PERIPHERAL SMEARS: THE PRIMARY DIAGNOSTIC TOOL PART II IRMA PEREIRA, MT(ASCP)SH

PERIPHERAL SMEARS: THE PRIMARY DIAGNOSTIC TOOL PART II IRMA PEREIRA, MT(ASCP)SH WORKSHOPS FOR LABORATORY PROFESSIONALS

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5783 Peripheral Smears: The Primary Diagnostic Tool Part II Course Director: Irma Pereira, MT(ASCP)SH Clinical Hematology Specialist and Consultant, Clinical Hematology Adjunct Lecturer in Hematology, Department of Medicine, Stanford University Hospital, Stanford, CA Although this workshop is a continuation of Part I (presented on May 14), the workshops may be taken individually. Improve your diagnostic ability that is crucial to properly evaluating a patient s hematologic status, by attending this intermediate-level workshop, geared to increase your confidence and productivity in the hematology laboratory! In Part II, more than four hundred images will be used to demonstrate techniques for the peripheral smear diagnosis of hematologic disorders. In addition, differential diagnosis exercises, using only peripheral smear findings, will challenge your knowledge and reinforce the information presented. During Part II, you will hear thought provoking discussions on: the WHO classification of lymphoid leukemia/ lymphoma and its rare leukemic variants, including detailed explanations of the morphological differentiation of B-cell and T-cell leukemia/lymphoma disorders peripheralized mature B-cell neoplasms, such as: CLL, SLL/PLL, mantle cell (splenic and nodal), Marginal, Burkitt, myeloma (secretory and nonsecretory), Waldenstrom, hairy cell, follicular aggressive transformation of CLL (Richer Syndrome) Sezary Syndrome reactive lymphocytosis vs. lymphoma miscellaneous disorders, such as the Chediak-Higashi Syndrome and certain mucopolysaccharide disorders intracellular organisms You also will learn techniques for peripheral smear differential diagnosis of microcytic anemias, macrocytic anemias (megaloblastic, megaloblastoid, macronormoblastic), and the extracorpuscular and intracorpuscular hemolytic anemias. Following this workshop, you will be able to: Apply improved morphologic skills to more accurate diagnoses of hematologic problems Assist non-hematologist physicians in understanding patient disorders Alert physicians to unknown hematologic situations Discuss patient diagnoses at a higher technical level

PERIPHERAL SMEARS The Primary Diagnostic Tool The Lymphoproliferative Disorders Irma Pereira, MT(ASCP)SH Clinical Hematology Specialist and Consultant Stanford University Hospital The Reactive Lymphs Lack of homogeneity Range in age from immunoblast to mature reactive lymph Found primarily in acute viral infections May be associated with autoimmune hemolytic anemia or thrombocytopenia Usually seen as activated B-Cells or LGLs for activated NK cells 1

The Reactive Lymphs May be associated with autoimmune hemolytic anemia or thrombocytopenia Usually seen as activated B-Cells or LGLs for activated NK cells Age is not a factor in the production of reactive lymphocytes, although neonates <3 months do not produce their own antibodies, and very aged people have a more difficult time. Infectious Mononucleosis or Other Viral Infections 2

Large Granular Lymphs (LGLs) Mott Cell 3

Pertussis The one exception to the heterogeneity rule in reactive lymphocytosis is Pertussis. Children present with high WBCs and a severe lymphocytosis. Characteristics are often a monotonous picture of cleaved small lymphocytes. Pertussis 4

WHO Histological Classification of Precurser B-cell and T-cell Neoplasms Precursor B lymphoblastic leukemia/lymphoblastic lymphoma Precursor T lymphoblastic leukemia/lymphoblastic lymphoma Malignant Lymphocytic Disorders Majority of Lymphocytic neoplasms are of B-Cell type. Represent about 85% of known lymphoid disorders. Other 2 types are T-Cell, Natural Killer, and unknowns The different types of B-Cell leukemia and lymphoma apparently occurs in the B-Cell life cycle. T-cell origination of malignancy is still in question. Many are associated with specific genetic gene translocations. 5

Stem Cell B-Cell Disorders by Cell Development ALL Lymphoblastic Lymphoma/ Leukemia Normal Malignant NORMAL MATURATION OF B-Cells Precursor Immature B-Cell B-Lymphoblast MALIGNANT PATH OF B-CELLS Pre B-Cell Leukemia/ Lymphoma Activated B-Cell Plasmacytoid Lymphocyte Myeloma and Waldenstrom s Burkitt s B-Immunoblast Mantle Cell Follicular Lymphomas Large Cell Lymphomas Marginal Zone & Monocytoid B-Cell Normal Malignant Naive B Cell CLL, Small Lymphocytic lymphoma A N T I G E N Clinical and laboratory features at diagnosis associated with outcome of children with ALL Age at diagnosis: The younger the worse prognosis. The risk of treatment failure being greatest for young infants (< 18 months) especially with t(4;11) translocation Older children and teenagers (>/= 10 years) have a poorer prognosis than young children Patients with higher WBC counts at diagnosis have a higher risk for treatment failure than do patients with lower WBC counts. The majority of children with recurrent ALL attain second remission, but the likelihood of cure is generally poor, particularly for those with bone marrow relapse following a short initial remission duration. 6

Clinical and laboratory features at diagnosis associated with outcome of children with ALL con t Approximately 80% of children with ALL have expression of CD19, HLA-DR, CD10 (calla - common acute lymphocytic leukemia antigen). The lack of calla expression has also been shown in some series to be associated with a worse prognosis. Infants less than 18 months usually have a poor prognosis, due to higher rates of CNS involvement, higher leukocytosis, more hepatosplenomegaly, shorter remissions, and more severe thrombocytopenia than their older counterparts. Infants are usually calla negative and have an absence of cytoplasmic or surface immunoglobulins Clinical and laboratory features at diagnosis associated with outcome of children with ALL..con t Girls have a better prognosis than boys. The probable reason is, boys often get testicular relapse. They also tend to get more bone marrow relapse than girls. Reason unknown. Young boys have a higher probability of presenting, clinically with a mediastinal mass, being of slightly older age, having a higher leukocytosis than girls, and typing as T-cell ALL. T- Cell ALL has expression of associated antigens CD2, CD7, CD5, or CD3. Prognosis, with appropriate chemotherapy, is much closer to that of the B-Cell ALLs. Race: Black children have a lower survival rate than white children. Reason unknown. FROM Smith MA, Ries LA, Gurney JG, et al.: Leukemia. In: Ries LA, Smith MA, Gurney JG, et al., eds.: Cancer Incidence and Survival Among Children and Adolescents: United States SEER Program 1975-1995. Bethesda, MD: National Cancer Institute, SEER Program, NIH Pub.No. 99-4649, 1999, pp 17-34. 7

General Concerns of ALL In all the acute lymphocytic leukemia/lymphomas, marked thrombocytopenia and a normocytic normochromic anemia are expected. WBCs range from marked leukocytopenia to a marked leukocytosis. Chromosomal translocations of any type, especially t (4:11), have a very poor prognosis. The incidence of ALL among children ages 2 to 3 years is approximately fourfold greater than that for infants and is nearly 10- fold greater than that for children who are 19 years old FAB Classification of ALL The FAB lymphocytic leukemias classified solely by morphology. No prognostic difference using this limited classification L1 and L2 ALLs are no longer separated as distinct leukemia types. L1 often has the cytogenetic translocations of t(10;14); t(11;14); t(9;22). 8

FAB Classification of ALL con t FAB L3 morphology is morphologically and cytogenetically the same as Burkitt's lymphoma Immunophenotyping results are now a criteria for classification, but morphology still plays an important role. Although, the morphological FAB classification is no longer used,we will describe the morphological features used in the past for differentiation, simply as a lesson on the morphological variations seen in precurser B-cell and T-cell leukemias. Precurser B-Cell Lymphoblastic Leukemia/Lymphoma (L1 and L2) Composed of medium sized blasts, high N/C ratio, usually round, coarser chromatin than myeloblasts and 1 or more distinct or indistinct nucleoli. Some of these cells may contain coarse azurophilic granules If cells have extensive involvement with bone marrow and blood, its called lymphoblastic leukemia If there is primary involvement with extranodal or nodal sites, then it is called lymphoblastic lymphoma, i.e. confined to a mass lesion with little to no bone marrow (< 25%) or blood involved 9

Pre B-Cell ALL (L1) Pre B-Cell ALL (L1) 10

GRANULAR PRE-B ALL 11

T-Cell ALL (L1) T-Cell ALL 12

Burkitt s (L3) Bi Lineal Leukemia 2 separate cell lines present consisting of lymphoblasts and myeloblasts Difficult to treat. Must be treated with therapy for both ALL and AML Can be composed of myeloblasts and either T or B cell lymphoblasts Prognosis is poor 13

Bi Lineal Leukemia T-Cell Lymphoblastic Leukemia/Lymphoma (L1) Small to medium (in some cases, large) cells with a very high N/C ratio, condensed, coarse chromatin, inconspicuous nucleoli, and usually with irregular nuclear borders Small to intermediate cells have the indistinct nucleoli. The large cells may have distinct nucleoli Presents with leukocytosis, anemia and thrombocytopenia, and often a mediastinal mass In children, now, the prognosis is similar to its B-cell counterparts 14

The Lymphomas 3 major categories of lymphomas Non Hodgkin's Lymphoma - B-Cell malignancies Non Hodgkin's Lymphoma - T and NK Cell malignancies Hodgkin's Lymphoma (not to be covered in this hematology peripheral blood session) 15

Mature B-Cell Neoplasms to be covered CLL/small cell lymphocytic lymphoma and mixed CLL B-Cell prolymphocytic leukemia (transition from CLL. Richter s transformation of CLL). DeNovo PLL Multiple Myeloma and Plasma Cell Leukemia Waldenstrom s Macroglobulanemia Hairy Cell Leukemia Splenic Marginal Zone (Splenic lymphoma with villous lymphocytes). Nodal Marginal Zone Lymphoma Follicular Lymphoma Mantle Cell Lymphoma Diffuse Large B-Cell Lymphoma Blastic Large B-Cell Lymphoma Immunoblastic Large B-Cell Lymphoma Burkitt s Lymphoma Mature B-Cell Neoplasms to be Covered ALL TdT NEGATIVE CLL/small cell lymphocytic lymphoma (SLL) and mixed CLL B-Cell prolymphocytic leukemia (transition from CLL) and Richter transformation of CLL) DeNovo PLL Multiple Myeloma and Plasma Cell Leukemia Waldenstrom s Macroglobulanemia Hairy Cell Leukemia Splenic Marginal Zone (Splenic lymphoma with villous lymphocytes). Nodal Marginal Zone Lymphoma Follicular Lymphoma Mantle Cell Lymphoma Diffuse Large B-Cell Lymphoma Blastic Large B-Cell Lymphoma Immunoblastic Large B-Cell Lymphoma Burkitt s Lymphoma Diffuse Large B-Cell Lymphomas ( DLBCL) 16

CLL/SCL CLL/SCL, the most common of the chronic lymphoproliferative disorders, is a B-cell leukemia. White blood counts can range from normal to greater than 300 K/µL. Patients are usually >55 years of age, (app. 40% are < 60 yrs.) with survival of a few weeks to over 20 years. CLL/SCL is defined as an accumulation and slow proliferation of normal appearing immunologically incompetent lymphocytes. Lymphocyte counts in the blood are usually > 5,000/mm3 with coexpressions of the B-cell antigens CD19, CD20 and CD23, along with the T-cell antigen CD5 Selected patients treated with allogeneic stem cell transplantation have achieved prolonged disease-free survival CLL/SLL con t Lymphocytes tend to have a coarse blocky chromatin pattern. The most common cytogenetic abnormality in CLL is a trisomy 12, although 14q+ or t(11;14)has been noted. Majority of patients with the 14q+, however, tend to transform into a prolymphocytic or Richter phase Patients with CLL who show prolymphocytic transformation maintain the classic CLL phenotype and have a worse prognosis than PLL patients 17

RAI Clinical Staging of CLL LOW RISK Median Survival (years) Stage 0: Absolute lymphocyte count in blood >15 >15 x 109/L, and >40% in the marrow with no organomegaly or lymphadenopathy INTERMEDIATE RISK Stage 1: Absolute lymphocytosis plus 9 enlarged lymph nodes. Usually no organomegaly Stage 2: Absolute lymphocytosis plus enlarged 5 liver and/or spleen HIGH RISK Stage 3: Absolute lymphocytosis plus anemia 2 (Hgb < 11 g/dl or PCV < 33% Stage 4: Absolute lymphocytosis plus 2 thrombocytopenia, (< 100 x 10 /L) regardless of the number of organs involved CLL/Small cell lymphocytic lymphoma 18

Mixed CLL CLL is often found with a mixed cellular phase of this disease, i.e. larger lymphocytes, with a more open chromatin pattern and indistinct nucleoli Important to distinguish between these larger lymphocytes and prolymphocytes Important not to accept smudge cells as part of a report, in case they are prolymphocytes. A 1:12 dilution, 22% albumin to blood is recommended to prevent smudging >10% prolymphocytes in CLL has a poorer prognosis than patients with <10% CLL-Mixed Cellularity 19

Prolymphocytic Transition of CLL CLL/PLL Prolymphocytic leukemia transformations start with a gradual onset of cytopenias with increasing lymphocytosis and >20 but <55% prolymphocytes. Lymphadenopathy and splenomegaly is also progressive along with a decreased responsiveness to therapy Median survival is about 1 year for stage III and IV Other types of transformations in CLL include the Richter syndrome, a blastic type transformation (in < 2% of patients), usually seen in tissues, and a plasmacytoid transformation, in less than 1% of cases Prolymphocytic Leukemia de novo Primarily affects older men Usually fail with standard CLL chemotherapy, however, Cladribine (2- chlorodeoxyadenosine) has a 60% complete remission rate for patients with de novo B-cell prolymphocytic leukemia ** Prolymphocytic leukemia (PLL) is rare with a short survival time if therapy fails Present with massive splenomegaly and little if any lymphadenopathy ** Adapted from NCI (PDQ) on Prolymphocytic Leukemia 20

Prolymphocytic Leukemia con t Phenotype is positive for CD19, CD20, and surfacemembrane immunoglobulin and negative for CD5 B-cell types have the cytogenetic aberrations of 14q ; t(11;14) 6q ; t(6;12) and frequent karyotypic evolutions Peripheral blood shows a marked leukocytosis with >55% prolymphocytes and thrombocytopenia Prolymphocytes are intermediate in size with an immature chromatin pattern and 1 distinct nucleolus. Nucleus tends to be central with a small to moderately high N/C ratio CLL/PLL 21

Acute Prolymphocytic Leukemia Richter Transformation of CLL Approximately 3-19% of patients with CLL will develop an aggressive phase of their disease Progression may be preceded by an intermediate prolymphocytic stage, which then progresses to the terminal phase of a large cell lymphoma picture Progression is usually a direct one, preceded by an abrupt onset of fever, weight loss, increasing organomegaly and lymphandenopathy Large tumor masses with GI tract involvement are common. Normally this phase is a tissue phase, and not seen n the peripheral blood, however, there are times when small numbers of these cells may circulate 22

Richter Transformation of CLL Cells are of either the large cleaved or convoluted type with a high N/C ratio and varying number of nucleoli Rapid downhill course to this disease, with a median survival of 1 year If these large cells are circulating in the peripheral smear of a recognized CLL patient, that a reference should be made to their presence CLL Richter s Transformation 23

Immunoglobulins seen in CLL Immunoglobulins may be seen as intracytoplasmic crystals or globules They are of no clinical significance, but should be included in the morphology of the cell 24

Atypical CLL (acll) Peripheral lymphocyte nuclei have nuclear folds an deep clefts. 10% atypical lymphs should be present to call acll Have a high mean WBC and lower platelet count than typical CLL Have significantly higher percent of disease progression May have significantly higher expression of CD23 Adapted from: Frater JL, McCarron KF, Hammel, JP, et al. Typical and Atypical Chronic Lymphocytic Leukemia Differ Clinically and Immunophenotypically. AM J Clin Path 2001; 116: 655-664 25

Multiple Myeloma Severe skeletal destruction by neoplastic plasma cells results in: Bone pain Bone fractures Anemia Hypercalcemia Renal dysfunction, caused by plugging of glomeruli by Bence Jones protein (monoclonal light chain proteinuria), is common Recurrent infections are common due to granulocytopenia and decreased normal immunoglobulins as a result of marrow replacement by the malignant clone Multiple Myeloma Common theory regarding the development of multiple myeloma is called the two-hit hypothesis an initial antigenic stimulus causes multiple benign monoclones. A second hit, accident or mutagenic event then causes the malignant transformation Plasma cells are rarely found in the peripheral smear, probably because of the increased cohesiveness (secretion) of the plasma cells. Heavy chain disease characterized by increased rouleaux formation, (as a result of a monoclonal IgA or IgG protein) 26

Multiple Myeloma con t Pseudo-spherocytic football-shaped RBCs, leukopenia and thrombocytopenia. Rarely peripheral plasma cells Patients frequently present with lower back pain. X-rays reveal lytic lesions in the bone Complications include anemia, hypercalcemia and renal failure due to plugging of glomeruli with Bence Jones protein. Associated with a t(11;14). Median survival is 3 years. (10% survival at 10 years) In rare instances the terminal phase is associated with a leukemic transformation Multiple Myeloma Bone Marrow 27

Plasma Cell Leukemia Rouleaux formation is missing, because the primitive plasma blasts are probably not secratory (synthesize but do not secrete Ig molecules). A light chain kappa or lambda disorder Patients show increased numbers of plasma blasts and proplasma cells in the peripheral blood. Marked thrombocytopenia and anemia is common. A leukocytosis is characteristic. Patients often present with renal failure and a moderate to marked hepatosplenomegaly. An aggressive disease with short survival. Plasma Cell Leukemia 28

WALDENSTROM S MACROGLOBULINEMIA Also known as lymphoplasmacytic lymphoma Usually a disease affecting older men Characterized by the presence of plasmacytoid lymphocytes. These lymphocytes synthesize large amounts of monoclonal IgM molecule Most patients have bone marrow, lymph node, and splenic involvement WALDENSTROM S MACROGLOBULINEMIA Hyperviscosity often caused by increased IgM can lead to congestive heart failure, confusion, continual mucosal bleeding and/or coma. Plasmapheresis is useful for temporary, acute symptoms (such as retinopathy, congestive heart failure, and CNS dysfunction), but should be combined with chemotherapy for prolonged control of the disease. Therapy may be required to correct hemolytic anemia in patients with chronic cold agglutinin disease; chlorambucil, with or without prednisone, is the mainstay. Rituximab, the anti-cd20 monoclonal antibody, shows 60% to 80% response rates in previously untreated patients 29

WALDENSTROM S MACROGLOBULINEMIA Hairy Cell Leukemia - Untreated An easily controlled lymphoma involving the red pulp of the spleen Disease may present with a mild pancytopenia Thrombocytopenia is usually present Hairy cell numbers may vary from 1-60% in the peripheral blood Splenomegaly and an inability to obtain a marrow aspirate (due to fibrosis) are common findings 30

Hairy Cell Leukemia - Progressive Some patients get advanced disease. Patients usually splenectomized, but characterized with progressive marrow involvement with hairy cells. Treatment with cladribine (2-chlorodeoxyadenosine, 2-CdA), pentostatin (2 -deoxycoformycin), or interferon alfa, the survival rate appears to be greater than 85% at 5 years after the initiation of any one of these therapies.** Clinical relapse is often reached because 2 CdA does not eradicate the malignant clone The cytochemistry used for identification of hairy cells is a tartrate-resistant acid phosphatase (TRAP). This condition is often associated with a 6q chromosomal defect **From: Frassoldati A, Lamparelli T, Federico M, et al.: Hairy cell leukemia: a clinical review based on 725 cases of the Italian Cooperative Group (ICGHCL). Italian Cooperative Group for Hairy Cell Leukemia. Leuk Lymphoma 13 (3-4): 307-16, 1994. Hairy Cell Leukemia 31

Hairy Cell Variant (HCLv) Rare disease, similar to hairy cells but CD 25 negative Leukocytosis is common HCLv cells have round or oval nucleus and a distinct nucleolus Cytoplasm has broad based villi Does not respond to drugs used in HCL, so prognosis is poor Hairy Cell Variant 32

Normal Reactive Lymph Node Splenic Marginal Zone Lymphoma (SLVL) Disease of the white pulp of spleen Cells are similar in morphology to the Hairy cell Splenomegaly is common, as well as chronic fatigue and mild weakness The characteristic cell is larger than CLL lymphs, with diameters comparable to those cells seen in prolymphocytic leukemia 33

SLVL con t The nucleus is round or oval, with a slightly clumped chromatin pattern, often containing a single prominent nucleolus Characteristic irregularity of the plasma membrane, made up of thin and short villi unevenly distributed and often with focal tufts of villous cytoplasmic projections, usually in one pole of the cell The lymphoid cells are positive with acid phosphatase and negative with tartrate resistant acid phosphatase (TRAP) stain. SLVL 34

Nodal Marginal Cell Lymphoma Morphology can look like monocyoid B-cells. Cells are small to intermediate in size. Plasma cell features may be seen. Nucleoli are often present. Transformation to large B-Cell lymphoma may occur Patients respond to chemotherapy but have a high relapse rate. Survival is similar to other indolent lymphoma, app. 5 years. Nodal Marginal Cell Lymphoma 35

Nodal Marginal Cell Follicular Lymphoma Neoplasm of follicle centre B-Cells, made up of centrocytes/cleaved follicle centre cells and noncleaved/centroblasts). Usually presents with wide spread disease involving thoracic and abdominal nodes and spleen. 40% of cases involve the bone marrow Morphology composed of 2 types Small to medium sized with extremely high N/C ratio, nuclear clefts and indistinct nucleoli (centrocytes) Large with very round, oval, or sl indented nuclei, smooth chromatin pattern, high N/C ratio and 1 to 3 nucleoli (centroblasts) 36

Follicular Lymphoma Mantle Cell Lymphoma The term Mantle Zone pattern or Mantle-Cell Lymphoma (centrocytic lymphoma) is used when there is a proliferation of the lymphocytes located in the mantle zones. The mantle zones become broad and expansive. Malignant cells of mantle zone lymphoma are usually light chain monoclonal cells. Nuclear irregularities are usually present in the lymphocytes, though sometimes they are round. Small to medium sized cells with irregular and indented nuclei, moderately coarse chromatin, inconspicuous nucleoli, and a moderate N/C ratio. 37

Mantle Cell Lymphoma Lymph nodes most commonly involved site Spleen and bone marrow (with or without blood involvemnt) other important sites Median survival of 3-5 years, but most cannot be cured The presence of small, round lymphs, (probably T cells), and lymphoid cells with cleaved nuclei are often present as well. Blastic transformations have a high WBC, with large blastic appearing cells. Chromatin has a relatively clumpy chromatin pattern. Nucleoli visibility is variable. They have a significantly high N/C ratio. Mantle Cell Lymphoma The drawings below illustrate the various subtypes of MCL Mantle zone: This pattern resembles a normal node with lymphoid follicles, germinal centers, and parafollicular zones. Only the dark-blue mantle zone (arrow) is composed of neoplastic cells Nodular: Ill-defined, nodular follicles of neoplastic cells blend with non-neoplastic, parafollicular cells. Germinal centers are absent. Diffuse: Small neoplastic lymphocytes replace the node. Blastic: Enlarged neoplastic lymphocytes replace the node. Taken from: pathhsw5m54.ucsf.edu/case11 38

Mantle Cell Lymphoma BLASTIC MANTLE 39

Diffuse Large B-Cell Lymphoma This a neoplasm, that as it s name implies, is a diffuse proliferation of very large cells. These are high grade and aggressive lymphomas, but can be cured. There are 3 distinct morphological variants which are not always reproducible by pathologists. Many pathologists choose simply to refer to any and all of them as diffuse large B- Cell lymphoma. Immunoblastic These cells have a morphology that is often seen in the younger appearing cells of infectious mononucleosis. Copious amounts of deep basophilic cytoplasm, with varying morphology Have a single central nucleolus May resemble plasma blasts DLBCL Morphologic variants of DLBCL. con t DLBCL centroblastic These cells are usually large, but may be of medium size. Nucleus is oval to round with fine chromatin pattern 2-4 nucleoli close to membrane High N/C ratio and pale to basophilic cytoplasm Polymorphic and/or polylobated appearance Anaplastic Very large round, oval nucleus Bizarre nuclei resembling Reed-Sternberg cells For purposes of this lecture, we will not try to teach the various types of large cell lymphomas. What is important is that a large cell lymphoma is recognized if it has peripheralized, not the morphologic variant it is in the blood Descriptions of DLBCL adapted from WHO classsification of Tumours. Tumours of Haematopoietic and Lymphoid Tissues 40

DLBCL DLBCL Blastic 41

DLBCL Immunoblastic Burkitt Lymphoma Highly aggressive lymphoma Rapid short doubling time of tumor cells Translocation involving MYC gene t(8;14) Associated with EB virus in a high percentage of cases. 42

Burkitt Lymphoma con t Small non-cleaved cell is classification description Appear as intermediate to large size cells in peripheral smear, bone marrow, and body fluids Nucelus is usually round, with a coarse sometimes clumpy chromatin pattern and multiple nucleoli. Cytoplasm description is a midnight velveteen blue, often containing lipid laden vacuoles. Non vaculated cells are also common Burkitt Lymphoma 43

The T Cell and NK cell Lymphomas Mature T-Cell and NK Lymphoma Arise from mature or post-thymic T-cells Less common than B-Cell lymphomas T-cell lymphoma more common in Asia and the Caribbean basin due to the virus HTLV-1. Japan most frequently affected in the Asian group as well as the Black population in the Caribbean. 44

Anaplastic Large T-Cell Lymphoma LARGE T-CELL LYMPHOMA NOS 45

Mature T-Cell and NK Lymphomas to be covered Leukemic/disseminated T-cell Prolymphocytic Leukemia T/LGL lymphoma Blastic precursor NK leukemia/lymphoma T/NK lymphoma Cutaneous Sezary Syndrome Other T or NK lymphomas not often found in the peripheral blood. 46

T-Cell Prolymphocytic Leukemia This is a rare condition. It is progressive with short survival time. Patients treated with monoclonal antibody,chempath-1h, shows promise. Patients present with hepatosplenomegaly and lymphadenopathy. Anemia and thrombocytopenia common Marked lymphocytosis T-Cell Prolymphocytic Leukemia con t Small to intermediate sized lymphocytes. Nucleus can be round, oval or irregular resembling Sezary cells Nucleoli are present Common feature is cytoplasmic blebs 47

T-Cell Prolymphocytic Leukemia T8-LGL Lymphoma T8-CLL (LGL leukemia) has a relatively good prognosis. It is considered a benign variant of T-Cell CLL WBCs are relatively low <30,000/µL), with moderate lymphocytosis containing varying numbers and sizes of cytoplasmic azurophilic granules Nucleus is either round of indented and the cytoplasm is a pale blue Neutropenia, however, is constant regardless of the degree of lymphocytosis 48

T8-LGL Lymphoma Approximately 25% of patients are asymptomatic at diagnosis, and approximately 40% present with infections secondary to severe neutropenia An occasional patient will have recurrent episodes of perirectal abscesses or pneumonia. Occasional patient will expire from an aggressive lymphoproliferative disease (with this group, chemotherapy has generally been unsuccessful) Natural Killer Cell (NK) leukemia also presents with LGL morphology of the lymphocytes, and distinction from the T8 leukemia is made solely by immunophenotypic marking LGL Leukemia 49

LGL Leukemia Aggressive T/NK-Cell Leukemia Patients present with fever, hepatosplenomegaly, thrombocytopenia, anemia and severe neutropenia Very aggressive disease. Patients often die within 1 to 2 years. Many die within days to weeks of presentation Highly associated with the EB virus Infection is often the cause of death 50

T/NK Lymphoma Adult T-Cell Leukemia / Lymphoma (ATLL) Several clinical variants are known Acute the most common Lymphomatous Chronic Smoldering Caused by the human retrovirus, HTLV-1 (human T- cell leukemia virus type 1) Found mainly in Japan, the Caribbean basin, and parts of Central Africa 51

ATLL Acute ATLL usually present with hepatosplenomegaly, skin rash, and generalized lymphadenopathy Leukocytosis and eosinophilia are common as well as hypercalcemia Cells are medium to large with a marked nuclear pleomorphism Chromatin is coarse, often with distinct nucleoli. They have deeply basophilic cytoplasm ATLL 52

Blastic Precursor NK Leukemia / Lymphoma Rare form of lymphoma Majority of patients are middle aged to elderly Disease involves multiple sites but has prediliction for the skin. Cells resemble lymphoblasts or myeloblasts, with a very fine chromatin. Few to many azurophilic granules of varying numbers can be found Blastic Precursor NK Leukemia/Lymphoma 53

Sezary Syndrome This is the leukemic phase of mycosis fungoides Sezary syndrom is one of the cutaneos T-cell lymphomas (CTCL) This is a helper T cell lymphoma, and is the only lymphoma that maintains its helper cell activity Usually are CD4 positive and have a reduced CD7. Treated Sezary cells often lose markers, so a morphological grading of the percent of Sezary cells is important. Sezary Cells con t Peripheral blood Sezary cells show single to multiple internal nuclear folds, with little membrane irregularity Sezary cells can be of the large or small variety, with characteristic cerebriform-shaped nucleus, however, the cerebriform shape can only be clearly seen on EM. The PAS positive vacuoles surrounding the nucleus in a pearlnecklace pattern are uncommon in the blood, but appear to be more obvious in pleural fluid There are hundreds of varieties of Sezary cells. Variation from patient to patient and cellular responses to therapy is responsible for the heterogeneous population of the same cell type. 54

Sezary Cells 55