Blood Diseases: Myeloproliferative Neoplasms and Essential Thrombocythemia

UNICAMILLUS MEDICAL UNIVERSITY

Lesson 4 @ UNICAMILLUS MEDICAL UNIVERSITY Blood Diseases Slide 1 Emiliano Fabiani, PhD emiliano.fabiani@unicamillus.org UNICAMILLUS INT CAM

Myeloproliferative Neoplasms (MPNs)

Lesson 4 @ UNICAMILLUS MEDICAL UNIVERSITY Myeloproliferative Neoplasms (MPNs) Slide 2 Myeloproliferative Neoplasms (MPNs): are a group of clonal myeloid neoplasms in which a genetic alteration occurs in a hematopoietic progenitor cell leading to its proliferation resulting in an increase of white blood cells (WBCs) and/or red blood cells (RBCs) and/or platelets (PLTs) in the peripheral blood MPNs are characterized by:

• Effective hematopoiesis
• Increased circulating maturing cells from one or more hematopoietic lineages (leukocytosis, thrombocytosis, erythrocytosis)
• Normal differentiation
• Chronic course
• Progression to Acute Myeloid Leukemia (AML) Splenomegaly and hepatomegaly are common and caused by the sequestration of excess blood cells and extramedullary hematopoiesis

Hematopoietic Progenitors and MPNs

Lesson 4 O UNICAMILLUS MEDICAL UNIVERSITY Hematopoietic Progenitors and MPNs Slide 3 All MPNs arise from precursors of the myeloid lineage in the bone marrow Genetic Mutation Blood stem cell Myeloid stem cell Lymphoid stem cell Myeloblast Lymphoblast Red blood cells Platelets White blood cells National Cancer Institute

Classification of MPNs (W. Demeshek classification)

Lesson 4 @ UNICAMILLUS MEDICAL UNIVERSITY Classification of MPNs (W. Demeshek classification) Slide 4 The type of disorder is often based on the predominant cell line that is affected, but because blood counts are often abnormal in more than one cell line, diagnoses based only on blood counts may be inaccurate

• Chronic Myelogenous Leukemia (CML)
• Essential Thrombocytosis (ET)
• Polycythemia Vera (PV)
• Primary Myelofibrosis (PMF) Polycythemia vera (PV), together with essential thrombocythemia (ET) and myelofibrosis (MF), belongs to the so-called "classic" t(9;22)-BCR-ABL1-negative myeloproliferative neoplasms (MPN)

Chronic Myeloid Leukemia, BCR-ABL1 Positive

Lesson 4 @ UNICAMILLUS MEDICAL UNIVERSITY Chronic Myeloid Leukemia, BCR-ABL1 Positive Slide 5 Chronic myelogenous leukemia (CML) is a myeloproliferative neoplasm that origines from an abnormal multipotent myeloid stem cell and is consistently associated with the BCR-ABL1 fusion gene (Philadelphia (Ph) chromosome) CML is characterized by a proliferation and progressive accumulation of mature granulocyte cells in bone marrow and peripheral blood Central for the pathogenesis of CML is the fusion of the Abelson Murine Leukemia Viral Oncogene Homolog 1 (ABL1) gene on chromosome 9 with the breakpoint cluster region (BCR) gene on chromosome 22; t(9;22)(q.34,1;q11,2) "Philadelphia (Ph) chromosome". This results in the expression of an oncoprotein termed BCR-ABL1 The oncoprotein BCR-ABL1 encodes for a constitutively active tyrosine kinase that promotes growth and replication of hematopoietic cells Atlas of Hematological Cytology. Masaryk University, Faculty of Medicine / University Hospital Bra. Available from: http://www.leukemia-cell.org/atlas

CML Features

Lesson 4 @ UNICAMILLUS MEDICAL UNMESTY Chronic Myeloid Leukemia, BCR-ABL1 Positive Slide 6

• In CML, the predominant feature is leukocytosis
• Mild anemia, normal to elevated platelet count, and peripheral blood basophilia are often observed Chronic Myelogenous Leukemia (CML)

CML Incidence and Survival

Lesson 4 O UNICAMILLUS MEDICAL UNIVERSITY Chronic Myeloid Leukemia, BCR-ABL1 Positive Slide 7 The annual incidence of CML is 1.5 cases per 100.000 individuals

• CML accounts for approximately 15% of newly diagnosed cases of leukemia in adults
• The median age at CML onset is 45-65 years
• No familial associations in CML
• The median survival in CML is 3-7 years, but in patients treated with the tyrosine kinase inhibitor (TKI) Imatinib a normal life expectancy has been achieved, even if patients require life-long therapy The annual mortality has been reduced from 10-20% to 2%
• Higher incidence in males compared to females 100 Chronic myelogenous leukemia Incidence rate (cases/100.000 population) 10 -Male Female 0.1- <1 1-4 5-9 10-1415-19 20-24 25-29 30-34 35-39 40-44 45-49 50-54 55-59 60-64 65-69 70-74 75-79 80-84 85+ Age

Philadelphia (Ph) chromosome t(9;22)

Lesson 4 @ UNICAMILLUS MEDICAL UNIVERSITY Philadelphia (Ph) chromosome t(9;22) Slide 8

• The Philadelphia chromosome (Ph) was the first recurrent genetic alteration found to be associated with a specific human cancer, chronic myeloid leukemia (CML)
• The Philadelphia chromosome has been identified in more than 95% of CML patients, but also in 5% or less of children with ALL (20% of adult ALL) and in 2% or less of children with AML
• The genetic mechanisms involved in t(9;22)(q34;q11) are well understood
• The fusion gene encodes an activated tyrosine kinase, which dysregulates cell proliferation, differentiation and apoptosis
• The breakpoint on the BCR gene characterizes different protein isoforms of the hybrid BCR/ABL fusion gene

Breakpoint Regions in BCR Gene

Lesson 4 @ UNICAMILLUS MEDICAL UNIVERSITY Philadelphia (Ph) chromosome t(9;22) Slide 9 The ABL gene contains a large breakpoint region (~200 kb), whereas three breakpoint regions have been found in the BCR gene:

1. m-bcr -> p190 (e1a2): is also frequently associated with Ph+ acute lymphoblastic leukemia (ALL)
2. M-bcr -> p210 (b2a2; b3a2): can be detected in more than 95% of chronic myeloid leukemia (CML)
3. - bcr -> p230 (e19a2): more rare a BCR gene (# 22q11) 1 2 34 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 utr utr m-bcr (~55 kb) p190 variant v-bcr p195 variant M-bcr (~2.9 kb) p210 variant p225 variant p-bcr (~1 kb) p230 variant ABL gene (# 9q34) 1a 1b 2 3 4 5 6 7 8 9 10 11 utr utr breakpoint region ~200 kb m-bcr, p190 proteins e1a2 1 2 3 4 e1a3 1 3 4 M-bcr, p210 proteins e14a2{ 11 12 13 14 2 3 4 V e13a2> 11 12 13 2 3 4 3 e14a3 11 12 13 14 3 4 3 e13a3 5 11 12 13 3 4 3 u-bcr, p230 proteins e19a2§ 17 18 19 2 3 4 3 e19a3 17 18 19 ---- 3 4 V rare variants v-bcr, p195 proteins e6a2 5 5 6 2 3 4 3 p200 proteins e8a2 5 7 8 2 3 4 5 p225 proteins e18a2 S 17 18 2 3 4 3 Weerkamp Fet al. Flow cytometric immunobead assay for the detection of BCR-ABL fusion proteins in leukemia patients. Leukemia. 2009 - p200 variant Л variable insertions

CML Diagnosis and Monitoring

Lesson 4 O UNICAMILLUS MEDICAL UNIVERSITY Philadelphia (Ph) chromosome t(9;22) Slide 10 The diagnosis of typical CML is based on the presence of the Philadelphia (Ph) chromosome abnormality Citogenetic test (Karyotyoe/FISH) Karyotype 5 10 11 12 17 18 3. . 15 19 20 21 22 x (FISH) BCR-ABL1 Chromosomes 9 22 ABL BCR Molecular studies (RT-PCR or QPCR) RT-PCR M nRT 778 ctrl+ ctrl- M nRT 778 ctrl+ ctrl- M 778 1 p190 p210 ABL QPCR High Concentration Moderate Concentration Low Concentration - 0 3 6 9 12 15 18 21 24 27 30 33 36 PCR Cycle Number Standardized and regular MRD monitoring in CML patients is essential for defining treatment response MRD Amount of Fluorescence 15 16 3€

Phases of CML

Lesson 4 @ UNICAMILLUS MEDICAL UNIVERSITY Phases of CML Slide 11 The natural history of untreated CML is bi- or triphasic:

• Chronic phase (CP) Initial asymptomatic period that can last from 5 to 6 years (blast <10%)
• chronic phase (blasts 10-15%) Accelerated phase (AP) Treatment failure, worsening anemia, progressive thrombocytopenia or persistent thrombocytosis, worsening of splenomegaly, clonal evolution. Blasts (15-30%)
• Blast phase (BP) Progressive accumulation of blasts in extramedullary sites (eg, bone, central nervous system, lymph nodes, skin) (Blasts ≥ 30%) Chronic Phase Accelerated Phase Blast Crisis Median duration without treatment 5-6 years 6-9 months 3-6 months BCR-ABL Expansion of myeloid compartment New cytogenetic abnormalities Increased genomic instability Blasts 10-15% >15%<30% 530% Symptoms Asymptomatic Increased tiredness Weight loss Enlarged spleen Increased blast cells in bone marrow & blood Extramedullary disease Secondary genetic and molecular abnormalities lead to an accumulation of mutations and genomic instability can led to progression, blast crisis and poor prognosis

Signs and Symptoms of CML

Lesson 4 O UNICAMILLUS MEDICAL UNIVERSITY Signs and Symptoms of CML Slide 12

• CML is in most cases an asymptomatic disease and leukocytosis is the principal clinical feature
• 50-60% of CML patients are diagnosed by routine blood tests
• WBCs count can range from 20.000 to 300.000/ml
• In about 1/3 of cases, leukocytosis is associated with an increased number of platelets The peripheral blood smear shows an increase in neutrophil and basophil granulocytes and the presence of myeloid precursors (myelocytes,metamyelocyte) At the diagnosis, CML patients often present: splenomegaly, anemia and related symptoms (30-40% of cases)
• Less common findings may include thrombotic events (cardiovascular complications or bleeding) neutrophil basophil myelocytes metamyelocyte Systemic Psychological Fatigue and Loss of appetite - Weight loss - Fever - Frequent infections Lungs Shortness of breath Lymph nodes Swelling Muscular Weakness Spleen Enlargement Bones or joints Pain or tenderness Skin Liver Enlargement Night sweats, Easy bleeding and bruising, Purplish, patches or spots

Clinical and Laboratory Features of CML

Lesson 4 @ UNICAMILLUS MEDICAL UNVESTY Clinical and Laboratory features of CML Slide 13 Clinical presentation of CML Asyntomatic Leucocytosis Splenomegaly Laboratory features and morphology

• Hb decreased
• Increased WBCs count
• Increased in neutrophil and basophil granulocytes count
• Increased myeloid precursors
• Platelet count normal or increased
• Increased levels of Lactate dehydrogenase (LDH)
• Hypercellular bone marrow
• BCR/ABL1 positivity

Treatment of CML

Lesson 4 @ UNICAMILLUS MEDICAL UNIVERSITY Treatment of CML Slide 14 CML therapy has been revolutionized since 2006 after the important results obtained with Imatinib, a drug capable of inhibiting the tyrosine kinase activity of the BCR/ABL1 fusion protein However, most patients have residual molecular disease and require life-long therapy Four tyrosine kinase inhibitors (TKIs) have been approved for first-line therapy:

• Imatinib (first generation TKIs)
• Dasatinib (second generation TKIs) Nilotinib (second generation TKIs)
• Ponatinib (third generation TKIs) During Imatinib treatment, different resistance mechanisms due to somatic mutations in the ABL1 domain can arise and in these cases second and third generation inhibitors can be used