MLAB 1415- HematologyKeri Brophy-Martinez

Introduction to Hematopoietic Neoplasms

Terms Neoplasm or tumor: “New growth”

Results from a dysregular prolieferation of a single transformed cell Can be malignant or benign

Malignant “Deadly” Clone of abnormal, proliferating cells, without function or

differentiation Have the potential to metastasize or get bigger “Cancer”

Benign Premalignant Originate from highly organized, differentiated cells Do not spread or invade surrounding tissues

Classification of Neoplasms in Bone Marrow

Lymphoid Only lymphocytic cells affected

Myeloid Granulocytes, monocytes, megakaryocytes,

erythrocytes affected Both Lymphoid and Myeloid lines can

include benign and malignant neoplasms

More Terms Leukemia

A malignant disease of hematopoietic tissue characterized by replacement of normal bone marrow elements with abnormal (neoplastic) blood cells. Abnormal cells are also seen in peripheral blood

Lymphoma Abnormal proliferation of lymphoid cells within the

lymphatic tissue or lymph nodes, results in a solid tumor

Leukemias

General ClassificationAcute Chronic

Age All ages Adults

Clinical onset Sudden Gradual

Course of disease Weeks to months Months to years

Predominant cell BlastsSome mature forms

Mature forms

Anemia Mild-severe Mild

Thrombocytopenia Mild-severe Mild

WBC Variable Increased

Organomegaly Mild Prominent

Blood Picture

Acute Chronic

How Do Leukemias Arise? Somatic mutation of a single

hematopoietic stem or progenitor cell

Unlimited self-renewal of the cancer-initiating cell

As the mutant cell line predominates, normal hematopoiesis is inhibited causing leukemic cells to spill into peripheral blood

Proto-oncogenes and Oncogenes Proto-Oncogene—normal unaltered gene that has the

potential to become an oncogene Oncogene–Altered cell genes that cause tumors Located at breakpoints of chromosomal aberrations, such as

translocations

Oncogene Activation Factors

Genetic susceptibility Fanconi’s anemia Down’s syndrome (18-20 fold increased incidence)

Somatic mutation Ionizing radiation, nuclear weapons Chemicals and drugs Benzene,Chloramphenicol, phenylbutazone Certain chemotherapy drugs that are cytotoxic, especially when used in conjuction with

therapeutic radiation Viral infection

Retrovirus-HIV-1, HTLVI, II EBV

Immunologic dysfunction Ataxia-telangiectasia - lymphoid leukemia or lymphoma Sex-linked agammaglobulinemia

Epidemiology Most new cases found in older adults ( > 67 yrs old) 50% of leukemias are acute More common in whites More common in males Age groups

ALL: children 2-5 years old: lymphoid CLL: Adults> 50: lymphoid AML: adults: myeloid CML: adults: myeloid

Evaluation of Leukemia Note onset of symptoms Analyzing CBC

Thrombocytopenia? RBC level/ anemia?

Observe cell lineage Lymphoid or Myeloid?

Assess maturity of predominating cells

Lab Features and Patient Symptoms Normochromic,normocytic anemia

Due to erythropenia

Thrombocytopenia Results in bleeding episodes Platelet morphology and function can be abnormal

Leukocyte count can be increased, decreased or normal If count decreased- results in infections

Immature leukocyte precursors seen Bone marrow hypercellular

Bone pain due to marrow expansion

Maturation abnormalities in all cell lines Uric acid increased

Due to cellular breakdown

Official Hematopoietic Neoplasm Classification

Two systems French-American British(FAB)

Historical World Health Organization (WHO)**

Widely accepted Important because..

Allows clinicians a way to compare therapeutic regimens System for ID and comparison of clinical features & lab findings Permit associations of cytogenetic abnormalities with disease

FAB classification

Consists of three groups Myeloproliferative Disorders(MPD) Myelodysplastic Syndromes(MDS) Acute leukemia (AL)

Based on morphological characteristic of Wright-stained cells in peripheral blood or bone marrow with supplementary cytochemical stains

WHO Classification Classification system uses morphology, cytochemistry and

immunophenotyping to determine cell lineage and degree of maturation(similar to FAB)

Additionally uses genetic and clinical features prior to therapy and history of MDS to define subgroups

Consists of three groups Myeloid

Further classified as MPD, MDS/MPD, MDS, AML

Lymphoid Further classified as B/T cell, T/NK cell, Hodgkin’s Disease, CLL

Histiocytic

Lab Techniques for Diagnosis and classification of neoplasms

Cytochemical analysis In vitro staining of cells to look at cells’ chemical

composition Evaluation of positivity in these stains must be

determined on the leukemic blast stage of the cell Usually performed on bone marrow slides Helpful in differentiating lymphoid or myeloid lineage of

blasts in AL Reactions are either enzymatic or nonenzymatic

Cytochemical stains Types

Myeloperoxidase (MPO) Activity is present in the primary granules and Auer rods of

myeloid cells Separates myeloid and lymphoid blasts Stains late myeloblasts, granulocytes, monocytes less intensely Differentiates AML from ALL Granules stain black to reddish-brown

Cytochemical stains: con’t Sudan Black B

Activity is present in phospholipids in the membrane of 1J (nonspecific) and 2J granules (specific)

Differentiates AML from ALL Granules stain black

Periodic Acid Schiff (PAS) Activity is in glycogen and related substances Stains lymphocytes, granulocytes, megakaryocytes Helpful in diagnosing erythroleukemia where there is strong reactivity

in normoblasts Stains red-purple in blocks in cytoplasm

Cytochemical stains: con’t Esterases

Specific Esterase (Naphthol AS-D Chloroacetate) Activity is in cytoplasm Stains neutrophilic granulocytes, differentiates monoblasts and

myeloblasts Granules of myeloblasts stain blue-black

Nonspecific Esterase (Alpha-Naphthyl Acetate) Activity is in cytoplasm Stains monocytes and also megakaryocytes Differentiates myeloblasts from monoblasts Granules stain orange red

Cytochemical stains: con’t

Leukocyte Alkaline Phosphatase (LAP) Enzyme within the 2O or specific granules of maturing

granulocytes Distinguishes leukemoid reactions ( ) from chronic

myelogenous leukemia ( ) Acid Phosphatase (Tartrate Resistant)

Present in lysosomes in normal leukocytes Helpful in diagnosing hairy cell leukemia because they

are NOT inhibited by TRAP

Cytochemical stains: con’t

Terminal Deoxynucleotidy transferase (TdT) Primitive cell marker found in cell nuclei Distinguishes ALL from malignant lymphoma

Toluidine Blue Positive marker for basophils and mast cells

Lab Techniques for Diagnosis and classification of neoplasms

Immunologic marker studies Cell surface markers

Monoclonal or polyclonal antisera is added to cell suspensions of fresh peripheral blood or bone marrow and an immunofluorescent method is used in a flow cytometry instrument to analyze the markers which are expressed as cluster designations (CD).

CD’s identify antibodies that are specific for certain cells and allow for a positive identification.

Lab Techniques for Diagnosis and classification of neoplasms

Molecular Genetics This newer method of diagnosing leukemia consists of DNA probes and

polymerase chain reaction (PCR)-based studies. They are rapid and precise and are used to confirm chromosomal abnormalities that are not detected by conventional studies. They are also used to monitor residual disease

following therapy. FISH (Fluorescence in situ hybridization) Cytogenetics (Chromosome studies)

Identifies chromosome translocations which are specific for certain leukemias

Philadelphia chromosome (t[9:22]) is associated with CML t[15:17] is associated with acute promyelocytic leukemia

Treatment Cures are not common except in childhood

leukemia. The best hope for a cure in adults lies in bone marrow transplantation. Cytoreductive chemotherapy

Reduces the leukemic cell mass Block DNA synthesis Block RNA synthesis Complications arise from marrow hypoplasia and

resulting cytopenia

Treatment Radiotherapy (radiation)

Kills focalized leukemic cells Usually used in addition to chemotherapy and for CNS

prophylaxis

Bone marrow transplantation Bone marrow is eradicated with chemo and radiation. Compatible donor cells are transfused and they travel to the

empty marrow where they engraft and repopulate the marrow with healthy cells.

Complications include graft vs host (GVH) disease which can be fatal.

References McKenzie, Shirlyn B., and J. Lynne. Williams. "Chapter 21."

Introduction. Clinical Laboratory Hematology. Boston: Pearson, 2010. Print.

http://www.lls.org/content/nationalcontent/resourcecenter/freeeducationmaterials/generalcancer/pdf/facts.pdf