Blood Cell Identification: 2010-C Mailing: Hemoglobin SC Disease CASE HISTORY DISCUSSION NORMAL ADULT HEMOGLOBIN

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1 Please Note: To view the Figures and Images contained within this education activity in color, access the electronic version of the reading. CASE HISTORY The patient is a 37-year old female with a history of multiple sickle cell crises. She now presents with avascular necrosis of the left hip. Laboratory data include: WBC = 12.8 x 10 9 /L; RBC = 3.05 x /L; HGB = 8.7 g/dl; HCT = 26.4%; MCV = 86.4 fl; MCHC = 33.0 g/dl; RDW = 16.9; and PLT = 331 x 10 9 /L. DISCUSSION This case illustrates some of the morphologic findings seen in the blood smear of a woman experiencing multiple sickle cell crises. The features illustrated here include several sickle cells (drepanocytes; see Figure 6. on page 8 and Figure 10. on page 12), target cells, and moderate anemia. These findings suggest a sickling disorder, although the morphology does not specifically point to the exact etiology. In this particular instance, the sickling disorder is secondary to Hemoglobin SC (Hb SC) disease, and the blood smear also shows some misshapen cells with crystalline material, often referred to as SC poikilocytes (see Figure 10. on page 12). Hemoglobin SC disease is a hemoglobinopathy, a term that refers to production of abnormal hemoglobin molecules. In contrast, the term thalassemia refers to decreased production of the normal globin chains that comprise hemoglobin. Hb SC disease is a compound heterozygote phenomenon, signifying that it is a genetic combination of two beta globin abnormalities, Hb S and Hb C. Both of these hemoglobin abnormalities are seen most frequently in the United States in African Americans where the hemoglobin S gene is present in ~8% of individuals (heterozygous Hb S; Hb S trait) and the hemoglobin C gene in ~2-3% (heterozygous Hb C; Hb C trait). The frequency of Hb SC disease in African Americans is ~0.05%, which is less common than Hb SS (0.16% of African Americans) and more common than Hb CC (0.02% of African Americans) disease. In order to better understand Hemoglobin SC disease, it is important first to review the structure of normal adult hemoglobin and methods used to identify abnormal hemoglobins, then discuss homozygous Hemoglobin SS and homozygous Hemoglobin CC in comparison to Hb SC disease. NORMAL ADULT HEMOGLOBIN Hemoglobins are each composed of heme groups and protein groups. The heme group contains an iron molecule bound within a porphyrin ring and is identical in all hemoglobins. The protein part of the molecule is a tetramer, comprised of 2 dimers. Each dimer contains an α like globin and a β-like globin, and each globin chain binds a heme group. 1 - Education

2 Hemoglobin A is the major normal adult hemoglobin (~97% of total normal hemoglobin). It consists of two identical α-chains, each comprised of 141 amino acids; and two identical β-chains, each comprised of 146 amino acids, and thus, can be referred to as α2β2. Each chain is linked to one heme group, with the heme groups at the surface of each molecule, thereby having the ability to combine reversibly with oxygen. In addition to Hb A, adult red cells also contain small quantities of Hb A2 (α2δ2; %) and Hb F (α2γ2; < 2%); see Table 1. for a comparison of hemoglobins present in a normal adult with those seen in Hb SS disease, Hb CC disease, and Hb SC disease. Table 1. Comparison of Degree of Anemia, Hb values, MCV, and HPLC Fractions Genotype Degree of Anemia Hb (mg/dl) MCV (fl) HbS (% of total Hb) HbC (%) HbA (%) Normal Normal >95 SS Severe > CC Asymptomatic to >90 - mild SC Mild to moderate ~ DIAGNOSIS OF HEMOGLOBINOPATHIES Several laboratory methods are available to evaluate hemoglobin composition, including electrophoresis (acid and alkaline), high performance liquid chromatography (HPLC), capillary electrophoresis, isoelectric focusing, and amino acid/dna sequencing. With the exception of DNA sequencing, these methods generally rely on differential charge of various hemoglobin types to identify abnormalities. In addition to these other methods, if a sickling disorder is a consideration, a solubility test for sickling hemoglobins may also be useful. Solubility Test for Sickling Hemoglobins This test is relatively simple to perform, but has significant limitations in overall interpretation. In this test, lysates of red blood cells are placed in a high phosphate buffer. Sodium hydrosulfite is added to the solution lowering the oxygen tension. If Hb S is present, a cloudy solution will form. Unfortunately, this test will be positive in both heterozygous and homozygous Hb S, as well as in other mixed disorders such as Hb SC disease. In addition, some hemoglobinopathies contain two amino acid substitutions, and if one of these is similar to that of Hb S, such as in Hb C-Harlem, the test may also be positive. So, while this is an easy to perform and rapid test, overall interpretive conclusions are limited. In addition, if Hb S represents less than 15-20%, a false negative test may occur. Hemoglobin Electrophoresis Hemoglobin electrophoresis is generally performed at both alkaline and acid ph. Using cellulose acetate or agar at an alkaline ph of 8.6, hemoglobins are negatively charged and will move on the gel towards the positive electrode (anode). Hemoglobins containing an amino acid substitution that changes the overall charge of the molecule will have differing mobility from Hb A (Figure 1. on the following page). Alkaline 2 - Education

3 electrophoresis is quite useful; however, many hemoglobin variants will migrate in a similar position. For example, hemoglobins S, D, and G migrate in the same location, as do hemoglobins C, E, O, and A2. Acid electrophoresis, run at a ph of 6.2, complements information obtained from alkaline electrophoresis. By this method, the most common abnormal hemoglobins, Hb S and Hb C, are effectively separated from Hb A, as well as most others that migrate in similar locations by alkaline electrophoresis (Figure 2.). Unfortunately, many of the other abnormal hemoglobins migrate in a pattern similar to Hb A, and thus, acid electrophoresis is often not useful for characterization of other abnormal hemoglobins. Figure 1. Figure 2. Hemoglobin electrophoresis performed at alkaline ph. Hemoglobins are negatively charged and move towards the positively charged electrode (anode). Mobility within the gel differs based on the overall charge of the hemoglobin. This method allows separation of many hemoglobins from normal Hb A; although multiple abnormal hemoglobins may migrate in the same position. For example, hemoglobins S, D, and G, all migrate in the S postion, while hemoglobins C, E, O, and A2 migrate in the same position. Hemoglobin electrophoresis performed at acid ph. By this method, the most common abnormal hemoglobins, Hb S and Hb C, are effectively separately from Hb A, as well as most others that migrate in similar locations by alkaline electrophoresis. Isoelectric Focusing Isoelectric focusing is an additional electrophoretic method in which a ph gradient from approximately 6-8 is established within the gel. This differential ph gradient allows hemoglobins to migrate within the gel to their isoelectric point, the point at which they contain zero charge. Isoelectric focusing allows better discrimination of hemoglobins that migrate in similar locations on alkaline electrophoresis. Isoelectric focusing has largely been replaced by high performance liquid chromatography (HPLC) as a method of hemoglobin analysis. High Performance Liquid Chromatography (HPLC) Although historically used in the research setting, more recently compact HPLC instruments designed for hemoglobin analysis have become available for routine use in the clinical laboratory. Generally, these instruments utilize a weak cation exchange column to which hemoglobins bind. As the ionic strength of the eluting liquid phase increases, hemoglobin variants will come off the column at a specific retention time, 3 - Education

4 thus allowing identification of the hemoglobin variant based on the overall charge characteristics of the protein. The pattern seen by alkaline electrophoresis demonstrates some correlation with retention time by HPLC since both methods are dependent on the charge of the hemoglobin molecule; although the specific retention time by HPLC is dependent on the column and eluting solution used in the instrument. In general terms, amino acid substitutions leading to more overall negative charge will result in faster migration by alkaline electrophoresis and a shorter retention time on the column by HPLC. One advantage of this method is that Hb C does not migrate with Hb A2 as it does on alkaline electrophoresis, thus allowing measurement of Hb A2 in a patient with heterozygous or homozygous Hb C. Unfortunately, Hb E does elute with Hb A2 by this method, precluding accurate measurement of Hb A2 when Hb E is present. Examples of a normal adult hemoglobin pattern by HPLC, as well as some common hemoglobinopathies, are shown in Figure 3. The proportions of the various hemoglobins in normal adults, sickle cell disease, Hb CC disease, and Hb SC disease are compared in Table 1. on page 2. Figure 3. HPLC in a normal patient, Hb SS disease, Hb SC disease, and Hb C trait. Elution times on these plots shows Hb F at minutes, Hb A at minutes, Hb A2 at minutes, Hb S at minutes, and Hb C at minutes. Note that in Hb SS disease a dominant peak in the S window is present, without detectable Hb A, while in Hb SC disease, similar peaks are noted in the S and C windows. In Hb C trait (heterozygous), normal Hb A is present, as is Hb C. 4 - Education

5 Capillary Electrophoresis Most recently, automated capillary electrophoresis (CE) instruments have been making their way in to the clinical laboratory for hemoglobin analysis. By this method, electrophoresis is performed by adding patient sample to a thin capillary tube containing a buffer, most often an alkaline buffer. Voltage is applied to allow separation of hemoglobins based on their charge, similar to the traditional gel electrophoresis methods mentioned above. This method has the advantage over HPLC of allowing accurate quantitation of Hb A2 in the presence of Hb E; although on some CE instruments adequate separation of Hb A2 from Hb C to allow accurate quantitation may not be possible in all cases. DNA Sequencing While most common hemoglobin variants can be identified by other methods, some uncommon variants require DNA sequencing for further identification. In particular, sequencing may be required for some clinically important variants, including unstable hemoglobins, low or high oxygen affinity hemoglobins, and M-hemoglobins, that fail to separate from Hb A by other methods, often due to their neutral charges. Generally, polymerase chain reactions (PCR) are performed to amplify the exons of the ß and/or α-globin genes. Then these PCR products are put through a sequencing reaction that determines the nucleotide sequence of these genes (Figure 4.). The nucleotide substition that results in the abnormal amino acid sequence is usually easily identified, and can be compared to known sequences for identification. In addition to its use in hemoglobinopathies, DNA sequencing can also be useful to identify nucleotide substitutions associated with ß-thalassemia, a group of disorders resulting in decreased production of a normal ß-globin chain. Figure 4. DNA sequencing of the ß-globin chain of hemoglobin in the region of codon 6. The reference gives the normal DNA sequence in this region, which is the same as that from a normal individual. In Hb SS disease, there is a single DNA nucleic acid substitution of a T instead of A (see box). This changes codon 6 from GAG that codes for the amino acid glutamic acid to GTG, encoding valine. Hb S differs from Hb A by only this single nucleic acid. 5 - Education

6 Now let s move on to discuss Hb SS disease, Hb CC disease, and Hb SC disease. The hemoglobin mutations in these disorders are compared in Table 2. and the laboratory findings and clinical features of these disorders are compared in Table 3. Table 2. Prevalence of Hemoglobin Disorders and Comparison of Mutations Prevalence in Genotype Mutation African Americans Hemoglobin SS 0.16 Homozygous; β6 glu val Glutamic acid in 6 th position on β- globin chain replaced by valine Hemoglobin CC 0.02 Homozygous; β6 glu lys Glutamic acid in 6 th position on β- globin chain replaced by lysine Hemoglobin SC 0.05 Heterozygous; one allele with β6 glu val, other allele with β6 glu lys Table 3. Laboratory Findings and Clinical Features Clinical Manifestations Sickling Test Blood Smear Findings Clinical Expression Life Expectancy Hemoglobin SS Hemoglobin CC Hemoglobin SC Severe anemia Normocytic Mild anemia Microcytic Mild to moderate anemia Normocytic Positive Sickle cells Polychromasia Nucleated RBCs Target cells, numerous Howell-Jolly bodies Pappenheimer bodies + Neutrophilia + Thrombocytosis Negative Hemoglobin C crystals (hexagonal or rodshaped) Target cells Microspherocytes Polychromasia, minimal Positive Sickle cells / S/C poikilocytes misshapen crystals Target cells, numerous Anisocytosis, mild to severe Poikilocytosis, mild to severe Severe sickling crises Often asymptomatic + Occasional jaundice + Occasional abdominal discomfort Usually mild hemolytic anemia Splenomegaly Less frequent sickling crises Less painful crises Normal to stocky body habitus More retinopathy than Hb SS 6 - Education Markedly decreased Fatal In the US, median survival ~45 years of age Normal life expectancy Slightly to moderately shortened

7 HEMOGLOBIN SS Hemoglobin S is comprised of two normal α-globin chains and two abnormal β-globin chains that contain a single amino acid substitution, from glutamic acid to valine, on the 6 th amino acid of the beta globin chain, hence the nomenclature of β6 glu val. Patients who are homozygous for Hb S (sickle cell disease) generally have debilitating illness with only partial compensation of their hemolytic anemia and baseline Hgb level of 6-10 g/dl. The mutation in the β- globin chain results in decreased solubility of de-oxygenated hemoglobin S that then forms rigid polymers that distort the red cells in to the characteristic sickled shape. Classically, these red cells appear in the shape of a thin crescent with two pointed ends and will lack central pallor. The polymerization of deoxygenated hemoglobin S may cause red cells to appear in one or more of the following forms: crescentshaped, boat-shaped, filament-shaped, holly-leaf form, or envelope cells (Figure 5.). Figure 5. Sickle cells can be seen in a variety of sickling disorders, including sickle cell anemia (homozygous Hb S), hemoglobin SC disease, SD disease, and S-beta-thalassemia. In addition to sickled forms, the blood smear findings include target cells, increased polychromasia, nucleated red cells, and findings associated with hyposplenism, such as Howell-Jolly and Pappenheimer bodies (Figure 6. on the following page and Table 3. on page 6). 7 - Education

8 Figure 6. Blood smears from Hb SS patients demonstrating sickled cells, as well as occasional target cells. As with other hemolytic anemias, these patients are prone to gallstones, and suffer consequences of microvascular occlusion due to the decreased deformability of the sickled red cells. Microvascular occlusion leads to pain crises, cerebrovascular accidents (strokes), aseptic necrosis of the hip, renal injury, acute chest syndrome with pulmonary compromise, and skin ulcers. Repeated splenic infarction results in autosplenectomy in adult patients and increased suseptibility to infection. See Figure 7. on the following page and Table 4. on page 10 for a summary of sickle cell anemia and complications due to sickling crises. Vascular occlusive crises can be precipitated by infection, acidosis, cold exposure, and hypoxia. Unfortunately, life expectancy is significantly decreased in patients with sickle cell disease with median age of death of approximately 45 years. 8 - Education

9 Figure 7. Sickle Cell Formation Summary of sickle cell disease, including the complications arising from red cell sickling. 9 - Education

10 Table 4. Complications of Sickling Crises Due to Ischemia Affected Manifestation Site/Organ Hand-foot Distal extremities Bilateral swelling of hands and syndrome, aka feet sickle cell dactylitis Splenic Spleen Sudden pooling of blood and sequestration rapid enlargement of spleen Functional asplenia Spleen Inadequate antibody production, impaired ability of reticuloendothelial system to clear bacteria from blood Renal papillary necrosis Vaso-occlusive Abdomen Abdominal pain crises Aseptic bone necrosis Consequence Pain Hypovolemic shock Increased risk of certain infections, like Salmonella or pneumococcal infection Kidney Blood in urine Chronic kidney disease Bone Bone pain Possible osteomyelitis; bone fracture Acute chest syndrome Lungs Shortness of breath Hypoxia HEMOGLOBIN CC Similar to hemoglobin S, Hemoglobin C results from a single amino acid substitution on the 6 th amino acid of the beta globin chain. Only, in hemoglobin C, the substitution is from glutamic acid to lysine (β6 glu lys). Patients who are homozygous for Hb C generally have mild red cell microcytosis (MCV of approximately fl) and a mild to moderate hemolytic anemia that is less severe than that seen in homozygous Hb S. In patients with homozygous Hb C the hemolysis is well compensated, and anemia may not even be present. These patients are largely asymptomatic; although some degree of splenomegaly may be present. Hb C is also less soluble than Hb A, and forms crystals in the oxygenated state. These crystals may contribute to hemolysis; although some authors attribute the hemolysis in these patients to potassium efflux from the red cells. Characteristic hemoglobin C crystals within red cells of patients with this homozygous mutant are composed of dense structures with rhomboidal, tetragonal, or rod shapes (Figure 8. on the following page). They often distort the cell and project beyond its rim. The classic crystal shape resembles the Washington monument. The crystals are often surrounded partly by a clear area or blister devoid of hemoglobin. Hemoglobin C crystals are readily seen after splenectomy in patients with hemoglobin C disease or SC disease. In addition to Hb C crystals, the blood smear frequently shows numerous target cells and occasional microspherocytes. Hb C appears identical to Hb E on alkaline electrophoresis but can be easily identified on acid electrophoresis since Hb E migrates with Hb A by this method Education

11 Figure 8. Blood smears from Hb CC patients demonstrating Hb C crystals. HEMOGLOBIN SC Hb SC disease can be viewed as a hybrid of hemoglobin S and C; an intermediate type of disease, if you will. The symptoms are not as severe as Hb SS, and yet its manifestations are not quite as benign as Hb CC. Diagnosis of Hemoglobin SC Hemoglobin SC disease is easily diagnosed, based primarily on the essentially equal amounts of HbS and HbC detected on hemoglobin electrophoresis (Figure 9.) and HPLC (Figure 3. on page 4). On alkaline electrophoresis, however, recall that hemoglobin C can migrate with hemoglobins A2, E, O-Harlem, and O- Arab. Hb C is easily distinguished on acid electrophoresis, thankfully, as it is the only hemoglobin that migrates to the C position. Figure 9. Alkaline and acid electrophoresis in a patient with Hb SC disease (see starred gel lanes) Education

12 Blood Smear Findings Anemia is frequently seen, albeit often a mild hemolytic anemia. On occasion, moderate, normochromic and normocytic anemia is present; although the Hb level usually remains above 10 g/dl. While true sickled cells are rare, as in the present case (Figure 10A.), irregularly shaped cells, appearing to contain crystalline material, are frequently present and often referred to as SC poikilocytes (Figure 10B. and C.). Other cells may have a boat shaped (resembling plump sickled cells) or clam shell appearance, and target cells are frequently seen. The typical polyhedral crystals characteristic of homozygous C disease are unusual in SC disease. Our current case is an excellent example where target cells predominate, affecting up to 85% of all red blood cells, and true sickled cells are fairly uncommon. Figure 10A. Figure 10B. Figure 10C. Photomicrographs from the blood smear in our patient with Hb SC disease. Panel A shows a sickled cell, an uncommon finding in SC disease. Panels B and C show crystalline material in SC poikilocytes. Pathophysiology of Hemoglobin SC The patholophysiology of sickling manifestations and hemolytic anemia in SC disease remain controversial. It is not clear that Hb S and Hb C co-polymerize to result in sickling in these patients. Two possible factors contribute to the sickling manifestations in SC disease, namely the increased proportion of Hb S in comparison to S trait (50% vs. 40%) and the cellular dehydration associated with the presence of Hb C Education

13 Clinical Implications Interestingly, Hb SC presents most often as a mild hemolytic anemia; although there is a wide spectrum of disease severity in these patients. Some individuals are largely asymptomatic while others suffer frequent sickling crises and resulting in severe consequences. Sometimes, splenomegaly may be the only sign of Hb SC. While this disorder typically presents in childhood, it may not be detected until later in life. In patients who suffer from sickling crises, their symptoms correlate with the sites affected by hypoxia: hip and low back pain may signal avascular necrosis of the femoral head. Blood in the urine may indicate infarcts in the kidney parenchyma. Abdominal pain may be a manifestation of splenic infarction. Acute chest syndrome, similar to that seen in Hb SS homozygotes, can present with fatigue, shortness of breath while resting and/or upon exertion. In contrast to Hb SS, life expectancy is only slightly to moderately shortened. CASE HISTORY, CONCLUSION As mentioned previously, the Case History and photographs (images) are from a patient with Hb SC disease. Although SC disease is generally less severe than homozygous Hb S, our patient demonstrates relatively severe clinical findings, including significant anemia, and a history of repeated sickling crises, the most recent of which was associated with avascular necrosis of the hip Education

14 References 1. McPherson RA, Pincus MR, eds. Henry s Clinical Diagnosis and Management By Laboratory Methods. 21st ed. Philadelphia, PA: Saunders Elsevier; Glassy EF, ed. Color Atlas of Hematology: An Illustrated Field Guide Based on Proficiency Testing. Northfield, IL: College of American Pathologists; Hoyer JD, Kroft SH, eds. Color Atlas of Hemoglobin Disorders: A Compendium Based on Proficiency Testing. Northfield, IL: College of American Pathologists: Nagel RL, Fabry ME, Steinberg MH. The paradox of hemoglobin SC disease. Blood Reviews. 2003:17; Education

15 Education Activity Authors Joan Etzell, MD, FCAP: Joan Etzell, MD, is a Professor of Clinical Laboratory Medicine and the Director of the Clinical Hematology Laboratory at the University of California, San Francisco (UCSF). She is AP/CP and Hematology Board certified by the American Board of Pathology. Dr. Etzell is actively involved in the education of medical technologists, medical students, residents, and fellows in hematology / hematopathology. She serves as the Hematopathology Fellowship Director and Associate Residency Program Director in Laboratory Medicine in UCSF. Dr. Etzell has authored over 50 papers, book chapters, educational activities and abstracts in the areas of hematology and hematopathology. Dr. Etzell currently serves as the Vice-Chair of the Hematology and Clinical Microscopy Resource Committee for the College of American Pathologists (CAP). Maria E. Vergara-Lluri, MD: Ria Vergara-Lluri, MD, is a Fellow in Surgical Pathology at the University of California, San Francisco (UCSF) Medical Center in San Francisco, California. She is in her fourth year of postgraduate training in anatomic pathology and clinical pathology. Dr. Vergara-Lluri is co-chief resident for the anatomic pathology department at UCSF ( ), serving as leader, liaison and advocate for resident education and training. She is the junior member of the Hematology and Clinical Microscopy Resource Committee for the College of American Pathologists (CAP) Education

16 Blood Cell Identification: 2010-C Mailing: Chronic Myelogenous Leukemia (CML) Please Note: To view the Figures and Images contained within this education activity in color, access the electronic version of the reading. DISCUSSION The most common of the myeloproliferative neoplasms, chronic myelogenous leukemia, BCR-ABL1 positive (CML), accounts for about 20 35% of all adult leukemias. It typically occurs at ages 40 60, with approximately 20 40% of patients being asymptomatic at diagnosis. The diagnosis of CML may be suggested by hepatosplenomegaly on physical examination or abnormal results leukocytosis, anemia, or thrombocytosis on routine hematologic testing. When symptoms occur, they usually relate to splenomegaly (left upper quadrant discomfort or early satiety), problems from increased white cell production (bone pain, mild fever, night sweats, weight loss), or anemia (dyspnea, fatigue, pallor); see Table 1 below. The median survival is now about 5 7 years, and data thus far from clinical trials with imatinib mesylate (Gleevec ) and similar targeted therapies suggest that this figure will continue to improve. Table 1. Signs and Symptoms of CML Symptoms Signs Fatigue Splenomegaly Weight loss Sternal tenderness Abdominal fullness and early satiety Lymphadenopathy Easy bruising or bleeding Hepatomegaly Abdominal pain Purpura Fever Retinal hemorrhage Adapted from Wintrobe s Atlas of Clinical Hematology. Chronic myeloproliferative syndromes. George TI. 2007:115. If untreated, the disease often progresses through three stages (Figure 1. on the following page): Chronic phase (CML-CP) Accelerated phase (CML-AP) Blast phase (CML-BP) 16 - Education

17 Blood Cell Identification: 2010-C Mailing: Chronic Myelogenous Leukemia (CML) Figure 1. Natural History of CML By definition, blasts represent less than 10% of marrow nucleated cells in the chronic phaseof the disease. In blast phase or blast crisis, 20% or more blasts are present in the bone marrow. Accelerated phase of the disease typically shows 10 19% marrow blasts. Most patients present in the chronic phase of the disease and if untreated progress to either accelerated phase or blast crisis. In the chronic phase, the blood studies typically show mild anemia and leukocytosis that usually exceeds 25 x 10 9 /L (median white count of about 170 x 10 9 /L), primarily composed of neutrophils in various stages of maturation, particularly due to increases in myelocytes and mature neutrophils (Figure 2. on the following page). Basophils are universally increased, and eosinophilia is common, which may be helpful in differentiating CML from a reactive neutrophilia. The platelet count is normal or elevated and may exceed 1000 x 10 9 /L, but resulting thrombosis is unusual. The serum lactate dehydrogenase (LDH) and uric acid are commonly increased, reflecting the underlying excessive marrow cell proliferation. The bone marrow shows hypercellularity in the neutrophil line, but myeloblasts usually constitute < 5% of the cells. Unlike other myeloproliferative diseases, megakaryocytes are often small and hypolobated (Figure 3. on the following page). Reticulin fibrosis may be increased, particularly in the 50% of patients where megakaryocytes are increased in number. Because of the increased and uncontrolled hematopoiesis, the number of cells undergoing proliferation and eventual cell death is increased, and macrophages containing the lipids from the dead cells may be visible as sea-blue histiocytes or pseudo-gaucher cells Education

18 Blood Cell Identification: 2010-C Mailing: Chronic Myelogenous Leukemia (CML) Figure 2. Peripheral Blood Smear Figure 3. Bone Marrow Biopsy Peripheral blood smear, chronic phase of CML. The blood smear shows a marked leukocytosis with a predominance of segmented neutrophils and myelocytes. (Wright Giemsa, original magnification x600). George TI, Arber DA. Pathology of the myeloproliferative diseases.hematol Oncol Clin North Am. 2003;17:1104, Fig 1. Bone marrow biopsy, chronic phase of CML. The biopsy is markedly hypercellular with very little fat. A myeloid hyperplasia is present with many segmented neutrophils. Increased numbers of dwarf megakaryocytes are present, some in a cluster. (Hematoxylin & eosin, original magnification x 400). George TI, Arber DA. Pathology of the myeloproliferative diseases. Hematol Oncol Clin North Am.2003;17:1104, Fig 1. The accelerated phase is defined by at least one of the following (also, see Table 2. below): 1. Persistent or increasing white count (> 10 x 10 9 /L) and/or spleen size despite therapy 2. Persistent thrombocytopenia (< 100 x 10 9 /L) or thrombocytosis (> 1000 x 10 9 /L) despite treatment 3. Cytogenetic evidence of clonal evolution 4. Peripheral blood basophilia 20% % blasts in the bone marrow or peripheral blood The first 3 criteria have been associated with the transformation from chronic phase to accelerated phase, with the last 2 criteria associated with the transition from accelerated phase to blast phase. Other bone marrow findings that are suggestive of, but not definitive for, CML-AP are marked dysplasia of neutrophil precursors or the appearance of abundant aggregates of small, dysplastic megakaryocytes associated with marked reticulin or collagen fibrosis. Table 2. Accelerated Phase of Chronic Myelogenous Leukemia Criteria Occurrence Blood/marrow blasts 10-19% Blood basophils 20% Cytogenetics Clonal evolution Persistent thrombocytopenia < 100 x 10 9 /L Persistent thrombocytosis* > 1,000 x 10 9 /L Splenomegaly, rising WBC* Morphology/histopathology Megakaryocyte proliferation, fibrosis and/or granulocytic dysplasia *unresponsive to therapy 18 - Education

19 Blood Cell Identification: 2010-C Mailing: Chronic Myelogenous Leukemia (CML) The blast phase is defined by one or more of the following features: 1. Blasts account for 20% of peripheral blood white cells or nucleated bone marrow cells 2. Blasts proliferate in extramedullary sites, such as the skin, lymph nodes, and spleen 3. Large aggregates of blasts occur in the bone marrow The blasts are usually myeloid, but in about 20 30% of cases they are lymphoid, usually precursor B lymphoblasts. The ability of CML to transform to either a myeloid or lymphoid leukemia confirms that this disorder arises from a pluripotent bone marrow stem cell that is capable of multilineage differentiation. Occasionally, both types of leukemic blasts are present simultaneously. Genetic abnormalities now define CML. All cases of CML have a BCR-ABL1 fusion gene resulting from a translocation involving chromosomes 9 and 22. In 90 95% of cases the characteristic translocation t(9;22)(q34;q11) results in the Philadelphia chromosome and is detected on routine cytogenetic studies. The Philadelphia chromosome refers to the abnormal chromosome 22 created from fusion of part of the ABL1 gene on chromosome 9 to part of the BCR gene on chromosome 22 (Figure 4. below). Figure 4. Cytogenetics Karyotype Arrows indicate the characteristic translocation involving chromosomes 9 and 22 of chronic myelogenous leukemia. In addition, this karyotype also has a translocation between chromosomes 4 and 5, as well as an extra Y chromosome. Clonal evolution, that is additional chromosomal abnormalities other than the t(9;22) translocation often signify acceleration of disease. Courtesy of Stanford Cytogenetics Laboratory, Stanford, CA Education

20 Blood Cell Identification: 2010-C Mailing: Chronic Myelogenous Leukemia (CML) Most of the remaining cases of CML have variant genetic abnormalities that result in the BCR-ABL1 fusion gene, but involve other chromosomes in addition to chromosomes 9 and 22. A small number of cases of CML have cryptic BCR-ABL1 translocations that cannot be identified by routine cytogenetic analysis. When this is suspected, molecular genetic studies, such as reverse transcriptase polymerase chain reaction (RT-PCR) analysis and FISH analysis, are indicated (Table 3. and Figure 5. below). In all cases the BCR-ABL1 fusion gene results in production of an abnormal BCR-ABL1 protein with enhanced tyrosine kinase activity that leads to the marked cellular proliferation characteristic of CML. Table 3. Genetic Evidence of Chronic Myelogenous Leukemia Technique Sensitivity (%) Cytogenetic karyotype RT-PCR 99 FISH > 99 RT-PCR = reverse transcriptase polymerase chain reaction FISH = fluorescent in situ hybridization Figure 5. FISH Interphase dual fusion fluorescent in situ hybridization (FISH) for detection of the BCR-ABL1 fusion gene resulting from the t(9;22) translocation. In interphase FISH, a probe directed at 9q34 (ABL1 gene) is labeled with a green fluorophore and a probe directed at 22q11.2 (BCR gene) is labeled with a red fluorophore. When a fusion gene is present, the red and green combine to form a yellow signal. The presence of two yellow signals indicates that both BCR-ABL1 and ABL1-BCR fusion genes are present. Courtesy of Stanford Cytogenetics Laboratory, Stanford, CA Education

21 Blood Cell Identification: 2010-C Mailing: Chronic Myelogenous Leukemia (CML) The differential diagnosis of chronic myelogenous leukemia includes both reactive and neoplastic disorders. The reactive disorders are called leukemoid reactions and mimic the features of CML. In leukemoid reactions the white blood cell count is typically below 50 x 10 9 /L, while CML often presents with much higher white blood cell counts. Leukemoid reactions can be due to any strong stimulation of the bone marrow, including bacterial infection, growth factors, carcinoma or other metastatic disease, tuberculosis and even certain viral infections. While a leukemoid reaction will show a neutrophilia and may show a leftshift in granulocytes, they do not show the typical predominance of segmented neutrophils and myelocytes seen in CML. Leukemoid reactions also lack the eosinophilia and basophilia found in CML. Leukemoid reactions often show features of neutrophil infection/activation (i.e., toxic granulation, vacuolated neutrophils, Dohle bodies). Neoplastic disorders that can mimic CML include chronic myelomonocytic leukemia (CMML) and atypical chronic myelogenous leukemia, BCR-ABL1 negative (acml), (Table 4. below). Table 4. Differential Diagnosis of Chronic Myelogenous Leukemia Feature CML acml CMML BCR/ABL WBC Basophils* 2% < 2% < 2% Monocytes* < 3% 3 10% > 10% Immature granulocytes* > 20% 10 20% 10% Blasts* < 2% > 2% < 2% Granulocyte dysplasia Marrow erythroid hyperplasia CML = chronic myelogenous leukemia BCR-ABL1 positive acml = atypical chronic myelogenous leukemia BCR-ABL1 negative CMML = chronic myelomonocytic leukemia *In peripheral blood Adapted from George TI, Arber DA. Pathology of the myeloproliferative diseases. Hematol Oncol Clin North Am. 2003;17: All can show elevated white blood cell counts, but the highest counts are typically seen in CML. CMML and acml can show basophilia, but typically only CML has peripheral blood basophilia more than 2%. All three disorders may show a monocytosis, but a monocytosis of more than 10% of white blood cells is usually only seen with CMML. Large numbers of immature granulocytes in the peripheral blood (i.e., more than 20%) are typical of CML. Whereas immature myeloid forms may be seen in CMML and acml, immature granulocytes are less than 20% of the blood leukocytes. Blasts may be observed in the peripheral blood of any of these disorders, but the highest blast counts are seen in acml (> 2%), while blasts counts less than 2% are more typical of chronic phase CML and CMML. Granulocytic dysplasia is most marked in acml and is seen to a lesser extent in CMML. Although granulocytic dysplasia can be seen in CML, it is less marked than in the other 2 neoplastic disorders. If dysplasia is seen in CML, it is usually manifested by hypogranular leukocytes, giant metamyelocytes, hypersegmented neutrophils, leukocytes with mixed basophil-eosinophil granules, and Pelgeroid-like neutrophils. Other morphologic abnormalities include nucleated red blood cells, target cells, giant platelets, mitotic cells and megakaryocyte nuclei. However, as noted above, the defining feature of CML is the presence of the BCR-ABL1 fusion gene, allowing for 21 - Education

22 Blood Cell Identification: 2010-C Mailing: Chronic Myelogenous Leukemia (CML) definitive diagnosis with molecular or cytogenetic testing in most cases. Review of cases that were previously called BCR-ABL1 negative CML has resulted in reclassification of many of these cases as either CMML or acml. In the past, CML was considered a progressive disease with allogeneic bone marrow transplant as the only potentially curative treatment. Median survival was approximately 5 years. The therapy of CML was markedly altered by discovery of imatinib mesylate, a tyrosine kinase inhibitor that directly targets the BCR- ABL1 tyrosine kinase fusion protein. This has allowed for targeted therapy specific for CML. Newly diagnosed patients have a 95% chance of reaching a hematologic response within 18 months (resolution of blood and marrow abnormalities with normalization of blood counts) or a 75% chance of complete cytogenetic response (loss of detectable t(9;22)). Responses are also seen in accelerated and blast phases of CML, although the response rate is not as high. Treatment of CML is not curative in most cases, and small numbers of abnormal cells remain that can be detected by molecular (RT-PCR) monitoring to detect the BCR-ABL1 transcript. All patients treated with imatinib mesylate have blood or marrow monitored on a routine basis by quantitative molecular testing using RT-PCR to identify changes in BCR-ABL1 transcript levels. Significant increases in the levels of the BCR-ABL1 transcript correlate with developing resistance to imatinib therapy, usually due to mutations in the drug-binding site in the neoplastic cells. Mutations in the drug-binding site may also be identified by gene sequencing in patients who do not respond initially to therapy. When drug resistance is suspected, increasing doses of imatinib or use of second-generation tyrosine kinase inhibitors often allows control of the disease. With this approach, patients with CML can be effectively treated and maintained for long periods of time without disease progression or transformation Education

23 Blood Cell Identification: 2010-C Mailing: Chronic Myelogenous Leukemia (CML) References 1. George TI. Chronic myeloproliferative syndromes. In: Tkachuk D, Hirschmann JV, eds. Wintrobe s Atlas of Clinical Hematology. Philadelphia, PA: Lippincott Williams and Wilkins, Inc; 2007: George TI, Arber DA. Pathology of the myeloproliferative diseases. Hematol Oncol Clin North Am. 2003;17(5): George TI. Pathology of the myeloproliferative diseases. In: Greer JP, Foerster J, Rodgers GM, Paraskevas F, Glader B, Arber DA, Means RT, eds. Wintrobe s Clinical Hematology. 12 th ed. Philadelphia, PA: Lippincott Williams and Wilkins, Inc; Education

24 Blood Cell Identification: 2010-C Mailing: Chronic Myelogenous Leukemia (CML) Education Activity Authors Tracy I. George, MD, FCAP: Tracy George, MD, is Director of Hematology for Stanford University Medical Center Clinical Laboratories, which serves Stanford Hospital and Clinics and Lucile Salter Packard Children s Hospital. She is an assistant professor of Pathology at the Stanford University School of Medicine in Stanford, CA. Dr. George has written over 70 papers, book chapters, books, educational activities and abstracts in the areas of hematology, hematopathology and surgical pathology. She teaches medical students, residents, and fellows, participates in clinical service work in hematopathology, and performs translational research in the areas of myeloproliferative neoplasms and laboratory hematology. Dr. George is currently Chair of the Hematology and Clinical Microscopy Resource Committee and a member of the Council on Scientific Affairs for the College of American Pathologists (CAP). Kyle T. Bradley, MD, MS, FCAP: Kyle T. Bradley, MD, is an Assistant Professor in the Department of Pathology & Laboratory Medicine at Emory University Hospital in Atlanta, GA. He is board certified in anatomic pathology, clinical pathology, and hematology by the American Board of Pathology. His primary responsibilities are in clinical service work and resident/fellow teaching in the areas of surgical pathology and hematopathology. Dr. Bradley has authored a number of original articles, abstracts, and educational activities in the fields of hematopathology and anatomic pathology and is a member of the Hematology and Clinical Microscopy Resource Committee for the College of American Pathologists (CAP). Sherrie L. Perkins, MD, PhD, FCAP: Sherrie L. Perkins, MD, PhD, is a professor of Pathology at the University of Utah Health Sciences Center and the Chief Medical Officer for ARUP Laboratories in Salt Lake City, UT. She is the Director of Hematopathology for ARUP Laboratories and has responsibilities in teaching, resident training, clinical service and research. Dr. Perkins has written over 140 peer-reviewed papers and 70 book chapters in the areas of hematology and hematopathology. Dr. Perkins is currently a member of the College of American Pathologists (CAP) Hematology and Clinical Microscopy Resource Committee Education