2. Hematology and Hematologic Malignancies Cancer of the formed elements of the blood

3. What is hematology? Hematology is the study of blood and is concerned mainly with the formed elements in the blood. The formed elements in the blood include: The white blood cells (leukocytes) which include the neutrophils, eosinophils, basophils, monocytes, and lymphocytes (. The red blood cells (erythrocytes) The platelets (thrombocytes) All of the formed elements in the blood are derived from same pluripotential stem cell in the bone marrow

4. What is hematology continued Erythrocytes function in the transport of oxygen to the tissues. Leukocytes function in both specific (immune responses) and non-specific defenses against foreign invasion. Thrombocytes function in hemostasis or blood clotting.

5. What is a hematological malignancy? A hematologic malignancy is a malignancy (or cancer) of any of the formed elements in the blood. The malignancies may be classified into Lymphomas Hodgekins versus non-Hodgekins B cell versus T cell Lymphoid leukemias Chronic versus acute B cell versus T cell

6. What is a hematological malignancy (continued) Acute myelogenous leukemia Myelodysplastic syndromes Myeloproliferative disorders (includes chronic myelogenous leukemias) For the purposes of this class we will concentrate only on the hematological malignancies of lymphocytes, i.e., lymphomas and lymphocytic leukemias. Remember that the malignant cell in a leukemia originates in the bone marrow and that the malignant call in a lymphoma originates in tissue other than the bone marrow.

7. What is a hematological malignancy (continued) (The word myeloid or the prefix myelo- refers to cells that are not lymphoid or lymphocytic (e.g., neutrophils, eosinophils, basophils; not T cells and B cells). The lymphoid malignancies are a heterogenous group of disorders that occur as a result of neoplastic transformation at different stages of B cell and T cell development. A hematologic malignancy may develop at any stage of development of B or T cells.

8. Notice the cell and stage-specific markers on this and the subsequent slide.

10. Some abbreviations from previous slides ALL – acute lymphocytic leukemia CLL – chronic lymphocytic leukemia CTCL – cutaneous T cell lymphoma PTCL – peripheral T cell lymphoma LGL – larger granular lymphocytic leukemia

11. What are the criteria for classification as a malignant proliferation of hematopoietic cells? Monoclonality – all malignant cells arose from a single clone Clonal progression – once started the proliferation does not stop, i.e. the malignant clone expands Clonal dominance – a proliferative advantage allows the malignant clone to replace normal cell lines Grows faster Secretes something that interferes with expansion of normal clones

12. Criteria for Classification continued Extinction of normal clones – early in disease progression normal clones are present, but suppressed. Later in the disease progression, all cells of normal clones die. Genetic instability – as the malignant clone proliferates, subclones arise with properties less and less like normal cells (well differentiated  less differentiated)

13. What is cancer? Cancer is a form of genetic disease. Cancer is the result of a multistep process. Cancer is characterized by an accumulation of multiple genetic mutations in a population of cells undergoing neoplastic transformation. After the first mutation, there is limited expansion After subsequent mutations, there is greater proliferation potential.

14. How does this happen? In the following slides: = a non- dividing cell 1, 2, 3 = successive mutations, each contributing in some way to an increased rate of cell division or decreased rate of cell death.

15. 1 Non-dividing cells

16. 1 1 2 Non-dividing cells

17. 2 11 1 1 1 22 2 3 Non-dividing cells

18. This process continues, with each successive mutation leading to a faster rate of cell division, slower rate of cell death, and eventually loss of cell adhesion. 123 1 1 1 2 1123 Non-dividing cells

19. A sort of summary of the previous four slides.

20. What is cancer, continued The progression of cancer is easily documented in some tumor systems: Benign – tumor is not recurrent Malignant – the tumor tends to become progressively worse Metastasis – the tumor is capable of spread to distant sites With hematological malignancies, the progression is often not as obvious.

22. Why is it important to determine the cell lineage of a leukemia or a lymphoma? Different types of leukemia and lymphoma are treated with different types of chemotherapy. Different types of leukemia and lymphoma have different prognoses.

23. How do you determine the cell lineage of a leukemia or lymphoma? Morphologic characteristics of the malignant cells (done by a pathologist) Cytochemistry (look for the presence of specific enzymes or lipids and glycogen associated with specific types of types of cells) Myeloperoxidase Esterase Sudan black Terminal deoxynucleotidyl transferase

24. How do you determine the cell lineage of a leukemia or lymphoma? Immunohistochemistry (look for the presence of cell surface markers) Immunoglobulin CD4 CD8 Cytogenetics (chromosome analysis) Chromosomal translocations Molecular tests Restriction digest of genomic DNA+Southern blotting PCR+/- Southern blotting of PCR products PCR for chromosomal translocations and overgrowth of any monoclonal lymphocyte population.

25. Molecular tests are expensive. Why would one use a molecular test for the diagnosis of a hematological malignancy? To prove that a malignancy is present when the cells are not morphologically malignant. To prove that a neoplastic population of B or T cells is monoclonal in origin To look for chromosomal translocations

26. How can one prove that a neoplastic population of cells is monoclonal in origin? Southern blot PCR (we will only discuss this method which has many advantages over Southern blotting) Cheaper and quicker Requires less initial DNA DNA can be of low quality Can detect a monoclonal population that comprises as little as.1% of the total population of cells (as compared to 5% for the Southern blot)

27. How is PCR used to establish the presence of a monoclonal population of malignant cells? Isolate or extract DNA (biopsy, bone marrow) PCR using consensus primers (i.e., primers that recognize all V or J segments) for the immunoglobulin heavy chain (for monoclonal B cells ) or TCR (for monoclonal T cells). In the germline DNA these primers are too far apart to give a good PCR result The only cells in which a PCR product will be generated are cells in which a DNA rearrangement has occurred to bring the V and J segments close enough to generate a PCR product using the consesus primers Run the PCR products on an agarose gel Remember that DNA rearrangement is a normal process that occurs during the normal maturation of B and T cells to immunocompetent B and T cells.

28. How is PCR used to establish the presence of a monoclonal population of malignant cells? From a normal individual, there should be a smear of DNA from the many VDJ rearrangements (a polyclonal population of cells) If an individual has an abnormal expanded monoclonal population of cells, a distinct band will be seen, even when the monoclonal subpopulation of cells is as low as.1% of the total population of cells.

29. PCR to identify a monoclonal population of cells

30. Remember Monoclonal malignancies may or may not involve a translocation as one of the mutations. When a chromosomal translocation is involved in either leukemia or lymphoma, a proto-oncogene is often transposed from one chromosome to another.

31. Review We have used PCR to detect a translocation. This lecture introduced use of PCR to detect a monoclonal lymphoma or leukemia that did not involve a translocation. What are the technical and biological differences?

33. How is PCR used to identify a chromosomal translocation? Perform PCR on patient tumor DNA using consensus primers. One primer will bind to a region on one chromosome while the other primer will bind to a region on the other chromosome. There is no product if a translocation between the two chromosomes has not occurred.