PATHOGENESIS OF LEUKEMIAS František Folber, Jiří Mayer Dept. of Internal Medicine, Hematology and Oncology University Hospital Brno and Masaryk University Outline •Epidemiology •Clinical signs •Key subtypes of leukemias •Model diseases •Elementary principles of pathogenesis •Implications of these facts for diagnostics and therapy •with time relationships of different discoveries •and original data from the literature •Including case reports • Cancer incidence In Czechia, 2010-2014 Source: National Cancer Registry, ÚZIS ČR Males Females other skin ca (C44) colon, rectum (C18–C21) breast, females (C50) prostate (C61) lung (C33, C34) kidney (C64) melanoma (C43) urine bladder (C67) pancreas (C25) uterus (C54, C55) stomach (C16) oral cavity (C00–C14) non-Hodgkin lymphoma (C82-C85,C96) acute leukemias and CLL (C91–C95) ovarium (C56) thyroid gland (C73) cervix uteri (C53) liver (C22) gallbladder (C23, C24) brain (C70–C72) esophagus (C15) larynx (C32) multiple myeloma (C90) testis (C62) connective tissue (C47, C49) Hodgkin lymphoma (C81) Chronic myeloid leukemia (C921) others ca in situ (D00–D09) nonmalignant and uncertain tumors (D10–D36, D37–D48) Average number of yearly diagnosed cases Virchow leukemie Virchow wirch04 WHO classification, 2022 upgrade •More than 50 types and subtypes •Leukemias – disturbances in the regulation of growth and differentiation of WBC, white blood cells •Key types: •CML, chronic myeloid leukemia •AML, acute myeloid leukemia •APL, acute promyelocytic leukemia •ALL, acute lymphoblastic leukemia •CLL, chronic lymphocytic leukemia •HCL, hairy cell leukemia • Key clinical signs •Leukocytes •leukocytosis, hyperviscosity •leukopenia, neutropenia •diminished cellular immunity, diminished humoral immunity (CLL) •infections •Thrombocytes •thrombocytopenia •bleeding •thrombocytosis (CML) •Erythrocytes •anemia •Organ infiltration •bone marrow, spleen, liver, lymph nodes, brain, testis, skin, … •myelosarcoma • • Number of analyzed cases (2004-2009) – 1007 patients 1000 leukemia cases AML CLL CML HCL ALL Ráčil et al., Am J Hematol 86, 2011, 800–803 Clinical symptoms Subjective complaints of patients according to the history at diagnosis ACUTE LEUKEMIAS CHRONIC LEUKEMIAS Objective findings Objective findings by the first visited physician 15% 26% ACUTE LEUKEMIAS CHRONIC LEUKEMIAS Blood count Lab exam by the first visited physician PERFORMED? ABNORMALITY? CML Chronic Myeloid Leukemia model disease of a single chromosomal abnormality http://www.leukemia-cell.org/atlas/res/photogallery/cml-3.jpg leukemia (1850) chemotherapy (1950) BMT IFN Ph1 (1960) BCR/ABL PCR imatinib, dasatinib, nilotinib others …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. P.C. Nowell, D.A. Hungerford, University of Pennsylvania in Philadelphia 1960 1973: translocation of chromosomal material •Rowley JD: A new consistent chromosomal abnormality in chronic myelogenous leukemia identified by quinacrine fluorescence and Giemsa staining. Nature, 243, 290-293, 1973 …suggesting that there may be a hitherto undetected translocation between the long arm of 22 and the long arm of 9, producing the 9q+ chromosome… http://www.leukemia-cell.org/atlas/res/photogallery/fig-1.jpg http://www.leukemia-cell.org/atlas/res/photogallery/fig-3.jpg 1982: abl localized on chromosome 9 •Heisterkamp N et al.: Chromosomal localization of human cellular homologues of two viral oncogenes. Nature 299, 1982, 747-749. •…we now show that the human equivalents of c-fes and c-abl are localized on human chromosomes 15 and 9, respectively. It is of interest that both of these chromosomes are involved in specific rearrangements found in certain forms of human cancer… •ABL gene = the human homologue of the v-abl oncogene of the Abelson murine leukemia virus. Abelson HT, Rabstein LS: Proc Am Assoc Cancer Res 10: 1, 1969 1985: fused protein BCR::ABL •Shtivelman E et al.: Fused transcript of abl and bcr genes in chronic myelogenous leukaemia. Nature 315, 1985, 550-554. •…characterization of an 8-kilobase RNA specific to chronic myelogenous leukaemia shows it to be a FUSED transcript of the two genes. The FUSED protein that would be produced is probably involved in the malignant process… Mechanismus STI z NEJM Kinase activity Rok Epidemiology of CML in Czechia Prevalence Source: National Cancer Registry, ÚZIS ČR trend of 2005-2014 Year Relationship between the number of malignant cells, therapy response, and BCR-ABL Baccarani et al.: Blood 108, 2006, 1809-1820 Crystal structure solved, but resistance described •Schindler T et al.: Structural mechanism for STI-571 inhibition of Abelson tyrosine kinase. Science 289, 2000, 1938-1942. •Gorre ME et al.: Clinical resistance to STI-571 cancer therapy caused by BCR-ABL GENE MUTATION or amplification. Science 293, 2001, 876-880. ze Science podrobněji Direct sequencing •BigDye v3.1 Termination kit (Applied Biosystems) R-ABL F-ABL ABL KD 914 bp (AA 235 – 505) ABI 3100 (Applied Biosystems) Mutation detection - MutationSurveyor® software (Softgenetics) T315I Nucleotide change Amino acid change reference sequence (GenBank M14752) sample sequence Apperley JF: Lancet Oncol 8, 2007: 1018 CML Case •older man, age 60 •history: hypertension, bronchial asthma •drugs: sartan, CaC blocker •occupation: office worker •abusus: no alcohol, smokes 5 c./day •current illnesses: •no symptoms •maybe a bit tired •sent by his GP – leukocytosis 26 found at a routine check-up CML Case •CBC •WBC 26 (4-10) •Hb 143 (135-176) •plt 207 (150-350) •microscopic WBC differential count •neutrophils 53% (50-70) •lymphocytes 23% (20-40) •eosinophils 2% (0-5) •basophils 6% (0-1) •metamyelocytes 5% (0-0) •myelocytes 6% (0-0) •blasts 0% (0-0) •immature granulocytes = myeloproliferative disorder suspected CML Case •bone marrow •very increased cellularity •increased granulocyte lineage •blasts 1% (0.1-3.5) •promyelocytes 2% (0.5-5) •myelocytes 28% (5-23) •metamyelocytes 6% (10-30) •bands 15% (10-30) •segments 27% (7-25) •= myeloproliferative disorder probable •mutations: BCR::ABL found by PCR •cytogenetics: 46,XY,t(9;22) = Philadelphia chromosome •Dg.: chronic myeloid leukemia in chronic phase CML Case •CML is not an emergency •except hyperleukocytosis (e.g. priapism) •standard treatment •tyrosine kinase inhibitors (TKIs) •1st: imatinib •2nd: dasatinib, nilotinib, bosutinib •3rd: ponatinib •one pill every day, but forever •patient alive in CR CML - Take home messages •chronic disease •usually found incidentally •Ph chromosome => BCR::ABL fusion gene •TKI = one pill a day •survival better than 95 % •comparable to normal population AML Acute Myeloid Leukemia model disease of heterogeneous abnormalities and clonal evolution http://www.leukemia-cell.org/atlas/res/photogallery/aml-m1-1.jpg AML: outline •Myeloid pattern of the malignant cells •Variable maturation grade •Amazing genetic heterogeneity significantly influencing the prognosis •Prognostic stratification used in clinical practice for selecting the best therapeutic strategy •New molecular pathogenesis findings lead to the development of new, targeted therapies very recently • • Jaiswal S et al. N Engl J Med 2014;371:2488-2498 Clonal hematopoiesis Jaiswal S et al. N Engl J Med 2014;371:2488-2498 Clonal hematopoiesis DNMT3A •DNA (cytosine-5)-methyltransferase 3A is an enzyme that catalyzes the transfer of methyl groups to specific CpG structures in DNA, a process called DNA methylation. The enzyme is encoded in humans by the DNMT3A gene. • •It is responsible for de novo DNA methylation. DNMT3A forms part of the family of DNA methyltransferase enzymes. • •While de novo DNA methylation modifies the information passed on by the parent to the progeny, it enables key epigenetic modifications essential for processes such as cellular differentiation and embryonic development, transcriptional regulation, heterochromatin formation, X-inactivation, imprinting and genome stability. • TET2 •TET2 tet methylcytosine dioxygenase 2 •The protein is a methylcytosine dioxygenase that catalyzes the conversion of methylcytosine to 5-hydroxymethylcytosine. •The encoded protein is involved in myelopoiesis, and defects in this gene have been associated with several myeloproliferative disorders. ASXL1 •The ASXL1 gene provides instructions for making a protein that is involved in a process known as chromatin remodeling. • •Through its role in chromatin remodeling, the ASXL1 protein regulates the expression of many genes, including a group of genes known as HOX genes. • •The ASXL1 protein may have an additional role in gene regulation by signaling to molecules to add a methyl group (a process called methylation) to an area near a gene called the promoter region, which controls gene activity. When a promoter region is methylated, gene activity is repressed, and when a promoter region is not methylated, the gene is active. • Genovese G et al. N Engl J Med 2014;371:2477-2487 Clonal hematopoiesis: more frequent than hematological malignancies, as well as cardiovascular diseases! Increase in total mortality. 2016, n=1540 Papaemmanuil E et al. N Engl J Med 2016;374:2209-2221 Papaemmanuil E et al. N Engl J Med 2016;374:2209-2221 Genovese G et al. N Engl J Med 2014;371:2477-2487 AML Case •young man, age 24 •history: none, no drugs taken •occupation: 1st factory worker, 2nd bartender •abusus: alcohol, 10-20 cigarettes/day •current illnesses: •weight loss 5 kg in last month •3 weeks fatigue •2 weeks fever •gum bleeding – stomatologist: gingivitis, insufficient hygiene •1,5 weeks cough – GP: bronchitis – started ATB •1 week ago blurry vision left eye, then both eyes – ophthalmologist: retinal hemorrhage – sent to infectious dep. •infectious: WBC 320 •hematologist: urgent transfer to our centre AML Case •CBC •WBC 321 x10e9/l (normal range 4-10) •Hb 66 g/l (135-176) •plt 89 x10e9/l (150-350) after transfusion •microscopic WBC differential count •neutrophils 0.5% (50-70) •lymphocytes 1.5% (20-40) •monocytes 0.0% (2-12) •blasts 98.0% (0-0) = Dg. acute leukemia •coagulation •D dimers 14 ug/ml (0-0.5), otherwise normal •biochemistry •LDH 9.88 ukat/l (2.25-3.75), otherwise normal AML Case •bone marrow assessment •increased cellulatity •low % of normal cells 0-1% •blasts 93% (0-5) •cytochemistry •POX (myeloperoxidase) positive •Diagnosis: acute myeloid leukemia •work-up: •mutations: none found •cytogenetics: several chromosome abnormalities (complex karyotype) •risk group: very high risk (of relapse) •hyperleukocytosis + adverse karyotype AML Case •hyperleukocytosis + leukostasis = hematological emergency •emergency treatment •leukapheresis •leuk- = leukocytes •-apheresis = removal •supportive measures •treatment •induction: 1 cycle – complete remission •Webster: to induce = to cause (something) to happen or exist •consolidation: 2 cycles •Webster: to consolidate = to make (something) stronger •allogeneic stem cell transplantation •died due to transplant related toxicity (infection) Licht JD N Engl J Med 2009;360:928-930 APL APL Case •elderly man, age 70 •history: non-STEMI, type 2 diabetes, cataract •drugs: ACE-i, BB, ASA, statin, 3 PADs, eye drops •occupation: retired •abusus: non-smoker, alcohol seldom •current illnesses: •2 weeks shortness of breath •thought of heart problems •cardiologist: CBC – pancytopenia •no other symptoms APL Case •CBC •WBC 2.10 (normal range 4-10) •Hb 74 (135-176) •plt 24 (150-350) •= pancytopenia •microscopic WBC differential count •neutrophils 8% (50-70) •lymphocytes 41% (20-40) •monocytes 0.5% (2-12) •blasts 50% (0-0) = Dg. acute leukemia •coagulation •D dimers 11.5 (0-0.5), INR 1.35 (0.8-1.2), fbg 1.7 (1.8-4.2) = DIC •biochemistry: normal APL Case •pancytopenia + DIC = APL suspected = hematological emergency •bone marrow assessment •increased cellularity •low % of normal cells •blasts 26% (0-5) •promyelocytes 43% (0.1-5) = APL probable •cytochemistry •POX positive •work-up: •mutations: PML::RARa detected PCR = Dg. APL •cytogenetics: 46,XY,t(15;17) •risk group: favorable = very low risk of relapse APL Case •emergency treatment •ATRA as soon as possible •All-Trans Retinoic Acid (vitamin A derivative) •standard treatment •induction: ATRA + chemo •consolidation: ATRA + chemo •maintenance: ATRA alone •no transplant necessary •option for chemo-free therapy: ATRA + ATO (As2O3) •remission ALL Acute Lymphoblastic Leukemia model disease of child malignancies with first and second hit • ALL: Clinical symptoms at diagnosis ALL: Time from first symptoms to diagnosis ALL at 4,1 y at 3,8 y chemotherapy, transplantation relapse, death is living identical germinal translocation hyperdiploidity IKZF1 deletion therapy, relapse, death ALL at 5 y no ALL is living identical germinal translocation from Guthri cards IKZF1 deletion normal IKZF1 According to: Cazzaniga G et al. Blood 118, 2011, 5559-5564 Monozygotic twins IKZF1 •DNA-binding protein Ikaros also known as Ikaros family zinc finger protein 1 is a protein that in humans is encoded by the IKZF1 gene. •This gene encodes a transcription factor that belongs to the family of zinc-finger DNA-binding proteins associated with chromatin remodeling. •Ikaros displays crucial functions in the hematopoietic system and its loss of function has been linked to the development of lymphoid leukemia. •Ikaros point mutant mice are embryonic lethal due to anemia; they have severe defects in terminal erythrocyte and granulocyte differentiation, and excessive macrophage formation. •The expression of this protein is restricted to the fetal and adult hemo-lymphopoietic system, and it functions as a regulator of lymphocyte differentiation. • ALL Case •young adult man, age 25 •history: none, no drugs •occupation: construction worker •abusus: alcohol sometimes, smokes 10/day •current illnesses: •weight loss 10 kg in last 6 months •several weeks fatigue •10 days feeling common cold, cough •3 days ago fainting – local internal dept.: orthostasis •yesterday at GP: „bad“ blood count – sent to local internal dept. •anemia, lymphocytosis, blasts: transferred to our centre ALL Case •CBC •WBC 7.49 (4-10) •automated diff: Lympho 49% (20-40) •Hb 75 (135-175) •plt 134 (150-350) •microscopic WBC differential count •neutrophils 31.5% (47-70) •lymphocytes 10% (20-45) •monocytes 0% (2-12) •blasts 55% (0-0) = Dg. acute leukemia •coagulation: normal •biochemistry: normal ALL Case •bone marrow •increased cellularity •low % of normal cells •blasts 60% (0-5) •cytochemistry •POX negative •flowcytometry (immunophenotyping) •CD10-19+20-22+34+38+c79a+cTdT+cIgM-sIgM- = pro-B immunophenotype •= Dg. Acute B-lymphoblastic leukemia •mutations: none found •cytogenetics: 46,XY = normal karyotype •risk group: high risk ALL Case •no emergency treatment necessary •standard treatment •prephase: steroids •induction: 2 cycles, achieved CR •consolidation: 1 cycle (now) •allogeneic stem cell transplant •no sibling donor •found 10/10 matched unrelated donor •still alive in CR Acute leukemias - Take home messages •acute leukemia: 20 or more % of blasts in blood and/or bone marrow •acute = short history, rapid progression: days or weeks • •acute myeloid leukemia: POX positive (or negative) •hyperleukocytosis: emergency • •acute promyelocytic leukemia: pancytopenia, DIC, emergency • •acute lymphoblastic leukemia: POX neg., immunophenotype • •survival depends on risk factors CLL Chronic Lymphocytic Leukemia model disease of complex pathogenesis involving signalling pathways and tumor microenvironment Obrázek 4 - CLL - průtoková cytometrie CD19 CD5 CLL - hallmarks •Different behavior than AML, ALL, or CML •Significant proportion of patients never require therapy (smoldering disease) •According to our data, in 60% of patients just observation, watchful waiting •The pathogenesis is extraordinarily complex, not yet fully understood, but big progress in recent years •Not just the malignant cells are involved, also the interactions with microenvironment are crucial (vs AML) •Due to recent progress, new therapies are emerging •However, still incurable (but treatable) disease Fabbri G, Dalla-Favera R, Nat Rev Cancer 16, 2016, 145-162 •MBL - monoclonal B cell lymphocytosis: •B lymfo < 5x109/L, monoclonal B lympho of CLL phenotype •± 5% in population over 40 y, risk of progression into CLL Fabbri G, Dalla-Favera R, Nat Rev Cancer 16, 2016, 145-162 Hallek M, Am J Hematol 92, 2017, 946-965 Döhner H et al.: N Engl J Med 2000; 343:1910-1916 Trbusek M et al., J Clin Oncol 29, 2011, 2703-2708 A, wild-type p53 and mutated IgVH B, p53 defect and mutated IgVH C, wild-type p53 and unmutated IgVH D, p53 defect and unmutated IgVH CLL Case •elderly man, age 80 •history: hypertension, chronic cardiac disease •drugs: ACE-i, BB, ASA, statin •occupation: retired •abusus: alcohol sometimes, non-smoker •current illnesses: •weight loss 10 kg per last year •a bit tired •night sweating •no other problems •1 month fever: up to 38 °C •sent by his GP = fever, lymph nodes and WBC 40 CLL Case •CBC •WBC 44 (4-10) •Hb 105 (130-176) •plt 182 (150-350) •lymphocytes 93% (20-40) •microscopic WBC differential count •neutrophils 5% (50-70) •lymphocytes 94% (20-40) •monocytes 0% (2-12) •blasts 0% (0-0) •coagulation and biochemistry: normal •lymphocytosis = lymphoproliferative disorder probable CLL Case •bone marrow •increased cellularity •no blasts •lymphocytes 53% (5-20) •immunophenotyping •CD5+10-19+20dim23+200+79a-FMC7- = typical for B-CLL •Dg.: Chronic lymphocytic leukemia •work-up: mutations, cytogenetics •indication for treament •symptoms: fever, sweating, weight loss •patient is alive, now 2nd relapse CLL - Take home messages •chronic disease •similar to indolent lymhomas •no symptoms for many months or years •leukocytosis with lymphocytosis •specific immunophenotype (CD5+19+23+) •no treatment when no symptoms nor problems •treatment necessary only in advanced cases •go go = younger, healthy patients •slow go = elderly patients •no go = frail patients with comorbidities •repeated remissions and relapses, incurable Summary •After more than 150 years, the term leukemia still survives •Leukemias have different clinical behavior, yet with some similar patterns •Where the pathogenesis is relatively simple, just one targeted therapy may show miraculous effect (CML) •Complex genetic changes in other types of leukemia, especially in advanced stages, preclude simple therapeutic strategy •In CLL, disrupting the interactions with tumor microenvironment seems to be very important •Classical non-specific chemotherapy, in combination with monoclonal antibodies, or BMT, still serve as therapeutic option in many cases The Emperor of All Maladies •$6 (e-book) •$12 (paperback) • • • • • • • • • •500 Kč (print)