Pathophysiology of hematopoietic system I– hematological malignancies Assoc. Prof. RNDr. Sabina Ševčíková, PhD. Babak Myeloma Group Department of Pathophysiology I. Hematological malignancies Targeted therapy • Treatment aiming at specific properties of cancer cells (growth, survival…) • Need to know specific changes in cancer cells • Trastuzumab – targeting HER2 positive breast cancer Signaling pathway End of 1 disease = 1 treatment • Time of precision and translational medicine • Precision medicine • Takes into account genetics, environment and lifestyle of patient • Patients with same diagnosis react vs do not react to treatment…why? • Translational medicine – bench to bedside • Genetic dispositions influence almost all diseases • There are different molecular subtypes in one diagnosis • 5 subtypes of breast cancer Agnostic approach to cancer treatment • Treatment based on mutation, not based on tumor • Mutation BRAFV600E – melanoma, non small cell lung carcinoma, colorectal cancer • New tyrosine kinase inhibitor vemurafenib targets BRAFV600E • The BRAFV600E – oncogenic „driver“ mutation associated with agresive phenotype, shorter survival than wt BRAF tumors • FDA approved treatment of other tumors I. Hallmarks of cancer Hallmarks of cancer 39 308 citations Hallmarks of cancer Carcinogenesis is individual Order, number of hits and specific genes are individual @Jana Šmardová Six hallmarks of cancer Sustaining proliferative signaling Evading growth suppressors Resisting cell death Enabling replicative immortality Inducing angiogenesis Activating metastasis Tumor microenvironment @Jana Šmardová Six hallmarks of cancer Sustaining proliferative signaling Evading growth suppressors Resisting cell death Enabling replicative immortality Inducing angiogenesis Activating metastasis Tumor microenvironment @Jana Šmardová • Homeostasis – cells are dependent on growth signals Sustaining proliferative signaling Sustaining proliferative signaling • Tumor cells – dysregulation of critical pathways for survival • Tumor cells • Uncontrolled proliferation • Invasivity • Resistance to death signals • Angiogeneiss • Metastasis • Resistance to apoptosis @Jana Šmardová Six hallmarks of cancer Sustaining proliferative signaling Evading growth suppressors Resisting cell death Enabling replicative immortality Inducing angiogenesis Activating metastasis Tumor microenvironment @Jana Šmardová • Basic principle for growth and reproduction – formation of new cells • End product: 2 daughter cells from 1 maternal cell • From mitosis to mitosis • Nuclear division followed by cellular division • G1 phase: cell is small and young, creating organelles • S phase: DNA synthesis, replication of DNA • G2 phase: cytoskeleton and protein development • M phase: mitosis, 2 new cells Cell cycle Mitosis Tumor as a cell cycle disease? • Loss of CC regulation critical for transformation • Loss of CC regulation is not the only part of carcinogenesis • Does not lead to full transformation Six hallmarks of cancer Sustaining proliferative signaling Evading growth suppressors Resisting cell death Enabling replicative immortality Inducing angiogenesis Activating metastasis Tumor microenvironment @Jana Šmardová Apoptosis • Programmed cell death • Cell survive for a limited time – then apoptosis • Tumor cells are resistant to proapoptotic signaling • Growth of tissues – balance between proliferation and cell death (homeostasis) • In tumors, balance dysregulated Dysregulation of apoptosis Six hallmarks of cancer Sustaining proliferative signaling Evading growth suppressors Resisting cell death Enabling replicative immortality Inducing angiogenesis Activating metastasis Tumor microenvironment @Jana Šmardová Replicative potential • Mammal cells have replicative potential of about 60-70 divisions (Hayflick limit) • Then senescence – change of morphology, metabolically active, do not divide • 10-7 – immortal cell • Most tumor cells – immortal – unlimited replicative potential Six hallmarks of cancer Sustaining proliferative signaling Evading growth suppressors Resisting cell death Enabling replicative immortality Inducing angiogenesis Activating metastasis Tumor microenvironment @Jana Šmardová Angiogenesis • Growth of blood vessels • Tumor – population of quickly and uncontrollably growing cells • Tumors cannot grow more than 1-2 mm3 - several million cells (not enough nutrients and oxygen) • Need angiogenesis – until then, tumor growth slowly and linearly, then exponentially • Growth of new vessels is key for metastasis and for nutrients and oxygen • In tumors, new vessels are not regular, blood flow irregularly, • Different cells in the tumor microenvironment Increasing angiogenesis Tumor vasculature is abnormal • Highly disorganized • Blood flow chaotic • Places of hypoxia in certain parts of tumors • Influences therapy effect • Selection and clonal expansion of tumor cells @Jana Šmardová Six hallmarks of cancer Sustaining proliferative signaling Evading growth suppressors Resisting cell death Enabling replicative immortality Inducing angiogenesis Activating metastasis Tumor microenvironment @Jana Šmardová • Tumor cells invade surrounding tissue • Migrate from primary tumor to other tissues forming secondary tumors Formation of metastasis Metastases – most common cause of death • Cause of about 90% of death of tumor patients • Less common – effect of primary tumors • Brain tumors • Leukemia, lymphoma Bad news about metastases • More than 70% of patients with invasive tumors have metastases at the time of diagnosis • Invasive character – early in tumor progression • Millions of tumor cells get into blood every day • Angiogenesis influences metastasis formation @Jana Šmardová Good news about metastases • Not very effective: less than 0.01% of circulating tumor cells is able to form metastasis • We can detect circulating tumor cells early • Leading to early therapy @Jana Šmardová Tumor – complex tissue Tumor microenvironment supports growth of tumor, other cell types @Jana Šmardová II. Hematological malignancies Important definitions • Incidence - number of new cases of a disease diagnosed each year • Prevalence - total number of people living with a certain disease during a given period of time • Overall survival - length of time from either the date of diagnosis or the start of treatment Important definitions • Remission – decrease of signs of cancer, including normalization of lab values (blood count) and imaging methods (X ray, ultrasound, CT) in response to treatment. • Complete remission - disappearance of all signs of cancer in response to treatment. This does not always mean the cancer has been cured. Also called complete response. • Partial remission - decrease of leukemic cells by at least 50% (observing total number of leukemic cells) • Relapse - return of a disease after a period of improvement. Reaching remission does not mean cure as there might be lesions that are impossible to detect and may become the source of new return of the disease. https://www.cancer.gov/publications/dictionaries/cancer-terms/search?contains=false&q=overall+survival Minimal residual disease - MRD • Tumor cells not eradicated by the treatment • Usually results in growth of these cells – resistance to treatment • Emerging component of CR assessment in MM patients • MRD negativity - associated with significantly longer OS Paiva et al, 2008; Rawston et al., 2013 Minimal residual disease Diagnosis Treatment Clinical remission Relapse Persistence Cure MRD DETECTION MRD MRD • Clonal expansion and selection • Presence of many subclones, sometimes undetectable at diagnosis • Different response to treatment • Change of treatment Clonal evolution of tumors Clonal tides Hematological malignancies Leukemia Lymphoma Multiple myeloma Hematological malignancies Leukemia Lymphoma Multiple myeloma • From Greek – leukos-white, hemos-blood • Symptoms known in the era of Hippokrates (460 - 370 BC) • R. Wirchow described in 1839 – 1845, named leukemia • „Omnis cellula e cellula“ Leukemia Leukemia • heterogeneous group of diseases • most common tumors in children • leukemic cells lose the ability to differentiate, high proliferation potential • two cell populations in the body - mature cells and immature cells = blasts Clinical features • Erythropenia – anemia • Thrombocytopenia – bleeding • Leukocytopenia – infections Prognosis of leukemia Morphology Chromosomal aberrations Age – worse prognosis B cells - worse prognosis Treatment of leukemia • Induction – treatment given with intent to induce complete remission • Consolidation – repetition of induction in a patient with induced complete remission to increase cure rate • Maintenance – long-term, low-dose treatment to delay regrowth of residual tumor cells • radiation and chemotherapy (combination) After chemotherapy • biopsy of bone marrow • further treatment if 5-10% of blasts • bone marrow transplantation Leukemia Acute Chronic Myeloidní Lymfoidní Leukemia Acute Chronic Myeloid Lymphoid Acute leukemia • fast proliferation of immature cells • bone marrow does not produce enough healthy cells • leukemic cells get into peripheral blood and infiltrate other organs (even CNS) • fast treatment needed – „medical emergency“ • most common in children Chronic leukemia • proliferation of relatively mature but abnormal cells • lasts for months or years • treatment not necessary at once in comparison to acute leukemia • mostly in older people ALL – more common in children AML – more common in elderly CLL – most common in adults CML – mostly in adults Hematopoesis Lymphoid leukemia Myeloid leukemia Risk factors for leukemia development • ionizing radiation • chemicals – benzene, cytostatics, alkylators and carcinogens • syndrome: Down (trisomy 21), Klinefelter (47, XXY) • viruses – HTLV-1 causes development of leukemia from T cells in adults • secondary leukemia - common after treatment for other malignancies Acute myeloid leukemia - AML Acute myeloid leukemia - AML • Fatigue, fever, bleeding • accumulation of blasts in bone marrow (> 20 %), bone marrow failure • Blasts in peripheral blood • Differentiation block at various stages of development • Most common leukemia in adults over 65 (80%) • about 20,000 of newly diagnosed patients in a year • Incidence 1.3/100 000 until 65, 12.5/100 000 over 65 • 70% of patients die within one year after diagnosis Auer rods • typical feature of AML • in cytoplasm of myeloblasts • negative prognostic marker • abnormal fusion of primary granules • Identified in 1905 Prognosis of AML Morphology Chromosomal aberrations Age at diagnosis Number of leukocytes at diagnosis FAB classification Classification of AML • 8 subtypes • based on morphology and cytochemistry FAB French American British classification • based on molecules, morphology and clinics WHO classification Subtype FAB Type Morphology Cytogenetic Abnl AML w/o maturation M0 no azurophil granules AML M1 few Aeur rods del(5); del(7); +8 AML w/ differentiation M2 maturation beyond promyelocytes; Auer rods t(8:21) t(6:9) Acute Promyelocytic Leukemia M3 hypergranular promyelocytes; Auer rods t(15:17) Acute Myelomonocytic Leukemia M4 > 20% monocytes; monocytoid cells in blood inv(16) del(16) t(16:16) t(4:11) Acute Monocytic Leukemia M5 monoblastic; promonocytic t(9:11) t(10:11) Acute Erythroleukemia M6 predominance of erythroblasts; dyserythropoiesis Acute Megakaryocytic Leukemia M7 dry' aspirate; biopsy dysplastic with blasts Classification of AML FAB Classification WHO classification Swerdlow 2016 Survival of young and older AML patients • upper graph shows survival of younger patients from 1970 (<60 years) • lower graph shows survival of older patients from 1970 • Kantarjian et al 2015 - MD Anderson Acute promyelocytic leukemia - APL the most malignant human leukemia APL • accumulation of promyelocytes (differentiation stage of granulocytes) • M3 classification based on FAB • treatment commenced immediately – medical emergency • for a diagnosis - detection of translocation necessary • median age at diagnosis 40 - same risk throughout lifetime • 1957 - subtype of leukemia • 1970 – identification of translocation - Dr. J. Rowley Molecular basis of APL • RARα – receptor pro all-trans retinoic acid • PML – promyelocytic gene • Translocation t(15;17) – reciprocal translocation APL treatment APL treatment Crespo-Solis 2016 APL survival Acute lymphoid leukemia - ALL Acute lymphoid leukemia - ALL • malignant transformation and proliferation of lymphoid progenitor in the bone marrow, peripheral blood and extramedullary sites • 80% ALL in children • Incidence 1.6/100 000 (USA) • 2016 - 6590 of newly diagnosed patients, 1400 deaths • bimodal distribution of incidence – children (4 years) and adults (50 years) • In children – survival 90% but only about 30-40% of adults reach long-term remission ALL etiology • significant correlation with Down syndrome, Fanconi anemia, Bloom syndrom, Ataxia Telangiectasia and Nijmegen breakdown syndrome • ionizing radiation, pesticides, smoking • Viruses - Epstein-Barr and HIV • Often de novo • Chromosomal aberrations t(12;21), t(1;19), t(9;22) and aberrations in MLL – not enough for ALL development – unknown origin • Induction (vincristin, corticosteroids, anthracyclins) • Transplantation of bone marrow Or • Consolidation • Maintenance 2-3 years ALL treatment Terwilliger 2017 Chronic myeloid leukemia - CML Chronic myeloid leukemia - CML first tumor linked to specific translocation between chromosomes 9 and 22 t(9;22) Philadelphia chromosome •1960 – Peter Nowell and David Hungerford described an abnormal chromosome in CML •First genetic cause of tumors •1972 – reason or consequence? Janet Rowley – t(9,22) CML • first tumor linked to specific aberration • CML chromosome described in 1960 in Philadelphia – Philadelphia chromosome • 1972 translocation described t(9;22) (Rowley) • 1983 kinase abl described on chromosome 9 (Heisterkamp) • 1984 bcr region described on chromosome 22 (Groffen) • 1990 bcr-abl reason for CML (Daley) • Bcr-abl- abnormal tyrosin kinase (Lugo, 1990) • Chronic phase, accelerated phase, blast crisis • Very bad prognosis (Less than 3 years) CML progression CML • Incidence 1-2/100 000 • 15% newly diagnosed patients with leukemia • 9000/year of new cases in USA • 1000/year die (since Gleevec – annual mortality 1-2%) • Prevalence – 25 000 (2000), 100 000 (2017), 180 000 (2030) CML treatment • Until 2000 – hydroxyurea, IFNα • Transplantation of bone marrow curative but high mortality Gleevec (1993) Novartis • Imatinib mesylate • Active against CML colonies (Druker 1996) • 2 years later – clinical study: 31 patients, 98% response rate • Clinical study phase III: 16 countries, 177 centers, 1000 patients – study stopped, all patients on Gleevec • Survival 95%, survival 65% in blast crisis (8 years) • Molecular positivity of bcr-abl a problem - leukemic cells survive - danger of relapse? Current treatment of CML • Imatinib (Gleevec) • Dasatinib (Sprycel) • Nilotinib (Tasigna) • Bosutinib (Bosulif) • Ponatinib (Iclusig) • Asciminib (Scemblix) FDA approved Current treatment of CML • Imatinib – in recent years even generics • Dasatinib • 350 More potent than imatinib • inhibition of Src pathway • five years survival similar to imatinib • Nilotinib • structural analogue of imatinib but binds better • Five-year survival better than imatinib • Bosutinib - Src/Abl inhibitor • for patients resistant to previous lines of therapy CML diagnosis • 50% patients asymptomatic • Anemia, splenomegaly, fatigue, weight decrease • Cytogenetics for diagnosis • 100% of patients - bcr-abl, but also other aberrations (trisomy 8, …) • bone marrow biopsy Chronic lymphocytic leukemia - CLL Chronic lymphocytic leukemia - CLL • 30% of all leukemias • the most common type of leukemia in Western countries • clonal expansion of B cells - CD5 positive in peripheral blood, bone marrow, lymph nodes and spleen • more common in men (1.7:1) • Incidence 4.1/100 000 • Median age at diagnosis 67 Hallek 2019 CLL etiology • Genetics • Viruses (EBV, HIV) • Radiation • Chemicals • Smoking CLL genetic changes • primary changes in multipotent hematopoietic stem cells • Deletion 13q, deletion 11q, trisomy of chromosome 12 • Del(13q14) primary change - 55% of cases • Del(11q) - 25% of patients – deletion 11q23- gene ATM – decreased OS • Trisomy 12- 10-20% of patients • Del(17q) – 5-8% of patients – resistence to chemotherapy CLL diagnosis • Blood smear, immunophenotyping • More than 5000 B cells/1 μl of peripheral blood • Clonality based on flow cytometry CLL risk factors • deletion or mutation of TP53 • IGHV mutation • Serum β2 macroglobulin • Age over 65 CLL treatment • Chlorambucil – alkylator • Purine analogues - fludarabin, pentostatin, cladribin • Monoclonal antibodies – antiCD20 (rituximab) • Ibrutinib – inhibitor of bruton tyrosine kinase, FDA approved • Venetoclax – inhibitor of BCL2, FDA approved CLL treatment CLL Hematological malignancies Leukemia Lymphoma Multiple myeloma Lymphoma • malignant proliferation of lymphatic tissue – B, T cells • Solid tumor of blood cells • 1832 described by Dr. Hodgkin • most common hematological malignancy • 5.3 % of all tumors • Diffusing into other lymph nodes and tissues • Histology: • Hodgkin (more common in men) • Non-Hodgkin (B,T, NK cells) Lymphoma • Diffuse large B cell lymphoma (30 %) • follicular lymphoma (22 %) • MALT-lymphoma (8 %) • chronic B lymphocytic leukemia (7 %) • mantle cell lymphoma (6 %) Most common lymphoma: • Weight loss (10 % / 6 months) • Fever, night sweats All malignant lymphoma may present as B-symptoms: Hodgkin lymphoma • Painless enlargement of nodes (neck, axillary) • Fever, sweating, fatigue, weight loss • Splenomegaly • Cough, emphysema • Infiltration of parenchymous organs • Etiology unknown – genetics, HIV, EBV • Common in adults between 20-30 and over 50 Hodgkin lymphoma •type I - lymphocyte-rich: majority of lymphocytes (few Reed-Sternberg cells, best prognosis) (5% of cases) •type II - nodular-sclerosis (nodular deposits, cells – reticular, lymphocytes, histiocytes) in collagen fibres (70%) •type III - mixed cellularity (20–25%) •type IV - lymphocyte-depleted (Reed-Sternberg cells increased, worst prognosis) (1%) Reed-Sternberg cells – abnormal lymphocytes, characteristic for lymphomas, multinucleated cells Hodgkin lymphoma Hodgkin lymphoma Non-Hodgkin lymphoma • Heterogeneous group of tumors (cca 40 types) • Arising from lymph nodes – fast migration into tissues and metastases in children • At the time of diagnosis – 2/3 of patients have advanced stage of the disease • in children highly malignant tumors - intense chemo treatment - successful in 80% of cases • In adults – less malignant Myelodysplastic syndromes - MDS Myelodysplastic syndromes - MDS • Heterogenous group of myeloid disorders characterized by cytopenia in peripheral blood and increased risk of progression into secondary AML • Incidence 3-4/100 000 (USA) • Prevalence increases with age • Diagnosis: bone marrow biopsy • Stratification: analysis of peripheral cytopenia, percentage of blasts in the bone marrow, cytogenetic analysis Cytogenetic classification of MDS • Mutations in TP53, RUNX1, ASXL1, JAK2 and RAS genes is connected to significantly shorter OS after allotransplantation of the bone marrow • TP53 mutations have a strong negative effect Survival of MDS patients depends on TP53 mutation Montelban-Bravo, 2018 Hematological malignancies Leukemia Lymphoma Multiple myeloma Multiple myeloma MM Hájek, 2012 Anderson, 2011 • second most common hematological malignancy • 10% of hematological malignancies • median age at diagnosis - 65 • Incidence 4/100 000 • more common in men • multistep pathogenesis Pathogenesis of MM - multistep process Jovanovic 2019 healthy bone marrow MM bone marrow www.pathologyatlas.com MGUS • Monoclonal gammopathy of undetermined significance • Benign, asymptomatic • accumulation of genetic changes in plasma cells leading to malignant transformation • bone marrow infiltrated by <10 % of malignant plasma cells • 15 % people with MGUS progress into MM • 1 % risk of progression to MM every year • Incidence 3 % of population over 50 (increases with age) MM • infiltration of bone marrow by malignant plasma cells • bone lesions • presence of monoclonal immunoglobulin (M-Ig) in serum and/or urine • Bone marrow niche supports proliferation and survival of malignant myeloma cells Plasma cell leukemia • loss of dependence of plasma cells on bone marrow microenvironment • > 20 % circulating plasma cells in peripheral blood • Incidence 4/ 10 000 000 • transformation from MM - 21 months • Very bad prognosis - 2 - 3 months https://www.labmedica.com/hematology/articles/294776427/new-definition-for-plasma-cell-leukemia-proposed.html MM symptoms • Effect on bone marrow: anemia, decrease of immune reactions, bleeding • Osteolytic lesion: pain, fractures • Presence of defective immunoglobulins: hyperviscosity, decrease of immunity MM diagnosis quite difficult – pain, fatigue, repeated infections common for other diseases 1) number of myeloma cells in the bone marrow 2) presence of abnormal protein in blood or urine 3) typical changes on the bones Treatment of MM ….this is what we tried Hájek, 2012 Anderson, 2011 Treatment of MM …and this is what we're currently using • chemotherapy • transplantation of bone marrow • immunomodulatory drugs • proteasome inhibitors • monoclonal antibodies Hájek, 2012 Anderson, 2011 Prognosis of MM • untreated patients survive 14 months • standard therapy 3 - 4 years • Transplantation 6 – 7 years • New drugs increase five-year survival for about 80% of patients Hájek, 2012 Treatment possibilities for MM IMIDs (immunomodulatory drugs) Proteasome inhibitors •1953- created by Chemie Grünenthal •1957- distribution (without prescription) •Sedative •Relieves morning sickness •Heavy teratogen •Insufficient testing in animals •About 10 000 children effected – around 40 % survived •FDA - Dr. Francis Kelsey – did not allow usage of thalidomide in the United States Thalidomide – first IMID White House Archive Dr. Francis Kelsey (1914-2015) Thalidomide children… today Thalidomide – continuation • 1964 – Jason Sheskin – patient with leprosy and complications • 1993- Judah Folkman – angiogenesis important not only for solid tumors but also hematological • 1994 – refractory MM patient – thalidomide – clinical study 1/3 of patients responded • 2006 – FDA – treatment of MM approved • unpleasant side effects - neuropathy Sedlaříková, 2012 Treatment possibilities for MM IMIDs (immunomodulatory drugs) Proteasome inhibitors Proteasome inhibitors •Proteasome – a proteolytic complex for degradation of ubiquitinated proteins •MM cells produce large amount of proteins - inhibition of proteasome leads to accumulation of proteins in the cells and apoptosis •Bortezomib – first proteasome inhibitor approved for treatment of MM III. Survival of patients with hematological malignancies Incidence and survival and that is all …. There are papers for your further studies in IS Thank you for your attention