Week 13 - Acute Leukemias -- Introduction to Hematologic Neoplasms

General characteristics of neoplasms:

• Either benign or malignant

• Only malignant cells are referred to as cancer

• Acquired genetic diseases — most patients not born with disease but acquire it sometime later (not always the case)

Neoplasm means?:

• New growth caused by dysregulated proliferation of a single transformed cell

Multiple ways to approach hematological neoplasms:

Characteristic hematologic findings in hematopoietic neoplasms:

Comparison of precursor and mature hematopoeitic neoplasms:

Neoplastic disorder categories:

• Can be grouped into 3 main categories

  • Acute leukemia

  • Myeloproliferative neoplasms (MPN): bone marrow makes too many cells

  • Myleodysplastic syndrome (MDS): bone marrow doesn’t produce enough healthy cells

Classification

• Various ways to categorize and classify

• FAB and WHO are two major systems

   FAB more straight forward,

   Trend leaning towards WHO classification

Why is it important to classifiy neoplasms:

 Can compare various therapeutic regimens

 System for diagnosis using clearly defined clinical features and

laboratory evaluation

 Permits meaningful associations of genetic abnormalities with

pathogenesis of disease

Two major classification systems:

• Before 2001, FAB (French-American-British)

  • Morphology, cytochemistry, immunophenotyping

   WHO (2001, 2008, 2016, 2022) (World Health Organization)

  • Morphology, cytochemistry, immunophenotyping, molecular analysis

  • Based on neoplastic cell lineage

   Three major groups

  – Myeloid, lymphoid, and histiocytic/dendritic

Diagnosing and Classifying Neoplasms: Initial evaluation

• Peripheral blood and BM samples

  • Aspirate and biopsy for BM

• Morphology and blast count

• Differentiation into cell lineage

  • Morphology

  • Immunophenotyping

  • Cytogenetic analysis

  • Molecular analysis

• MDS and MPN

  • Immunophenotyping usually not necessary

Inital evaluation, differential diagnosis, prognosis, monitoring includes:

• cytochemistry

• Immunological analysis

• Genetic analysis

• Cytogenetic analysis

• Molecular analysis

Hematologic remission:

• Absence of neoplastic cells in PB and BM

• Return to normal levels of hematologic parameters

Cytogenic remission:

• Absence of recognized cytogenetic abnormalities associated with a given neoplastic disease

Molecular remission:

• Absence of detectable molecular abnormalities using PCR or

  related molecular technologies

Minimal residual disease:

• Negative “traditional” tests (PB and BM cell counts,

  cytogenetics)

• Positive molecular tests (PCR/FISH)

Therapeutic success rates:

• Differ by disease and the patient’s condition at time of

  diagnosis

• Often obtain remission and then relapse

• Most treatment regimens

  • Target actively proliferating cells and not the hematopoietic stem cell

  • Reason for relapse – because the stem cell is responsible for propagation of neoplasm

Chemotherapy:

• Goal is to eradicate all malignant cells

• Allows for repopulation by residual normal precursors

• Problem

  • Not specific for leukemic or cancerous cells

  • Kills many normal cells

• Complications

  • Bleeding, infections, anemia

Three groups of chemotherapy drugs:

• Antimetabolites

• Alkylating agents

• Anti-tumor antibiotics

Molecular treatment:

• Target genetic mutations

Epigenetic therapies:

• Drugs attempting to alter gene expression

Bone marrow transplant/stem cell transplant:

• Try to replace with healthy stem cell

Hematopoietic growth factors:

• Supportive care of acute leukemic (AL) patients

• Erythropoeiten, G-CSF, GM-CSF, Interleukin-11

Complications of Treatment:

• Aggravate patient’s clinical situation

• ↑ uric acid from cell turnover

  • Precipitates in renal tubules

  • Leads to renal failure

• Lysed cells

  • Release procoagulants

  • Disseminated intravascular coagulation (D I)C) and hemorrhage

• Chemo destroys normal and leukemic cells

  • Causes infection, bleeding, anemia

Acute Leukemias:

• AML,ALL

Chronic Leukemia (CL):

• CLL, PLL, HCL, CLTC, Mature B, Mature T

Myeloproliferative Neoplasms (MPN)

• CML, Myelofibrosis, Polycythemia, ET, other MPN’s

Myelodysplastic Syndrome (MDS):

• Group of syndromes

Lymphoma’s and Plasma Cell Disorders:

• Hodgkin’s, Burkitt's, Mantel Cell, Marginal B-Cell, Sezary Syndrome,

• Multiple Myeloma, Waldenstroms, PCL

Acute Leukemia (AL): ALL and AML general characteristics:

• stem cell disorders characterized by malignant neoplastic proliferation and accumulation of immature and nonfunctional hematopoietic cells in the bone marrow

Two major categories of AL are classified according to the origin of the cell with the primary defect:

• acute myeloid leukemia (AML): defect primarily affects common myeloid progenitor

• acute lymphoblastic leukemia (ALL): defect primarily affects common lymphoid progenitor

Definition of Leukemia:

• Mutation of stem cells in the bone marrow, causing impaired production of normal RBC’s, WBC’s and Platelets

• Characterized by unregulated proliferation of cells with replacement of normal bone marrow cells

• A gap in normal maturation form of cells

• Alterations in BM = production issues, spleen becomes site of extramedullary hematopoiesis

Leukemias are categorized based on cell lineage and maturity:

• Acute myeloid leukemias (AML)

• Acute lymphoblastic leukemias (ALL)

• Chronic myelocytic leukemias (CML)

• Chronic lymphocytic leukemias (CLL)

2 Major cell types with both acute and chronic forms:

• Myelogenous or Lymphocyte

• Acute or chronic

Cell maturity: Acute:

• Immature cell types predominate

Cell maturity: Chronic

• More mature cell types predominate

  • Usually seen in adults

Characteristics of Acute Leukemia

• Rapid onset

• Short duration

• Disease seen at any age

• Variable WBC count

• PLT count usually decreased

• Anemia: mild

  • Normocytic/Normochromic with decreased amount of RBC

• Fatal within months if not diagnosed and treated aggressively

• Blasts and immature cells are observed in the bone marrow and peripheral smear

• FAB Classification

• >30% blasts in bone marrow

• WHO Classification

• >20% blasts in bone marrow

Leukemia sub classification:

• Morphology, cytochemical stain, immunologic criteria, molecular analysis

FAB Classification of Leukemia:

• AML – Acute Myeloid Leukemia

  • M0 – M7

• CML – Chronic Myeloid Leukemia

• ALL – Acute Lymphoblastic Leukemia

  • L1 – L3

• CLL – Chronic Lymphocytic Leukemia

• ANLL – Acute Non lymphocytic Leukemia

Acute Leukemia: General Features:

• N/N Anemia

  • Fatigue, malaise, pale appearance

• Thrombocytopenia

  • Gingival bleeding, epistaxis, ecchymosis, petechiae

• Fever

• WBC variable

• Hepatosplenomegaly, lymphadenopathy, or bone pain

  and tenderness

Acute Leukemia defect:

• Blasts do not mature

• Leukemic cells are arrested at an immature stage

  • Leukemia Hiatus: presence of blast cells and mature cells without an orderly morphologic progression between the 2

• Lack of normal differentiation antigens and are unable to respond to normal regulatory feedback control

• Neoplasia in Common Myeloid Progenitors AML

French-American-Britich (FAB) Classification of AML:

• 

2016 WHO Classification of Myeloid Malignancies:

• Histopathological features

• Genetic features

• Pathogenesis of neoplasms

• Standardization of nomenclature

• Advances in therapy

• The large mononuclear cells are myeloblasts. The cell at the left (arrow) is a myeloblast with an Auer rod. Note the high nuclear-to-cytoplasmic ratio, the fine lacy chromatin, and the prominent nucleoli. (Wright-Giemsa stain, 1000× magnification)

Genetic Susceptibility:

• Congenital abnormalities associated with karyotype abnormalities (Down’s Syndrome)

Somatic Maturation:

• Acquired change in genetic material of cells (radiation, chemicals i.e. benzene)

Viral Infection:

• Retroviruses are proven to be responsible for leukemia in lab animals (Human T Cell Leukemia/Lymphoma)

Immunological Dysfunction:

• Increased incidence of lymphocytic leukemia has been observed in both congenital and acquired immunological disorders. Breakdown in cell mediated immune self surveillance

Classification of any type of leukemia:

• Morphology alone is ineffective

  • Call blasts “blasts” on peripheral smear, don’t differentiate

  • Definition of Blast: earliest, least differentiated stage of hematopoietic maturation that can be identified by its morphology in a wright stain (typically in) bone marrow - for example: Myeloblast, Pronormoblast (Rubriblast), Lymphoblast

• Other methods: cytochemical stains, CD markers, chromosomal analysis, molecular testing

Cytochemical Analysis:

• Used to identify the chemical composition of cells without

  altering the cell morphology

• Often performed on bone marrow slides

Enzymatic reactions:

• Peroxidase, Esterases, Phosphatases, Terminal deoxynucleotidyl transferase (TdT)

Non-enzymatic reactions:

• Sudan Black, Periodic Acid Shift (PAS), Toluidine Blue

Staining profiles: Myeloid:

• Peroxidase and Sudan Black POSITIVE

Staining profiles: Lymphocytic:

• PAS and TdT POSITIVE

Staining profiles: Basophil:

• Toluidine POSITIVE

Peroxidase:

• Myeloperoxidase (MPO) stain used as a marker for primary neutrophilic granules

• Not found in lymphs

• NEED: freshly prepared blood or bone marrow

• Peroxidase activity disappears

  • Enzyme dissipates after awhile = need fresh sample

• ONLY evaluate the BLAST cell

• Myeloid +

• Lymphoid -

• Peroxidase present in primary granules of myeloid cells.

• Enzyme not present in lymphocytes (or precursors)

• Useful to differentiate between AML and ALL

• More specific then Sudan Black B

• Image shows + peroxidase in M3 (APL)

Esterase:

• Differentiate granulocytic from monocytic

• 2 Types: specific and non-specific

• Specific Esterase:

  • Naphthol AS-D Chloroacetate (CAE)– granulocytic marker (primary granules)

• Non-Specific Esterase:

  • Alpha-Naphthyl-Acetate (ANAE)

  • Alpha-Naphthyl-Butyrate

  • These are useful markers for monocytes

• Combined Esterase:

  • Combination of both

• Specific

  • Naphthol AS-D Chloroacetate

• Granulocytic

• +AML - M2

• Non-specific

  • Alpha-naphthyl butyrate

• Monocyte

• +AML - M5

Phosphotase:

• Acid phosphatase: present in all hematopoietic cells.

• When tartaric is added all cells become negative EXCEPT hairy cell lymphoma.

• Known as TRAP stain (Tartrate Resistant Acid Phosphatase)

Leukocyte Alkaline Phosphatase (LAP):

• enzyme activity found in the secondary granules of neutrophils.

• Used to differentiate CML from Leukemoid reaction

  • Decreased with CML

  • Increased with Leukemoid reaction

LAP Score (used to grade granulation of neutrophil):

• Normal 13-130 (may vary slightly from lab to lab)

• Greater then 160 usually considered increase

• Lower then 13 usually considered decreased

• Range of assay (0-400)

• 100 segmented neutrophil/bands are counted.

• Each cell is graded using scale 0 - 4+ according to appearance (amount and intensity of precipitated dye)

Example of LAP Calculation:

• 

Terminal Deoxynucleotidyl Transferase (TdT):

• Enzyme marker for primitive lymphocytes

• A DNA polymerase found in immature lymphocytic cells

• High levels have been found in 90% of all cases of ALL

• Can use in conjunction with various immunological techniques (immunofluorescence, flow cytometry, immunohistochemistry)

Periodic Acid Schiff (PAS):

• Non enzyme reaction that stains glycogen, mucoproteins, glycoproteins, glycolipids, and polysaccharides.

• Stains lymphocytes, granulocytes, monocytes, and megakaryocytes.

• Glycogen found in all leukocytes, but patterns differ

  • Granulocytes and myeloblasts: diffuse pattern

  • Monos: weak stain

  • Lymphs of ALL (80%) chunky or block like

• Not frequently used for classification or identification of Acute leukemia’s

• PAS positive in Acute Lymphoblastic Leukemia (ALL)

• Note the “block staining” pattern

Sudan Black B (SBB):

• Non-enzyme reaction which stains intracellular lipids

• Also stains neutral fats and phospholipids

• SBB most sensitive for granulocytic precursors

• More stable

  • Because it doesn't depend on enzymes

  • Particularly useful for specimens that aren’t fresh

• Closely parallels myeloperoxidase (MPO)

Toluidine Blue:

• Basic dye that reacts with mucopolysaccharides

• Specific for the basophil and mast cell

• Special Stains Chart

CD Markers: Cluster of Differentiation:

• A system of nomenclature developed in order to label membrane proteins or complexes of proteins in which physical properties or interactions with monoclonal antibodies are known

• Designations are most commonly seen on hematopoietic cells

• Can test for cell surface markers using monoclonal antibodies (against specific marker)

  • Use immunohistochemistry or flow cytometry analysis

Flow Cytometry:

• Yields information regarding biophysical and biochemical nature of a cell

• Device design

• Analysis is by light scatter and fluorescence dyes that have been tagged with monoclonal antibodies

  • Look at side scatter and forward scatter to investigate cell population

  • Gating: defined boundary that can be used to identify a refined cellular population

• Flow Cytometry to study cell populations

• Gating created to distinguish cell populations of interest

• Flow cytometry and fluorescence to analyze cell surface markers

• Quadrants in scatter plot designate presence or absence of markers

• Common CD markers

• Characteristic Immunophenotype of Lymphoproliferative Disorders

• Flow Cytometric Requirements for Assigning Blast Lineage

• Commonly analyzed Markers

• AML Phenotype

Acute Myelocytic Leukemia:

• Occurs primarily in the adult or infants (less then one year)

• Myeloid stem cell that mutates and losses ability to proliferate and differentiate

• Relatively immobile cells confined to the bone marrow

  • n/n anemia, PLT count decreased, WBC vary high – low

  • Increase in pseudo pelger-huet, Eos and Baso

• Helpful when Auer Rods are found

  • never in lymphs

AML subtypes:

• M0, M1, M2, M3, M4, M4EO, M5a, M5b, M6, M7

AML - M0:

• Minimally Differentiated

• Blast are cytochemically unreactive

• Myeloid and Monocytic antigens can be detected

  • CD11, CD14, CD34, CD117

• Frequency around 5%

• Poor Prognosis

AML - M0 Immunophenotype:

• CD13+, CD33+, CD117+, HLA-DR±, CD34±, CD38+

M1: Myeloid Leukemia without Maturation:

• Seen in young to middle age adults

• Predominant cell myeloblast, +/- auer rods

• Frequency 10%

• MPO+, SBB+, CAE+

• CD13, CD33 myeloid markers

• High WBC count

• Poor Prognosis

M1 Immunphenotype:

• CD13+, CD33+, CD34±, CD117+, HLA-DR±

Without maturation:

• Myeloblasts are large with scant amount of gray blue cytoplasm

• Nucleus can be round to oval with fine chromatin

• Usually very distinct nucleoli

• Auer rods present in about 50% of cases

M2 - With Maturation:

• More mature then the blast seen

• 50% blasts and 50% are beyond the blast

• Auer rods usually not present

• Blasts tend to look monocytic

• Frequency of occurrence ~ 30-45%

• Most common in adults

• MPO+, SBB+, CD13 and CD33

• Diagnostic translocation chromosome 8;21

• (q22;q22)

• Favorable prognosis

M2 Immunophenotype:

• CD13+, CD33+, CD65+, CD11b+, CD15+, HLA-DR±

M3 - Promyelocytic:

• Acute Promyelocytic Leukemia (APL)

• More common in young adults (males)

• Often massive bleeding due to thrombocytopenia and DIC

• Bi-lobed promyelocytes containing primary granules (heavy) Auer rods in bundles cigar shaped (faggot) cells

• Chromosome abnormality t(15;17) (q22;q12)

• Frequency of occurrence ~ 10%

• MPO+, SBB+, SE+, PAS-

M3 Immunophenotype:

• CD13±, CD33+, CD34−, HLA- DR-

M3v - Acute Promyelocytic Leukemia: microgranular variant:

• Very tiny or non-visible granulation

• Bi-lobed nucleus resembling a monocyte

• Often confused with monocytic leukemia

• Higher WBC count than M3

• Frequency ~ 20 – 30% of cases

• Abnormal karyotype t(15;17)

• MPO+, SBB+

• Prognosis good with the t(15;17)

M3v Immunophenotype:

• CD13±, CD33+, CD34−, HLA- DR−, CD64+, CD117±

M4 Myelomonocytic:

• Predominant cells: myeloblast and monoblast

• More common in males >50% or occurring in the first few months of life

• Soft tissue infiltrates (gums)

• Highest of WBC

• Frequency of occurrence ~ 20%

• Lysozome and muramidase +

• MPO+, SBB+, NSE+, SE+

• CD 13, 33 – Myeloid

• CD14, 4, 11c, 64 - Monocytic

M3 Myelomonocytic Immunophenotype:

• CD13+, CD33+, CD14+, CD4+, CD11b+, CD64+, CD15+, CD36+

M4EO - Eosinophilia:

• Acute myelomonocytic leukemia with eosinophilia

• Also referred to as AML with inv(16) (13.1;q22)

• Variant type characterized by increase of eosinophils

• Bone marrow Eos dysplastic with many large granules

• Frequency 4%

• Chromosomal abnormality: inversion of chromosome16

• Has Central Nervous System involvement

M4EO - Immunophenotype:

• CD34+, CD117+, CD13+, CD33+, CD15+, CD4+, CD11b+, CD11c+, CD14+, CD64+, CD36+, CD65+

M5 - Monocytic:

• Very high WBC counts

  • Predominance of monoblasts

• More prevalent in males and younger patients

  • Adult males >49 years of age

• Gum and skin involvement

• CNS and renal involvement

• Frequency of occurrence 5-15%

• MPO-, SBB-, NSE+

• Chromosome abnormality: translocation 9q to 23

• CD14, 4, 11c, 36, 64, 88

• Unfavorable prognosis

• 2 subtypes 5a and 5b

M5 Immunophenotype:

• CD33+, CD13+, CD4+, CD14+, CD11b+, CD64+,

  CD15+, CD65+, CD11c+, CD36+, CD68+, HLA-DR+

M5 Subgroups: M5a - Poorly Differentiated:

• 1-3 nucleoli

• Cytoplasm more abundant, tends to be large and irregular in shape

• Monoblasts account for about 80% of all monocytic cells

• Remaining 20% are monocytes

• Patients typically younger and have poor prognosis

M5 Subgroups: M5b - Well Differentiated:

• All stages of monocytes seen

• More than 80% of monocytic cells located in nonerythroid

  marrow

• Remaining cells are promonocytic and monocytic

• Less then 30% blasts

• Gum infiltrates, skin rashes

• MPO-, SBB-, NSE+