Tabuk University 1 3 rd Year – Level 5 – AY Faculty of Applied Medical Sciences Department Of Medical Lab. Technology

2 Dr. Walid ZAMMITI Msc, PhD. MLT

 To recognize the presenting symptoms and signs of multiple myeloma.  To identify criteria for diagnosis of multiple myeloma and related disorders.  To relate the clonal expansion of immunoglobulin-producing plasma cells and accompanying destructive skeletal changes that occur with multiple myeloma in terms of manifestations and clinical course for this disorder.  To explain how expansion of a single clone of plasma cells that produce immunoglobulins results in the pathologic changes seen in multiple myeloma.  To discuss risk factors, prevalence, diagnosis, clinical manifestations, and treatment for multiple myeloma. 3 Objectives

Definition Multiple Myeloma is a plasma-cell neoplasm characterized by the proliferation of a single clone of plasma cells engaged in the production of a monoclonal immunoglobulin. ( M protein ): Multiple myeloma (myeloma or plasma cell myeloma) is a cancer of the plasma cells in the bone marrow. There are different clones of plasma cells in the bone marrow, which make the numerous types of immunoglobulins (antibodies) needed for the immune system. In myeloma, the cancerous plasma cells are monoclonal and overcrowd the bone marrow, causing some of the complications associated with the disease.

Definition These abnormal cancerous plasma cells make a similar immunoglobulin (monoclonal immunoglobulin, also called an M- protein). This can be of any type: IgG, IgA, IgD or IgE; IgG is, however, most common. Overall, approximately 70% of patients with myeloma will have elevated IgG, 20% IgA, and 5%-10% light chains only (Bence Jones protein). About 1% will have IgD, IgE, IgM or nonsecretory disease (cancerous plasma cells that do not secrete immunoglobulin). About 30% of the time there is an imbalance in the production of light and heavy chains resulting in excess light chains along with the monoclonal antibody.

In healthy bone marrow (A), B-cells develop into antibody-producing plasma cells when foreign substances (antigens) enter the body. Normally, plasma cells make up less than 1 percent of the cells in the bone marrow. In multiple myeloma (B), genetic damage to a developing B cell transforms the normal plasma cell into a malignant multiple myeloma cell. The malignant cell multiplies, leaving less space for normal blood cells in the bone marrow, and produces large quantities of M protein.

Myeloma cells in the bone marrow cause osteolytic lesions, which appear as “holes” on an x-ray. Weakened bones increase the risk of fractures, as shown in this x-ray of a forearm. DeVita VT Jr, Hellman S, Rosenberg SA, eds. Cancer: Principles and Practice of Oncology. 5th ed. 1997:2350. Adapted with permission from Lippincott Williams & Wilkins.

 There is no known cause, although, patients with MGUS are at increased risk for developing multiple myeloma.  Radiation.  Recently viruses like HHV-8 and SV-40, have been linked to myeloma development.  MGUS: Monoclonal Gammopathy of Undetermined Significance= is a condition in which a paraprotein is found in the blood or urine.paraprotein  The paraprotein is an immunoglobulin or light chain; produced by a single clone of plasma cells. The paraprotein 8 Causes

Immunoglobulin Molecular Structure

Paraprotein = M protein The plasma cells in myeloma are identical ('clonal'), because they originate from a single abnormal cell that starts to multiply out of control. The protein produced is, therefore, also identical ('monoclonal' meaning the product of a single clone). So Paraprotein is:a monoclonal immunoglobulin observed as a result of an isolated increase in a single immunoglobulin type as a result of a clone of plasma cells arising from the abnormal rapid multiplication. Examples of such proteins : Myeloma proteins, Bence-Jones protein, amyloid proteins, Waldenstrom's macroglobulinemia, or cryoglobulins.

Clinical Presentation The clinical presentation of multiple myeloma is variable; approximately 30% of patients are asymptomatic at diagnosis. Diagnosis is usually made because a healthcare practitioner notices an elevated protein on routine blood tests and follows up. Clinical findings suggest a possibility of multiple myeloma include: Bone disease Hypercalcemia Anemia Renal Insufficiency Infections Venous Thromboembolism Hyperviscosity

Clinical Presentation (cont,)

Venous Thromboembolism  Patients with multiple myeloma are at a high risk of developing venous thromboembolism (VTE).  This risk is increased by several of the agents used to treat it such as thalidomide and lenalidomide. Clinical Presentation (cont,)

 ESR > 100 ( Marked Rouleux formation in blood films)  anaemia, thrombocytopenia  marrow plasmacytosis (usually > 20%)  Hyperproteinemia : elevated total protein levels  Hypercalcemia : elevated blood Ca ++ levels ; in 45% of patients.  Proteinuria: protein in urine  Serum Alkaline Phosphatase is normal  Serum β 2 -microglobulin is often raised  Immunophenotyping : malignant plasma cells phenotypically expressing CD38, CD56, and CD138. In addition, approximately 20% of malignant plasma cells express CD20 18 Laboratory tests

Diagnosis Initial work-up  If multiple myeloma is suspected, the initial work-up should include: CBC Chemistry, including calcium, BUN, and creatinine Serum protein electrophoresis and immunofixation Quantitative immunoglobulin levels 24-hour urine protein electrophoresis and immunofixation  If a monoclonal protein is detected, a bone marrow aspirate and biopsy should be performed and sent for pathological examination to assess the amount of plasma cells, as well as for chromosomal studies and flow cytometry.

1-Increased plasma cells in the bone marrow.(more than10%)* 2- Monoclonal protein in the serum or urine (usually > 3g/dl) This is done using Serum Protein Electrophoresis (SPE). 3- Evidence of end-organ damage hypercalCemia, Renal insufficiency, Anemia, or Bone lesions (a group of findings referred to as CRAB) * If the bone marrow plasma cell Count is >10% but there is no evidence of tissue damage the disease is termed asymptomatic or smouldering myeloma. 20 Diagnosis of Multiple Myeloma

21 pathologic fractures in MM

22 A skull x-ray taken from the side shows typical findings of multiple myeloma and multiple "punched-out" holes. The arrow is pointing at one of the larger holes.

23 Plasma cells : basophilic cytoplasm and an eccentric nucleus. The cytoplasm also contains a pale zone.

24 Plasma cells

25 ROULEAUX FORMATION IN BLOOD FILMS

26 Background staining and rouleaux formation in myeloma. The excessive immunoglobins produced by the neoplastic plasma cells in myeloma are acidic and take up the basophilic stains used in blood smears. (slide on right) compared with normal (slide on left). The abnormal immunoglobins also cause the RBCs to adhere. This resulting rouleaux (“stacking of coins”) formation may be visible on those portions of the smear where erythrocytes are normally apart.

Diagnosis  The quantitative immunoglobulin test will show if there is an increase in any particular type of immunoglobulin, but it does not determine if immunoglobulin is monoclonal.  Electrophoresis is an essential test in the work up of myeloma. Electrophoresis will identify if the elevated antibody level is monoclonal or polyclonal.  Other tests are used to help determine prognosis including: B2 microglobulin, lactate dehydrogenase (LDH), albumin, and C- reactive protein. An elevated level of B2 microglobulin, LDH, and C-reactive protein, and a low level of serum albumin are all markers of a poor prognosis.

 Electrophoresed serum is separated to: albumin, alpha 1, alpha 2, beta, and gamma regions.  The gamma region contains the 5 types of Immunoglobulin's (Ig’s): G, A, M, E, and D  Any increase in this region is considered as an increase in 2 or more of the immunoglobulin's (polyclonal) which shows as an expansion of the whole gamma region (in the electrophoresis).  On the other hand, if the increase shows as a sharp peak in the gamma region, this is conclusive of an increase in one of the Ig's only, i.e., “monoclonal”. 28

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Genetics and Prognosis  Certain chromosomal abnormalities are associated with prognosis in multiple myeloma.  A translocation between chromosome 4 and 14 (t(4;14)), translocation between chromosome 14 and 16 (t(14;16)), or deletions in chromosome 13 are poor prognostic factors in myeloma. Patients with these abnormalities are considered high risk.  Translocations between chromosomes 11 and 14 (t(11;14)) may be associated with an improved survival.

Bence Jones proteins Bence Jones proteins are immunoglobulin light chains (paraproteins) and are produced by neoplastic plasma cells. They can be kappa (most of the time) or lambda. They are found in the blood or urine.immunoglobulin light chainsparaproteinsneoplasticplasma cells The light chains have traditionally been detected by heating or electrophoresis of concentrated urine. More recently serum free light chain assays have been indicated over the urine tests. electrophoresisserum free light chain assays Causes of Bence Jones proteins: Amyloidosis, benign monoclonal gammopathy, cryoglobulinemia, Fanconi syndrome, hyperparathyroidism, multiple myeloma, osteomalacia, Waldenström's macroglobulinemia. 32

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