hemopoietic malignancy Dr. ahmad hassaneen

1. hemopoietic malignancy Dr. ahmadhassaneen

2. ■ The hemopoieticmalignancies are clonal diseases that derive from a single cell in the marrow or peripheral lymphoid tissue that has undergone genetic changes. Haematological malignancies represent approximately 7% of all malignant diseases. There are major geographical variations in occurrence of these diseases; for example, chronic lymphocytic leukaemia(CLL) is the most common leukemia in the West but rare in the Far East.

3. The etiology of Hemopoietic malignancy: Exactly how genetic mutations accumulate in haemopoietic malignancies is largely unknown. It is the combination of genetic background and environmental effect that determines the risk of developing a malignancy. However, in the majority of cases neither a genetic susceptibility nor an environmental agent is apparent.

4. Inherited factors The incidence of leukemia is greatly increased in some genetic diseases : 1- Down’s syndrome (Most important , where acute leukemia occurs with a 20 to 30 fold increased frequency) 2- Bloom’s syndrome (Mutations in the BLM gene , characterized by genome instability. The most prominent features are short stature and a rash on the face) 3- Fanconi ’s anemia 4- Ataxia telangiectasia 5- Klinefelter ’ s syndrome 6- Wiskott – Aldrich syndrome (X-linked disorder characterized by the triad of thrombocytopenia, eczema, and recurrent infections)

5. Environmental effects • Chemicals Chronic exposure to benzene is a cause of myelodysplasia and AML. • Drugs The alkylating agents (e.g. chlorambucil and melphalan) predispose to AML , especially if combined with radiotherapy. • Radiation Radiation, especially to the marrow, is leukaemogenic. This is illustrated by an increased incidence of all types of leukaemia (except CLL) in survivors of the atom bomb explosions in Japan.

6. Infection ►Some cases of childhood acute lymphoblastic leukemia (ALL) are initiated by chromosomal abnormality, e.g. t(12; 21) that occur during development in uteroandcaused by environmental exposure during pregnancy (first event or first hit). ►The mechanism of the (second genetic hit) within the tumour cell is suggested to be an abnormal response of the immune system to infection. ►Children with a high level of social activity, notably those attending early nursery , have a reduced incidence of ALL, whereas those living in isolated communities and who have a reduced exposure to common infections in the first years of life have a higher risk.

7. ● Viruses Viral infection is associated with several types of hemopoieticmalignancy, especially lymphoma: ►The retrovirus human T - lymphotropic virus type 1 (HTLV-1) is the cause of adult T - cell leukemia/lymphoma. ►Epstein – Barr virus (EBV) is associated with: 1-Almost all cases of endemic (African) Burkittlymphoma. 2-Some cases of Hodgkin lymphoma. 3-Post-transplant lymphoproliferative disease (which develops during immunosuppressive therapy after solid organ transplantation). ►Human herpes virus-8 is associated with Kaposi ’s sarcoma.

8. ► HIV infection is associated with an increased incidence of lymphomas at unusual sites such as the central nervous system. • Bacteria • Helicobacter pylori infection has been implicated in the pathogenesis of gastric mucosa B - cell lymphoma

9. The genetics of haemopoieticmalignancy 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: Oncogenesand Tumour‐suppressor genes. • Oncogenes Oncogenes arise because of gain‐of‐function mutations or inappropriate expression pattern in normal cellular genes called proto‐oncogenes. Oncogenic versions are generated when the activity of proto‐oncogenes is increased or they acquire a new function. This can occur in a number of ways including translocation, mutation or duplication.

10. These mutations affect the processes of cell signaling, cell differentiation and survival. • One of the striking features of hematological malignancies, in contrast to most solid tumors, is the high frequency of chromosomal translocations. • Several oncogenes are involved in suppression of apoptosis → the best example is BCL‐2 which is overexpressed in follicular lymphoma.

11. Tyrosine kinases These are enzymes which phosphorylate proteins on tyrosine residues and they are important mediators of intracellular signaling. Mutations of tyrosine kinases underlie a large number of hematological malignancies and they are the targets of many effective drugs. ►Common examples include: ABL1in chronic myeloid leukaemia(CML). JAK2in myeloproliferativeneoplasms. FLT3in acute myeloid leukemia (AML) Brutonkinase in chronic lymphocytic leukaemia (CLL)

12. Tumour‐suppressor genes ●Tumour‐suppressor genes may acquire loss‐of‐function mutations, usually by point mutation or deletion, which lead to malignant transformation. ●Tumour‐suppressorgenes commonly act as components of control mechanisms that regulate entry of the cell from the G1 phase of the cell cycle into the S phase or passage through the S phase to G2 and mitosis. ●The most significant tumour‐suppressor gene in human cancer is p53 which is mutated or inactivated in over 50% of cases of malignant diseases.

13. Important genetic abnormalities within hematological tumors

14. Clonal progression Malignant cells appear to arise as a multistep process with acquisition of mutations in different intracellular pathways. This may occur by : 1- Linear evolution, in which the final clone harbors all the mutations that arose during evolution of the malignancy, or 2-Branching evolution, in which there is more than one clone of cells characterized by different somatic mutations but which share at least one mutation traceable back to a single ancestral cell. During this progression of the disease, one subclone may gradually acquire a growth advantage.

16. Progression of subclinical clonal haematologicalabnormalities to clinical disease ►Immunological and molecular tests have shown that many healthy individuals (Especially the elderly) harbor clones of cells which have acquired somatic mutations and from which overt hematological clinical disease may arise. ►Examples include: 1-Clones of cells identical to those of chronic lymphocytic leukaemia, which can be present in the blood of individuals with a normal lymphocyte count. 2-Clones of cells harboring mutations, such as of TET 2, which are characteristic of myeloid malignancy may be present in a normal appearing bone marrow in nearly 20 % of elderly healthy subjects.

17. Chromosome structure: • Each chromosome has two arms: the shorter called ‘p’, the longer called ‘q’. These meet at the centromereand the distal ends of the chromosomes are called telomeres. • On staining each arm divides into regions numbered outwards from the centromereand each region divides into bands. • When a whole chromosome is lost or gained, a − or + is put in front of the chromosome number. If part of the chromosome is lost it is prefixed with del(for deletion). If there is extra material replacing part of a chromosome the prefix add(for additional material) is used.

19. Telomeres ●Telomeres are repetitive sequences at the ends of chromosomes. They decrease by approximately 200 base pairs of DNA with every round of replication. When they decrease to a critical length, the cell exits from cell cycle. ● Germ cells and stem cells, which need to self‐renew and maintain a high proliferative potential, contain the enzyme Telomerasewhich can add extensions to the telomeric repeats and compensate for loss at replication, and so enable the cells to continue proliferation. ● Telomerase is also expressed in malignant cells but this is probably a consequence of the malignant transformation rather than an initiating factor.

20. Specific examples of genetic abnormalities in haematologicalmalignancies: The genetic abnormalities underlying the different types of leukaemiaand lymphoma include the following: ◘Point mutation ◘ Translocations ◘ Deletions ◘ Duplication or amplification ◘ Epigenetic alterations

22. Point mutation: • 1-Mutation in the JAK2 gene, which leads to activation of the JAK2 protein in most cases of myeloproliferative diseases. • 2-Mutations within the RASoncogenes or p53tumour‐suppressor gene are common in many haemopoietic malignancies. • 3-The nucleophosmin gene shows an insertion of four base pairs in 35% of cases of AML . • 4-Internal tandem duplication (The occurrence of two identical sequences, one following the other, in a chromosome segment) occurs in the FLT3gene in 30% of cases of AML.

23. Translocations • These are a characteristic feature of haematologicalmalignancies and there are two main mechanisms whereby they may contribute to malignant change: 1-Fusion of parts of two genes to generate a chimeric fusion gene that encodes a new « fusion protein » , e.g. • BCR - ABL1 in t (9; 22) in CML . • RAR α – PML t (15; 17) in acute promyelocyticleukamia • TEL - AML1 in t (12; 21) in B - ALL. 2- Overexpression of a normal cellular gene, e.g. ● Overexpression of BCL-2 in the t (14;18) of follicular lymphoma. ● Overexpression of MYC in t (8; 14)in Burkitt lymphoma.

24. Deletions • Chromosomal deletions may involve a small part of a chromosome, the short or long arm (e.g. 5q–) or the entire chromosome (e.g. monosomy 7). • The critical event is probably loss of a tumour suppressor gene or of a microRNA as in the 13q14 deletion in CLL . • Loss of multiple chromosomes is termed hypodiploidy and is seen frequently in ALL. Note: The 13q14 gene deletion means deletion of a region present on the q (large arm) of 13th chromosome and 14 refers to “region 1, band 4”.

25. Duplication or amplification Examples : ♦Trisomy 12 in CLL. ♦ Amplification of MLL gene.

26. Epigenetic alterations Gene expression in cancer may be dysregulated 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 epigenetic and are stably inherited with each division of the malignant cell. The most important mechanisms: 1-Methylation of cytosine residues in DNA. 2-Acetylation or methylation of the histone proteins that package DNA within the cell.

27. Micro RNAs Chromosomal abnormalities, both deletions and amplifications, can result in loss or gain of short (micro) RNA sequences. These are normally transcribed but not translated. MicroRNAs (miRNAs) control expression of adjacent or distally located genes. Deletion of specific microRNAs have been described in CLL and AML.

28. Diagnostic methods used to studymalignant cells: ● Karyotyping: Direct morphological analysis of chromosomes from tumor cells under the microscope. The cells are cultured first and examined while in the metaphase . ● Fluorescence in situ hybridization (FISH) It involves the use of fluorescent - labelled genetic probes which hybridize to specific parts of the chromosomes. This sensitive technique can detect extra copies of genetic material in both metaphase and interphase (non - dividing) cells (e.g. trisomy12 in CLL) and chromosomal translocations e.g. t(9; 22) in CML.

29. ● Polymerase chain reaction (PCR) ▪It 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 detect ‘clonal’ cells of B - or T - cell lineage by immunoglobulin or T-cell receptor (TCR) gene rearrangement analysis. ▪ It is extremely sensitive (detecting one abnormal cell in 100,000 – 1000,000 normal cells).

30. ● Flow cytometry • In this technique, antibodies labeled with different fluorochromes recognize the pattern and intensity of expression of different antigens on the surface of normal and leukemic cells. • Normal cells each have a characteristic profile but malignant cells often express an aberrant phenotype that can be useful in their detection . • In the case of B-cell malignances (e.g. CLL), expression of only one light chain, κ or λ by the tumor cells distinguishes them from a normal polyclonal population which express both κ and λ chains, usually in a κ : λ ratio of 2 : 1

32. Value of detection of genetic abnormalities in management of haematological malignancy 1-Initial diagnosis • Many genetic abnormalities are so specific for a particular disease that their presence determines that diagnosis. • An example is the t(11; 14) translocation which defines mantle cell lymphoma. • Clonal immunoglobulin or TCR gene rearrangements are useful in establishing clonality and determining the lineage (B or T) of a lymphoid malignancy.

33. 2- For establishing a treatment protocol • Each major type of hematological malignancy can be further subdivided on the basis of genetic information. • For example, AML is a diverse group of disorders where individual subtypes respond differently to standard treatment →The t(8; 21) and inv(16) subgroups have a favorable prognosis, whereas monosomy 7 carries a poor prognosis. • Knowledge of a genetic abnormality can lead to more rational treatment, e.g. 1-The use of all - trans retinoic acid (ATRA) in acute promyelocyticleukaemia with t (15; 17) 2-For BCR – ABL1 +ve CML and ALL, drugs are now available that target the fusion protein and improve the survival.