Week 10 Malignancies Cytogenetics 244 Vs 2024.pdf

CANCER GENETICS & THE CYTOGENETICS OF HAEMATOLOGICAL MALIGNANCIES REBECCA DE KRAA Cytogenetics Dept, Haematology Pathwest Laboratories, Fiona Stanley Hospital Learning Objectives 1) Understand that cancer is a disease of abnormal gene function & expression. Describe Knudson’s two-hit hypothesis and...

CANCER GENETICS & THE CYTOGENETICS OF HAEMATOLOGICAL MALIGNANCIES REBECCA DE KRAA Cytogenetics Dept, Haematology Pathwest Laboratories, Fiona Stanley Hospital Learning Objectives 1) Understand that cancer is a disease of abnormal gene function & expression. Describe Knudson’s two-hit hypothesis and understand the stem cell model of carcinogenesis. 2) Understand the concept of the cancer-associated genes; oncogenes & tumour suppressor genes. What are the mechanisms involved in oncogenic activation & their potential to cause cancer? What are the hallmarks of cancer? 3) Be familiar with the cytogenetics of CML. 4) Understand the importance of the diagnostic karyotype in conferring prognosis. What are the main prognostic markers of AML ? Give examples of these cytogenetic markers & the associated genetic mechanisms of oncogenesis. Lecture Outline § Definition of cancer Overview of History and Timeline of Significant Events Carcinogenesis – Knudson’s Two-Hit Hypothesis & Stem cell model § Cancer-associated genes Proto-oncogenes and oncogenes – mechanisms of activation Tumour suppressor genes Hallmarks of cancer § Cytogenetics of haematological malignancies FISH CML AML – predictive markers & prognostic groups Definition of Cancer Disease of multicellular organisms Abnormal proliferation of cells Invasion of local tissue can metastasize Can cause significant morbidity/death Abnormal gene function Dysregulation of proliferation, differentiation & cell death Clonality or tumour cell population is favoured Theoi Project Copyright © 2000 - 2008, Aaron J. Atsma, New Zealand History of Cancer (Karkinos) ~ 1600BC Heracles, the Hydra & the Crab, Athenian black-figure lekythos C5th B.C., Musée du Louvre, Paris ~ 300BC Cancer – Timeline of advances in cancer research 1960 Philadelphia chromosome 1970 First oncogene, src 1971 Knudson’s “2-Hit” Hypothesis 1979 TP53 gene 1980’s The role of cyclins & cyclin-dependent kinases 1983 Polymerase chain reaction (PCR) 1984 Epstein Barr virus (EBV) 1990 FDA-approved gene therapy 1994 - 2000 Human Genome Project Advancements in technology 1920 Squash & smear techniques – plant cytogenetics 1970 Banding techniques (Casperson) 1988 Fluorescence in situ hybridization (Lichter et al) Sky-FISH/M-FISH Comparative genomic hybridization Microarray Terminology § “hereditary” cancers...v rare, germline origin § “familial”... clusters in families, combination germline & acquired § “acquired”...not germline, all acquired mutations Gene Locus position on chros Chromosomes Alleles variant forms at same locus Terminology Transcription/gene expression = process by which a protein is formed from the genes that encode it Proliferation – cell growth & division Dysregulation – impairment of a physiological regulatory mechanism Cell cycle regulation Cyclins Growth factors & growth factor receptors Signalling transduction pathways Transcriptional regulation Apoptosis Checkpoints Telomeres and telomerase Extracellular matrix DNA Mismatch repair Signal transduction pathways.svg Carcinogenesis Process by which normal cells are transformed into cancer cells. Theories of Carcinogenesis § Knudson’s Two-Hit Hypothesis - 1971 § The Multi-Step Nature of Cancer – Vogelstein B and Kinzler KW 1993 § Somatic Mutation Theory (SMT) vs. Tissue Organization Field Theory (TOFT) § Variant theories – Stem cell Model Epigenetic variations Knudson’s Two-Hit Hypothesis (single) 1)Is especially true for the recessive nature of tumour suppressor genes 2) Multi-step process that involves > 1 mutation Multi-step nature of carcinogenesis Theories of Carcinogenesis Metabolism & repair Processes altered [Irreversible but not yet cancer] § Knudson’s Two-Hit Hypothesis § The Multi-Step Nature of Cancer Promoters contribute cells acquire more mutations [Form benign or precancerous lesions] Vogelstein B and Kinzler KW 1993 Selection/growth advantage § Somatic Mutation Theory (SMT) vs Tissue Organization Field Theory (TOFT) § Variant theories – Stem cell Model Epigenetic variants eg factors & mechanisms DNA instability increases with each step Principles of Cancer Biology. Kleinsmith LJ. 2006. Pearson Benjamin Cummings Cancer Stem cell theories National Institute of Health resource for stem cell research Stem Cell - myeloid lineage Lecture Outline § Definition of cancer Overview of History and Timeline of Significant Events Carcinogenesis – Stem cell model § Cancer-associated genes Proto-oncogenes and oncogenes - mechanisms of activation Tumour suppressor genes Hallmarks of cancer § Cytogenetics of haematological malignancies FISH CML AML – predictive markers & prognostic groups Cancer-associated genes § Proto-oncogenes and oncogenes Mechanisms of oncogene activation § Tumour suppressor genes § DNA mismatch-repair genes Proto-oncogenes and oncogenes Proto-oncogene – regulates cell growth & differentiation potential to become cellular oncogene Involved in signal transduction & execution of mitogenic signals e.g. myc involved in cell regulation - codes for transcription factor, eg’s ras, wnt Oncogene or cellular oncogene c-onc – potential to increase the malignancy of a cell, once it becomes activated, constitutively expressed eg’s c-myc, k-ras Classification of Oncogenes Function Mechanism of Action Examples Growth factors Overexpression – an oncogene may cause a cell to secrete growth factors even though it usually doesn’t. This induces uncontrolled proliferation (autocrine loop) and proliferation of neighbouring cells. c-sis Growth Factor Receptors (Receptor Tyrosine Kinases) Overexpression or amplification – receptor kinases add phosphate groups to the amino acid tyrosine in target proteins that can cause cancer by switching the receptor permanently on without signals from outside the cell. Epidermal growth factor receptor (EGFR) or erb-B1 in lung, breast, stomach cancers, platelet-derived growth factor receptor (PDGFR), and vascular endothelial growth factor receptor (VEGFR), HER2/neu Cytoplasmic tyrosine kinases Translocations leading to fusion hybrid protein Src-family, Syk-ZAP-70 family, and BTK family of tyrosine kinases, the Abl gene in CML - Philadelphia chromosome Cytoplasmic Serine/threonine kinases and their regulatory subunits Point mutations, amplifications or translocations Raf kinase, Cyclin D1 or CDK4 Regulatory GTPases Point mutations leading to deregulated overactivity Ras in many common cancers, lung, colon, pancreas Transcription factors Point mutations, amplifications or translocations c-myc amplification in dmins in AML, Mechanisms of oncogene activation 1) Mutations 2) Gene amplification 3) Chromosomal rearrangements Mutations § alter structure of proto-oncogene oncogene § dominant gain-of-function § involve protein regulatory regions uncontrolled continuous activity of the mutated protein § types of mutations: Point mutations Deletions Amplification Integration of proviral DNA from a retrovirus Example of a point mutation DNA sequence analysis of K-ras gene at codon 12 g g t g wild type of K-ras g c g g a t g g c G to A mutation Sharaf HM, El- Kinawy NS, Mahmoud AO, Ali MA. Detection of a Point Mutation at Codon 12 of the Kirsten-Ras (K-ras) Oncogen in Myelodysplastic Syndrome. WebmedCentral HAEMATOLOGY2012;3(5):WMC003357 Example of a deletion Chros 9 If 1 copy is deleted, it leads to expression/change of function (due to dominant gain-of function) Eg. “Oncogenic activation of the Notch1 gene by deletion of its promoter in Ikaros-deficient T-ALL” Robin Jeannet et al BLOOD, 16 DECEMBER 2010 VOLUME 116, NUMBER 25 https://www.ncbi.nlm.nih.gov/pubmed/20829372 Note: gene dosage effect associated with deletions where obvious deletions in chromosomes represent an increased number of genes deleted eg 5q- syndrome in MDS Notch1 gene Gene Amplification = oncogene Mechanism: repeated copying in DNA replication process expansion in copy number increase in gene expression onc deregulated cell growth present Example of gene amplification resulting in dmins Amplification can result in: double minutes (d-mins) homogenously staining regions (hsrs) Amplified regions can contain > 100’s copies e.g. c-myc is amplified in small-cell lung ca, breast/ovarian ca and leukaemias. FISH image of c-myc amplification in d-mins Example of gene amplification resulting in hsr’s Amplification can result in: homogenously staining regions (hsr’s) Amplified regions can contain > 100’s copies Metaphase image and associated karyotype of a hsr in a ring 3 Example of gene amplification resulting in hsr’s Amplification can result in: homogenously staining regions (hsr’s) Amplified regions can contain > 100’s copies Directed metaphase FISH image of hsr in ring 3 with amplified MECOM using a breakapart probe for MECOM MECOM (codes for a transcription factor) well documented in AML, MDS and CML with a poor prognosis Chromosomal rearrangements Recurrent chromosomal rearrangement are often detected in haematological malignancies & some solid tumours Types of recurrent rearrangements: chromosomal translocation – reciprocal exchange Inversions – segment reversed end to end When these rearrangements happen, oncogenes can be activated by: 1) proto-onco/onc is moved next to an immunoglobulin gene IGH and falls under its control deregulated expression neoplastic transformation OR 2) juxtaposition of 2 different genes (other than IGH to form a novel fusion gene codes for chimeric protein transforming activity. Example of de regulated expression of oncogene via regulatory control of an immunoglobulin gene IGH CCND1(aka BCL1) involved in G1 to S transition Norback H et al, Cytogenetics at theWaisman Center, Atlas of Genetics and Cytogenetics in Oncology and Haematology Example of formation of novel fusion gene that codes for chimeric protein with transforming activity BCR-ABL1 Classic e.g. of t(9;22) in CML ABL1 gene encodes tyrosine kinase activity Involved in cellular differentiation BCR located at 22q11 BCR-ABL1 fusion gene encodes chimeric protein Atlas of Genetics and Cytogenetics in Oncology and Haematology Cancer-associated genes § Proto-oncogenes and oncogenes Mechanisms of oncogene activation § Tumour suppressor genes § DNA mismatch-repair genes Tumour Suppressor genes Suppress cellular growth/survival when needed to prevent tumours forming. Outcomes triggered by tumour suppressor activation: Arrest cell cycle to inhibit cell division Induce cell cycle to DNA damage repair mechanisms Promote apoptosis if damage cannot be repaired Induce senescence Two most important tumour suppressor genes; TP53 and RB Other eg’s: APC, BRCA1, BRCA2 Follow Knudson’s 2-Hit Hypothesis e.g. RB Usually recessive nature– both copies mutated before function is affected § § The exception is TP53 it can be; recessive or dominant negative Recessive and Dominant Negative TP53 mutations Two normal TP53 genes. Homozygous recessive mutation No TP53 produced TP53 protein Heterozygous recessive mutation. No TP53 produced TP53 protein Dominant negative mutation. Altered TP53 Non-functional TP53 Adapted from Principles of Cancer Biology. Kleinsmith LJ. 2006. Pearson Benjamin Cummings. Hallmarks of Cancer 2000 Hanahan and Weinberg 6 biological capabilities acquired by cancer cells: (1) Sustaining growth signalling (2) Evading growth suppressors (3) Resisting cell death (4) Enabling replicative immortality (5) Inducing angiogenesis (6) Activating invasion and metastasis 2011 sequel of 4 more 1) abnormal metabolic pathways 2) evading the immune system 3) chromosome abnormalities & unstable DNA (4) inflammation Hallmarks of Cancer (1) Sustaining growth signalling (2) Evading growth suppressors (3) Resisting cell death (4) Enabling replicative immortality (5) Inducing angiogenesis (6) Activating invasion and metastasis Hallmarks of cancer Sustaining Growth Signalling Cancer cells can sustain growth by: 1) producing their own growth factor molecules e.g. glioblastomas – PDGF 2) send their own growth signals send their own signals to normal cells in ECM around tumour react and supply tumour with GF e.g. E-cadherin/catenin complex receptor proteins at the cancer cell surface hyper responsive to usually limited supply growth signalling e.g. increased HER2/neu receptors in some breast cancers Outcome: circumvent limited pathway & keep growth signalling switched on Hallmarks of Cancer (1) Sustaining growth signalling (2) Evading growth suppressors (3) Resisting cell death (4) Enabling replicative immortality (5) Inducing angiogenesis (6) Activating invasion and metastasis Hallmarks of cancer Evading Growth Suppressors § § § Function of a growth suppressor is to control growth (regulatory pathways/factors) Many of these are dependent on tumour suppressor genes eg RB and TP53 Defects in pathways/genes cancer cells able to resist inhibitory signals that would usually stop their growth cyclin D- CDK4(6) cyclin E-CDK2 cyclin A-CDK2 ATP Growth suppression pRB active pRB - Phos inactive PP1 Mutated RB gene ADP Cell proliferation PP1 phosphatase pRB inactivated growth suppressor evaded, proliferates Retinoblastoma Protein phosphatase type 1, the product of the retinoblastoma susceptibility gene, and cell cycle control. Rubin E et al. Frontiers in BioScience 3 d1209-19219, Dec 1998. Hallmarks of Cancer (1) Sustaining growth signalling (2) Evading growth suppressors (3) Resisting cell death (4) Enabling replicative immortality (5) Inducing angiogenesis (6) Activating invasion and metastasis Hallmarks of cancer Signal transduction pathways.svg Apoptosis = programmed cell death Different pathways regulating/effecting apoptosis (e.g. TP53 mediated/BCL2 regulated) Opportunities for cancer cells to resist apoptosis through defects in these pathways Hallmarks of cancer TP53/BCL2 regulatory pathway to Apoptosis § BCL2 can: anti cell death function(BCL2 hyperphos’d) promotes cell death cell lives cell dies § TP53 can: promote cell death promote DNA repair Apotosis acts to control cancer cells but it can be overcome if: 1) Over expression of BCL2 by translocation, controlled by IGH 2) Mutation/loss of TP53 OR Hallmarks of cancer Resisting Cell Death P P § Normal situation: normal cell normal regulation § Normal situation: DNA damaged cell (e.g. stressed/aged) P P Bcl-2 cell lives apoptosis induced cell death Bcl-2 P § BUT........ Scenario 1) Over expression BCL2::IGH Translocation proliferate Bcl-2 Bcl-2 Bcl-2 Bcl-2 Bcl-2 Damage sensor Scenario 2) No TP53 or altered mutation/loss TP53 Usually multistep TP53 mutated >50% cancers Hallmarks of Cancer (1) Sustaining growth signalling (2) Evading growth suppressors (3) Resisting cell death (4) Enabling replicative immortality (5) Inducing angiogenesis (6) Activating invasion and metastasis Hallmarks of cancer Enabling Replicative Immortality § Multiply forever! § Normally: cells have limited # growth/division cycles before senescence is reached or a crisis phase leads to cell death So what causes some cells to bypass this? § Telomeres involved immortalization Telomeres protect ends of chromosomes As cells reach end of lifespan, telomeres shorten genome instability/apoptosis Telomerase, maintains telomere length is almost absent in normal cells but in 90% immortalized cells, including cancer cells Genetic mechanisms are unclear, but a combination of changes occur: loss of TP53 and RB pathway function& activation of RAS or myc telomerase genomic stability multiply forever Hallmarks of Cancer (1) Sustaining growth signalling (2) Evading growth suppressors (3) Resisting cell death (4) Enabling replicative immortality (5) Inducing angiogenesis (6) Activating invasion and metastasis Hallmarks of cancer Inducing Angiogenesis § Formation of new blood vessels. § Balanced by inducers and inhibitors § e.g. Inducers VEGF-A which bind to receptors on endothelial cells Inhibitor TSP-1 regulated by TP53 § For cancer cells to grow they need a blood supply. During carcinogenesis an “angiogenic switch” is tripped and remains on. Inducers & inhibitors control this switch. § e.g. TP53 loss or mutation can dysregulate TSP-1 and induce angiogenesis. as seen in growth of breast and melanoma cancers Hallmarks of Cancer (1) Sustaining growth signalling (2) Evading growth suppressors (3) Resisting cell death (4) Enabling replicative immortality (5) Inducing angiogenesis (6) Activating invasion and metastasis Hallmarks of cancer Activating invasion and metastasis § Tissue invasion: localized § Metastasis: distant areas § attach to ECM/ conscript normal cells for support Activated by changes in molecules needed for cell adhesion: - cadherins & integrins e.g. E-cadherin – assemble epith cells sheets & maintain integrity mutation or of E-cadherin by some cancer cells cells to detach activates invasion and metastasis e.g. integrins - mediate cell attachment/integrity & send signals to regulate this, - involved in the motility of cells. expression of integrins have been correlated with metastatic progression in breast, prostate and lung ca. Genetic alteration in cadherins/integrins or factors that regulate/effect their pathways activation of invasion/metastasis Lecture Outline § Definition of cancer Overview of History and Timeline of Significant Events Carcinogenesis – Stem cell model § Cancer-associated genes Proto-oncogenes and oncogenes - mechanisms of activation Tumour suppressor genes Hallmarks of cancer § Cytogenetics of haematological malignancies FISH CML AML – predictive markers & prognostic groups Importance of cancer genetics in malignant haematology § Cancer – abnormal gene function § Genetic mutations § Epigenetic changes § In malignant haematology: genetic mechanisms associated with chromosomal rearrangements transforming activity that lead to cancer being developed. Laboratory investigations haematological malignancies Morphology Immunophenotyping Molecular Haematology Cytogenetics Coagulation studies HLA Typing Samples for malignant cytogenetics Bone marrow aspirate Peripheral blood Bone marrow trephine Lymph nodes Solid tumours Dividing cells Principles of cell culture § Normal pb contains NO dividing cells. But if you add a mitogen eg PHA, lymphocytes transform mitosis. 1st division metaphases at 24 hrs, 2nd division at 48 hrs § Bone marrow and leukaemic blood – blasts, spontaneously dividing and require no mitogens (stimulants added IL6 lymphoid, IL3 myeloid) § Lineage specific cultures; lymphoid/myeloid cultures, chromosomes/no chromosomes (interphase cells) § Short term O/N cultures for improved metaphase quality and higher MI’s § The next few slides demonstrate the steps performed in the lab chromosomes for analysis. 1) Perform WCC → Sample 10x10⁶ cells Cell Culture Process 2) RPMI + FCS + CSFs RPMI + FCS + CSFs 3) Culture flasks incubated 24 hrs at 37⁰C (block/unblock) 4) Arrest mitosis with colchicine CLASS II lamina flow hood Sterile conditions Cell cycle & the stages of mitosis Principles of conventional cytogenetic culture Dividing cells Metaphase chros’s highly condensed Mitotic arrest at metaphase Block(synchronize) at S phase to ↑number metaphase chros’s, morphology & banding 5-Fluorodeoxyuridine acts as an antagonist to thymidylate synthetase interrupting the purine pathway (A&G) in the ‘S’ phase. Thymidine bypasses block in a salvage pathway This releases the arrested cells to continue on throughout the cell cycle. Next day Unblock cells Inactivate spindle form/n. Ready for harvesting Harvesting and slide preparation § Hypotonic treatment to swell cells § Fixation with methanol/ acetic acid § Slide making temperature/humidity/ important for spreading of metaphase chromosomes and banding quality. § Can use a temp/humidity controlled chamber/room. Conventional (classical) cytogenetic analysis Metaphase Cells, Solid stained and G-banded under Oil Immersion The role of Molecular Haematology Detect fusion transcripts generated by novel fusion genes & monitor Monitors engraftment post BMT Amplify & detect small mutations FLT3, NPM & CEBPA Sensitivity levels (good for MRD): Conventional cytogenetics ~1:20 FISH ~1:100 – 500 Immunophenotyping ~1:1000 – 1:10,000 Molecular (PCR) ~1:100,000 If this is so sensitive, what is the advantage of doing cytogenetics? The role of clinical cytogenetics in haematological malignancies Confirm diagnosis Classify haematological malignancies and the subsets Determine disease status MRD Predictive factors for prognosis Stratify patients for treatment protocols & clinical trials Chromosomal Rearrangements Numerical Structural Intrachromosomal Interchromosomal Certain rearrangements characterize subgps of AML FISH Principles DNA probes bind to specific target sequences Probes labelled with fluorescent dyes Hybridization process Single stranded DNA anneals to complementary DNA Hybridization of the probe to target DNA Visualized as a brightly coloured signal by fluorescence microscopy. Target sequences on: Metaphase chromosomes Interphase nuclei Tissue sections (fixed, paraffin embedded) FISH probes used in malignant cytogenetics Whole chromosome paints WCP Resolving complex changes Repetitive sequence probes CEP Centromeric - enumeration of chromosomes Locus specific indicators LSI Detecting structural rearrangements Specific genes – deletions, amplifications Single fusion (not used as much) Dual fusion - translocations Break apart – inversions, translocations FISH probes used in malignant cytogenetics Whole chromosome paints WCP – case study example Resolving complex changes wcp19 t(1;19)(q23;p13) FISH probes used in malignant cytogenetics Repetitive sequence probes CEP – case study example Centromeric - enumeration of chromosomes 17 10 4 MetaSystems 4 10 17 enumeration probes Cep 4 10 17 FISH FISH probes used in malignant cytogenetics Locus specific indicators LSI – case study examples Detecting structural rearrangements Specific genes – deletions, amplifications Single fusion (not used as much) Dual fusion - translocations Break apart – inversions, translocations RUNX1-ETV6DF Probe Amplification of RUNX1 TP53 and cep17 probes deletion of TP53 RUNX1 BAP Probe FISH probes used in malignant cytogenetics Locus specific indicators LSI Detecting structural rearrangements Specific genes – deletions, amplifications Single fusion (not used as much) Dual fusion - translocations Break apart – inversions, translocations KMT2A breakapart probe ? ? Mmm.... looks like a leukaemia ? I suspect it’s … leukaemia CLL LEUKAEMIA CHRONIC CML ALL ACUTE AML Chronic Myeloid Leukaemia t(9;22)(q34;q11.2) Most common of the myeloproliferative disorders 15-20% of all leukaemias Occurs at any age Med age at diagnosis 50 -60 yrs 90-95% of cases: t(9;22)(q34;q11.2) Philadelphia chromosome - der(22q) 5-10% variant translocation involving 3rd or 4thchros or cryptic insertion ABL1 gene on 9q34 BCR gene on 22q11 BCR::ABL1 fusion gene – encodes chimeric protein with increased tyrosine kinase activity Cytocell BCR-ABL1 Translocation, Dual Fusion Probe t(9;22) BCR::ABL1 FISH der(9)t(9;22) normal 9 normal 22 der(22)t(9;22) Targeted Therapy for CML STI571 - Imatinib Mesylate Mechanism of action - Tyrosine Kinase inhibitor I suspect it’s … leukaemia CLL LEUKAEMIA CHRONIC CML ALL ACUTE AML Disease Presentation Clinical history Pancytopaenia – decrease in cell blood lines causes of pancytopaenia Leucocytosis Peripheral blood film Definition - AML Accumulation of clonal immature cells from the myeloid lineage in the bone marrow that interferes with normal production. ≥ 20% blasts * WHO 5th Edition – classification of AMLs *AML with defining genetic abnormalities AML defined by morphology Epidemiology Globally 60 cases of AML per million population per year. Most cases of AML are in adults. Median age of 66 yrs. The incidence rate rises with age. M:F 1:1 Risk factors There are few known proven risk factors for AML and it is relatively unknown what causes AML to develop. Smoking Chemicals Radiation Viruses Congenital syndromes (some - increase risk) Certain blood disorders How can we classify haematological malignancies? Biological genetic classification - World Health Organization Classification of Tumours (WHO) clinical indications, morphology, biological behaviour, immunophenotyping, cytogenetics, molecular haematology Prognostic classification outcomes – discriminating factors 5th Edition of the World Health Organization Classification of Haematolymphoid Tumours: Myeloid Revised MRC prognostic classification based on multivariable analyses (5876 patients) - 2010 Cytogenetic abnormality Favourable t(15;17)(q24;q21) t(8;21)(q22;q22) inv(16)(p13q22)/t(16;16)(p13;q22) Comments Irrespective of additional cytogenetic abnormalities* Intermediate Normal karyotype Trisomy 8 t(9;11)(p22;q23) Entities not classified as favourable or adverse Adverse inv(3)(q21q26)/t(3;3)(q21;q26) -5/ del(5q) -7/del(7q) abn(17p) Complex ( ≥3 unrelated abnormalities) *All favorable-risk abnormalities. †All adverse-risk abnormalities. Excluding cases with favorable karyotype† UK Medical Research Council Blood 2010, 116:354-365 AML prognostic groups Revised MRC prognostic classification based on multivariable analyses (5876 patients) - 2010 Cytogenetic abnormality Favourable t(15;17)(q24;q21) t(8;21)(q22;q22) inv(16)(p13q22)/t(16;16)(p13;q22) Comments Irrespective of additional cytogenetic abnormalities* Intermediate Normal karyotype Trisomy 8 t(9;11)(p22;q23) Entities not classified as favourable or adverse Adverse inv(3)(q21q26)/t(3;3)(q21;q26) -5/ del(5q) -7/del(7q) abn(17p) Complex ( ≥3 unrelated abnormalities) *All favorable-risk abnormalities. †All adverse-risk abnormalities. Excluding cases with favorable karyotype† UK Medical Research Council Blood 2010, 116:354-365 AML with favourable prognosis Acute promyelocytic leukaemia with PML::RARA fusion Acute ProMyelocytic Leukaemia DIC t(15;17)(q24;q21) PML gene on 15q24 RARα gene on 17q21 PML::RARα fusion – product has enhanced affinity to sites on the cell’s DNA, This blocks transcription and differentiation of granulocytes. ATRA (all-trans-retinoic acid) t(15;17)(q24;q21) MetaSystems PML::RARα(RARA) Translocation, Dual Fusion Probe PML-RARα Dual Fusion FISH ( Metaphase) t(15;17)(q24;q21)PML::RARA FISH normal 15 derivative (15) der(15)t(15;17) normal 17 derivative (17) der(17)t(15;17) AML with favourable prognosis Acute myeloid leukaemia with RUNX1::RUNX1T1 fusion Mostly associated with Acute myeloblastic leukaemia with maturation Most common structural abnormality in AML Occurs predominantly in younger patients t(8;21)(q22;q22) RUNX1T1 (ETO) gene on 8q22 codes for a transcription factor RUNX1 (AML1) gene on 21q22 codes for a CBFA transcription factor RUNX1::RUNX1T1 fusion – product interferes with normal activity of CBFA transcription factor produced by RUNX1 t(8;21)(q22;q22) AML with favourable prognosis AML with CBFB::MYH11 fusion Acute MyeloMonocytic leukaemia (AMML) AMML Eo Detected in 5% of AMLs Can occur in all age groups, predominates in younger group inv (16)(p13q22) – majority cases assoctd with AMML Eo t(16;16)(p13;q22) MYH11 gene at 16p13 chemical energy -> mechanical energy CBFB gene at 16q22 CBFβ::MYH11 fusion – product binds to RUNX1 to inhibit it’s function in haematopoiesis Tinv(16)(p13q22) Break Apart Probe 16q22 region Centromere Telomere CBFB gene 150 kb Exon 6 Exon 5 Exon 1 50 kb 170 kb Inversion (16) breakpoint region CBFB::MYH11 Inverted 16 CBFB-MYH11 novel hybrid fusion gene CBFβ::MYH11 M YH 11 MYH11 Normal 16 unrearranged CBFB gene q22 CBFβ CBFβ MYH11 CBFβ Revised MRC prognostic classification based on multivariable analyses (5876 patients) - 2010 Cytogenetic abnormality Favourable t(15;17)(q24;q21) t(8;21)(q22;q22) inv(16)(p13q22)/t(16;16)(p13;q22) Comments Irrespective of additional cytogenetic abnormalities* Intermediate Normal karyotype Trisomy 8 t(9;11)(p22;q23) Entities not classified as favourable or adverse Adverse inv(3)(q21q26)/t(3;3)(q21;q26) -5/ del(5q) -7/del(7q) abn(17p) Complex ( ≥3 unrelated abnormalities) more *All favorable-risk abnormalities. †All adverse-risk abnormalities. Excluding cases with favorable karyotype† UK Medical Research Council Blood 2010, 116:354-365 Normal karyotype Trisomy 8 AML with KMT2A rearrangement t(9;11)(p22;q23) KMT2A::MLLT3 AF9 or MLLT3 KMT2A (MLL) KMT2A (MLL) break apart probe Revised MRC prognostic classification based on multivariable analyses (5876 patients) - 2010 Cytogenetic abnormality Favourable t(15;17)(q24;q21) t(8;21)(q22;q22) inv(16)(p13q22)/t(16;16)(p13;q22) Comments Irrespective of additional cytogenetic abnormalities* Intermediate Normal karyotype Trisomy 8 t(9;11)(p22;q23) Entities not classified as favourable or adverse Adverse inv(3)(q21q26)/t(3;3)(q21;q26) -5/ del(5q) -7/del(7q) abn(17p) Complex ( ≥3 unrelated abnormalities) *All favorable-risk abnormalities. †All adverse-risk abnormalities. Excluding cases with favorable karyotype† UK Medical Research Council Blood 2010, 116:354-365 deletion of 7q AML with MECOM rearrangement inv(3)(q21.3q26.2) GATA2::MECOM MECOM (EVI1) triple colour breakapart FISH Break in distal red region of MECOM Normal 3 Inverted 3q MECOM (EVI1) rearrangement Chromosome 3 Proximal EVI1 Inv(3q) Distal EVI1 Complex AML Monosomal karyotype Monosomal karyotype ≥ 2 autosomal monosomies or 1 autosomal monosomy with at least one structural abnormality excluding rings or marker chromosomes Kayser et al 2012 MK+ ≥3 abs 9% 4yrs MK+ ≤2 abs 13% 4yrs Breems DA et al JCO 2008;126:4791-4797 Complex Monosomal AML Outcome after alloHSCT in patients harboring a monosomal karyotype compared to other cytogenetic abnormalities. Angelique V.M. Brands-Nijenhuis et al. Haematologica 2016;101:248-255 ©2016 by Ferrata Storti Foundation Monosomal karyotype as an adverse prognostic factor in patients with acute myeloid leukemia treated with allogeneic hematopoietic stem-cell transplantation in first complete remission: a retrospective survey on behalf of the ALWP of the EBMT Lecture Outline § Definition of cancer Overview of History and Timeline of Significant Events Carcinogenesis – Knudson’s Two-Hit Hypothesis & Stem cell model § Cancer-associated genes Proto-oncogenes and oncogenes – mechanisms of activation Tumour suppressor genes Hallmarks of cancer § Cytogenetics of haematological malignancies FISH CML AML – predictive markers & prognostic groups