Making Blood: The Good, the Bad and the Ugly Kate Kolibaba, M.D. Northwest Cancer Specialists Vancouver, WA [email protected]

Objectives Normal hematopoiesis Anemia • Evaluation and management

Myelodysplasia Leukemia • Subtypes • Management considerations

the good

the good

the bad

the ugly

Blood Cells Red blood cells • Carry oxygen

White blood cells • Fight infection - Lymphocytes - viruses

Make antibodies - Granulocytes - bacteria

Platelets • Stop bleeding

Red blood cells • Easy to transfuse • Live for weeks

White blood cells • Cannot be transfused • Lymphocytes live for months

• Granulocytes live for hours

Platelets • Limited ability to transfuse • Live for days

Anemia Decreased hemoglobin concentration and decreased red blood cell number Finding as part of workup for fatigue, pallor, generalized weakness, failure to thrive Incidental finding

Anemia Classification of anemia • Size of the red cells • Mechanism of the anemia

An approach to the diagnosis of anemia Treatment of anemia Indications for referral to a specialist

Mechanisms That Cause Anemia Decreased red blood cell production Increased red blood cell destruction Sequestration • Hypersplenism-usually apparent on physical exam

Dilution • Usually iatrogenic--IV fluids, plasma

Blood loss

Effects of Plasma Volume: Dilution

Plasma Volume

Packed Cell Volume Normal

Anemia From Dilution

“Normal” Dehydration

Anemia After Hydration

Diagnostic Problems in Anemia Differentiating between • Blood loss • Production problems - Reticulocyte count

• Red cell destruction - Difficult to measure

Approach to Anemia History • Symptoms or signs of blood loss • Diet: Iron, folic acid, B12 • Symptoms of low oxygen delivery - Fatigue, chest pain, SOB

• Rate of symptom development

Normal Hematopoiesis

Anemia: Clinical Findings Physical examination • • • • • •

Pallor Jaundice Tongue Splenomegaly Heme+ stools Lymphadenopathy

Peripheral Blood Smear Red blood cell morphology • • • • •

Size Shape (Fragments, spherocytes, etc.) Amount of hemoglobin Heterogeneity Polychromasia (reticulocytes)

White blood cell number and morphology Platelets number and morphology Pathologist review

Anemia and Red Cell Production High reticulocyte count = increased red cell production • Corrected reticulocyte count > 2% • Absolute reticulocyte number is elevated

Low or “Normal” Reticulocyte count = Production problem • Corrected reticulocyte count is < 1% • Normal or reduced reticulocyte number

Reticulocytes Reticulocytes are “young” red blood cells RNA in the cytoplasm Identifiable with “supravital” stains In a normal individual with a normal hematocrit and normal hematopoiesis, • 1% of red blood cells are identified as reticulocytes

Reticulocyte Counting

Reticulocyte Counting

The Corrected Reticulocyte Count In anemia, a given number of reticulocytes in the blood will have a higher percentage if the total number of red blood cells is diminished Corrected for the (low) number of red blood cells in anemia: Corrected retic count = measured retic % x Hct Normal Hct

Absolute Reticulocyte Number An alternate (better) means of assessing bone marrow production of reticulocytes Units are thousands per mm3 Normal: 10 - 90 k/ mm3 • Normal hematocrit, normal hematopoiesis

In an anemic patient, a “normal” reticulocyte number represents an abnormal, hypoproliferative response

Hyperproliferative Anemia 10% of anemia cases Normal compensation • • • •

Bleeding Nutritional recovery Recovery after treatment Hematopoietic growth factors

Hemolysis

• Elevated LDH, indirect bilirubin • Coombs positivity • Decreased haptoglobin

Hyperproliferative Anemia Identify cause of anemia • Bleeding, drugs, autoimmune etc

Ensure “raw” materials are available • Iron • Folate

Hypoproliferative Anemia 90% of cases Blood cell size • Macrocytic • Microcytic • Normocytic

Decreased Red Cell Production Nutritional deficiency • Iron • Folic acid • B12

Marrow empty

• Aplastic anemia

Marrow sleeping

• Erythropoietin deficiency • Hypothyroid

Decreased Red Cell Production: The Bad Marrow broken • Myelodysplasia • Hematopoietic malignancies or disorders

Marrow full of something else • Infection

Decreased Red Cell Production: The Ugly Marrow full of something else • Cancer • Hematopoietic malignancies

Classification of Anemia by Red Blood Cell Size Automated red cell counters

• MCV = mean corpuscular hemoglobin volume • Physically measured • Misleading in the setting of populations of red cells of varying sizes

Morphologic review

• Mechanism of anemia often apparent • Trained human eye

Normocytic Anemia: Red Cells of Normal Size Blood loss Bone marrow failure Mixed nutritional deficiencies Anemia of chronic disease • May be microcytic -- iron utilization impaired

Renal insufficiency • May be microcytic -- iron utilization impaired

Microcytic Anemia: Small Red Blood Cells Red blood cells are bags of hemoglobin Less hemoglobin = small red cells Problems • Globin • Heme

Microcytic Anemia: Heme Problems Iron deficiency • Less heme

Lead poisoning

• Interferes with heme synthesis

Sideroblastic anemia • Heme defect

Anemia of chronic disease (sometimes normocytic) • Impaired iron utilization, • Impaired heme synthesis

Causes of Microcytic Anemia: Globin Thalasemia Globin gene defect • Alpha • Beta • May or may not cause - Microcytosis - Anemia

Laboratory Evaluation of Microcytic Anemia

MCV Serum iron TIBC Iron Saturation Ferritin

Iron Deficiency

Anemia of Chronic Disease

Low Low >350 Low Low (100)

Bone Marrow and Laboratory Evaluation of Microcytic Anemia Iron Deficiency

Anemia of Chronic Disease

Erythropoietin

Very High

Low, for degree of anemia

Marrow sideroblasts

Absent

Absent

Marrow iron

Absent

Present

Treatment of Iron Deficiency Identify and treat the source of the blood loss! Oral iron therapy If oral iron is poorly absorbed or tolerated, parenteral iron may be given • Risk of anaphylaxis

Oral Iron Therapy 325 mg ferrous sulfate po TID • Provides 150-200 mg elemental iron daily • Absorption is increased if taken between meals with an acidic beverage or food (fruit) • Hemoglobin should rise 1-2 gm/dl per month • After anemia is fully resolved, continue iron therapy for 4 – 6 months to replete iron stores

Anemia of Chronic Disease Block in iron utilization by red cells Shortened red cell survival Direct inhibition of erythropoiesis • Cytokines: IL-1, TNFα

Relative deficiency of erythropoietin • Stage III kidney disease

Treatment of the Anemia of Chronic Disease Iron alone will not help! Recombinant erythropoietin • Renal insufficiency • Concerns about safety - Black box warnings: thromboembolism

Macrocytic Anemia: Large Red Blood Cells Increased membrane around the bag of hemoglobin • Round macrocytes

Defective nuclear division -- the bag of hemoglobin continues to grow while waiting for the nucleus to be “finished” • Oval macrocytes

Round Macrocytes: Large Round Red Cells Liver disease Thyroid disease Alcohol

Oval Macrocytes: Large Oval Red Cells Megaloblastic anemia

• Accompanied by hypersegmented neutrophils • B12 or folate deficiency

Myelodysplasia Drugs

• AZT • Hydroxyurea • Chemotherapy

Relationship Between B12 and Folate Metabolism DNA

Thymidilate

Deoxyuridilate

N5-Methyl FH4 ← N5N10-Methylene FH4 ← FH4 ← FolateH2

MethylCobalamin

Homocysteine

Cobalamin (B12) Methionine

Methylmalonic Acid

Megaloblastic Anemia Folate Deficiency

B12 Deficiency

Most common cause

Diet

Malabsorption

Time to development

Months

Years

Response to B12 therapy

No

Yes

Response to Folic Acid

Yes

Heme: Yes Neuro: No

Laboratory Evaluation of Megaloblastic Anemia If vitamin B12 deficiency or folic acid deficiency are suspected, draw: • homocysteine • methylmalonic acid • ferritin

Laboratory Evaluation of Megaloblastic Anemia “Normal” B12 levels have been reported in 30% of patients with hematologic and/or neurologic disease from B12 deficiency 20% of elderly patients are B12 deficient Methylmalonic acid levels are more sensitive than B12 measurement 30% of patients with folate deficiency are also iron deficient

Laboratory Evaluation of Megaloblastic Anemia

Folate Deficiency

Homocysteine Methylmalonic Acid

B12 Deficiency

Elevated

Elevated

Normal

Elevated

Treatment of Megaloblastic Anemia Draw labs Give 1 mg B12 IM • –5 doses during first month, then monthly thereafter • 1000 μg po daily as effective as 1000 μg IM in 2 randomized trials (specific settings)

Folic acid 1 mg po QD • (up to 5 mg per day)

Treatment of B12 Deficiency Must use parenteral B12 for • Compliance questions • Malabsorption -

Crohns Whipple’s disease Celiac disease Gastrectomy Resection of final 80 cm of small intestine Chronic pancreatitis, pancreatectromy Cholestyramine, cholchicine

Andres et al, CMAJ 2004; 171: 251-259

Referral to a Specialist When in doubt, obtain a “curbside” consult Don’t use the name of the physician in the chart unless you will have them see the patient Bone marrow aspirate and biopsy

Indications for Referral to a Hematologist Pancytopenia Circulating primitive cells (blasts) Very low reticulocyte count, i.e. 2.5

Median Survival 5.7 years 3.5 years 1.2 years 0.4 years

Dysplastic Hematopoiesis

Acute Leukemia

Leukemia

the ugly

Defining Leukemia Hematologic malignancy arising in the bone marrow Clonal neoplasm arising from a single pluripotent stem cell • Clonal: progeny of a single cell

Incidence of Leukemia in USA

Number of Cases

10000 9000 8000 7000 6000 5000 4000 3000 2000 1000 0

Adults Children

AML

ALL

CML

CLL

Types of Leukemia Acute

Chronic

Myeloid

acute myelogenous (AML)

chronic myelogenous (CML)

Lymphoid

acute lymphocytic (ALL)

chronic lymphocytic (CLL)

Leukemia 28,000 cases diagnosed annually in the USA Many patients can be cured Acute lymphoblastic leukemia is the most common childhood cancer • Recurrent ALL is the 6th most common childhood malignancy

Some leukemia results from toxic exposures or therapeutic drugs or radiation

Normal Hematopoiesis vs. Leukemia Heterogeneous Tightly regulated, orderly proliferation Polyclonal Capacity for renewal and proliferation on demand (stem cells) Capacity for function by production of mature cells

Monotonous Unregulated proliferation Disorderly maturation Clonal expansion and dominance Loss of renewal Loss of functional capacity

Problems Caused by Leukemia Bone marrow failure Infiltration of organs by leukemic cells Systemic effects Immune dysfunction in CLL

Bone Marrow Failure Anemia • fatigue or lethargy • pallor

Thrombocytopenia • bruising • bleeding

Neutropenia • Infection • ANC < 500ul

Infiltration of Organs Sites • • • •

bone marrow liver, spleen enlargement in up to 75% lymph node enlargement in up to 80% meninges, testes

Interference with function • Compression

Pain

Systemic Effects of Tumor Burden Result from hypermetabolism, cytokines • fever • sweats • malaise • anorexia, weight loss

Diagnosing Leukemia Bone marrow aspirate and biopsy • Cytogenetics • Immunophenotyping - Cell surface proteins

• Molecular analysis - PCR (polymerase chain reaction) - FISH (fluorescent in situ hybridization)

• Morphologic review

Normal Hematopoiesis

Acute Leukemia

Acute Leukemia Accumulation of immature cancer cells Differentiation is blocked Production of cancer cells is excessive, uncontrolled Low or elevated WBC in untreated patients

Types of Leukemia Acute

Chronic

Myeloid

acute myelogenous (AML)

chronic myelogenous (CML)

Lymphoid

acute lymphocytic (ALL)

chronic lymphocytic (CLL)

ALL

vs.

Lymphoblast Scant cytoplasm No cytoplasmic granules Cytochemical stains • PAS

Immunophenotype • CD10 • CD7 • tdT

AML Myeloblast Abundant cytoplasm Cytoplasmic granules • Auer rods

Cytochemical stains • Myeloperoxidase

Immunophenotype • CD33 • CD13

Presentation of Acute Leukemia Bone marrow failure • anemia: pallor, fatigue • thrombocytopenia: bleeding, petichiae, ecchymoses • neutropenia: infection

Systemic symptoms • malaise, anorexia • sweats

Presentation of Acute Leukemia Organ infiltration • • • • •

Bone pain Mild hepatosplenomegaly – ALL Gingival hypertrophy – AML CNS involvement Leukostasis- high myeloid blast count - Medical emergency

Acute Leukemia Emergencies Leukostasis • Extremely high myeloid blast count • CNS - Headache, vision problems, slowing

• Pulmonary - Chest pain, dyspnea

Tumor lysis syndrome • Hyperuricemia, hyperkalemia, hyperphosphatemia • Renal failure

Therapy for Acute Leukemia Cure is possible Chemotherapy • Remission induction • Post-remission therapy

Bone marrow transplantation Supportive care • IV access, TPN, transfusion

Acute Leukemia: Complications Bleeding Infection

Chemotherapy for AML Induction • Nearly myeloablative, 3-4 weeks of cytopenias • induce remission, return of normal blood cells • Induction death rate 20% in adults

Consolidation (post remission) • eradicate residual leukemia cells

In some cases, stem cell transplantation • high dose chemotherapy • infusion of bone marrow to “rescue” patient

Prognosis in AML 2/3 of patients achieve remission Of those who achieve remission, half will relapse and die of their disease or of complications Bottom line for treated patients: • Patients 65: 10-20% disease-free at 5 yrs

Chemotherapy for ALL Induction • induce remission and the return of normal blood cells

Consolidation Maintenance CNS prophylaxis In some cases, stem cell transplantation

Prognosis in ALL Childhood ALL • 80% cure • Risk-stratified therapy

Adult ALL • 75% achieve remission • Majority relapse • 20-30% disease-free at 5 yrs

Types of Leukemia Acute

Chronic

Myeloid

acute myelogenous (AML)

chronic myelogenous (CML)

Lymphoid

acute lymphocytic (ALL)

chronic lymphocytic (CLL)

CLL Excess mature lymphocytes “smudge cells” Flow cytometry: • CD5/CD19 +

vs.

CML Full spectrum of maturing myeloid cells basophila cytogenetics: t(9;22) -- the Philadelphia chromosome

Chronic Leukemia Accumulation of mature-appearing cancer cells Bone marrow differentiation is intact Cancer cell production is excessive, uncontrolled Elevated WBC in untreated patients

Normal Hematopoiesis

Chronic Leukemia

What Problems Are Caused by Chronic Leukemia? Infiltration of organs by leukemic cells Systemic effects CLL--increased susceptibility to infections Bone marrow failure only with advanced disease

Types of Leukemia Acute

Chronic

Myeloid

acute myelogenous (AML)

chronic myelogenous (CML)

Lymphoid

acute lymphocytic (ALL)

chronic lymphocytic (CLL)

Chronic Lymphocytic Leukemia (CLL) Disease of the elderly Incurable without stem cell transplantation Chronic disease Accumulation of mature lymphocytes • Monoclonal B-cells

Immune dysregulation • Autoimmune anemia and thrombocytopenia • Infection

Immune Dysfunction in CLL CLL (Chronic Lymphocytic Leukemia) • Autoimmunity - Hemolytic anemia - Immune thrombocytopenia

• Anergy • Hypogammaglobulinemia - Susceptibility to encapsulated bacteria

CLL at Diagnosis 20% asymptomatic -- incidental finding Organ infiltration • lymphadenopathy • hepatosplenomegaly

Systemic symptoms • night sweats, fever, weight loss, malaise

Bone marrow failure • only with advanced disease

Treatment of CLL Treatment prolongs survival if intensive Chemotherapy is indicated for palliation • • • •

painful organomegaly systemic symptoms cytopenias recurrent infections

Treatment • chlorambucil -- oral • Fludarabine, cyclophosphamide, bendamustine • Rituximab, ofatumomab

Prognosis of CLL Incurable with standard chemotherapy Chemotherapy to palliate symptoms, prolong survival Median survival predicted by extent of disease and molecular features: • > 12 yrs: lymphocytosis only • 6 yrs: lymphocytosis + lymphadenopathy and/or hepatosplenomegaly • 4 yrs: lymphocytosis + anemia and/or thrombocytopenia

Types of Leukemia Acute

Chronic

Myeloid

acute myelogenous (AML)

chronic myelogenous (CML)

Lymphoid

acute lymphocytic (ALL)

chronic lymphocytic (CLL)

CANCER TREATMENT OF THE FUTURE: Identify what’s broken and target that defect CML: The first targeted approach in leukemia treatment. Tyrosine kinase inhibitors stop the activity of the oncoprotein BCR-ABL.

Evolving concepts in cancer biology Preclinical studies Clinical trials New therapies

Chronic Myelogenous Leukemia Philadelphia chromosome

• Balanced reciprocal translocation between the long arms of chromosome 9 and 22 • c-bcr from 22 • c-abl from 9

Ph1 c-bcr

c-abl 9

22

bcr-abl 9q+

22q-

Molecular Consequences of the Philadelphia Chromosome Translocation Chromosome 22 1

2

1

4

3

2

Chromosome 9 5

c-Bcr

1

2-11

c-Abl

+/ 3 -

2-11

p210 Bcr-Abl

1

2-11

p185 Bcr-Abl

Exons

Introns

CML Breakpoints ALL Breakpoints

Chronic Myelogenous Leukemia Chronic phase • Massive expansion of white blood cells • No increased risk of infection

Accelerated phase • increased blood counts, symptoms

Blast crisis • terminal stage resembling acute leukemia

CML at Diagnosis Asymptomatic: • 15-30% of chronic phase patients

Organ infiltration • massive splenomegaly

Systemic symptoms • weight loss, malaise

Prognosis of CML Untreated median survival of about 5 yrs Long term control with tyrosine kinase inhibitors is possible Median survival >20 yrs However: Adherence Resistant disease

Treatment of CML Imatinib (Gleevec) • Competitive inhibitor of the bcr-abl, kit, PDGFR kinases • Oral therapy of indefinite duration • Hematologic remission- normal CBC • Cytogenetic remission - Suppresses the leukemic (Philadelphia +) clone

• Molecular remission - detection by PCR • Durability of remission unknown - Loss of control is about 3% per year initially

Mechanism of Action of Imatinib Mesylate

Treatment of CML Newer tyrosine kinase inhibitors are effective in imatanib resistance or as initial therapy Dasatanib Nilotinib For resistant disease: Bosutinib Ponatinib Oral therapy of indefinite duration

• Hematologic remission – normal blood counts • Cytogenetic remission • Molecular remission – suppression of the leukemic clone

Stem Cell Transplantation Myeloablative therapy • High-dose chemotherapy • + radiation

“Rescue” infusion of hematopoietic stem cells • Bone marrow • Peripheral blood (apheresis) • Umbilical cord blood

Non-myeloablative = “mini”

Stem Cell Sources RISK OF RELAPSE

Autologous Allogeneic • Syngeneic (twin) • HLA-matched related • Unrelated volunteer

GRAFT VS HOST DISEASE RISK

New Therapies for the Future Antisense therapy Proteasome inhibitors Small molecule inhibitors Gene expression profiling Cytokines Vaccines Monoclonal antibodies

Questions?

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