Hematology and Hematologic Malignancies

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

2. 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

3. 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.

4. 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

5. 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.

6. 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.

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

9. 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

10. 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

11. 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 differentiatedàless differentiated)

12. 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.

13. 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.

14. 1 Non-dividing cells

15. 1 1 Non-dividing cells 2

16. 1 1 1 1 1 2 2 2 3 Non-dividing cells 2

17. Non-dividing cells 1 2 1 2 1 1 1 2 1 2 1 123 123 123 123 123 123 123 123 123 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.

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

19. 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.

21. 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.

22. 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

23. 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.

24. 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

25. 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)

26. 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.

27. 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.

28. PCR to identify a monoclonal population of cells

29. 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.

30. 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?

32. 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.