PATHOGENESIS OF LEUKEMIAS J. Mayer Dept. of Internal Med., Hematology and Oncology Univ. Hospital and Masaryk University, Brno, CZ …what I am going to say… • Epidemiology, frequency • Model diseases, prototypes • What leukemias are, clinical signs • Key subtypes • Elementary principles of pathogenesis • Implications of these facts for diagnostics and therapy • Emphasize to • time relationships of different discoveries • original data from the medical literature 22 753 8 227 6 904 6 904 6 506 3 068 2 216 2 195 2 134 1 956 1 541 1 519 1 476 1 235 1 064 1 049 934 916 869 864 619 538 535 474 309 271 95 3 115 7 016 2 353 0 2 000 4 000 6 000 8 000 10 000 12 000 14 000 16 000 18 000 20 000 22 000 24 000 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) others leukamias (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 others ca in situ (D00–D09) nonmalignant and uncertain tumors (D10–D36, D37–D48) Average number of yearly diagnosed cases WHO classification, 2016 upgrade • More than 50 types and subtypes • Leukemias – simply, 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, pathogenesis • 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 11% 4% 12% 29% 44% 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 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% ALL AML APL CLL CML HCL %ofcases FATIGUE PAIN DYSPNEA LOW GRADE FEVER PYREXIA LYMPHADENOPATHY BLEEDING INFECTION B SYMPTOMS OTHERS ACUTE LEUKEMIAS CHRONIC LEUKEMIAS Clinical symptoms 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% ALL AML APL CLL CML HCL %ofcases B SYMPTOMS DYSPNEA BLEEDINGS INFECTIONS LYMPHADENOPATHY SPLENOMEGALY HEPATOMEGALY Objective findings by the first visited physician 15% 26% ACUTE LEUKEMIAS CHRONIC LEUKEMIAS Blood count YES 80% Lab exam by the first visited physician PERFORMED? ABNORMALITY? YES 100% 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% CML time leukemia (1850) chemotherapy (1950) BMT IFN Ph1 (1960) BCR/ABL PCR imatinib, dasatinib, nilotinib others alotof„nothing“ Rok Epidemiology of CML in Czechia 0 1 2 3 4 5 6 7 8 19 9019 9119 9219 9319 9419 9519 9619 9719 9819 9920 0020 0120 0220 0320 0420 0520 0620 0720 0820 0920 1020 1120 1220 1320 14 Prevalence Numberoflivingpersonswiththe diseaseper100000inhabitants Source: National Cancer Registry, ÚZIS ČR trend of 2005-2014 Year …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… 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 cfes 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… Kinase activity 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. Relationship between the number of malignant cells, therapy response, and BRC-ABL Baccarani et al.: Blood 108, 2006, 1809-1820 Direct sequencing • BigDye v3.1 Termination kit (Applied Biosystems) R-ABLF-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 AML 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, Xinactivation, 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 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 Licht JD N Engl J Med 2009;360:928-930 APL ALL ALL 4,1 y3,8 y chemotherapy, BMT relapse, deathis living identical BCR ABL translocation hyperdiploidity IKZF1 deletion therapy, relapse, death 5 y no ALL is living identical BCR ABL translocation from Guthri cards slightly positive BCR ABL in blood 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 hemolymphopoietic system, and it functions as a regulator of lymphocyte differentiation. CLL 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 < 5.109/L, monoclonal B lympho of CLL phenotype • ± 5% in population over 40 y, risk of progression into CLL 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 Fabbri G, Dalla-Favera R, Nat Rev Cancer 16, 2016, 145-162 Hallek M, Am J Hematol 92, 2017, 946-965 Summary • After more than 150 years, the term leukemia still survive • 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