Version 1.0 10/99

Recommended Reading

Babior and Stossel: pp. 370-373, Chapters 23, 25-26, 29 and/or
Robbins (6th edition): pp. 654-659, 663-666, 675-685

Lecture objectives

After the lecture (and appropriate laboratories) the student should be able to:

1. Describe the differences between acute and chronic leukemias
2. Describe the ways by which AML is differentiated from ALL
   
3. Describe the pathophysiology of ALL, AML, CML, CLL, and multiple myeloma
4. Describe the pertinent clinical facts about the leukemias and multiple myeloma
5. Describe the fundamental principles of treatment for leukemias and multiple myeloma
   

The leukemias ("white blood") are neoplastic proliferations of white blood cells. In distinction to the lymphomas, which primarily involve the lymph nodes, the leukemias are principally found in the peripheral blood and bone marrow. As the neoplastic cells grow in the bone marrow, they crowd out the normal hematopoietic cells, giving rise to the clinical triad of leukemia: (1) anemia (lack of RBC production), (2) infection (lack of normal WBCs), and (3) bleeding (lack of platelets).

Leukemias are classified into four fundamental types based on the cell of origin (lymphoid vs "myeloid") and clinical presentation (acute vs. chronic).

The treatment of ALL is different from that of AML. Therefore, in a patient with an acute leukemia, it is critically important to determine whether the cell of origin is lymphoid or myeloid. This must be done as rapidly as possible so that appropriate therapy can be administered. The following information is important.

• Patient age and clinical features – ALL is primarily a disease of children (66% of cases), while most cases of AML are found in adults (~80% of cases). However, since exceptions occur, laboratory studies must be performed.
  
• Cell morphology (light morphology) – Examination of the peripheral blood and bone marrow under the microscope confirms the diagnosis of acute leukemia but cannot reliably differentiate ALL from AML in the majority of cases (unless Auer rods are present, see below). Therefore, the diagnosis must be confirmed by other tests.
  
• Cytochemistry (chemical stains for cell enzymes) – Some of the enzyme of lymphoid cells are different from those of myeloid cells. The cells of AML, being derived from myeloid cells, usually contain some of the enzymes (e.g. myeloperoxidase) found in the primary granules of mycloid cells. In contrast, the neoplastic cells of ALL always lack myeloperoxidase. Similarly, the nuclei of immature lymphoid precursors contain an enzyme called terminal deoxytransferase (TdT) which is usually not found in immature myeloid cells. Therefore, the cells of ALL should be positive for TdT, whereas those of AML will be negative.
  
• Immunophenotype (antigen expression) – Some of the antigens on the surface of lymphoid cells are different from those found on myeloid cells. The presence of these cell surface antigens can be determined by laboratory techniques (flow cytometry and immunoperoxidase staining) which use monoclonal antibodies against the antigens prepared in animals. The most important antigens include CD2 and CD3 (characteristic of T cells), CD10 and CD19 (characteristic of B cells), and CD13 and CD33 (characteristic of myeloid cells).
  
• Cytogenetics (cell chromosomal content or karyotype) - Certain characteristic chromosomal changes are characteristic of ALL or AML. Finding these changes to confirm the diagnosis of ALL or AML, and also provides with some prognostic information.
  

Pathogenesis – Acute leukemia derived from the proliferation of lymphoblasts (immature lymphocytes).

Patient age – Primarily in children (2-10). Most common malignancy in children, and second or third leading cause of death in children. One-third of cases occur in adults (usually > 65 years). About 3,000 new cases occur in the United States each year.

Clinical presentation – Characteristic abrupt stormy onset, sometimes proceeded by a viral prodrome. Anemia, fever, and/or bleeding due to the suppression of normal hematopoiesis in the bone marrow. Bone pain may occur due to expansion of leukemic cells in bone marrow. Lymphadenopathy, hepatomegaly, and/or splenomegaly may occur, since the leukemic cells may home to other organs, which contain lymphoid cells. Involvement of the CNS and testes may occur. CNS involvement is of particular concern since many of the chemotherapeutic agents used to treat ALL do not cross the blood/brain barrier.

Laboratory features – Lymphoblasts are found in large numbers in the bone marrow, and usually in the peripheral blood. The white blood count is variable, but usually high. Anemia and thrombocytopenia is usually found. Lymphoblasts are small cells, with a high nuclear: cytoplasm ratio (the nucleus takes up almost all the cell). The nuclear chromatin pattern is delicate, characteristic of blast cells. Lymphoblasts are negative for myeloperoxidase, positive for TdT, and express lymphoid (B-cell or T-cell) cell surface antigens. 85% of ALLs have the immunophenotype of pre-B cells.

It is important to understand that some types of lymphoma and ALL are different ends of a clinical spectrum. In some lymphomas, the neoplastic cells may also appear in the blood and bone marrow, while the neoplastic cells of some leukemias will infiltrate the lymph nodes, simulating lymphoma. Lymphomas caused by a proliferation of immature lymphocytes, the same cells that appear as ALL in other patients, are caused lymphoblastic lymphoma. In the lymphoblastic lymphomas, the detection of TdT is necessary for diagnosis, just as in patients who present with ALL.

Another example of the "overlap" between leukemia and lymphoma is an uncommon subtype of ALL, the L3 subtype. In contrast to other ALLs, this subtype is comprised of more mature B-cells, which express monoclonal light chains on their surface. Morphologically, these cells are large with abundant blue vacuolated cytoplasm. ALL L3 is the leukemic counterpart to Burkitt's lymphoma (Burkitt’s leukemia/lymphoma). The cells contain the t(8;14) translocation, involving the c-myc oncogene on chromosome 8 and the immunoglobulin heavy chain gene on chromosome 14, and the disease is associated with EBV infection.

Small lymphocytic lymphoma (SLL) and chronic lymphocytic leukemia (CLL) is a third example of two "different" diseases caused by the same cell.

Cytogenetics – Over 90% of ALLs will have a chromosomal abnormality, either numerical or structural. For example, hyperdiploidy (>50 chromosomes) is found in many cases and is a good prognostic sign. In contrast, a few ALL patients will have the Philadelphia chromosome [t(9;22)- see below]; this is a bad prognostic sign in ALL.

Treatment – Chemotherapy (drug treatment) is the mainstay for the treatment of patients with ALL. Three phases of treatment (induction, consolidation, and maintenance) are used. Induction therapy, lasting about a month, is used to rid the body of leukemic cells. Since cholesterol released from rapidly dying tumor cells is metabolically degraded to uric acid, gout may occur unless the patient is also treated with a drug such as allopurinol during induction. Prophylaxis to the brain and spinal cord (CNS) with intrathecal chemotherapy +/- radiation is usually given to destroy ALL cells that may be present. Although > 98% of patients have no evidence of disease (i.e., are in remission) after induction, the disease rapidly recurs in many unless a second phase of high-dose chemotherapy (consolidation or intensification) is given over a period of about six months. Low dose maintenance chemotherapy is then used for a period of several years. Although the present cure rate (> 5 year survival) is approximately 70-80% for children with ALL, the prognosis is much worse for adults, where a two-year continuous remission of about 35-40% is achieved. Bone marrow transplantation is considered for patients younger than 55 years of age if a suitable donor is available.

Pathogenesis – AML is a proliferation of immature "myeloid" cells derived from the myeloid stem cell. An older and probably better term is acute non-lymphoblastic leukemia, ANLL Since the myeloid stem cell can differentiate into myeloid, monocytic, or erythroid cells, or into megakaryocytes. Therefore, AML can be derived from myeloblasts (MI-M2), promyelocytes (M3), a mixture of myeloblasts and monoblasts (M4), monoblasts (M5), erythroblasts (M6), or megakaryoblasts (M7). Although intellectually interesting, it is not clinically important to make this subclassification, except for acute promyelocytic leukemia (APL, FAB M3), which has unique biologic and clinical features.

Patient age – AML is principally a malignancy of adults. It comprises about 45% of all leukemias, 80% of all adult acute leukemias, and less than 20% of childhood acute leukemia. In addition to primary cases, AML develops in approximately 5% of patients receiving chemotherapy and/or radiotherapy for hematologic or other malignancies (acute and chronic leukemia, Hodgkin's disease, non-Hodgkin’s lymphoma, multiple myeloma, ovarian cancer, etc.). There is also an increased incidence of AML in patients with other hematologic diseases, such as paroxysmal nocturnal hemoglobinuria (PNH), or genetic diseases with chromosomal instability. A history of exposure to chemical agents or radiation is present in some patients with AML.
Clinical presentation – The clinical features of AML are similar to those of ALL, with marrow replacement causing the classic triad of profound anemia, leukopenia, and thrombocytopenia. Adenopathy, splenomegaly, hepatomegaly and CNS involvement are not as common as in ALL, but extramedullary involvement is seen in some patients. This includes gingival and cutaneous involvement, and infiltration of the ovaries, testes, breast, orbital region, perianal area, and other sites.

Laboratory features – The peripheral blood of AML patients shows anemia and thrombocytopenia with a variable (but usually greatly elevated) white blood cell count, usually with many blast cells. Myeloblasts are generally larger than lymphoblasts, and usually have more cytoplasm. An important morphologic characteristic of AML is the Auer rod. This is comprised of crystallized primary granules, and appears as a small red rod in the cytoplasm of the myeloblast. Although it is not present in every case of AML, when you do see it, it is pathogomonic for AML. Sheets of blast cells replace the bone marrow.

The cells of AML (at least those of FAB M 1 -4) will be positive for myeloperoxidase. All types of AML will be negative for TdT. By flow cytometry, the cells of AML will have myeloid lineage markers (e.g. CD13, CD33), and will be negative for B- and T-lineage markers.

Cytogenetics – Most cases of AML will have some cytogenetic abnormality, such as t(8;21) or trisomy 8.

An important subtype of AML to recognize (because its course and treatment is different than the others) is acute promyelocytic leukemia (APL). Morphologically, this is comprised of a proliferation of malignant promyelocytes. The cells contain multiple Auer rods; these cells are sometimes referred to as faggot cells (faggot= bundle of sticks). Clinically, this type of leukemia is associated with disseminated intravascular coagulation (DIC). The characteristic chromosomal abnormality seen in APL is t(I 5; 17). The breakpoint on chromosome 17 contains the gene for the retinoic acid receptor gene. Because of this, the treatment of choice for APL is not standard chemotherapy, but retinoic acid; this differentiates the cells into more mature (and less harmful) cells.

Treatment – Patients with AML are treated with chemotherapy. AML chemotherapy (non-M3) consists of an induction and consolidation phase. APL is treated preferentially with retinoic acid. 65% of patients will go into initial remission; the overall survival in AML is not as good as that seen in ALL. Bone marrow transplantation should be considered in AML patients during first remission if a suitable donor is available.

Pathogenesis – CML is due to a chronic proliferation of the myeloid stem cell, which results in the production of excess myeloid cells. CML is one of a family of similar diseases of stem cell origin (myeloproliferative diseases) which also result in chronic proliferations of megakaryocytes (essential thrombocythemia), or red blood cells (polycythemia vera). In a fourth type, myclofibrosis, the marrow becomes fibrotic.

CML is comprised of proliferation of myeloid cells in the peripheral blood and bone marrow. This proliferation is primarily of mature myeloid cells, i.e. myelocytes, metas, bands, and polys. Although an occasional blast cell may be seen in CML, it is just that, occasional to rare; we do not see the sheets and sheets of blast cells (usually > 80%) seen in AML.

Patient age - CML is primarily a disease of young and middle-aged adults but can occur in children (2%-3% of all childhood leukemias are CML) or older adults.

Clinical presentation and course – CML usually has an insidious onset and is often discovered incidentally on a routine peripheral blood analysis or when the patient seeks medical help because of nonspecific findings, such as anemia, weight loss, and/or malaise. Fever and night sweats (due to marked granulocytic cell turnover) may occur and excessive bleeding or bruising is seen in the late stages of the disease. Lymphadenopathy is often present but rarely prominent, but splenomegaly is progressive and often massive. Splenomegaly may cause abdominal discomfort and splenic infarcts can produce left upper quadrant pain.

The clinical course of CML (and the other myeloproliferative diseases) can be subdivided into several different stages (pre-treatment CML, chronic phase, accelerated phase, and blast phase). Transitions between these phases are usually gradual and indistinct, but abrupt transitions can also occur. During the chronic phase of CML, the anemia, elevated white count, and splenomegaly are usually easily controlled for a number of years, but the disease eventually goes into an accelerated phase, where the blast cell count increases, the neoplastic cells acquire additional chromosomal abnormalities (e.g. trisomy 8), and the disease becomes resistant to treatment. The disease may then progress to blast crisis. In blast crisis, the predominant cell is a blast cell (most of the time myeloid, but sometimes lymphoid), and the disease behaves like an acute leukemia, requiring acute leukemia type of chemotherapy. The disease may go into a spent phase, where the marrow becomes fibrotic, and the patient becomes pancytopenic, because the normal marrow elements are pushed out of the bone marrow.

Laboratory features – The peripheral blood white count is increased, sometimes to a marked degree. Most of the white cells are mature, and few if any blast cells are present. Basophils are characteristically increased, and the platelet count is usually high. The bone marrow is hypercellular due to a proliferation of myeloid cells (high M:E ratio) and resembles the peripheral blood. Megakaryocytes are usually increased.

Several other disease entities enter into the differential diagnosis of CML, of which the most important is a benign leukemoid reaction. The body's response to infection is to release WBCs from the bone marrow, sometimes a bit too premature. This will give rise to a peripheral blood picture that may be indistinguishable morphologically from CML. History often helps. A laboratory test that can be ordered to make this distinction is a leukocyte alkaline phosphatase (LAP) score. For some (unknown) reason, the neoplastic granulocytes of CML contain no LAP, compared to reactive polys, which have lots of LAP. Therefore, a low LAP score is most consistent with CML, whereas a high LAP score indicates a reactive process.

Cytogenetic features - The molecular hallmark of CML is the Philadelphia chromosome. This is a balanced translocation involving the abl oncogene on chromosome 9 and the bcr oncogene on chromosome 22 [t(9;22)]. Demonstrate of this translocation, either by classic cytogenetics or by molecular techniques (i.e., RT-PCR) is necessary for the diagnosis of CML. The other myeloproliferative diseases may simulate CML. However, these will lack the t(9;22) translocation (and will have a high LAP score). Morphologically, one should be able to make the distinction between CML and AML by determining how many of the cells are blast cells.

Treatment and prognosis - Bone marrow transplantation (BMT) is the only "cure" for CML. However, this is limited to those patients who have a compatible donor, and carries a high up-front mortality rate (about 10% of patients die of infection and other problems soon after transplantation). For those patients who are not suitable candidates for bone marrow transplantation, chemotherapy with hydroxyurea, alkylating agents, or interferons is the only alternative. With chemotherapy the disease can often be controlled for several years, but many patients eventually progress.

Myelofibrosis can be one of the myeloproliferative diseases by itself, or a stage of another myeloproliferative process. It is characterized by collagen deposition in the bone marrow. This fibrosis squeezes out the normal hematopoietic precursors. The stem cells circulate through the blood, and finally take up residence in the spleen (remember that the spleen was a hematopoietic organ during fetal development). Extramedullary hematopoiesis ensues, resulting in massive splenomegaly.

In the peripheral blood, we see anemia and leukoerythroblastosis (i.e., nucleated red blood cells and myeloblasts in the peripheral smear) due to the early release of blood cells. The RBCs in the peripheral blood are classically teardrop shaped; this is a result of the cells squeezing through the reticulin fibers. The bone marrow is hypercellular. Increased reticulin fibers are seen in the bone marrow.

Pathogenesis – Chronic lymphocytic leukemia (CLL) is a malignant clonal disorder of relatively mature, indolent B lymphocytes (95%) or T lymphocytes (5%) which multiply slowly and do nothing useful. In CLL, the abnormal clone either replaces normal B cells or inhibits their growth and maturation, depressing immunoglobulin levels and impairing humoral immunity. It is the leukemic counterpart to small lymphocytic lymphoma (SLL); since they are different manifestations of the same neoplastic cell. No specific risk factors have been identified for CLL, not even a history of radiation.

Patient age – CLL is a disease of older (middle-aged and elderly) individuals, with an increasing disease frequency with age. Most cases occur in patients over the age of 60 and is rare before the age of 40. It is twice as common in males than females.

Clinical presentation and course – CLL is not uncommonly discovered during a routine peripheral blood examination because systemic symptoms are much less common than in other types of chronic leukemias. However, a history of weakness, fatigue, anorexia, and weight loss may be elicited in some patients, and physical examination may reveal generalized nontender adenopathy and splenomegaly. Generalized lymphadenopathy and pancytopenia are common with later progression of the disease. Complications of pancytopenia, including hemorrhage and infection, are a major cause of death in these patients.

Laboratory features - An absolute lymphocytosis (> 4000 lymphocytes/mL is present. The lymphocytes are small and have mature cytologic features, resembling those normally present in the peripheral blood. However, the lymphocytes in CLL are more fragile than normal lymphocytes, and disintegrate during smear preparation, forming "smudge cells," Immunologically the cells are also atypical. In addition to being monoclonal, they also express CD5, an antigen otherwise characteristic of T lymphocytes. Either kappa or lambda immunoglobulin is weakly expressed by CLL cells. Variable numbers of similar lymphocytes are present in the bone marrow, where they can "crowd out" normal hematopoietic cells and lead to anemia, leukopenia, and/or thrombocytopenia in severe cases.

Cytogenetics - Trisomy 12 is a common cytogenetic abnormality.

Treatment and prognosis – The median 5-year survival in CLL patients is approximately 40-60%, but many patients for ten years or longer, and "die with CLL rather than of it." However, cytopenias can result from bone marrow replacement, and hypogammaglobulinemia may predispose to infection. In addition, some patients develop autoimmune hemolytic anemia or autoimmune thrombocytopenia. The disease transforms to a large cell lymphoma in a minority of patients. This is called Richter's syndrome, and is a bad prognostic development There is no curative therapy for CLL. Indeed, antileukemic therapy is usually unnecessary in uncomplicated early disease and may lead to the patient’s demise from infection and other complications. Patients are usually treated symptomatically.

Pathogenesis - Multiple myeloma ("plasma cell myeloma", "malignant plasmacytoma") is a cancer of the plasma cells throughout the skeletal system. Plasma cells are B-cells that are differentiated enough to secrete an immunoglobulin and/or an immunoglobulin light chain (kappa or lambda, though of course never both).

The term "multiple myeloma" comes from its tendency to make multiple "lytic lesions" in the bone marrow ("myelo-") and nearby cortex. Cancer of plasma cells always involves bone, but only about one-half of cases feature good "punched-out" X-ray lesions. The remaining patients have diffuse disease and suffer precocious osteoporosis. The vertebral column, skull, ribs, and pelvis are most often involved.

In addition to occupying marrow space, replacing normal marrow cells, causing lytic lesions, and weakening bones, plasma cells also produce immunoglobulin, sometimes in very large quantities. Since the plasma cells in multiple myeloma are clonal (i.e., derived from the same progenitor cell, the immunoglobulin produced by all cells will be the same, giving rise to monoclonal immunoglobulin production. Monoclonal immunoglobulins are also termed paraproteins. The monoclonal protein may give rise to renal failure (see below) or a hyperviscosity syndrome.

Patient age- Multiple myeloma is only slightly less common than leukemia or lymphoma. It is a disease of older individuals, and is rare under the age of forty.

Clinical presentation and course – Some patients are asymptomatic at presentation, and the disease is first detected on a routine chemistry panel as an elevated total serum protein level (due to the increased immunoglobulin). However, many patients become anemic from decreased red blood production, develop bone fractures from the tumorous masses and lytic lesions in the bone, complain of bone pain, or develop renal failure from the effects of the monoclonal protein on the kidney. In addition, the production of normal immunoglobulins is usually suppressed in multiple myeloma patients, leading to infection, while hypercalcemia may result for bone resorption. Occasionally, the increased protein in the blood may lead to increased blood viscosity, which causes sluggish blood flow to the brain and other organs ("hyperviscosity syndrome").

Renal failure is a serious complication of multiple myeloma. Several mechanisms are responsible, but the formation of tubular casts from monoclonal protein leaking from the glomerulus is the most important. A monoclonal protein in the urine is called a Bence-Jones protein, and was first recognized by its abnormal precipitation upon heating the urine (by Dr. Henry Bence-Jones). In addition, a special form of immunoglobulin light chains (amyloid protein) may be deposited in the renal parenchyma, giving rise to renal failure. Hypercalcemia and renal infection (pyelonephritis) may aggravate the underlying renal insufficiency.

Laboratory features - It is a common misconception among physicians that neoplastic plasma cells can be seen in the blood of patients with multiple myeloma. This only rarely happens, and mostly in patients with very advanced disease. However, the monoclonal protein may disrupt the negative electrical charge that normally keeps red blood cells apart, so that they stick to each other like "stacks of coins" in patients with multiple myeloma. This phenomenon is known as rouleaux formation. It is not specific for multiple myeloma, but can also be seen in patients with infections, pregnancy, and other diseases who have increased fibrinogen levels.

The laboratory demonstration of a paraprotein by protein electrophoresis of the serum or urine (SPEP, UPEP) is a critical part of the diagnosis of multiple myeloma. In this procedure, a small quantity of the serum or urine specimen is placed on a thin gel (usually agarose), which is placed in an electric field for a short period of time. Different types of proteins separate by charge and migrate to different regions of the gel. Normal immunoglobulins appear as a broad "blurred" area on the electrophoresis gel, since many thousands of antibodies are present. However, since all of the protein molecules forming a monoclonal protein are identical, and migrate identically in an electric charge, a narrow band (M spike) will appear in the immunoglobulin region of the gel. A different technique, immunofixation electrophoresis, is used to confirm the monoclonality of the band and to determine its composition (i.e, IgG, IgA, IgM, IgD, or IgE heavy chain, kappa or lambda light chain). A few individuals with multiple myeloma have plasma cells which secrete only heavy chains, only light chains, immunoglobulin fragments, or no immunoglobulin at all). Bone marrow evaluation reveals increased numbers of plasma cells, which often occur in sheets and clusters. Radiographic studies demonstrate the characteristic punched out lesions. In the end, the diagnosis of multiple myeloma is a clinical, not a pathological, one, combining the clinical presentation with the diagnostic tests.

Treatment - Multiple myeloma patients with early disease are usually not treated but are carefully monitored for evidence of disease progression. Localized radiotherapy and chemotherapy are options for older patients with more advanced disease, while bone marrow transplantation is an option for younger patients (i.e., < 55 years) with aggressive disease. Localized radiotherapy is used to treat affected bones. The median survival is approximately 3 years.