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 • myeloid cells: neutrophils, eosinophils, basophils, monocytes • 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? (cont’d.) • Erythrocytes (red blood cells) function in the transport of oxygen to the tissues. • Leukocytes (white blood cells) 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.

5. What is a hematological malignancy? • The malignancies may be classified into • Lymphomas • Hodgkins versus non-Hodgkins • B cell versus T cell • Lymphoid (or lymphocytic) leukemias • Chronic versus acute • B cell versus T cell • Acute myelogenous leukemia • Myelodysplastic syndromes • Myeloproliferative disorders (includes chronic myelogenous leukemias)

6. What is a hematological malignancy (continued) • 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. • (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).

7. What is a hematological malignancy (continued) • The lymphoid malignancies are a heterogeneous group of disorders that occur as a result of neoplastic transformation at different stages of B cell and T cell development. • A lymphoid 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. The maturity of the cell at the time the cancer developed can be determined by doing a molecular assay for which Ig (BCR) genes have rearranged.

9. What are these various cell surface “CD” markers? • All cells carry various functional proteins in their membranes which are at least partly exposed on the cell surface. • CD molecules are cell surface molecules expressed on various cell types in the immune system. • They are designated by the “cluster of differentiation” or CD nomenclature, which includes “CD” followed by a number, as in CD4. • The various CD molecules and their known or proposed functions are listed in Abbas and Lichtman “Basic Immunology.”

10. The maturity of the cell at the time the cancer developed can be determined by doing a molecular assay for which TCR genes have rearranged.

11. 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

12. Determining the origins and classifying B and T cell malignancies • Cancers arising from cells that are already immunocompetent or that are undergoing antigen-independent maturation will have characteristic rearrangements of their B cell receptor (Ig) or T cell receptor genes. • The DNA is examined to determine whether or not a rearrangment has occurred, and whether it is in a B cell or T cell gene • This method is very accurate. • In cytopathology, the level of cellular differentiation is used as a measure of cancer progression. “Grade” is a marker of how differentiated a cell in a tumor is. Often the less differentiated, the more readily cell division takes place. • Cell surface markers of maturity on lymphocytic cells are more likely to be accurate early in the development of the cancer. (Sharp’s best guess.)

13. What are the criteria for classification as a malignant proliferation of hematopoietic cells? • Monoclonality • all malignant cells arose from a single cell • 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 cells and their descendants • Grows faster • Secretes something that interferes with expansion of normal clones

14. 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)

15. What is cancer? • Cancer is a form of genetic disease. • This does not mean that you can inherit cancer. • 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.

16. 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.

17. 1 Non-dividing cells

18. 1 1 Non-dividing cells 2

19. 1 1 1 1 1 2 2 2 3 Non-dividing cells 2

20. 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.

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

22. 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 • Metastatic – the tumor is capable of spread to distant sites • With hematological malignancies, the progression is often not as obvious.

23. Features Causes of Change

24. 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.

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

26. How do you determine the cell lineage of a leukemia or lymphoma? • Immunohistochemistry (look for the presence of cell surface markers with antibody probes) • Immunoglobulin • CD4 • CD8 • Molecular tests for rearrangement of the T or B cell receptor (antibody) gene

27. How do you diagnose a leukemia or lymphoma? • Cytology – microscopic examination • Cytogenetics (chromosome analysis, chromosome painting) • 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.

28. 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 do not appear morphologically malignant. • To prove that a neoplastic population of B or T cells is monoclonal in origin • If the entire population is from one clone, it is in late stage (see earlier slides) • To look for chromosomal translocations

29. How can one prove that a neoplastic population of cells is monoclonal in origin? • Southern blot without PCR • 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 0.1% of the total population of cells (as compared to 5% for the Southern blot without PCR)

30. How is PCR used to establish the presence of a monoclonal population of malignant cells? Note: this approach works even when there is no translocation involved in the development of the hematological malignancy.

31. How is PCR used to establish the presence of a monoclonal population of malignant cells? • Isolate or extract DNA (biopsy, bone marrow) • Next slide

32. PCR to identify a monoclonal population of cells

33. How is PCR used to establish the presence of a monoclonal population of malignant cells? (cont’d.) • PCR using consensus primer pairs (i.e., primer pairs 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, because rearrangement has not occurred. • 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

34. How is PCR used to establish the presence of a monoclonal population of malignant cells? (cont’d.) • 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.

35. How is PCR used to establish the presence of a monoclonal population of malignant cells? • From a normal individual, there should be fragments of many different lengths – which will look like a smear of DNA from the many different VDJ rearrangements (a normal 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 0.1% of the total population of cells.

36. PCR to identify a monoclonal population of cells

37. 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 and a portion of an IgG gene are often transposed from one chromosome to another and located next to each other.

38. 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 necessarily involve a translocation. • What are the technical and biological differences?

40. 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. • Confirm with specific probe.

41. Answer to naming leukemias and lymphomas • From: Wilczynski, Sharon [mailto:SWilczynski@coh.org]Sent: Wed 5/20/2009 4:30 PMTo: Glackin, CarlottaCc: Sharp, SandraSubject: RE: Question about cancer

42. The hematopathologists have made it their life work to classify and then reclassify and change the names of theses cancers.  It is a special club and only they seem to understand the nuances.  However, as a general surgical pathologist, I can tell you that site the tumor arises in is not really that important although historically it was used.  Now we are tending to classified by the cell of origin (usually immunohistochemistry is used to identify the lineage of the cell) but more and more we are looking at the underlying molecular defect.   Lymphoma is a a neoplastic proliferation of lymphoid cells arise in lymphoid tissue anywhere in the body (usually that is lymph node but it can be organs like stomach or cervix or in the bone (osteolymphoma).  These are B-cell lymphomas (origin from precursors to immunoglobulin secreting cells and will have gene rearrangments of the immunoglobulin heavy and/or light chains).  T-cell lymphomas have rearrangments of the T-cell receptors.    Leukemia is from the hematopietic cells (precursors to granulocytes or polymorphonuclear leukocytes, red blood cells, or  platelets).  But it isn't that straight forward.   To confuse the issue you do have acute lymphoblastic leukemia (ALL)  which usually presents with elevated blasts in blood and is considered a leukemia,  Sometimes it presents with a mass and then it is termed lymphoblastic lymphoma.    There is also chronic lymphocytic leukemia/small lymphocytic lymphoma which are really  the same entity but now are classified as B-lymphoblastic leukemia/lymphoma and then characterized by the type of translocation such as (9:22).   As we learn more of the molecular we are splitting and combining diagnosis.  For example the EWS/FLI-1 translocation has united into one diagnostic category Ewings sarcoma and peripheral primitive neurectodermal tumor.  With the leukemias and lymphomas the underlying molecular fundamental alteration is altering how we name them.  I would recommend that you use the World Health Organization Classification for leukemia and lymphomas.  They just redid it last year again.    If you don't have acess to it I can send you the short classification scheme.   Sharon