Thalassemia syndromes: - Anemia: severity of anemia is dependent on the degree of residual chain synthesis - Splenomegaly (extramedullary hematopoiesis, RBC

destruction) - Secondary hemochromatosis (iron overload) - Skeletal deformities - Definitive confirmation: hemoglobin electrophoresis

Erythroid cell death, decreased ox transport, splenomegaly, crew-cut skull. Anemia anoxia skeletal deformities.

Above: Basophilic stippling in thalassemia. Peripheral blood film demonstrating microcytic hypochromic RBCs and basophilic stippling (arrows). Basophilic stippling occurs in thalassemia as well as in other hematologic disorders.

Above: High performance liquid chromatography (HPLC) sample demonstrating increased hemoglobin A2 (arrow) in a case of thalassemia trait. HPLC is an automated way of separating and identifying variant hemoglobins and is more accurate at quantifying hemoglobin A2 than is Hb electrophoresis. It can separate HbA2 from certain hemoglobins, which is not possible using hemoglobin electrophoresis alone.

HbH inclusions. Peripheral blood stained w/supravital stain brilliant cresyl blue. The RBC near the top central area (red arrow) demonstrates numerous inclusions in an evenly diffuse distribution, creating a golf ball pattern. This cell is an HbH inclusion body seen in thalassemia. The difference between the HbH bodies that appear like dimpled golf balls w/diffuse even involvement can be seen from reticulocytes w/uneven reticulin deposits (black arrows). The HbH inclusions are precipitated globin tetramers. Reticulocytes, Heinz bodies, and Howell-Jolly bodies stain positive w/brilliant cresyl blue. Reticulocytes are darker, more reticular, clumped, and uneven in distribution. Heinz bodies are larger and not so numerous. Howell-Jolly bodies are usually single inclusions. Rare HbH inclusion bodies may be seen in one or two -gene deletions in -thalassemia trait, but there the absence of identifying these inclusion bodies does not exclude the disorder, which may require molecular studies for definitive diagnosis. In HbH disease (three -gene deletion), HbH bodies are frequent and easily identifiable.

Above: Hydrops fetalis at autopsy in hemoglobin Bart disease. Hepatosplenomegaly in a newborn with hemoglobin Bart disease. The loss of all four -globin genes results in severe anemia, highoutput heart failure, splenomegaly, edema, and intrauterine or immediately postpartum death for the affected fetus. Dystocia, eclampsia, and hemorrhage can occur in the mother carrying the affected fetus.

Above: skeletal deformity and splenomegaly resulting from thalassemia.

Macrocytosis pattern Normal in neonates (MCV 110-130 fL) Secondary to increased reticulocytes (severe hemolytic anemia with marked increase in erythropoiesis) True macrocytosis: Alterations in lipid content of RBC membrane (liver disease, post-splenectomy) Alteration in DNA synthesis (B12/folate deficiency) Malignant primary bone marrow disorder (myelodysplastic syndrome, MDS/MPD) Liver disease/Post-splenectomy Biliary obstruction leads to increase in cholesterol and phospholipids in plasma, with increase in the RBC membrane

Macrocyte: >8 mm MCV >95-100 fL

B12/folate deficiency: Impaired DNA synthesis Bone marrow: Megaloblastic erythropoiesis Megaloblastic changes in granulocytic series (giant bands diagnostic??) Peripheral blood: Macrocytosis (>100 fL) Macroovalocytes Hypersegmented neutrophils Neurologic changes seen only in B12 deficiency (demyelinization, paraparesis, paresthesias)

B12/folate deficiency: Bone marrow aspirate smear Hypercellular bone marrow Erythroid hyperplasia Megaloblastic erythropoiesis Megaloblastic changes in granulocytic series (giant bands) giant granulocytic precursors Open chromatin Nuclear-cytoplasmic asynchrony Peripheral blood smear Macrocytosis (>100 fL) Macroovalocytes Hypersegmented neutrophils

Test q: A 45F has experienced fatigue and tingling in her extremities for the past year, more pronounced in the last month. On phys exam, she has pale 3 3 conjunctivae. Her liver and spleen are of normal size. The following lab data are obtained: Hgb 7.5 g/dL, MCV 125m , WBC 3000/mm , Seg 25%, 3 Lymph 73, Mono 1, Eos 1; Plt 77,000/mm . A peripheral smear contained granulocytes w/4, 5, and 6 nuclear lobes. Which of the following lab tests would be most helpful in establishing a diagnosis? Serum B12 and folate levels.

Role of B12/folate in DNA synthesis:

Megaloblastic anemias: VITAMIN B12 DEFICIENCY Decreased Intake Inadequate diet, vegetarianism Impaired Absorption Intrinsic factor deficiency Pernicious anemia /Gastrectomy Malabsorption states Diffuse intestinal disease (e.g., lymphoma, systemic sclerosis) Ileal resection, ileitis Competitive parasitic uptake, Fish tapeworm infestation Bacterial overgrowth in blind loops and diverticula of bowel

Test q: A 39F sees her physician because she has experienced abdominal pain and intermittent low-volume diarrhea for the past 3 months. On phys exam, she is afebrile. A stool sample is positive for occult blood. A colonoscopy is performed, and biopsy specimens from the terminal ileum and colon show microscopic findings consistent w/Crohn disease. Because she has failed to respond to medical therapy, surgery is warranted, and part of the colon and terminal ileum are removed. She is transfused w/2 units of packed RBCs during surgery. Several weeks later, she appears healthy but complains of easy fatigability. On investigation, CBC findings show Hgb 3 10.6 g/dL, hematocrit 31.6%, RBC count 2.69 million/L, MCV118m , 3 3 platelet count 378,000/mm , and WBC count 9800/mm . The reticulocyte count is 0.3%. Which of the following is most likely to produce these findings? Vitamin B12 deficiency. Test q: A 40F vegetarian experienced fatigue. On phys exam, she is pale and has poor balance. What might you expect to see on a peripheral blood smear? Macrocytosis and hypersegmented neutrophils.

Megaloblastic anemias: FOLIC ACID DEFICIENCY Decreased Intake Inadequate diet, alcoholism, infancy Impaired Absorption Malabsorption states, Intrinsic intestinal disease Anticonvulsants, oral contraceptives Increased Loss -Hemodialysis Increased Requirement Pregnancy, infancy, disseminated cancer, markedly increased hematopoiesis Impaired Utilization Folic acid antagonists

B12/folate deficiency:Pernicious anemia Autoimmune destruction of gastric mucosa Antibodies blocking B12 binding to IF Antibodies blocking interaction of B12-IF complex with ileal mucosa
Test q: A 67F complains of gradually increasing fatigue. On phys exam, she is found to be anemic and has a peripheral neuropathy characterized by loss of position and vibratory sense. Lab studies document a macrocytic anemia and decreased WBC and platelet counts. What pathological mechanism accounts for these findings? Autoantibodies against intrinsic factor.

Confirmation of B12/folate deficiency: Decreased serum B12 Increased methylmalonic acid and total homocysteine (early indicators) Parietal cell antibodies Serum gastrin levels (elevated in PA) Decreased folate Demyelination and Subacute combined degeneration:

Myelodysplastic syndromes (MDS): Clonal stem cell disorder characterized by maturation defect and ineffective hematopoiesis Patient presents with pancytopenia, however bone marrow is hypercellular. Morphology is heterogeneous with all lineages present and showing dysplastic features
Test q: A 70M undergoes bone marrow biopsy. The biopsy shows dysplasia, 1% myeloblasts, and numerous ringed sideroblasts. All lineages are dysplastic. Deletion of 5q 32-33.3 is identified. Diagnosis? Myelodysplastic syndrome.

Normocytic normochromic pattern: Anemia of chronic disease Most common anemia in hospitalized patient Reduced erythroid proliferation Reduced iron utilization Anemia in renal failure Impaired EPO secretion Reduced erythroid production Shortened survival of RBCs Paroxysmal nocturnal hemoglobinuria

Test q: A 42M has been hospitalized in the burn unit for three months. He tires easily during daily walks. CBC shows a normocytic anemia. Serum iron is low. A bone marrow biopsy shows increased iron stores. You suspect: Anemia of chronic disease. Test q: A 50M has experienced chronic fatigue and weight loss for the past 3 months. There are no remarkable findings on phys exam. Lab studies shows 3 3 Hgb 11.2 g/dL, hematocrit 33.3%, MCV 91 m , platelet count 240,000/mm , 3 WBC count 7550/mm , serum iron 80 g/dL, total iron binding capacity 145 g/dL, and serum ferritin 565 ng/mL. The ANA test result is positive. Which of the following is the most likely diagnosis? Anemia of chronic disease.

Paroxysmal Nocturnal Hemoglobinuria: Acquired clonal disorder of hematopoiesis resulting from the mutation in PIG-A gene encoding GPI anchor Mutation in PIG-A gene Located on Chr. X Encodes for 60kDa protein glucosyl transferase (first step in synthesis of GPI anchor) More than 100 different mutations (deletions, insertions) described producing abnormal or truncated protein (non-functional) leading to lack or diminished expression of GPI anchored proteins Complement pathway: CD59. Lack of proteins hypersensitivity to complement-mediated lysis of RBCs. Thrombocytopenia. No anchor dysfunctional protein.

PNH: Clinical Features Cytopenias: all lineages can be involved Often diagnosis delayed due to complex clinical presentation 25% patients survive longer than 25 years Spontaneous remissions reported Treatment: symptomatic, prednisone, immunosuppression (targeted at abnormal clone), EPO, BMT Cause of death: Venous thromboses Complications of cytopenias Studies showed higher incidence of AML CD55 Expression in PNH: CD59 Expression in PNH:

Test q: A 17M reports passage of dark urine, especially at night, to his physician. He has a history of multiple bacterial infections and venous thromboses for the past 10 years, including portal vein thrombosis in the previous year. On phys exam, his right leg is swollen and tender. CBC shows 3 3 3 Hgb of 9.8 g/dL, hematocrit 29.9%, MCV 92m , platelet count 150,000/mm , and WBC count 3800/mm with 24% segmented neutrophils, 1% bands, 64% lymphocytes, 10% monocytes, and 1% eosinophils. He has a reticulocytosis, and his serum haptoglobin level is very low. A mutation affecting which of the following gene products is most likely to give rise to his clinical condition? Phosphatidylinositol glycan A (PIGA). (Other choices: Spectrin, G6P dehydrogenase, -globin chain, and Factor V mutation)

Classification of Immunohemolytic Anemias: WARM ANTIBODY TYPE (IgG ANTIBODIES ACTIVE AT 37C) Primary (idiopathic) Secondary Autoimmune disorders (particularly systemic lupus erythematosus) Drugs Lymphoid neoplasms COLD AGGLUTININ TYPE (IgM ANTIBODIES ACTIVE BELOW 37C) Acute (mycoplasmal infection, infectious mononucleosis) Chronic Idiopathic Lymphoid neoplasms COLD HEMOLYSIN TYPE (IgG ANTIBODIES ACTIVE BELOW 37C) Rare; occurs mainly in children following viral infections Aplastic Anemia: ACQUIRED Idiopathic Acquired stem cell defects Immune mediated Chemical Agents Dose related- Alkylating agents, Antimetabolites Benzene, Chloramphenicol,Inorganic arsenicals Idiosyncratic- Chloramphenicol, Phenylbutazone, Organic arsenicals, Methylphenylethylhydantoin, Carbamazapine, Penicillamine, Gold salts Physical Agents- Whole-body irradiation Viral Infections INHERITED Fanconi anemia, telomerase defect

Pathophysiology of aplastic anemia:

Morphology:

No hematopoietic element in BM. Have some stromal cells

Parvovirus B19 Pathology:

Test q: A 29F has had malaise and a low-grade fever for the past week. On phys exam, she appears very pale. She has a history of chronic anemia, and spherocytes are observed on a peripheral blood smear. Her hematocrit, which normally ranges from 35% to 38%, is now 28%, and the reticulocyte count is very low. The serum bilirubin level is 0.9 mg/dL. Which of the following events is most likely to have occurred in this patient? Reduced erythropoiesis from parvovirus infection. (Other choices: Development of anti-RBC antibodies, DIC, accelerated extravascular hemolysis in the spleen, and superimposed iron deficiency.)

Giant Pronormoblast Intranuclear Inclusion

Infected Pronormoblast IHC for Viral Capsid Ag

Cause for the figures above can be infection of the stem cells by parvovirus B19.

Malignant Lymphomas WHO Classification of Tumors of Hematopoietic and Lymphoid Tissues: First classification in 2001 2008 classification, October 2008 Lymphomas: Malignant proliferations of lymphoid cells: Mirror stages of B or T cell differentiation Morphology, phenotype, and genetics Are clonal proliferations: one bad cell that keeps producing. Immunoglobulin heavy chain rearrangement Light chain restriction T cell receptor gene rearrangement If these (above) are all rearranged the same way, it is a clonal proliferation. Present as discrete tissue mass Hodgkin Lymphoma vs. non-Hodgkin
Test q: Which of the following lymph node architectures suggests lymphoma: Follicles w/a single cell morphology.

Fri. 10/22/10

Lymph Node Structure:

Lymph node has several parts capsule, subcapsular sinus, etc. Follicles = B cell areas. Follicles have two separate sections mantle zone and germinal center. Surrounding the follicles is the paracortex. Normal lymph nodes: light areas of B cells, darker surrounding areas of T cells. Immunophenotype:
Test q: Paracortical hyperplasia is: histologic changes in lymph node T cells. Test q: In a normal lymph node, the B cell and T cell areas should stain with __ and __ respectively: CD20 and CD3.

B cell marker: CD20 (follicular pattern)

T cell marker: CD3 (surrounding follicles)

WHO classification: Identify specific entities using a multiparametric approach: morphology immunophenotype (CD20 vs CD3) cytogenetics molecular analysis clinical features/presentation (age group is especially important) First requirement: Morphology Low power evaluation (2-4x microscopic): Is the lymph node architecture preserved? Or is it effaced? If it is effaced, what is the pattern of the proliferation? Nodular, diffuse, both

Diffuse:

Nodular:

Diffuse = no nodules.

Nodular look like B cell follicles, but this is actually very highly atypical.

Cellular Morphology: High power evaluation (20 or 40x): Do all the cells look similar? Are there multiple types of cell types? Size: Small, Intermediate, Large Nucleus: Irregular, regular and round Chromatin: Clumped, vesicular, open? Cytoplasm: abundant/scant, color

Above: Can see larger cells w/white in nucleus = vesicular open chromatin pattern. See red objects eosinophils.

Above: Pretty much every cell looks the same. More grayish/purplish nuclei = more coarsely clumped chromatin.

Above: Different sizes of cells predominant cell type is dark purple (represents coarse chromatin pattern). Can see much larger cells w/white areas vesicular open chromatin pattern.

B cells and T cells are hard to distinguish based on morphology. Immunophenotype is important for distinguishing mature B cells from immature B cells. Immature B cells are involved in acute lymphoblastic leukemias. Mature B cells are involved in lymphomas. CD34 and TdT are immature B cell markers. CD20 typically found in mature B cell lymphomas. Big distinguishing factor immunoglobulin on surface. We look at part of the immunoglobulin receptor, known as the light chain, which is seen in lymphomas. Immature cells have not developed surface Ig expression. CD34 and TdT are also markers for immature T cells. TdT+ = immature T OR B cell.
Test q: Mature T-cells are characterized by expressions of: CD4 or CD8.

Evidence of Clonality: B-cell lymphoma: PCR: Immunoglobulin Heavy chain gene rearrangement is the same. IHC: Only one type of immunoglobulin light chain produced: light chain restriction T-cell lymphoma: PCR: T cell receptor gene rearrangement Loss of specific CD antigen Lymphomas: Hodgkin lymphoma B-cell lymphomas T-cell lymphomas CASE: 18 yo male, broke his clavicle during football practice, presents to the ER. X-Ray showed a large mediastinal mass. Chest CT showed a large anterior mediastinal mass. Patient then said he was short of breath more often then usual. Hodgkin Lymphoma: Classical Hodgkin lymphoma (more common) subclassified based on morphology: Nodular sclerosis classical Hodgkin lymphoma Mixed cellularity classical Hodgkin lymphoma Lymphocyte-rich classical Hodgkin lymphoma Lymphocyte-depleted classical Hodgkin lymphoma Nodular lymphocyte predominant Hodgkin lymphoma 18 yo male: Mass was taken out: can see pink dense areas of fibrosis/sclerosis. The lymph nose has a thickened capsule. The sclerosis is separating the lymph node into nodules. Higher power very distinctive cell: 2 nuclei, VERY prominent nucleolus (almost same size as small lymphocytes in the background). Small lymphocytes are also accompanied by small reddish cells (eosinophils). Classical Hodgkin Lymphoma: Clinical features: - Bimodal age of presentation: - 15-35yo and later adult life - 75% of cases involve cervical region - Mediastinal, axillary and paraaortic region - Primarily a nodal based disease: peripheral adenopathy, B symptoms (night sweats) 40%

Test q: An 18M who broke his clavicle during football practice presents to the ER. Chest CT showed a large anterior mediastinal mass. Biopsy of the mass shows lymphoid tissue w/large nodular areas separated by fibrosis. Large cells w/prominent nucleoli are present. Numerous eosinophils and plasma cells are present. Diagnosis? Classic Hodgkin disease.

Modified Ann Arbor Staging: Stage Definition I Single nodal region (spleen, thymus, Waldeyer ring) II 2 or >regions, same side diaphragm. # of anatomic sites should be indicated by suffix (II3) III LN on both sides of diaphragm III1 W/ or w/o splenic, hilar, celiac or portal nodes III2 W/ paraaortic, iliac or mesenteric nodes. IV Extranodal sites: BM, liver and > 1 single extranodal site
Test q: A 22M has been diagnosed w/classical Hodgkin Disease. Workup reveals involvement of several lymph nodes on both sides of the diaphragm. There is no history of fever or weight loss. Bone marrow and liver are not involved. The stage is? III-A. Test q: A 33F reports having generalized fatigue and night sweats for 3 months. Phys exam shows nontender right cervical lymphadenopathy. Biopsy of one lymph node shows a microscopic pattern of thick bands of fibrous connective tissue w/intervening lymphocytes, plasma cells, eosinophils, macrophages, and occasional Reed-Sternberg cells. An abdominal CT scan and bone marrow biopsy specimen show no abnormalities. Which of the following is the most likely subtype and stage of this patients disease? Nodular sclerosis, stage IB.

Nodular sclerosis classical Hodgkin lymphoma (NS HL) ~70% of classical HL in Europe and USA Higher risk in high socioeconomic status Peak age is 15-34yo Clinical features: Mediastinal involvement in 80% Most present with stage II disease Histology: Nodular growth pattern surrounded by collagen bands
Test q: An enlarged lymph node is removed from the right neck of a 24y/o male w/fever and night sweats. H&E stained sections show a nodular appearance. Cellular areas alternate w/thick fibrous bands. The cellular areas contain plasma cells, eosinophils, small lymphocytes, and large cells w/prominent nucleoli which stain positive for CD30. Lacunar cells are identified. Diagnosis: Classical Hodgkins Disease (Other choices: Follicular lymphoma, Small lymphocytic lymphoma, Non-classical Hodgkins Disease, Benign reactive lymph node.)

HRS (Hodgkin, Reed-Sternberg) cells: Large, pleomorphic, owls eye look, inucleation. Characteristic milieu: eosinophils, plasma cells, small lymphocytes and histiocytes Modified cells: lacunar/mummified variant cells

Immunophenotype:

Reed-Sternberg/Hodgkins cells. HRS cells: Positive: CD15, CD30, focal/weak: CD20 Negative: CD3

CD15

CD30

CD30, CD15 stains membrane, sometimes Golgi apparatus. All Hodgkin, Reed-Sternberg cells are + for CD30 not all for 15, but a majority of them are.

Test q: A 45M has experienced recurrent fevers and a 6kg weight loss over the past 5 months. On phys exam, his temp is 37.5C, and he has cervical lymphadenopathy. A lymph node biopsy specimen shows effacement of the nodal architecture by fibrosis and a population of small lymphocytes, plasma cells, eosinophils, and macrophages. Which of the following additional cell types, which stain positively for CD15, is most likely to be found in this disease? Reed-Sternberg cell. (Other choices: immunoblast, epithelioid cell, neutrophils, and mast cell)

CASE: 65 yo male presents to his PCP with weakness and generalized lymphadenopathy. Exam: Hepatosplenomegaly. CBC: Lymphocytosis composed of small-intermediate sized mature lymphocytes

B-cell lymphomas: Comprise over 90% of lymphoid neoplasms worldwide. Incidence rate per 100,000 is 26.13 and is increasing. Lymphomas involving mature B cells: Negative for CD34 and TdT Recapitulate stages of B cell development

Three main stages of B cell maturation: earlier stage involves cells that have not yet reached the follicle, one stage involves the follicle, late stage involves cells that have found their antigen and are making antibodies/becoming memory cells.

Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL): Is a CHRONIC leukemia mature leukemia cell type. (Acute leukemias have immature cells.) Most common leukemia of adults in western countries, incidence rate is 12.8/100,000 at age 65yo. Increasing incidence in younger pts. Clinical features: Peripheral blood and bone marrow usually involved Lymph nodes, liver and spleen Many are asymptomatic, fatigue, anemia Much different than Hodgkin lymphoma monotonous population of small lymphocytes w/dark nuclei (coarsely clumped chromatin pattern)
Test q: A 65M has lymphadenopathy and a biopsy shows small lymphocytes w/rare mitoses and dense, regular nuclei. There are no follicles present. Diagnosis? Small Lymphocytic Lymphoma.

Diffuse infiltrate but vaguely nodular CLL/SLL immunophenotype: Flow cytometry

Use flow cytometry to look at antigenic expression of lymphoid cells look w/graph analysis. Can tell there is an aberrant B cell proliferation expressing CD5, negative for T cell marker CD3. CD20 is here dimly positive. 22 is another B cell marker. CD23 is positive.

Positive: CD20, CD5 *, CD23* Negative: CD3, cyclin D1

Test q: Small lymphocytic lymphoma is associated with: Clonal proliferation of mature CD5+ B-cells.

More CLL/SLL Flow Cytometry:

CLL/SLL: Prognosis: Genetics, IGH (immunoglobulin heavy chain rearrangement), Immunophenotype 2-8% Diffuse large B-cell lymphoma (Richters syndrome/transformation very aggressive) Median survival is < 1 year

CLL/SLL involve mature B cells, so they should express surface light chain Ig on these cells. Looking at lambda vs kappa light chain, all of these cells are kappa light chain positive (which is atypical). Should see a mixture of both.

Follicular lymphoma: Lymphoma composed of follicle center (germinal center) cells: centroblasts and centrocytes (B cells) Pattern recapitulates lymphoid follicles. 20% of all lymphomas, highest incidence in USA and western Europe th Median age in 6 decade (same as CLL) Clinical: Widespread disease at diagnosis: Peripheral and central lymphadenopathy Splenomegaly BM 40-70% Only 1/3 are Stage I or II at dx. Figure: Low power distinctly nodular infiltrate based on color differences. Grading depends on morphology: High power view is important determines grading. Low grade/indolent will have more centrocyte (raisin) cells crinkly, cleaved. Grade 3 more aggressive mostly centroblasts (large, prominent nucleoli). Greater than 15 centroblasts in view = Grade 3. Follicular Lymphoma: Immunophenotype: Positive: CD20, CD10, bcl-2 bcl-2 seen here because there is a specific genetic aberration translocation between 14;18 (involves BCL2 anti-apoptotic gene). Get increase in bcl-2 protein that normally would not be there cells do not die. Negative: CD5, CD3 Genetics: t(14;18): BCL2, IGH (immunoglobulin heavy chain locus) Grade 1,2: Indolent, usually not curable Grade 3: More aggressive but curable by high-dose chemotherapy agents.
Test q: A 40F presents w/generalized lymphadenopathy. A cervical lymph node is biopsied and shows a follicular architecture. Germinal centers w/macrophages are not present. The lymph node is positive for CD20, CD10, and bcl-2. The expected translocation is: t(14;18). Test q: A t(14;18) translocation in lymphoid malignancies causes the activation of which gene? BCL-2. Test q: A 40y/o man notices an increasing number of lumps in his groin and armpits. On physical exam, he has generalized nontender lymph node enlargement and hepatosplenomegaly. An inguinal node biopsy shows a malignant tumor of lymphoid cells. Immunoperoxidase staining of the tumor cells w/antibody to BCL2 is positive in the lymphocytic cell nuclei. Which of the following mechanisms has most likely produced the lymphoma? Inhibition of apoptosis. REPEATED x2 (once, was lack of apoptosis) Test q: Follicular lymphoma is often associated with: presence of t(14;18) chromosome translocation. Test q: Characteristics of B cell Follicular Lymphoma include: BCL-6 overexpression. (Other choices: t(8;14), dysregulated c-MYC expression, increased MYC/MAX, and T(11;18)). 2007, #99 Should the answer be BCL-2 overexpression?

CASE: 12 yo female w/ h/o non-specific abdominal pain and distention for ~1 week presents to ER. Abdominal imaging large ileo-cecal mass. Burkitt Lymphoma: another germinal center-type lymphoma Endemic BL: Equitorial Africa, most common childhood malignancy 4-7 yo, EBV+ in majority Sporadic BL: Throughout the world, children/young adults 30-50% of all childhood lymphomas. Median age of adult 30yo Immunodeficiency BL: HIV infection, often is initial manifestation of AIDS. Clinical presentation: All at risk for CNS involvement Endemic BL: 50%: Jaws, facial bones Ovaries, kidneys and breast: frequently Sporadic BL: Majority: Abdominal masses, ileo-cecal region. Ovaries, kidneys and breast: frequent Rarely Jaw Immunodeficiency-associated BL: Nodal localization and BM is frequent

Figure: Atypical lymphoid cells, intermediate size. See white, punched-out areas = TINGIBLE BODY MACROPHAGES. Looks like starry sky. Punchedout areas are macrophages engulfing lymphoid cell debris because this is highly proliferative.
Test q: A 12F presents w/an abdominal mass. CT imaging shows a large mass at the ileo-cecal junction. The mass is excised and is positive for CD20, CD10, and bcl-6. Bcl2 is negative. On histology, you would expect to see: Tingible body macrophages surrounded by atypical lymphocytes.

Burkitt Lymphoma: Immunophenotype: Positive: CD20, CD10, bcl-6, 100% proliferation rate (every cell is proliferative). CD10, bcl-6 are germinal center markers. Negative: bcl-2 (follicular lymphoma marker BIG distinguishing factor) Genetics: t(8;14) MYC, IGH t(8;22) MYC , lambda lc t(8;2) MYC, kappa lc Prognosis: 90% cure low stage, 60-80% - high stage Burkitts will ALWAYS and ONLY have the MYC gene (oncogene). Can be translocated on B cell receptor either the heavy chain on 8;14 or one of the light chain genes (8;22 or 8;2).
Test q: Characteristics of Burkitt Lymphoma include: t(8;14). Test q: A 12M is taken to the physician because he has had increasing abdominal distention and pain for the past 3 days. An abdominal CT scan shows a 7cm mass involving the region of the ileocecal valve. Histologic exam of the mass shows sheets of intermediate-sized lymphoid cells, w/nuclei having coarse chromatin, several nucleoli, and many mitoses. Cytogen analysis of the cells from the mass shows a t(8;14) karyotype. Which of the following is the most likely diagnosis? Burkitt lymphoma. Test q: Burkitt lymphoma is: bcl-2-; bcl-6+. Test q: Which of the following tumors is involved in the overexpression of c-myc due to chromosomal translocation (Chromosome 8 to 14)? Burkitts Lymphoma. Test q: Prognosis in Hodgkin Disease is best predicted by: Stage. Test q: Prognosis in Hodgkin Disease can be described as: Stage is important but grade is not.

Diffuse Large B cell Lymphoma: Most common lymphoma, 25-30% of adult non-Hodgkin lymphoma in western countries. Heterogenous group of lymphomas (can look different) which are nodal and extranodal based Morphology: Large pleomorphic cells which have a diffuse infiltrative pattern Immunophenotype: + CD20, +/- CD10, +/-bcl-2, +/- bcl-6
Test q: In the US, the majority of non-Hodgkin lymphomas in adults are derived from: B lymphocytes. Test q: A 60M has experienced vague abdominal discomfort accompanied by bloating and diarrhea for the past 6 months. On phys exam, there is a midabdominal firm mass. The stool is positive for occult blood. An abdominal CT scan shows a 5x12cm mass involving the wall of the distal ileum and adjacent mesentery. A laparotomy is performed, and the mass is removed. Microscopically, the mass is composed of sheets of large lymphoid cells w/large nuclei, prominent nucleoli, and frequent mitoses. The neoplastic cells mark w/CD19+ and CD20+ and have the BCL6 gene rearrangement. Which of the following prognostic features is most applicable to this case? Aggressive disease that can be cured by aggressive chemotherapy. (Other choices: Indolent disease w/survival of 7-9yr w/o treatment; Aggressive disease that does not respond to chemotherapy and transforms to acute leukemia; Indolent disease that can be cured by chemotherapy; Indolent disease that often undergoes spontaneous remission.) Robbins explanation: This patient has the clinical and morphologic features of diffuse large-cell lymphoma of B cells. These tumors often involve extranodal sites, show large anaplastic lymphoid cells that involve the tissues diffusely, and contain BCL6 gene arrangements. These clinical course is aggressive, and they become rapidly fatal if untreated. Test q: A 37M known to have been infected w/HIV for the past 10yr is admitted to the hospital w/abdominal pain of 3 days duration. Phys exam shows abdominal distention and absent bowel sounds. An abdominal CT scan shows a mass lesion involving the ileum. He undergoes surgery to remove an area of bowel obstruction in the ileum. Gross exam of the specimen shows a firm, white mass 10cm long and 3cm at its greatest depth. The mass has infiltrated through the wall of the ileum. Histologic studies show a mitotically active population of CD19+ lymphoid cells w/prominent nuclei and nucleoli. Molecular analysis is most likely to show which of the following viral genomes in the lymphoid cells? Epstein-Barr virus. (Other choices: HIV, HHV-8, HTLV-1, Cytomegalovirus) Robbins: This HIV-positive patient has an extranodal infiltrative mass, composed of B cells (CD19+) in the ileum. This is a diffuse large cell lymphoma of B cells. These tumors contain the Epstein-Barr virus (EBV) genome, and it is thought that immunosuppression allows unregulated proliferation and neoplastic transformation of EBV-infected B cells.

See very blue-staining cells can even see on low power. Pink area at top = necrosis.

Large cells, nucleoli, lighter-staining chromatin pattern, some of them are very large and pleomorphic (but not ReedSternberg cells). Very atypical cells.

T cell lymphoma: Mature T cell (so no CD34 or TdT) which loses normal antigen expression and shows a positive T-cell receptor gene rearrangement. Peripheral T cell lymphoma, NOS ~30% of T cell lymphomas in western countries. Adults, LAN and B symptoms Anaplastic large cell lymphoma, ALK-positive: D/Dx of Hodgkin lymphoma 10-20% of childhood lymphomas. Involves LN and extranodal sites, mediastinal disease is less frequent then HL. 70% present with stage III-IV disease, B symptoms Morphology: Variable, Hallmark cells (comma-shaped but hard to find) Immuno: ALK+, CD30+, EMA+; CD2, CD5, CD4 are positive 70% CD3 is negative in 75% Genetics: also involves translocation t(2;5) ALK , nucleophosmin 84% Overall 5 yr survival rate ~80% (in kids, lower in adults) All cells are 30+, as opposed to Hodgkins, where scattered large cells will be 30+ and the background reactive cells will be negative. CD2, 5 = T cell markers, but it loses CD3 in most cases.
Both figures above: Look like diffuse large B cannot differentiate based on morphology. Have large cells, prominent nucleoli, intermediate-size cells, mitotic figures, open chromatin.

Adult T-cell leukemia/lymphoma: MATURE T cell neoplasm. Is caused by the Human T-cell Leukemia virus type 1 (HTLV-1) human retrovirus Long latency, Japan incidence is 2.5% of HTLV-1 carriers. Occurs only in adults. Central Africa, Caribbean, Southwestern Japan.
Test q: A 25F immigrant from Japan develops lymphadenopathy. Lymph node biopsy shows features consistent with T cell lymphoma. It is likely she is infect with: HTLV-1.

Figure: HTLV-1. Nuclei are lobulated flower cells

Test q: A 51M visits his physician because the skin of his face, neck, and trunk has become scaly red. He also complains of intense itching and a 3kg weight loss over the past 2 months. On phys exam, his temp is 37.6C and he has a generalized exfoliative erythroderma. There is generalized 3 3 nontender lymphadenopathy. Lab studies shows Hgb 12.9 g/dL, hematocrit 42.0%, platelet count 23,000/mm , and WBC count 7940/mm with 57% segmented neutrophils, 3% bands, 26% lymphocytes, 5% monocytes, and 9% eosinophils. A skin biopsy specimen shows the presence of lymphoid cells in the upper dermis and epidermis. These cells have cerebriform nuclei w/marked infolding of nuclear membranes. Similar cells are seen on the peripheral blood smear. Which combo of the following phenotypic markers is most likely to be expressed on his abnormal lymphocytes? CD3+, CD4+. REPEATED x2 From Robbins: Cutaneous T cell lymphomas: The involvement of skin and the presence of lymphocytes w/complex cerebriform nuclei in the skin and the color are features of cutaneous T-cell lymphomas. These are malignancies of CD4+ and CD3+ T cells that may produce a tumor-like infiltration of the skin (mycosis fungoides) or a leukemic picture w/o tumefaction in the skin (Sezary syndrome). Cutaneous T-cell lymphomas are indolent tumors, and patients have a median survival of 8-9 years. Test q: A 53F has experienced nausea w/vomiting and early satiety for the past 7 months. On phys exam, she is afebrile and has no lymphadenopathy 3 3 or hepatospelnomegaly. CBC shows Hgb 12.9 g/dL, hematocrit 41.9%, platelet count 263,000/mm , and WBC count 8430/mm . An upper GI endoscopy shows loss of the rugal folds of the stomach over a 4x8 cm area of the fundus. Gastric biopsy specimens reveal the presence of Helicobacter pylori organisms in the mucus overlying superficial epithelial cells. There are mucosal and submucosal monomorphous infiltrates of small lymphocytes, which are CD19+ and CD20+ but CD3-. After treatment of the H. pylori infection, her condition improves. What is the most likely diagnosis? MALT (marginal zone) lymphoma. Robbins: These lymphomas arise in middle-aged adults at sites of autoimmune or infectious stimulation. If the lesion is associated w/lymphoid tissue, it is sometimes called a mucosa-associated lymphoid tissue tumor (MALT lymphoma, or MALToma). The most common sites are the thyroid (in Hashimoto thyroiditis), the salivary glands (in Sjogren syndrome), or the stomach (in H. pylori infection). Although monoclonal (similar to a neoplasm), these MALT lesions can regress w/antibiotic therapy for H. pylori. A MALT lesion can transform to diffuse large B-cell lymphoma. The cells correspond to the marginal B-cells found at the periphery of stimulated lymphoid follicles.

Post-Transplant Lymphoproliferative D/O: Lymphoid or plasmacytic proliferations that develop as a consequence of immunosuppression in a post transplant setting: solid organ, bone marrow or stem cell allograft Incidence: Kidney 1% Liver 2% Heart 1.8-9.8% Lung 4.6-9.4% BMT 0.6-24% Early lesions: in early development of lymphoproliferation, not in early time after transplant. Infectious Mononucleosis (IM)-like Plasmacytic hyperplasia lots of plasma cells, but are polyclonal. Polymorphic PTLD Effaced architecture Lots of different cell types: Immunoblasts, plasma cells and lymphocytes IGH clonal rearrangement, +/- EBV Monomorphic PTLD (= lymphoma) B or T cell lymphoma +/- EBV (If a patient has a transplant and develops PTLD within 3 years, is probably EBV+. If 5+ years, will probably be EBV-.
Test q: Post-transplant lymphoproliferative disease is most commonly associated with infection by: EBV. Test q: Polymorphic post-transplant lymphoproliferative disorders are frequently: EBV positive.

Leukemia

Fri. 10/22/10

Leukemias: Clonal expansion of hematopoietic cells Originate in bone marrow and involve blood Test q: All acute leukemias originate in the: bone marrow. Depending on clinical presentation leukemias are divided into: o Chronic and acute Depending on the lineage of origin, leukemias are divided into: o Lymphoid and myeloid Leukemias are further classified according to cell of origin, morphology, immunophenotype, molecular features, clinical behavior and prognosis

Leukemias: Myeloid leukemias: Acute Chronic myeloid neoplasms

Lymphoid leukemias: Acute Chronic

Myeloid neoplasms: Involve bone marrow and secondary hematopoietic organs (spleen, liver) Broadly subdivided into: o Acute (acute myeloid leukemias) o Chronic: myelodysplastic syndromes (manifesting as cytopenias) chronic myeloproliferative disorders (manifesting as increased numbers of cells in blood) Both acute and chronic myeloid leukemias arise as a result of transforming events at the level of hematopoietic stem/progenitor cells, which affect hematopoietic differentiation and proliferation in a manner specific for each individual entity Acute myeloid leukemias (AML): Most common form of acute leukemia in adults displacement/suppression of normal hematopoiesis Symptoms are related to the accumulation of immature myeloid cells due to aberrant differentiation Presentation: Within weeks to months of onset (acute onset) Fatigue, fever and mucocutaneous bleeding related to anemia, neutropenia and thrombocytopenia These symptoms are related to the replacement of normal bone marrow cells by leukemic blasts
Test q: A 35F presents w/fatigue, fever, anemia, thrombocytopenia, and mucocutaneous bleeding. You suspect: Acute leukemia.

Classification of acute myeloid leukemias: Past and present Past: French-American-British (FAB) classification Present: 2008 WHO classification o Classification according to morphologically defined o Classification according to the stage of stage of differentiation and hematopoietic lineage differentiation, hematopoietic lineage and genetics o Based on morphology and aided by enzyme o Based on morphology, flow cytometry, cytogenetics cytochemistry and molecular studies Acute myeloid leukemia and cytochemical stains o Myeloperoxidase and non-specific esterase are the most commonly used cytochemical stains o Myeloperoxidase is seen in granulocytic lineage and leukemias with myeloid blasts and granulocytic differentiation (red staining) o Nonspecific esterases are seen in monocytic cells (acute myelomonocytic and monocytic leukemia)
Test q: The cytochemical stain non-specific esterase may be positive in: Acute myeloid leukemia.

WHO retains AML subtypes from original FAB classification: (20% blasts required for the diagnosis) Type of leukemia
M0 M1 M2 M3 M4 M5 M6 M7 Minimally differentiated AML AML without maturation AML with maturation Acute promyelocytic leukemia AML with (15;17) [RARa/PML fusion gene] Acute myelomonocytic leukemia Acute monocytic leukemia Acute erythroleukemia Acute megakaryocytic leukemia

Myeloperoxidase

Normal marrow counterpart

Myeloblast Myeloblast Myeloblast Promyelocyte Myeloblast and monocytic precursors Monocyte and its precursors Erythroid precursors Megakaryocytic precursors

Select AML categories added in WHO classification: Do not memorize, for reference purposes (Note: this material has appeared in test qs some of it was mentioned in the heme preview lecture.)
Subtype AML with t(8;21) AML with inversion of chromosome 16 AML with t(15;17) Acute Promyelocytic Leuk. AML with involvement of chromosome 11q23 AML with deletions of chromosome 7 and 5 Comment favorable prognosis with intensified treatment regimen favorable prognosis with intensified regimens formerly AML M3, treated with retinoic acid, good prognosis includes some therapy related leukemias, intermediate prognosis therapy related, preceded by myelodysplastic syndrome adverse outcome

Test q: A diagnosis of leukemia has been made on a 23M. A bone marrow aspiration specimenwas sent to the cytogenetics lab. Culture cells showed a karyotype w/t(15;17) (q21;21). This is most consistent with: Acute promyelocytic leukemia (FAB, M3). Test q: A 35F presents w/acute leukemia. She exhibits microangiopathic hemolytic anemia and a translocation. She is successfully treated w/retinoic acid. Diagnosis? AML (M3). Test q: A 45M presents w/many blasts in the peripheral blood. The patient develops DIC rapidly but was successfully treated w/retinoic acid. The FAB classification for this AML is: M3.

Acute myeloid leukemias (2008 WHO classification) Do not memorize, for reference purposes Red box = FAB classification

Diagnostic work-up of patient with suspected acute leukemia: 1. Review of clinical history, CBC, differential count and peripheral blood smear: o 47 year-old male o No prior medical history o Routine CBC: WBC 3.3 K/ul hemoglobin 8.4 g/dL platelet count 49 K/ul Thrombocytopenia differential count: 7% blasts o No organomegaly 2. Bone marrow exam (biopsy)

Normal bone marrow

Acute myeloid leukemia