Participants Identification No. % Evaluation

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1 BMD-02 Cell Identification Participants Identification No. % Evaluation Erythrocyte precursor, normal Educational Erythrocyte, normal Educational Erythrocyte precursor with megaloblastic changes/maturation Educational The arrowed cells are normal erythrocyte precursors, as correctly identified by 95.8% of the participants, and are at the late polychromatophilic normoblast stage of maturation. Polychromatophilic normoblasts are round cells that are smaller than earlier erythroid precursors (pronormoblasts and basophilic normoblasts). The nucleus is often centrally located and round with clumped chromatin. Nucleoli are absent. The cytoplasm is relatively abundant and stains as mixtures of blue-gray (early polychromatophilic normoblasts) to pink-gray (late polychromatophilic normoblasts) depending on the relative proportions of RNA and hemoglobin present. By comparison, orthochromic normoblasts, which represent the final stage of nucleated erythrocytes, have markedly dense, pyknotic chromatin and pink-orange cytoplasm with little or no basophilia. The arrowed cells are morphologically normal and lack evidence of dysplasia such as nuclear irregularity, multinucleation, or dyssynchronous nuclear/cytoplasmic maturation. Together, polychromatophilic and orthochromic normoblasts represent approximately 90% of nucleated erythroid precursors in normal bone marrow aspirate smears. 3

2 BMD-03 Participants Identification No. % Evaluation Neutrophil, segmented or band Educational Neutrophil with hypersegmented Educational nucleus The arrowed cell is a normal segmented neutrophil, correctly identified by 98.3% of the participants. Neutrophils have three to five nuclear lobes connected by thin filaments. The chromatin is densely clumped. The cytoplasm is pale pink and has subtle specific granules. The arrowed neutrophil lacks evidence of toxic granulation, cytoplasmic vacuoles, or Döhle bodies. 4

3 BMD-04 Participants Identification No. % Evaluation Neutrophil, metamyelocyte Educational Neutrophil, segmented or band Educational Neutrophil, giant band or giant Educational metamyelocyte Neutrophil, myelocyte Educational The arrowed cell is a metamyelocyte, correctly identified by 97.5% of the participants. Metamyelocytes are characterized by a nucleus that is indented to less than half of the potential round nucleus (i.e., the indentation is less than half the distance to the farthest nuclear margin). By comparison, myelocytes have a round to oval nucleus, and band forms have a nuclear indentation that is greater than half of the potential round nucleus. The nuclear chromatin in metamyelocytes is clumped. The cytoplasm is amphophilic, containing rare azurophilic or pink (primary) granules and many fine bluish or specific granules. The arrowed metamyelocyte in this case has a subtle perinuclear hof (Golgi apparatus), which is more characteristic of myelocytes; however, the nuclear indentation and significant chromatin clumping indicate maturation beyond the myelocyte stage. Metamyelocytes typically constitute 10-15% of the marrow cellularity. 5

4 BMD-05 Participants Identification No. % Evaluation Lymphocyte Educational Lymphoma cell (malignant) Educational The arrowed cells are lymphocytes, and in this case represent CLL/SLL lymphoma cells, as correctly identified by 100% of the participants. Normal lymphocytes are small, round to ovoid cells ranging in size from 7 to 15 µm with an N:C ratio ranging from 5:1 to 2:1. Most have round nuclei that may be slightly indented or notched. The chromatin is diffusely dense or coarse and clumped without distinct nucleoli. The cytoplasm is scant, pale blue to moderately basophilic, and agranular. The neoplastic cells of CLL/SLL are often indistinguishable from normal small lymphocytes, although CLL/SLL cells may be slightly larger and have more of a block-like ( cracked ) chromatin pattern. Reliable morphologic distinction between lymphoma cells of CLL/SLL and normal lymphocytes is often not possible, particularly in a bone marrow aspirate smear. The subtle morphologic features of CLL/SLL cells are often more easily discerned in peripheral blood smears; frequent smudge cells in blood smears is an additional clue. The marked increase in lymphocytes in the bone marrow aspirate smear in the current case should raise the possibility that these lymphocytes are indeed CLL/SLL lymphoma cells, which should then be confirmed by immunophenotyping studies (e.g., flow cytometry). 6

5 BMD-06 Participants Identification No. % Evaluation Eosinophil, any stage Educational Eosinophil, any stage with Educational atypical/basophilic granulation The arrowed cell is a normal mature eosinophil, correctly identified by 98.7% of the participants. Eosinophils are round to oval leukocytes that are the same size as neutrophils. The most characteristic feature of an eosinophil is the coarse, orange-red, refractile cytoplasmic granules. The nuclear features follow the same maturation sequence as seen in neutrophils. Kyle T. Bradley, MD Hematology and Clinical Microscopy Resource Committee 7

6 BMD-07 Bone Marrow Interpretation Questions 1. Which of the following most accurately describes the typical appearance of the neoplastic cells in CLL/SLL? Response No. % Medium-sized with evenly dispersed chromatin, one or more nucleoli, and scant agranular cytoplasm. Small cells with high nuclear cytoplasmic ratio, round nuclei, condensed blocky chromatin, and inconspicuous nucleoli. Medium to large cells with eccentric nuclei, clumped chromatin, prominent perinuclear hof, and moderate amount of basophilic cytoplasm. Medium-sized cells with round nuclei, moderately clumped chromatin, a single prominent nucleolus, and scant cytoplasm Intended Response: Small cells with high nuclear:cytoplasmic ratio, round nuclei, condensed blocky chromatin, and inconspicuous nucleoli. The correct answer is the second option ( small cells with high nuclear:cytoplasmic ratio, round nuclei, condensed blocky chromatin, and inconspicuous nucleoli ), which is the description of a typical neoplastic lymphocyte in CLL/SLL. This description is essentially identical to that of a normal mature lymphocyte. In fact, a single typical CLL/SLL cell cannot be reliably distinguished from a normal lymphocyte based on morphology alone. In practice, the diagnosis of CLL/SLL in a blood or marrow aspirate smear should be suspected when small, matureappearing lymphocytes are markedly increased with accompanying frequent disrupted cells ( smudge cells). Overall, CLL/SLL cells are often slightly larger than normal lymphocytes and the chromatin shows more of a block-like or cracked pattern due to the presence of apparent cracks in between the clumped blocks of dense chromatin. The first option is a description of a blast cell. The third option is a description of a plasma cell. The fourth option is a description of a prolymphocyte. 2. Which of the following are typical CBC findings in patients with chronic lymphocytic leukemia? Response No. % High WBC count with an absolute lymphocytosis Low WBC count with an absolute neutropenia and relative lymphocytosis. - - Normal CBC including normal WBC count, WBC differential, RBC count, and - - platelet count. High WBC count with absolute lymphocytosis and frequent plasma cells Normal or high WBC count with a predominance of variably-sized blasts with scant cytoplasm and inconspicuous nucleoli. - - Intended Response: High WBC count with an absolute lymphocytosis. The correct answer is the first option ( High WBC count with an absolute lymphocytosis ). Patients with CLL/SLL are often asymptomatic at the time the disease is identified. In these patients, the disease is usually discovered incidentally when a CBC is performed for another reason. The WBC count is variable, but usually elevated. Absolute lymphocytosis is present in all patients, as the diagnosis requires at least 5 x 10 9 /L monoclonal lymphocytes with a typical CLL phenotype as a minimum criterion for the diagnosis. Cases with fewer than 5 x 10 9 /L of the same cells are currently considered to have monoclonal B-cell lymphocytosis (MBL). Whether MBL is a precursor of overt CLL/SLL is a topic of current research. A relative lymphocytosis (i.e., increased percentage of lymphocytes in the absence of an absolute lymphocytosis) may be seen in patients with absolute neutropenia (the second option listed). As discussed above, in patients with CLL/SLL the CBC is never normal (the third option) given that an absolute lymphocytosis is a required diagnostic criterion. The presence of plasma cells and blasts (the fourth and fifth options, respectively) are not features of CLL/SLL. 8

7 3. True or False: Occasional prolymphocytes (<10% of nucleated cells) are a typical finding in the blood smear and/or bone marrow aspirate smear in patients with CLL/SLL. Response No. % True False Intended Response: True The correct answer is True. Most cases of CLL/SLL have at least occasional prolymphocytes, usually <2%. Prolymphocytes are neoplastic B-cells that are about µm in diameter, which is 1.5 to 2 times larger than normal lymphocytes or typical CLL/SLL cells. They have a round nucleus with a characteristic single prominent, centrally-located nucleolus. The chromatin is clumped and mature, allowing distinction from a blast cell. Cases of CLL/SLL with >10% prolymphocytes have been shown to correlate with more aggressive disease and are referred to as CLL/SLL with increased prolymphocytes. 4. Which of the following statements regarding CLL/SLL is FALSE? Response No. % Compared to diffuse large B-cell lymphoma, CLL/SLL is more likely to involve the bone marrow at the time of disease presentation. The typical immunophenotype of CLL/SLL is CD5+, CD19+, CD20 dim+, CD23+, dim surface immunoglobulin light chain restriction. Due to the fragility of the neoplastic cells, flow cytometry is not a reliable means of identifying CLL/SLL. Patients with CLL/SLL are often asymptomatic, and the disease is often detected as an incidental finding on a CBC performed for another reason Intended Response: Due to the fragility of the neoplastic cells, flow cytometry is not a reliable means of identifying CLL/SLL. The correct answer (the false statement) is the third option, Due to the fragility of the neoplastic cells, flow cytometry is not a reliable means of identifying CLL/SLL. Although CLL/SLL cells are relatively fragile as evidenced by the presence of frequent smudge cells on smear preparations, they survive flow cytometry interrogation with excellent reliability. Flow cytometry is very helpful in making the diagnosis, particularly because most cases have a characteristic phenotype. Although the phenotype may be determined using immunohistochemistry in solid tissue sections, flow cytometry performed on a blood or marrow aspirate specimen is a more sensitive technique and has the added benefit of reliably demonstrating immunoglobulin light chain restriction (i.e., kappa or lambda light chain restriction) on the neoplastic lymphocytes, which is often not possible using immunohistochemistry. The other answer choices are all true statements. The second answer choice lists the typical immunophenotype in CLL/SLL. Kyle T. Bradley, MD Hematology and Clinical Microscopy Resource Committee 9

8 Case History Patient is a 54-year-old male with a history of chronic lymphocytic leukemia/small lymphocytic lymphoma diagnosed in He has not received therapy. He currently presents with fatigue and an elevated white blood count. Laboratory data include: WBC= 61.1 x109/l; HGB= 13.0g/dL; HCT= 38.1%; MCV= 87.7 fl; PLT= 183 x109/l. Flow cytometry studies performed on the bone marrow demonstrated B-cells with the following aberrant phenotype: Kappa + (dim), CD19+, CD5+, partial CD20+(dim), CD10(-), CD23+, FMC7(-), zap-70+, CD38+. INTRODUCTION Mature B cell lymphomas are neoplastic proliferations of mature B cells. B cells are lymphocytes which play a significant role in the immune system whose function it is to produce a unique antibody (i.e. immunoglobulin), which binds specifically to an antigen. B cells begin their maturation process in the bone marrow, where immature B cells are known as hematogones. During this phase, the immature B cells variably express CD34 and TdT (terminal deoxynucleotidyl transferase). A mature B cell is denoted by the loss of CD34 and TdT expression and acquisition of surface immunoglobulin (IgM). (Figure 1) Figure 1. B-cell maturation. Early B precursor and Pre-B stages show variable expression of the immature markers CD34 and TdT. The mature B cell stage occurs with the loss of TdT and CD34 expression and expression of surface immunoglobulin (SIg). Adopted from Robbins and Cotran Pathologic Basis of Disease, 7th ed. The 2001 and 2008 World Health Organization Classification of Tumours of Haematopoietic and Lymphoid Tissues classify lymphomas based on a multiparametric approach. Currently lymphomas are diagnosed based on clinical findings, morphology, immunophenotype, cytogenetics and molecular analysis. Prior to the WHO classification of 2001, classifications were based predominantly on morphology and/or clinical features. Today, morphological analysis may initially place lymphoid neoplasms into small, intermediate and large size categories. This review will focus on the differential diagnosis of small B cell lymphomas encountered in bone marrow aspirate specimens. 1- Surveys

9 SMALL B CELL LYMPHOMAS CHRONIC LYMPHOCYTIC LEUKEMIA/SMALL LYMPHOCYTIC LYMPHOMA Epidemiology Our current case is a classic and common example of a type of small B cell lymphoma known as chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL). CLL/SLL is the most common leukemia of adults in western countries. It is a chronic leukemia and is thus composed of mature B cells, not immature B cells, i.e. lymphoblasts. The median age of onset is 65 years; however, there is increasing incidence in younger patients. The male:female ratio is 1.5-2:1. Classification CLL and SLL are grouped together because they are indistinguishable, the difference being whether there is primarily involvement of the bone marrow and peripheral blood (CLL) versus primarily nodal based disease (SLL). In order to diagnose CLL in the absence of lymph node or extranodal tissue involvement, there must be > 5 x 10 9 /L monoclonal lymphocytosis with the appropriate immunophenotype (see below). SLL is diagnosed in cases with lymphadenopathy, no cytopenias on CBC and <5x10 9 /L clonal B-cells in the peripheral blood. A small number of healthy individuals may have <5x10 9 /L clonal B-cells in the peripheral blood with the phenotype of CLL but lack additional findings indicative of CLL/SLL. These cases are classified as monoclonal B cell lymphocytosis. Clinical/Morphological Findings Most patients initially present with involvement of lymph nodes, bone marrow and peripheral blood. Commonly, leukocytosis with an absolute lymphocytosis is detected on routine complete blood count (CBC). Manual differential analysis shows a lymphocytosis composed of small to intermediate sized lymphoid cells which are similar in size to slightly larger size than a normal lymphocyte. (Figures 2A and 2B on the next page) 2- Surveys

10 2A 2B Figure 2. CLL involvement of peripheral blood. A. Low-power view of Wright-Giemsa stained peripheral blood smear showing marked leukocytosis due to absolute lymphocytosis. B. High power evaluation demonstrates a lymphocytosis composed of small lymphoid cells with coarse chromatin and round to irregular nuclei. Scattered smudge cells are also seen 3- Surveys

11 The nuclei may be round to lobulated, mimicking other types of small B cell lymphomas. Due to the fragility of the lymphoid cells, smudge cells may be numerous especially on slides lacking albumin. CLL/SLL cells have coarse, clumped hyperchromatic chromatin which appears smoother on albumin treated slides. Small CLL/SLL cells typically lack prominent nucleoli, however a few prolymphocytes may be readily seen and contain distinct nucleoli. These cells may be mistaken as blasts, especially in albumin treated slides. The cytoplasm of typical CLL/SLL cells is a similar basophilic color as a normal lymphocyte. Bone marrow involvement is usually appreciated on aspirate smear slides as well as the bone marrow biopsy. (Figures 3A, 3B) The bone marrow differential demonstrates an increased number of lymphoid cells. 3A 3B Figure 3. CLL/SLL involvement of the bone marrow. A. Low power analysis of the Wright-Giemsa stained aspirate smear slide shows a homogenous cellular population composed of small sized lymphoid cells. B. Higher power shows small lymphoid cells with hyperchromatic, coarse chromatin. 4- Surveys

12 CLL/SLL involvement of lymph nodes show similar morphology. (Figures 4A, 4B) 4A 4B Figure 4. CLL/SLL involvement of the lymph node. A. Low power analysis of the hematoxylin and eosin stained formalin fixed paraffin embedded lymph node tissue shows effacement of the nodal architecture by an atypical lymphoid proliferation with proliferation centers. B. Higher power shows a monomorphic population of small lymphoid cells with hyperchromatic, coarse chromatin. Immunophenotypic Analysis Additional studies must be performed in order to fully characterize the atypical lymphoid population since CLL/SLL cells may morphologically mimic other small cell lymphomas. Thus, the next step is to analyze the protein expression of the lymphoid cells, i.e. immunophenotype the cell population. One method utilized for immunophenotypic analysis is flow cytometry. Basic flow cytometric principles of side and forward scatter are used on automated hematology analyzers. In multi-colored flow cytometric analysis a similar single cell suspension is used but is first added to and incubated in a number of tubes which contain fluorescently labeled antibodies. These antibodies bind to specific proteins on the cell surface which allow us to immunophenotype the cell. In one tube, one cell can potentially be labeled with 4 to fluorescently labeled antibodies. The fluorescence of each antibody is then analyzed for each cell and thus a complete immunophenotypic profile can be performed for the cell population of question. Another method used for immunophenotyping is immunohistochemistry. Formalin fixed paraffin embedded tissue is cut on glass slides which are then stained with an antibody which recognized a specific protein antigen. Immunophenotyping in this methodology is limited to usually one antigen per cell, as opposed to flow cytometry which can analyze a number of antigens per cell simultaneously. (Figure 5, next page) 5- Surveys

13 5A 5B Figure 5. Immunohistochemistry of a normal lymph node. A. Immunohistochemical staining for CD20 highlights B cells in their appropriate compartment (brown staining). B. Immunohistochemical staining for CD3 highlights T cells in their appropriate compartment (brown staining). CLL/SLL Immunophenotype Flow cytometric analysis of our current case demonstrated a lymphoid population expressing the B cell antigens CD20 and CD19. (Figure 6) The cells were mature B cells in that they lacked expression of the immature markers CD34 and TdT, and showed dim expression of surface kappa immunoglobulin light chain. Additionally, our case expressed the CD23 and CD5 markers, characteristic for CLL/SLL. CD5 is an antibody that normally recognizes T cells, but is aberrantly expressed in CLL/SLL. CLL/SLL shows a characteristic flow cytometric immunophenotype which includes dim expression of CD20, CD22 and light chain restriction and moderate expression of CD19. CD5 and CD23 are positive and negative antigens include FMC-7 and CD10. A few cases do show variation of the typical immunophenotypic profile. 6- Surveys

14 A B C D Figure 6. Flow cytometric analysis of CLL/SLL. A. CD19 versus CD5 shows a CD19/CD5 co-expressing B cell population in the upper right hand quadrant. B. CD19 versus CD23 shows dim expression of CD20 and expression of CD23 as highlighted in pink. C. CD19 versus kappa light chain shows a kappa light chain restricted (monoclonal) B cell population in the upper right hand quadrant. Kappa light chain expression is dim. D. CD19 versus lambda light chain demonstrates a lack of lambda light chain expression with very few cells in the upper right hand quadrant. Prognosis CLL/SLL is considered an indolent lymphoproliferative disorder with a variable clinical course. Prognosis is based on clinical staging of the patient, chromosomal aberrations, immunoglobulin gene mutational status and microrna expression (Table 1). Patients with mutated (somatic hypermutations) immunoglobulin gene CLL have a better prognosis than CLL patients with unmutated immunoglobulin gene (IGHV). Deletion 11q22-23, deletion 17p, and deletion 6q are chromosomal aberrations associated with a worse prognosis. Isolated deletion of 13q14.3 is associated with a more favorable prognosis. A small percentage of CLL/SLL patients may transform into an aggressive lymphoma, most commonly diffuse large B cell lymphoma. Clinically these patients usually present with a rapid onset of lymph node enlargement. Morphologically, the atypical lymphoid cells will be large with irregular nuclei and many with prominent nucleoli. 7- Surveys

15 Immunophenotypically, the cells retain expression of B cell markers, but may lose expression of CD5 and/or CD23 and gain expression of additional markers. This transformation is known as Richter s syndrome and portends a poor prognosis. MANTLE CELL LYMPHOMA Epidemiology/Clinical Findings CLL/SLL may be difficult to differentiate from another small B cell lymphoma known as mantle cell lymphoma. Clinically, both present in a similar age range and typically involve the lymph nodes with additional sites of involvement including bone marrow and peripheral blood. Mantle cell lymphoma is also known for involvement of the bowel mucosa known as lymphomatous polyposis. Morphological Findings CLL/SLL and mantle cell lymphoma contain small to intermediate sized mature appearing lymphoid cells with coarse chromatin, irregular nuclei which lack a nucleoli (Figure 7). Figure 7. Mantle cell lymphoma involvement of peripheral blood. Wright-Giemsa stained peripheral blood smear shows a small to intermediate sized population of lymphoid cells with predominantly irregular nuclei and coarse chromatin. A large granular lymphocyte can be seen in the center of the panel at left. 8- Surveys

16 Mantle cell lymphoma cells may not show this typical morphology; recognized morphologic variants include the following: Blastoid variant Atypical lymphoid cells resemble lymphoblasts due to immature chromatin features and typically are associated with a high mitotic rate. Pleomorphic variant - Pleomorphic lymphoid cells, many large, with oval to irregular nuclei, pale cytoplasm and some cells with prominent nucleoli. Small cell More closely resembles CLL/SLL Immunophenotypic / Cytogenetic Findings Mantle cell lymphoma expresses the B cell antigens CD19 and CD20. Similar to CLL/SLL, mantle cell lymphoma also uniquely expresses the T cell antigen CD5. Mantle cell lymphoma typically expresses FMC-7 and lacks CD23. There are a number of CLL/SLL cases which mimic the immunophenotype of mantle cell lymphoma and thus further testing would be required. Mantle cell lymphoma can be differentiated from CLL/SLL on the basis of cyclin D1 protein expression. Mantle cell lymphoma typically contains the chromosomal translocation t(11;14)(q13;a32) involving the CCND1 gene which produces the cyclin D1 protein. The cyclin D1 protein allows the cells to continue to proliferate. Immunohistochemical analysis of the cyclin D1 protein demonstrates positivity in the mantle cell lymphoma cells. (Figure 8) CLL/SLL would, however, be negative for this protein. 8A 8B Figure 8. Mantle cell lymphoma involvement of a lymph node. A. Hematoxylin and eosin stained formalin fixed paraffin embedded lymph node tissue shows effacement of the nodal architecture by an atypical monomorphic lymphoid proliferation. B. Immunohistochemical staining for cyclin D1 (brown staining) shows extensive nuclear positivity in the atypical lymphoid cell population. 9- Surveys

17 Prognosis The prognosis for mantle cell lymphoma is overall worse than for CLL/SLL patients. Most cases of mantle cell lymphoma cannot be cured and the median survival is 3-5 years. Adverse prognostic factors include a high mitotic rate, blastoid/pleomorphic morphology, trisomy 12, complex karyotype and TP53 mutation/overexpression/loss. Gains of chromosome 3q and deletions of 9q are also associated with a poor prognosis independent of the proliferation rate. FOLLICULAR LYMPHOMA Epidemiology Another small B cell lymphoma which may be seen in the bone marrow is follicular lymphoma. This lymphoma occurs mainly in adults at a slightly younger onset compared to CLL/SLL. Follicular lymphoma typically involves lymph nodes with a high percentage of cases also involving the bone marrow. However, unlike CLL/SLL, follicular lymphoma patients do not typically present with cytopenias or lymphocytosis on CBC testing. Follicular lymphoma typically involves the bone marrow in well-defined lymphoid aggregates which often press against the bony trabeculae. Although many cases show adequate residual hematopoiesis, a few patients may have extensive and/or diffuse involvement of the marrow space. (Figure 9) 9A 10- Surveys

18 9B Figure 9. Follicular lymphoma involvement of peripheral blood and bone marrow. A. Wright-Giemsa stained peripheral blood smear shows a small to intermediate sized population of lymphoid cells with deeply cleaved nuclei and coarse chromatin. B. Hematoxylin and eosin stained formalin fixed paraffin embedded bone marrow biopsy specimen shows effacement of the marrow space by an atypical lymphoid proliferation composed mostly of cleaved centrocytes with scattered larger centroblasts with prominent nucleoli. Morphological Findings Follicular lymphoma mimics its normal follicle center B cell counterpart both morphologically and immunophenotypically. Follicular lymphoma is subclassified into low grade (grade 1-2) and a higher grade (grade 3), the difference is based on the number of large non-cleaved cells (centroblasts) seen in a hematoxylin and eosin stained formalin fixed paraffin embedded tissue section. (Figure 10, next page) Low grade follicular lymphoma is most often encountered in the bone marrow and is composed mostly of small lymphoid cells known as centrocytes. Centrocytes have coarse chromatin and characteristically have cleaved nuclei. (Figures 9,10) Although CLL/SLL typically contains more regular nuclear membranes, they may also show deep cleavage and mimic follicular lymphoma cells. (Figure 2B, lower left hand corner) 11- Surveys

19 10A 10B Figure 10. Follicular lymphoma grading A. Hematoxylin and eosin stained formalin fixed paraffin embedded lymph node specimen shows effacement of the marrow space by an atypical lymphoid proliferation composed mostly of cleaved centrocytes with scattered larger centroblasts with prominent nucleoli. This is consistent with low grade, grade 1-2 follicular lymphoma. B. Lymph node specimen shows increased numbers of centroblasts consistent with grade 3 follicular lymphoma. Immunophenotypic Findings The immunophenotype of the cells distinguishes follicular lymphoma from CLL/SLL. Follicular lymphoma is a B cell lymphoma and thus expresses CD19 and CD20. Unlike CLL/SLL and mantle cell lymphoma, follicular lymphoma is negative for CD5. Since follicular lymphoma stems from a follicle center derived cell, it typically expresses germinal center markers such as CD10 and bcl-6. A majority of follicular lymphoma cases also have a chromosomal translocation t(14;18) which involves the BCL2 gene. BCL2 is an antiapoptotic (anti-death) protein which allows the neoplastic cells to survive. Prognosis Histologic grading is performed because it correlates with prognosis. Follicular lymphoma grade 1-2 are typically indolent but are not curable. Higher grade follicular lymphoma (grade 3) is associated with a more aggressive clinical course but may be more amendable to aggressive therapy. In 25-35% of cases transformation or progression to a higher grade lymphoma, typically diffuse large B cell lymphoma, occurs. 12- Surveys

20 MARGINAL ZONE LYMPHOMA AND LYMPHOPLASMACYTIC LYMPHOMA Additional examples of small B cell lymphomas which may be seen in bone marrow aspirates include marginal zone lymphoma and lymphoplasmacytic lymphoma. Marginal zone lymphoma may be subclassified based on the organ of involvement and includes: extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma), nodal marginal zone lymphoma and splenic B cell marginal zone lymphoma. Marginal zone lymphoma cells may show a variety of appearances including centrocytic which would mimic follicular lymphoma cells, small mature forms which would closely resemble a CLL/SLL cell, a monocytoid form with more abundant pale cytoplasm and a plasmacytic morphology which resembles a mature plasma cell. Lymphoplasmacytic lymphoma is composed of small lymphoid cells, plasmacytic lymphocytes and plasma cells. Lymphoplasmacytic lymphoma is typically distinguished from the previous lymphomas discussed due to varying numbers of plasma cells present, but may be difficult to distinguish from marginal zone lymphoma with plasmacytic differentiation. Unlike the previous three lymphomas discussed, marginal zone lymphoma and lymphoplasmacytic lymphoma do not have a characteristic immunophenotype. These are B cell lymphomas and thus express the pan-b cell markers CD19 and CD20 to a varying degree and express the plasma cell marker CD138 on plasma cells. They are typically negative for CD5 (in contrast to CLL/SLL and mantle cell lymphoma) and CD10 (in contrast to follicular lymphoma). Bone Marrow Evaluation Bone marrow evaluation plays an important role in patients with CLL/SLL. A bone marrow biopsy is initially performed to characterize the atypical lymphoid population typically detected on routine CBC analysis. Bone marrow biopsy may also be performed in patients initially diagnosed with CLL/SLL on lymph node examination. Subsequent bone marrow biopsies may be performed to evaluate for response to therapy, development of new cytopenias during or after therapy, and possible relapse/progression. Thus it is important to document the lymphocyte count in the bone marrow aspirate differential as well as examining for therapy related myelosuppression. CLL/SLL is a common small B cell lymphoma which utilizes bone marrow examination for diagnosis and staging. Thus it is important to recognize the morphological and immunophenotypical features of this lymphoma in order to fully classify this mature B cell neoplasm. 13- Surveys

21 REFERENCES 1. Ho, AK, Hill S, Preobrazhensky SN, Miller ME, Chen Z, Bahler DW. Small B-cell Neoplasms with Typical Mantle Cell Lymphoma Immunophenotypes often Include Chronic Lymphocytic Leukemias. Am J Clin Pathol. 2009;131(1): Arber DA, George TI. Bone marrow biopsy involvement by non-hodgkin lymphoma: frequency of lymphoma types, patterns, blood involvement and discordance with other sites in 450 specimens. Am J Surg Pathol. 2005;29(12): Fabbri M, Bottoni A, Shimizu M, et al. Association of a microrna/tp53 Feedback Circuitry with Pathogenesis and Outcome of B-cell Chronic Lymphocytic Leukemia. JAMA. 2011;305(1): Foucar K, Reichard K, Czuchlewski D. Bone Marrow Pathology, Vol 2. 3rd ed. Chicago, IL: ASCP Press; Glassy EF, ed. Color Atlas of Hematology: An Illustrated Field Guide Based on Proficiency Testing. Northfield, IL: College of American Pathologists; 1998: Hallek M, Cheson BD, Catovsky D, et al. Guidelines for the diagnosis and treatment of chronic lymphocytic leukemia: a report from the International Workshop on Chronic Lymphocytic Leukemia updating the National Cancer Institute-Working Group 1996 guidelines. Blood. 2008;111: Kumar V, Abbas AK, Fauston N, et al. Robbins and Cotran Pathologic Basis of Disease. 7th ed. Elsevier Saunders, Philadelphia, PA; Swerdlow SH, Campo E, Harris NL, et al. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. 4th ed. Lyon, France: IARC Press; AUTHOR BIOGRAPHY Kathryn A. Rizzo, DO, PhD, FCAP: Kathryn Rizzo, DO, PhD is the Medical Director of the Hematology, Flow cytometry and Molecular laboratories for Professional Pathology Services at Palmetto Health, Columbia SC. Dr. Rizzo is also a Clinical Assistant Professor of Pathology in the Department of Pathology, Microbiology & Immunology of the University of South Carolina, School of Medicine. She is board certified in Anatomic pathology, Hematology and Cytopathology by the American Board of Pathology. Her primary responsibilities are in Hematopathology, Cytopathology and Surgical pathology. Dr. Rizzo has authored a number of original articles, abstracts and educational activities in the fields of Hematopathology and Molecular biology and is a member of the Hematology and Clinical Microscopy Resource Committee for the College of American Pathologists (CAP). 14- Surveys