der(1),+3,+3,+3,-4,-5,-6,-6,-8,-10,-12,-13,+14,-15,-16,+18,-22,+mar,+mar Multiple myeloma: hyperdiploid subgroup non hyperdiploid subgroup Summary of whole genome profiles in 51 patients with multiple myeloma - hyperdiploid subtypes - "odd chromosome" trisomy array-CGH Trisomy of chromosomes 3, 5, 7, 9, 11, 15, 17, 19 often occurs in MM - better prognosis ! Consequences of individual chromosomal aberrations in the process of carcinogenesis lTranslocations ngenes directly involved in the tumour process identified at the breakpoints l lThere are two principal consequences of translocations and inversions: n na break site within genes on each chromosome, rearranging them to create a fusion gene encoding a chimeric protein involved in the malignant process n nderegulation of gene expression by translocation to a strong promoter region (gene for T-cell receptor or immunoglobulin protein near proto-oncogene) ...often transcription factors n Translocations and tumors Discovered fusion genes - counts Hemat. l malignancies - 1231 Solid tumours - 9641 Consequences of individual chromosomal aberrations in the process of carcinogenesis Translations associated with creation of chimeric proteins l t(9;22) – chronic myeloid leukemia (CML) l „PH“ chromosome lthe cellular proto-oncogene ABL is localized on chromosome 9q34, the BCR gene on chromosome 22q11 lduring translocation, mutual exchange of parts of both chromosomes cration of a BCR/ABL fusion gene is created which encodes the hybrid protein p210, which initiates the malignant process...several breakpoints lt(15;17) – acute myeloid leukemia (AML) lPML/RARA fusion protein is likely the target protein in trans retinoic acid therapy ldirect relationship between treatment of genetic defect and malignancy Consequences of individual chromosomal aberrations in the process of carcinogenesis Translocations associted with protooncogen activation lt(8;14) - Acute Lymphoblastic Leukemia lc-MYC oncogene from 8q24 moved to the immunoglobulin heavy chain gene region on 14q32 lthe relocation results in deregulation of c-MYC proto-oncogene expression l deregulation triggers the process of carcinogenesis lt(2;8), t(8;22) lvariant translocation with a breakpoint in the c-MYC gene ...regions of the kappa (2p11) and lambda (22q11) light chain genes Examples of translocations in B cell lymphomas – involvement of IgH locus 14q32! lMALT lymphomas (mucosa-associated lymphoid tissue) nrecurrent translocations associated with include t(11;18)(q21;q21); t(1;14)(p22;q23); t(14;18)(q32;q21) and t(3;14)(p14.1;q32) l lFollicular lymphoma (FL) nthe recurrent translocation associated with FL is characterised by t(14;18)(q32;q21) l lMantle cell lymphoma (MCL) qthe recurrent translocation associated with is t(11;14)(q13;q32) q lBurkitt lymphoma (BL) qcharacterised typically by t(8;14)(q24;q32) Consequences of individual chromosomal aberrations in the process of carcinogenesis Deletions - examples nloss of part of a chromosome, often affecting tumour-suppressor genes (RB1, p53) or genes for stimulatory and growth factors nLOH- loss of heterozygosity due to gene deletion nspecific changes in haematological malignancies ldel 5q - acute myeloid lekuemia, myelodysplatic syndrome lin AML and MDS, the deletion is interstitial, the extent is highly variable, l5q31-critical region is always affected, multiple genes controlling normal hematopoiesis are mapped in the region ldel 11q23 lin AML and ALL, MLL gene is mapped in the 11q23 region, moreover MLL involved in numerous translocations (6q27, 9p21, 10p15, 17q11, 19p13) besides deletions lMonosomy lloss of whole chromosomes, changes are rather secondary, frequent monosomy 5, 7 in MDS RB1 gene deletion - loss of constitutive heterozygosity in retinoblastoma - LOH Knuston hypothesis – (the double hit theory) retioblastom Upper image presents tumor suppressor gene mutation in a normal cell leading to sporadic cancer. Two chromosomes with wild type tumor suppressor gene are presented. Upon new mutation in one of the presented chromosomes the cell acquires first hit. Then a second hit is introduced in the form of deletion of the part of chromosome hosting tumor suppressor gene. This leads to tumor formation since no tumor suppressor genes are present. Lower image presents tumor suppressor gene mutation in a cell with germline mutation, leading to familial cancer. Two chromosomes are presented - one with wild type allele and one with inherited germline mutation in tumor suppressor gene. The cel already has a first hit. Second hit is introduced in the form of deletion of the part of chromosome hosting tumor suppressor gene. This leads to tumor formation since no tumor suppressor genes are present. „The most tumor suppressor genes require both alleles to be inactivated, either through mutations or through epigenetic silencing, to cause a phenotypic change“ Alfred G. Knudson, 1971 Examples of significant deletions in tumor diseases lRetinoblastoma - malignant eye tumor of children del(13)(q14) - RB1 gene - blocks progression to S phase l lWilms tumour - kidney tumour l del(11)(p) 11p13 - 11p15 l del(16)(q) WT1 gene l lNeuroblastoma - del(1)(p36) l lTP53 gene deletion (17p13) - many tumors !!! l l Consequences of individual chromosomal aberrations in the process of carcinogenesis Aberrations with gain of genetic material lDuplication, trisomy lmultiplication of whole or parts of chromosomes lfrequent secondary changes in tumor cells lmultiplication of gene dosage lcancer progression lin leukemias common +8, in CLL specific +12, other Ph copies n lAmplification lfrequent in solid tumors but also in leukemias lmostly amplification of proto-oncogenes (N-myc, l c-myc, Her 2) ldouble minutes, homogeneously staining areas (DMs, HSRs) are seen in mitosis !!! lDetection: I-FISH, array-CGH lexamination for the presence of amplification is of diagnostic and prognostic importance !!! l Amplifikace img044 regions of amplified genes in the karyotype (absence of banding) DMs Double minutes DMs during mitosis DMs are formed by HSR decay - they contain several thousand copies of a certain gene !!! Complex karyotypes in tumor diseases lComplex karyotype - we detect numerical or numerical changes involving three or more chromosomes or structural changes involving three or more breaks 7COLOR Poor prognosis!!! Chromothripsis - new view of tumour formation (new mechanism of complex changes - discovered in 2011) l lPacient WITH CLL lNGS - 42 genomových přestaveb ! Chromothripsis lchromothripsis (from Greek chromos - chromosome and thripsis - division into pieces) lthese are tens or hundreds of rearrangements on one or more chromosomes, occurring suddenly during a single catastrophic event la chromosome or a part of it is broken into small pieces (multiple double-strand breaks....) and these are then randomly put back together by reparative mechanisms lhowever, this assembly is not completely accurate, some parts may be assembled in a different order, missing or duplicated l lchromothripsis has been demonstrated in various types of tumours (2-3%), but also in congenital genetic diseases Chromothripsis Cytogenetic consequences - complex rearrangements, deletions, duplications, insertions, inversions... Meyerson and Pellman 2011 Chromothripsis is proposed to involve the shattering of a single chromosome, a small group of chromosomes, or a single chromosome arm. The fragments, or a subset of the fragments, are then stitched together by nonhomologous end-joining. Chromothripsis and tumor diseases lincidence of 2 - 3% of all cancers l lchromothripsis has been demonstrated in leukemia, multiple myeloma, colon cancer, medulloblastoma, neuroblastoma, etc. l lfrequent occurrence in bone tumors (up to 25%) - osteosarcoma, Ewing's sarcoma, chondrosarcoma l lchromothripsis - worse prognosis !!! - extensive rearrangements - deletions, duplications, inversions, insertions - deregulation of a large number of genes.... G-banding, I-FISH, M_FISH, M-BAND array-CGH Alghortim of cytogenetic/genomic analyses in cancer diseases: Comprehensive analysis of cytogenetic changes in tumors individualization of medical therapy, biological therapy" Prognostic changes of chromosomal aberrations associated with negative prognosis in hematoncologic and solid cancer diseases Chronic myeloid leukemia (CML) 1:100 000 nPh is usually present in 95% of patients at the time of diagnosis, (where it is not, we usually find a masked Ph chromosome as insertion) nthere are 3 breakpoint regions in the BCR gene l the best prognosis is for patients who have a l Ph chromosome as the only change at the time of diagnosis !!! nat the time of diagnosis, presence of other chromosomal changes besides Ph, is an unfavourable prognostic feature nat the time of blastic reversal, up to 70% of patients have additional chromosomal changes, most often +8, Ph duplication, +19, i(17q) nFISH detects BCR/ABL rearrangement, specific probe allows to investigate also interphase nuclei I – FISH translocation DNA probes Translocation t(9;22) - bcr/abl – interphase With additional signal DUAL fusion s – double fusion signal Translocation positive cells can be or Acute myeloid leukemia (AML) 3/100 000 n60-70% of patients have a clonal chromosomal aberration nthe aberrant clone is often accompanied by a normal clone nChromosomal changes present at the time of diagnosis disappear after achieving remission, reappear in relapse, often with other secondary changes nsome changes specific to FAB subtypes of AML, diagnostic and prognostic significance, disease monitoring ! nThe French-American-British (FAB) classification of AML: l M1 - M7, M0 according to aberrations FAB classification of Acute Myeloid Leukemia | Download ... leukemie AML subgroups AML aberrations lAML-M2 - t(8;21) lmapped genes ETO, AML1 lmainly in younger patients, often in children lsecondary changes -Y, -X, 9q-, 7q-, +8 lgood prognosis l lAML-M3: t(15;17) lPML/RARA genes mapped lLess frequently variant translocations lmost common additive change +8 lgood prognosis t15,17 AML aberrations lAML-M5 disruption 11q23 DNA probe l l11q23 aberrations involving l deletions or translocation in l the break region of the MLL gene lup to 50% of patients have these changes lmost common t(1;11), t(6;11), t(9;11), l t(10;11), t(11;19) and others (40 l translocations) loften also in paediatric AML lgenerally unfavourable prognosis MLL2 disrupce MLLa Disruption in MLL genu Myelodysplastic syndrome (MDS) 15/100 000 lrare disease, occurs in the elderly, pancytonemia in the blood, often transitioning to acute leukemia lchromosomal changes in about 60% of patients ldel(5q) - 5q-syndrome linterstitial deletion, extent of deletion varies, missing 5q31 band lother changes: -5, del(7q), -7, +8, del(11q), 12p-, lfrequent complex changes lbest prognosis in patients with normal karyotype, single 5q- favourable prognosis, l combination with other changes unfavourable, l -7 very unfavourable, patients with complex changes have the shortest survival !!! l 5q rare disease, occurs in the elderly, pancytonemia in the blood, often transitioning to acute leukemia Acute lymphocytic leukemia (ALL) nALL is the most common childhood malignancy; 3 : 100 000 n70-90% of patients have an acquired chromosomal aberration lt(12;21) - TEL/AML1 - better prognosis ! l lNumerical chromosome changes la) hyperdiploidy nmost common, more than 46 chromosomes, moderate prognosis nmore than 50 chromosomes, good prognosis nsupernumerary chromosomes 4, 6, 10, 14, 17, 18, 20 and 21 lb) hypodiploidy nnumber less than 46, unfavourable prognosis nthe importance of I-FISH in detecting hyperdiploid and hypodiploid clones, the algorithm uses a panel of the 10 most frequently included chromosomes Chronic Lymphocytic Leukemia nlittle cell proliferation activity in vitro, often normal karyotype found nCHAs found in more than 40% of CLL patients with FISH l lTrisomy 12 nmost common, in B-CLL, more than 20% of patients noften combined with del(13q), del(6q), del(17p), or complex rearrangements nmedian survival 3-4 years, better response to chemotherapy ldel(13q) nRB1 gene, the most common alteration after trisomy 12 l ldel(17p) nTP53 gene, prognostic significance, survival 1.5-2 years RB-1 Multiple myeloma lA clonal disease that results from a malignant mutation in the development of the B-lymphocytes l lIt accounts for about 10% of all oncohematological diseases l lAffects mainly the elderly, rare before the age of 40 l lCurrently incurable, with variable survival and patient response to treatment l lWide spectrum of clinical manifestations: bone destruction, pancytopenia, anaemia, impaired antibody immunity, myeloma nephropathy ... l lOptimization of treatment requires determination of prognostic factors Mnohočetný myelom a cytogenetika The multiple myelomas — current concepts in cytogenetic classification and therapy | Nature Reviews Clinical Oncology https://www.nature.com/articles/s41571-018-0018-y Multiple myeloma and CHAs lCytogenetic changes - an important prognostic factor in MM !!! l lHypodiploidy: most often monosomy of chromosome 8, 13, 14, X unfavourable prognosis lHyperdiploidy: most often trisomy of chromosome 3, 5, 7, 9, 11, 15,19, 21 favorable prognosis lTranslocation involving the IgH gene (14q32): l t(11;14) (q13;q32) favourable prognosis l t(4;14)(p16;q32), t(14;16) (q32;q23) unfavourable prognosis lRB1 gene deletion (13q14) - intermediate prognosis lDeletion of p53 gene (17p) poor prognosis l gain 1q21 - poor prognosis l l Cell sorting in MM pacients fluorescence immunophenotyping and interphase in situ hybridization amca https://www.researchgate.net/profile/Tae-Dong-Jeong/publication/234134859/figure/fig2/AS:2029998896 98820@1425410353851/Flow-cytometric-immunophenotyping-of-2-cases-A-and-B-with-CD138-negative-neopla stic_W640.jpg FICTION technique in MM fluorescence immunophenotyping and interphase in situ hybridization Simplified flow cytometric immunophenotyping panel for multiple myeloma, CD56/CD19/CD138(CD38)/CD45 Status 5 Year Survival (%) Pseudodiploid 49.9 Near-tetraploid 34.6 Hyperdiploid 33.5 Hypodiploid 10 Převzato z Debes –Marun et al., 2003, Blood Prognosis of disease in MM patients Effect of presence of aneuploidies Solid tumors Classification of solid tumors according to tissue of origin (modified... | Download Table https://www.researchgate.net/profile/Annette-Gylling-lindroos/publication/47931966/figure/tbl1/AS:6 69431457918989@1536616305664/Classification-of-solid-tumors-according-to-tissue-of-origin-modified- from.png Cytogenetics of solid tumors lBiological material: leffusion (ascitic tumors) - epithelial and tumor cells lculture of tumor cells ltumor imprints (FISH) ....confirmation by pathologist ! lparaffin sections (FISH) lcytospins l Cytogenetics of solid tumors lProblems: l lDifficulty of long-term cultivation ( will it outgrow non-tumor material ?) llack of mitoses, quality of slides lcomplexity of karyotype (unbalanced changes) lheterogeneity of tumors l l individualization of patient treatment - cytogenetic and genetic testing is essential ! l Cytogenetické změny u některých solidních nádorů dětí lRetinoblastoma - malignant eye tumour in children del(13)(q14) - RB1 gene lWilms tumor - kidney tumor l del(11)(p) 11p13 - 11p15 - WT1 gene l del(16q) lEwing's sarcoma - t(11;22)(q24;q12) l EWS-FLI1 fusion gene lNeuroblastoma - amplification of N-myc gene, del(1)(p36), del 11q, gain(17)(q) lMedulloblastoma - c-myc gene amplification Fig. 1 N-myc amplification del1p36 Neurobalstoma – FISH probes Prognosis and therapy of neuroblastoma based on genomic profiles of tumor cells! High-risk neuroblastoma tumors with 11q-deletion display a poor prognostic, chromosome instability phenotype with later onset | PNAS https://www.pnas.org/cms/10.1073/pnas.0910684107/asset/8abb56dc-6792-42d3-b860-42a65347b35e/assets/ graphic/pnas.0910684107fig04.jpeg https://www.researchgate.net/profile/Bjorn-Menten/publication/6674618/figure/fig2/AS:26791676765800 4@1440887743724/ArrayCGH-visualisation-of-representative-neuroblastoma-tumors-each-belonging-to-a_W 640.jpg https://www.researchgate.net/profile/Bjorn-Menten/publication/6674618/figure/fig2/AS:26791676765800 4@1440887743724/ArrayCGH-visualisation-of-representative-neuroblastoma-tumors-each-belonging-to-a_W 640.jpg Examples of FISH analyses usedin solid tumors l Personalized medicine and HER -2 gene amplification in breast cancer lBreast CA...6000 women per year in the Czech Republic lHer 2 - an important prognostic and therapeutic marker lOccurs in 25-30% of breast cancers lGene amplification is associated with an unfavorable prognosis (rapid proliferation, shortened survival time) l treatment with Herceptin ...transtuzumab - blocking Her 2 receptor ! erbb Prediction of the course of the disease HER-2/neu Overview lHuman Epidermal Growth Factor Receptor-2 lAlso known as: nc-erbB2 nneu (rat homolog) lCodes for a 185 Kd transmembrane cell surface receptor lMember of the tyrosine kinase family Her2/neu signaling pathway. The her2/neu heterodimer is a growth factor... | Download Scientific Diagram https://www.researchgate.net/profile/Parham-Jabbarzadeh-Kaboli-2/publication/264976148/figure/fig2/ AS:295978197372932@1447578109798/Her2-neu-signaling-pathway-The-her2-neu-heterodimer-is-a-growth-fa ctor-receptor-that.png Image from Genetech Indicators of Increased HER-2 production !CHR17 HER-2/neu DNA Probes HER-2/neu & chr. 17 17p11.1-q11.1 17 alpha 17q11.2-q12 HER-2/neu Large checker board ~ 400 kb Centromere Telomere 17q11.2 -q12 HER-2/neu gene HER-2/neu DNA Probes and Gene Amplification Chr 17 Her-2/neu probe Aneuploidy: multiple copies of chr. 17 True gene amplification: HSRs True gene amplification: dmin’s ALK gene (2p23) disruption in Non-small cell lung pcancer (NSCLC) •Lung cancer diagnosed annually in 1.6 million people worldwide ... • •NSCLC - 80% of cases; 5-year survival - about 15% • •KRAS, EGFR, ALK mutations • •Patients with ALK gene and disruption and EML/ALK fusion gene (5-7% of cases) benefit from targeted treatment with anaplastic lymphoma kinase inhibitor - crizotinib (Xalkori) • •for EGFR gene mutations - gefitinib and erlotinib... Disruption of ALK