Etiology, genetics and classification of hematological malignancies

2. Etiology, genetics and classification of hematological malignancies By/ Mr. WaqqasElaas; M.Sc; MLT

3. Objectives At the end of this lecture, the student should understand: • Definitions of some terminologies regarding Hematologic malignancies. • Understand the role of proto-oncogenes & tumor suppresser genes in the formation of blood cancers. • List the etiological factors behind hematologic malignancies. • Relate some viral, bacterial and protozoan infections to blood cancers. • List Genetic abnormalities associated with haematological malignancies. • Understand the two types of classification used to categorize hematologic neoplasms.

4. Definition • The hemopoietic malignancies are clonal diseases that derive from a single cell in the marrow or peripheral lymphoid tissue which has undergone a genetic alteration. • Cancer cells are cells that are engaged in uncontrolled mitosis.

5. Carcinogenesis or oncogenesis is literally the creation of cancer. • Cell division is a physiological process that occurs in almost all tissues and under many circumstances. Under normal circumstances, the balance between proliferation and programmed cell death, usually in the form of apoptosis, is maintained by tightly regulating both processes to ensure the integrity of organs and tissues. Mutations in DNA that lead to cancer (only certain mutations can lead to cancer and the majority of potential mutations will have no bearing) disrupt these orderly processes by disrupting the programming regulating the processes.

6. Carcinogenesis is caused by this mutation of the genetic material of normal cells, which upsets the normal balance between proliferation and cell death. This results in uncontrolled cell division and the evolution of those cells by natural selection in the body. The uncontrolled and often rapid proliferation of cells can lead to benign tumors; some types of these may turn into malignant tumors (cancer).

7. Benign tumors do not spread to other parts of the body or invade other tissues, and they are rarely a threat to life unless they compress vital structures or are physiologically active, for instance, producing a hormone. • Malignant tumors can invade other organs, spread to distant locations (metastasis) and become life-threatening.

8. Cancer is a genetic disease: In order for cells to start dividing uncontrollably, genes that regulate cell growth must be damaged. • Proto-oncogenes are genes that promote cell growth and mitosis, whereas tumor suppressor genes discourage cell growth, or temporarily halt cell division to carry out DNA repair. Typically, a series of several mutations to these genes is required before a normal cell transforms into a cancer cell.

9. Etiology • Exactly how genetic mutations accumulate in haemopoietic malignancies is largely unknown. • As in most diseases it is the combination of genetic background and environmental influence that determines the risk of developing a malignancy. However, in the majority of individual cases neither a genetic susceptibility nor an environmental agent is apparent.

10. 1. Inherited factors • The incidence of leukaemia is greatly increased in some genetic diseases, particularly Down's syndrome (where acute leukaemia occurs with a 20 -30- fold increased frequency), Bloom's syndrome, Fanconi'sanaemia, and others. • There is also a weak familial tendency in diseases such as acute myeloid leukaemia (AML), B-cell Chronic lymphocytic leukaemia (CLL).

11. 2. Environmental influences a. Chemicals : Chronic exposure to benzene may cause bone marrow hypoplasia, dysplasia and chromosome abnormalities and is an unusual cause of myelodysplasia or AML. Other industrial solvents and chemicals less commonly cause leukaemia. b. Drugs : The alkylating agents (e.g. chlorambucil) predispose to AML, especially if combined with radiotherapy or if used to treat patients with lymphocytic or plasmacytic disorders.

12. c. Radiation : Radiation, especially to the marrow, is leukemogenic. This is illustrated by an increased incidence of all types of leukemia (except CLL) in survivors of the atom bomb explosions in Japan. d. Infections

13. Appr. : HHV-8, human herpes virus 8; HIV, human immlmodeficiency virus; HTLV-I, human: T-lymphotropic virus type 1; MALT, mucosa-associated lymphoid tissue; PTLD, post-transplant lymphoproliferative disease.

14. The genetics of hemopoietic malignancy • Malignant transformation occurs as a result of the accumulation of genetic mutations in cellular genes. The genes that are involved in the development of cancer can be divided broadly into two groups: oncogenes and tumour-suppressor genes. • Oncogenes : An oncogene is a gene that when mutated or expressed at abnormally-high levels contributes to converting a normal cell into a cancer cell.

15. An oncogene is a gene that has the potential to cause cancer • Tumor-Suppressor Genes : The products of some genes inhibit mitosis. These genes are called tumor suppressor genes. The suicide of damaged cells — apoptosis — provides an important mechanism for ridding the body of cells that could go on to form a cancer. • It is not surprising then that inhibiting apoptosis can promote the formation of a cancer.

16. Genetic abnormalities associated withhematological malignancies • Point mutation: Mutation is defined as an alteration in the nucleotide sequence of DNA (deoxyribonucleic acid) of an organism. When a single base in the nucleotide sequence is replaced by another, then it is known as point mutation. Point mutations also include insertion and/or deletion of a single base in the DNA strand. • Translocations :These are chromosomal abnormalities which occur when chromosomes break and the fragments rejoin to other chromosomes. • Deletions :Chromosomal deletions may involve a small part of a chromosome, the short or long arm or the entire chromosome (e.g. monosomy 7). Losses most commonly affect chromosomes 5, 6, 7, 11, 20 and Y. The critical event is probably loss of a tumour suppressor gene or other mechanisms.

17. Duplication or amplification:In chromosomal duplication (e.g. trisomy 12 in CLL) or gene amplification, gains are common in chromosomes 8, 12, 19, 21 and y. Gene amplification is not a common feature in haemopoietic malignancy. • Epigenetic alterations:Gene expression may be dysregulated in cancer, not only by structural changes to the genes themselves but also by alterations in the mechanism by which genes are transcribed. These changes are called epigeneticand are stably inherited with each cell‘ division so they are passed on as the malignant cell divides. The most important mechanisms are methylation of cytosine residues in DNA and enzymatic alterations, such as acetylation or methylation, of the histone proteins that package DNA within the cell.

18. Diagnostic methods used to study malignant haemopoietic cells • The most common and frequent methods used can be classified as follow: • Karyotyping (Karyotype analysis) • FISH (Fluorescent In Situ Hyperdization) • FlowCytometry (FCM) • Immunohistology/Immunohistochemistry (IHC) • Cytochemistry • Molecular Diagnosis: PCR (Polymerase Chain Reaction)

19. Conventional cytogenetics:Karyotype analysis • Karyotype analysis; involves direct morphological analysis of chromosomes from the hematopoietic cells; (either bone marrow BM or lymph nodes) under the microscope This requires hematological cells to be in METAPHASE and so cells are cultured to encourage cell division prior to chromosomal preparation. • (Philadelphia “Ph” Chromosome/Chronic Myeloid Leukemia (CML)), in 1960, the 1st observed human hematological cancer, The Ph arises from a reciprocal translocation, t(9;22)(q34;q11).

20. G-banded karyogram with the translocation (8;21)(q22;q22) as the only anomaly. This change is seen exclusively in (AML/M2)

21. Molecular cytogenetics:Fluorescent in situ hybridization analysis (FISH) *Involves the use of fluorescent-labeled genetic probes which hybridize to specific parts of the genome. * a sensitive technique that can detect extra copies of genetic materials in both METAPHASE and INTER-PHASE (non-dividing) cells (e.g. trisomy 8 in some CML) or by using two different probes reveal chromosomal translocations, for example, t(9;22) in CML or reduced chromosome numbers or segments (e.g. monosomy7 or 5 in myelodysplasia).

22. Tetrasomy 10 Trisomy 8

23. Immunophenotyping:Flow cytometry *Antibodies labeled with different fluorochromes recognize the pattern and intensity of expression of different antigens on the surface of normal and leukemic cells. * A rapid technique. • AML high density of CD34, CD33, CD13 & MPO. • AML worse prognosis co-expression of CD56. • APL  lack of CD34, CD33, MPO & variable amount of CD13. CD : Cluster of Differentiation

24. Basic principle of FCM

25. Immunohistology/Immunohistochemistry (IHC) • Antibodies can also be used to stain tissue sections with fluorescent markers and this is known as immuno-histologtyor immuno-histo-chemistrty. The presence and architecture of turnout cells can be identified by visualization of stained tissue sections under the microscope. • IHC is an excellent detection technique and has the tremendous advantage of being able to show exactly where a given protein is located within the tissue examined, This technique is even more widely used in diagnostic surgical pathology; CD15 and CD30: used for Hodgkin's disease, Identification of B-cell lymphomas using CD20 and Identification of T-cell lymphomas using CD3.

26. Cytochemistry • During the development and maturation of hematopoetic precursors, certain enzymes and associated substances, such as glycogen, are produced in the developing cells which, if detected can provide important clues to the lineage and classification of leukemias, this performed by using cytochemical stains like: • (Sudan Black SB & Myeloperoxidase MPO stains; Myeloblasts+ve vs. Lymphoblasts –ve) • (Periodic Acid Schiff PAS stain, erythroleukemia (M6); a leukemia of immature red blood cells; these cells stained with a bright fuchsia).

27. Polymerase chain reaction(PCR): *Is used to amplify specific region of DNA (target), can be performed on blood or bone marrow for a number of specific translocations such as t(9;22) and t(15;17). It can also be used to detect clonal cells of B- or T-cells line. *Sensitive method (why?): (detects one abnormal cell in 10⁵- 10⁶ n.c)

29. Classification of Hematological Malignancies • Two types of classifications : • The French–American–British (FAB) classification. • The WHO classifications. • Application of the WHO criteria depend on the results of immunophenotyping and cytogenetic analysis. • The FAB classifications therefore continue to have a place: (a) when these techniques are not available, and (b) in making a provisional morphological diagnosis while awaiting the results of further tests (immunophenotypingandcytogenetics).

30. The World Health Organization (WHO) Classification includes tumors of lymphoid, myeloid, histiocytic, and dendritic cell lineages.• Each disease is defined as a distinct entity based on morphological, clinical, and biological features but the cell of origin is the starting point of disease definition.• Some lymphomas and leukemias can be identified by routine morphological approaches. However, for many diseases, knowledge of the immunophenotype and molecular genetics/cytogeneticsplays an important role in differential diagnosis.

31. Classification of myeloid malignancies • Myeloproliferativediseases (MPD) • Myelodysplastic/myeloproliferative diseases • Myelodysplastic syndromes (MDS) • Acute myeloid leukemias (AML) • Classification of lymphoid malignancies -Precursor Lymphoid Neoplasms -Mature B-Cell Neoplasms -Mature T-Cell and NK-Cell Neoplasms -Hodgkin Lymphoma -Histiocytic and Dendritic Cell Neoplasms -Post-Transplant Lymphoproliferative Disorders

32. Thank u