1. MLAB 1415- Hematology Keri Brophy-Martinez
Chapter 21:
Introduction to Hematopoietic Neoplasms

2. 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

3. 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

4. Further Classifications
Premalignant myeloid neoplasm
Myeloproliferative disorders (MPD)
Myelodysplastic syndromes (MDS)
Premalignant lymphoid disorders
Chronic lymphoproliferative disorders
Plasma cell disorders
Lymphoid and myeloid malignant bone marrow neoplasms
Leukemia

5. 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

6. Leukemias

7. General Classification
Acute
Chronic
Age
All ages
Adults
Clinical onset
Sudden
Gradual
Course of disease
Weeks to months
Months to years
Predominant cell
Blasts
Some mature forms
Mature forms
Anemia
Mild-severe
Mild
Thrombocytopenia
WBC
Variable
Increased
Organomegaly
Prominent

8. Blood Picture
Acute
Chronic

9. 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

10. 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

11. 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
Immunologic dysfunction
Ataxia-telangiectasia - lymphoid leukemia or lymphoma
Sex-linked agammaglobulinemia
From studies on laboratory animals, several factors have been proposed as playing roles in the activation of oncogenes including Genetic susceptibility, somatic mutation, viral infection, and immunologic dysfunction.
Strong evidence suggests that hereditary factors and abnormal genetic material have significant impact on oncogene activation. A number of individuals who have congenital abnormalities associated with karyotypic abnormalities have a markedly increased risk of developing acute leukemia. Down syndrome is the best known of the genetic abnormalities associated with leukemia, but Fanconi’s is another.
A somatic cell mutation is an ACQUIRED change in the genetic matierial of cells OTHER THAN those involved in reproduction. Mutations in the chromosome NEAR the proto-oncogenes likely play a role in the development of neoplasms. More than 50% of patients with leukemia show acquired abnormal karyotypes. Radiation, some chemicals, and drugs can cause chromosome mutations. Ionizing radiation has long been recognized as capable of inducing leukemia, which is evident from observations of human exposure to radiation from nuclear reactions, therapeutic radiation, and occupational exposure.
An increase in leukemia has be observed after treatmetn with alkylating agents and other chemotherapies. Benzene, Chloramphenicol, and phenlybutazone can cause somatic mutations as well.
Retroviruses contain a reverse transcriptase that allows them to produce a DNA copy of the viral RNA core. The DNA can then be copied to produce more viral cores or can be incorporated INTO the HOST’s cells nuclear DNA. The strongest support for the existence of a leukemia causing virus in humans come fro the isolation of several retroviruses known as HUMAN T CELL LEUKEMIA/LYMPHOMA virus ONE , TWO and FIVE. The Acronym is HTLV 1, two or five. The other retrovirus that causes leukemia is the human immunodeficiency virus or HIV-one.
Hereditary immunologic disorders such as Wiskott-Aldrich syndrome or Bruton’s type x-linked agammaglobulinemia, and ataxia-telangietasia can lead to leukemia due to the bodies inability to recognize and destroy neoplastic cells.

12. Epidemiology Most new cases found in older adults ( > 67 yrs old)
50% of leukemias are acute
More common in whites
Age groups
ALL: children 2-5 years old: lymphoid
CLL: Adults> 50: lymphoid
AML: adults: myeloid
CML: adults: myeloid

13. Clinical Findings Anemia Due to erythropenia Infection
Due to neutropenia
Bleeding Episodes
Due to thrombocytopenia
Bone pain
Due to marrow expansion
Weight loss

14. Evaluation of Leukemia
Note onset of symptoms
Analyzing CBC
Thrombocytopenia?
RBC level/ anemia?
Observe cell lineage
Lymphoid or Myeloid?
Assess maturity of predominating cells

15. Lab Features Normochromic,normocytic anemia Thrombocytopenia
Platelet morphology and function can be abnormal
Leukocyte count can be increased, decreased or normal
Immature leukocyte precursors seen
Bone marrow hypercellular
Maturation abnormalities in all cell lines
Uric acid increased

16. 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

17. Consists of three groups
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

18. 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 four groups
Myeloid
Further classified as MPD, MDS/MPD, MDS, AML
Lymphoid
Further classified as B cell, T/NK cell, Hodgkin’s Disease
Mast cell
Histiocytic

19. 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

20. 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 reddish-brown
Smears must be fresh

21. Cytochemical stains: con’t
Sudan Black B
Activity is present in phospholipids in the membrane of 1̊ (nonspecific) and 2̊ granules (specific)
Parallels myeloperoxidase but smears do not have to be fresh
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

22. 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)
Stains monocytes and also megakaryocytes
Differentiates myeloblasts from monoblasts (can use a double staining technique to view both specific and nonspecific stains on one smear)
Addition of Na fluoride to this stain inhibits activity in monocytes
Granules stain orange red

23. 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
Present in lysosomes in normal leukocytes
Helpful in diagnosing hairy cell leukemia because they are NOT inhibited by TRAP

24. 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

25. 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.

26. 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

27. 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

28. Treatment Radiotherapy (radiation) Bone marrow transplantation
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.
READ FROM SLIDE

29. References
McKenzie, Shirlyn B., and J. Lynne. Williams. "Chapter 21." Introduction. Clinical Laboratory Hematology. Boston: Pearson, Print.