HEMATOPATHOLOGY DIAGNOSIS & SUBTYPING. Use of IHC. Use of Polymerase Chain Reaction (PCR) Use of Flow Cytometry

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1 HEMATOPATHOLOGY DIAGNOSIS & SUBTYPING HEMATOPATHOLOGY DIAGNOSIS & SUBTYPING The 2008 WHO classification system for tumors of hematopoietic and lymphoid tissues specifies that various combinations of immunophenotypic studies (e.g., IHC and/or flow cytometry) and molecular studies (e.g., cytogenetics and/or fluorescence in situ hybridization [FISH] and/or PCR) are required for the diagnosis of virtually all lymphomas, leukemias, plasma cell neoplasms, and other less common hematolymphoid neoplasms. Use of IHC Classifying hematopoietic malignancies: As Table 1 (page 54) suggests, in addition to identifying mature and immature B and T cell neoplasms and Hodgkin lymphoma, IHC can identify virtually all other hematolymphoid malignancies, including NK cell neoplasms, precursor B- and T-lymphoblastic leukemia/ lymphoma, plasma cell neoplasms, myeloid leukemia/sarcoma, blastic plasmacytoid dendritic cell neoplasms, Langerhans cell histiocytosis, and mastocytosis. Helping establish certain benign diagnoses: A number of benign diseases can cause histologic changes that mimic hematolymphoid malignancies. IHC and related techniques can be very helpful in preventing the misdiagnosis of such diseases as neoplastic. For example, infectious mononucleosis (IM) can produce a highly atypical interfollicular hyperplasia in affected lymph node or tonsil that can mimic non-hodgkin or Hodgkin lymphoma. Special studies to detect Epstein-Barr virus (EBV)-infected cells (particularly EBER-1 in situ hybridization and occasionally EBV LMP IHC) are critical to making the diagnosis of IM in the appropriate clinical and serological setting (i.e., detection of an IgM antibody against the viral capsid antigen). Several years ago, PhenoPath made an important improvement to the reliability of our EBER-1 assay, by always performing it in parallel with in situ hybridization to detect the ubiquitous small nuclear U6 RNA. This serves as a positive internal control to document adequate mrna preservation in the tissue in order for the EBER-1 result to be interpretable. Other infections that can be documented by antibodies validated for clinical use at PhenoPath include: cytomegalovirus (CMV), herpes virus (HSV) I and II, varicella zoster (VZ), adenovirus, SV40/polyoma virus, toxoplasma, and chlamydia. HHV8 infection, which is often associated with malignancy, can also be documented by IHC at PhenoPath. A recently recognized, benign lymphoplasmacytic proliferation, IgG4-related sclerosing disease, can histologically mimic low-grade B cell lymphomas with plasmacytic differentiation. This process is almost always identified as benign by kappa/lambda immunostains demonstrating polytypic plasma cells, while the unique, IgG4-predominant nature of the plasma cells is demonstrated by showing a significantly increased proportion of IgG4-positive plasma cells among the total IgG-positive plasma cells. In tissues affected by IgG4-related sclerosing disease, this proportion is typically greater than 50%, while in unaffected tissues this proportion is typically 10% or less. Both IgG4 and generic IgG IHC are validated for clinical use at PhenoPath. Note that IgG4-related sclerosing disease is reported to be highly responsive to corticosteroid therapy. Use of Flow Cytometry Hematopoietic cell populations can be readily characterized by flow cytometric methods using antibodies to a large number of cell surface and intracellular antigens (see PhenoPath Science and Technology section of this guide). Flow cytometry allows for the rapid identification of immunophenotypic abnormalities associated with malignancy, including aberrant loss or gain of antigen expression, as well as monoclonality. Specifically, monoclonality can usually be established for B cells (light chain restriction), plasma cells (light chain restriction), alpha-beta T cells (restricted TCR-beta expression), and NK cells (restricted KIR expression). In addition, for both acute myeloid and lymphoid leukemias, the flow cytometric immunophenotype often gives strong clues to an underlying recurrent cytogenetic abnormality. Flow cytometric analysis is performed on fresh specimens including peripheral blood, bone marrow, body fluid, and tissues, and can be used to diagnose the full range of hematolymphoid neoplasms, plus a subset of non-hematopoietic tumors such as small cell carcinomas. PhenoPath utilizes state-of-the-art flow cytometers with three lasers and ten fluorescence detectors, providing 10-color flow cytometric evaluation. By determining the number of unique pairwise antigen comparisons possible with different flow cytometric methodologies (each pairwise antigen comparison is seen graphically as a dot-plot ), one can demonstrate that 10-color clinical flow cytometry provides 7.5 times the information of 4-color flow, 3 times the information of 6-color flow, and 61% more information than 8-color flow. The great efficiency of 10-color flow improves case turnaround time, and is of particular benefit when only a small amount of material is available or when minimal residual disease is being sought in the post-therapy setting. See Table 1 (page 54) for a list of antigens evaluated in the various PhenoPath flow cytometry panels. Importantly, in all cases the PhenoPath hematopathologist reviews a Wright-stained smear or cytocentrifuge preparation in parallel with the flow data, to confirm the flow findings are appropropriate. In many cases, the PhenoPath hematopathologist notifies the referring physician (by phone or ) at the time the diagnosis is finalized, to provide additional confirmation that the flow findings are concordant with the sample morphology at the referring institution. PhenoPath s flow cytometry reports include both a detailed comment describing the key findings leading to the interpretation, and flow cytometric histograms ( dot-plots ) showing the key data on which the diagnosis was based. These histograms enable easy identification of the neoplastic phenotype, for following the patient s disease in subsequent specimens. Situations in which 10-color flow cytometry is particularly useful include: Diagnosis and subclassification of leukemia, lymphoma and plasma cell dyscrasia when there is only a small amount of specimen (3 to 6-color flow cytometry may yield incomplete information) Aid to the diagnosis of myelodysplastic syndromes and chronic myeloproliferative neoplasms Lymphoma staging (e.g., blood, bone marrow, and occasionally cerebrospinal fluid) Monitoring of residual or relapsed disease after therapy Identification of prognostic markers or therapeutic targets Use of Polymerase Chain Reaction (PCR) Histologic and immunophenotypic methods have been essential in diagnosing B cell and T cell neoplasms in paraffin-embedded or fresh tissue, by identifying cell lineage and confirming aberrant antigen expression. In some B cell lymphomas, particularly those showing plasma cell differentiation, clonality can be demonstrated by IHC via restricted immunoglobulin light chain expression. However, for all T cell neoplasms and many B cell neoplasms in paraffin-embedded or fresh tissues, clonality determination is not currently possible by IHC, and therefore may require molecular studies. Assays that detect monoclonal rearrangement of B cell immunoglobulin heavy chain (IgH) or kappa light chain (IgK) genes, and T cell receptor gamma chain (TCR-gamma) genes, are essential in the evaluation for B and T cell lymphomas, respectively. This is especially true when only formalin-fixed, paraffin-embedded material is available, or when the lymphoid population is atypical but lacks definitive histologic and/or immunophenotypic features of lymphoma. These molecular studies are particularly helpful in the diagnosis of lymphomas in small biopsies involving extranodal sites, such as the gastrointestinal tract or skin, and can often be applied successfully to decalcified material containing atypical lymphoid infiltrates. Polymerase chain reaction (PCR) technology, optimized for use in formalin-fixed, paraffin-embedded tissue, has become a rapid, sensitive, and specific way to detect clonality in atypical lymphoid proliferations. Importantly, interpretation of molecular studies in the context of the histologic features, IHC findings, and clinical history is required to establish a definitive diagnosis in atypical lymphoid proliferations. At PhenoPath, our validated gene rearrangement assays target the IgH, IgK, and TCR-gamma genes, and use methodology based on the landmark European Biomed-2 consortium recommendations. All gene rearrangement PCR assays are performed in duplicate, to minimize the possibility of false-positives. Diagnosing myeloproliferative neoplasms: The great majority of myeloproliferative neoplasms are defined by one of two different molecular abnormalities: 1) the t(9;22)(q34;q11.2), which produces the BCR-ABL fusion gene that gives rise to chronic myelogenous leukemia (CML); and 2) the JAK2 gene V617F mutation, which is described in >90% of polycythemia vera cases, and 50-60% of cases of essential thrombocytosis and primary myelofibrosis. PhenoPath offers PCR-based assays to detect both of these molecular abnormalities. The BCR-ABL assay is a quantitative reverse transcription PCR (RT-PCR) assay with sensitivity down to 1 in 100,000 cells. The JAK2 V617F assay is a real-time, DNA-based PCR assay with sensitivity down to 1% involvement. Both assays can be performed on fresh specimens of any type. For the quantitative BCR-ABL assay, which is the gold standard for monitoring response to therapy in CML, our reports include both current and previous results (performed at PhenoPath), to document trends in the patient s disease

2 HEMATOPATHOLOGY DIAGNOSIS & SUBTYPING HEMATOPATHOLOGY DIAGNOSIS & SUBTYPING Use of Fluorescence In Situ Hybridization (FISH) FISH studies to confirm chromosomal translocations or gene rearrangements: Leukemias, lymphomas, and plasma cell dyscrasias frequently bear specific chromosomal abnormalities that can be detected with high sensitivity and specificity by fluorescence in situ hybridization (FISH). Identification of these translocations can provide crucial confirmatory diagnostic information in problematic cases, and can also help to guide therapy. For example, the identification of a t(15;17) in an AML patient confirms the diagnosis of acute promyelocytic leukemia (AML-M3 under the French-American-British classification system), and suggests that this patient will benefit from the therapeutic use of all-trans-retinoic acid, in addition to cytotoxic chemotherapy. Similarly, the identification of a chromosomal rearrangement involving the c-myc gene confirms the diagnosis of Burkitt lymphoma in the appropriate clinical, histologic, and immunophenotypic context, while identifying the t(11;18) in a patient with gastric marginal zone B cell lymphoma (MALT lymphoma) confirms this diagnosis and predicts poor response to Helicobacter pylori eradication antibiotic therapy. Many of the FISH studies performed at PhenoPath are amenable to either fresh cells or formalin-fixed, paraffinembedded tissue sections. PhenoPath s FISH studies appropriate for hematopoietic neoplasms are listed in Table 2 (page 55). Use of Cytogenetic Studies (Standard Karyotyping) PhenoPath offers standard karyotyping for the full range of specimens amenable to this technology, including fresh bone marrow, blood, tissue (including products of conception), and body fluid (including amniotic fluids). If clinically indicated, reflexive FISH studies can be performed on material originally submitted for standard karyotyping. Use of Multimodality Approaches to Hematopathology Diagnoses Distinguishing reactive follicular hyperplasia from follicular lymphoma: Typically, reactive lymphoid follicles contain CD10-positive, bcl-6-positive, germinal center-type B cells that lack bcl-2 expression and have a high Ki-67-defined cell proliferation index with frequent polarization. In follicular lymphoma (FL), the germinal center B cells usually overexpress bcl-2 as a consequence of the t(14;18)(q32;q21), and usually display a lower Ki-67-defined cell proliferation index. The presence of significant numbers of germinal center-type B cells outside of follicles also supports the diagnosis of FL. A minority of cases of t(14;18)(q32;q21)-positive FL lack detectable bcl-2 overexpression using the most common anti-bcl-2 monoclonal antibody (clone 124), but can often be shown to overexpress bcl-2 with a second monoclonal antibody (clone C2) that has been validated for clinical use at PhenoPath. In rare FL cases lacking detectable bcl-2 overexpression with either antibody, FISH will identify the t(14;18)(q32;q21). In other cases, FISH may identify a rearrangement of the bcl-6 gene. bcl-6 bcl-2 clone 124 bcl-2 clone C2 In cases that remain equivocal after both IHC and FISH, immunoglobulin heavy chain or kappa light chain gene PCR studies can be used to document clonal gene rearrangements in order to prove malignancy. Additional markers of germinal center origin that can be evaluated by IHC at PhenoPath include GCET1 and LMO2. Reproducible clonal peak Small biopsy with follicular lymphoma (FL), evaluated with two different bcl-2 antibodies. The first two rows of figures include summary dot-plots of 9-color flow cytometric (FC) evaluation of a small needle core biopsy. The neoplastic B cell population is colored black, and expresses normal/high-level CD45 and CD20, abnormally decreased CD19 (compared to the residual normal B cells colored blue and red in the histograms), lambda-restricted light chains, intermediate CD10, and low-intermediate CD38. FC evaluation with anti-bcl-2 clone 124, the most common diagnostic antibody, shows that the tumor cells do NOT overexpress bcl-2 when compared to the light green-colored T cells or lavender-colored residual benign B cells. IHC on the small amount of paraffin-embedded tissue (see images) shows that the bcl-6+, CD10+ follicle center B cells are bcl-2-negative with clone 124, but uniformly bcl-2+ with alternative anti-bcl-2 clone C2, confirming the diagnosis of FL. The different IHC results likely reflect a mutation in the 14 amino acid clone 124 epitope (the clone C2 epitope is thought to be much longer). We use clone C2 in the IHC workup of any suspected FL that appears bcl-2-negative with clone 124. Note the IgH chain gene PCR (on the same case) using the FR2 primer set (blue, with control DNA ladder shown in red), showing a reproducible clonal peak of ~272 base pairs, amid a few minor, non-reproducible peaks

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5 HEMATOPATHOLOGY DIAGNOSIS & SUBTYPING HEMATOPATHOLOGY DIAGNOSIS & SUBTYPING Anaplastic large cell lymphoma (ALCL), ALK positive, identifiable by both flow cytometry and IHC. The two rows of flow histograms show a cerebrospinal fluid containing a high proportion of abnormal large CD30+ T cells (colored black in these images), consistent with involvement by the patient s known ALK+ ALCL. The neoplastic cells show increased forward scatter (FSC) indicating large cell size, plus high-level coexpression of CD30, CD25, and HLA-DR, and expression of only CD4 among the T cell antigens evaluated (negative for CD2, CD3, CD5, CD7, CD8, and CD56). The lower row of flow histograms shows aberrant tumor cell expression of the myeloid-associated antigen CD13, which is common in ALK+ ALCL. However, the lack of CD64 and CD33 (not shown) argues strongly against this representing a monocytic population. The three photomicrographs are from a different, lymph node-based case of ALK+ ALCL. The bottom left image shows combined nuclear and cytoplasmic ALK expression, suggesting the t(2;5). The central figure shows the characteristic high-level expression of the potential therapeutic target CD25. The bottom right photomicrograph shows cytoplasmic expression of clusterin, an antigen expressed in both systemic and cutaneous ALCLs, and useful in distinguishing these neoplasms from CD30+ peripheral T cell lymphoma, not otherwise specified. Peripheral T cell lymphoma, not otherwise specified (PTCL, NOS), of gamma-delta origin. By flow cytometry, the neoplastic T cells (colored black in the two rows of flow histograms) are a markedly expanded, abnormal gamma-delta population with uniform aberrant loss of CD5 and acquisition of CD56 and CD16. The gamma-delta lineage of these cells is confirmed by IHC on the paraffin-embedded tissue, which shows that the CD3+ tumor cells (bottom left) lack TCR-beta (central image, showing a few residual benign alpha-beta T cells), and uniformly express TCR-gamma (bottom right). Note the TCR-γ chain PCR (in the same case) using Vγ1f-Jγ (green) and Vγ10-Jγ (blue) primer sets (DNA ladder shown in red). Reproducible clonal peak ALK CD25 Clusterin CD3 TCR-ßF1 TCRγ

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7 H EMATOPATHOLOGY D IAGNOSIS & S UBTYPING H&E CD117 (c-kit) H EMATOPATHOLOGY D IAGNOSIS & S UBTYPING CD71 CD123 HLA-DR TCL1 Blastic plasmacytoid dendritic cell neoplasm. This is a very rare neoplasm of putative plasmacytoid dendritic cell origin. The images are from a single case involving the bone marrow. The two rows of flow histograms show a fairly characteristic immunophenotype, with no significant expression of the lymphoid and/ or myeloid blast-associated antigens CD34 or CD117. Instead, the neoplastic cells express uniform HLA-DR (at lower-than-usual levels in this case), CD123, CD4, and CD56. As in a subset of these cases, the cells in this case express the myeloid-associated antigen CD33, but lack other myeloid antigens including CD13, CD64, and cytoplasmic myeloperoxidase. The photomicrographs, taken from the bone marrow core biopsy, show the characteristic uniform CD123 and the diagnosisdefining expression of TCL1. HLA-DR shows only variable weak positivity on the tumor cells (mature B cells and monocytes are favored to represent the strongly positive cells). E-cadherin Glycophorin A CD33 Myelodysplastic syndrome, unclassifiable (MDS-U), in which IHC on the bone marrow core biopsy is the key to the diagnosis. In this complex case from an elderly male with pancytopenia, 9/10-color flow cytometric evaluation of the bone marrow aspirate showed essentially normal immunophenotypes of the myeloid blasts (colored red in the first row of flow histograms) and maturing granulocytes and monocytes (colored dark green and lavender, respectively, in the second row of flow histograms). While flow evaluation of the myeloid cells did not help render a diagnosis, the combined cytogenetic detection of a complex abnormal karyotype in the marrow aspirate, plus the morphologic features of the bone marrow core biopsy, enabled definitive diagnosis. The H&E-stained core biopsy showed markedly hypercellular marrow replaced by a frankly neoplastic hematolymphoid infiltrate including collections of enlarged, blast-like cells. By IHC, these cells expressed uniform CD117, CD71, and E-cadherin, with variable glycophorin A, and no CD33, indicating a proerythroblastic proliferation estimated at 30% of the marrow cellularity. In contrast, CD34+ myeloid blasts represented only ~1% of the marrow cellularity. Because the proportion of erythroid cells did not satisfy criteria for the diagnosis of pure erythroid leukemia, the overall findings were most consisent with MDS-U

8 HEMATOPATHOLOGY DIAGNOSIS & SUBTYPING HEMATOPATHOLOGY DIAGNOSIS & SUBTYPING Table 1: Hematolymphoid neoplasms identified by IHC and flow cytometry at PhenoPath Laboratories Table 2: FISH studies for hematopoietic neoplasms performed at PhenoPath Laboratories TUMOR B cell non-hodgkin lymphoma (generic B-NHL) Chronic lymphocytic leukemia/ small lymphocytic lymphoma Mantle cell lymphoma B cell NHL with plasmacytoid differentiation Follicular lymphoma Hairy cell leukemia Diffuse large B cell lymphoma Burkitt lymphoma T cell non-hodgkin lymphoma Angioimmunoblastic T cell lymphoma Anaplastic large cell lymphoma (systemic) NK/T cell lymphoma, nasal type Plasma cell neoplasms Precursor B lymphoblastic lymphoma/leukemia Precursor T lymphoblastic lymphoma/leukemia Acute myeloid leukemia / granulocytic sarcoma Blastic plasmacytoid dendritic cell tumor Paroxysmal nocturnal hemoglobinuria (PNH) Langerhans cell histiocytosis ANTIGENS EVALUATED BY FLOW AND/OR IHC (in addition to the proliferation marker Ki-67, which is evaluated in most cases) Pan-B cell antigens (CD19, CD20, CD22, CD79a, PAX-5, Oct-2, Bob-1, HLA-DR, CD45, kappa, lambda) Pan-B cell antigens, CD5, CD43, CD23, cyclin D1, CD200, CD79b, CD38, ZAP-70, FMC7, LEF-1 Pan-B cell antigens, CD5, CD43, CD23, cyclin D1, CD200, CD79b, CD38, ZAP-70, FMC7 Pan-B cell antigens, CD138, kappa/lambda light chains, CD5, CD10, CD11c, CD25 Pan-B cell antigens, CD10, CD38, bcl-6, bcl-2; CD21+ follicular dendritic cell (FDC) networks Pan-B cell antigens, CD103, CD11c, CD25, CD123, CD10, cyclin D1, DBA.44, TRAcP Pan-B cell antigens, CD10, bcl-6, MUM-1, GCET1, FOXP1, LMO2, p53, EBER1 mrna Pan-B cell antigens, CD10, bcl-6, bcl-2, CD43, c-myc, EBER1 RNA T cell-associated antigens (CD2, CD3, CD4, CD5, CD7, CD8, CD43, CD45, CD56, TCR-β, TCR-γ) (TCR-β isoforms evaluated by flow only) T cell-associated antigens, CD10, bcl-6, PD-1; expanded CD21+ FDC networks; EBV+ B cells (EBER-1 RNA) T cell-associated antigens, CD30, ALK, TIA-1, EMA, clusterin, CD25 T cell-associated antigens, TIA-1, EBER1 RNA CD138, CD79a, kappa/lambda light chains; CD20, CD56, CD117, CD45, cyclin D1 Pan B cell antigens, TdT, CD10, CD34, aberrant myeloid antigens (CD13, CD15, CD33), CD45 T cell-associated antigens, TdT, CD10, CD34, CD1a, CD99, aberrant myeloid antigens, CD45 Myeloid-associated antigens (CD45, CD11b, CD15, CD33, CD34, CD117, CD38, CD71, CD19, CD43, MPO, CD68, lysozyme, HLA-DR, CD36, CD64, CD13, CD14, CD123, CD16); Erythroidassociated antigens (hemoglobin A, glycophorin A, E-cadherin, CD36, CD71); Megakaryocyteassociated antigens (vwf, CD9, CD36, CD41, CD42b, CD61) Myeloid-associated antigens, CD43, CD4, CD56, CD123, HLA-DR, TCL1, TdT CD59 (erythrocytes), CD14 (monocytes), CD16, CD24, CD66b (neutrophils), FLAER (monos. and neuts.), plus CD45, CD33, HLA-DR, CD10, and CD235a as gating antigens S100, CD1a, CD68, CD43, langerin B-LYMPHOID NEOPLASMS Chromosomal Abnormality Genes Involved Diagnosis t(14;18)(q32;q21) IGH / bcl-2 Follicular lymphoma, DLBCL t(11;14), t(4;14), t(14;16), deletions of 13q14, 13q34, 17p13, and hyperploidy CCND1/IGH, FGFR3/MMSET/IGH, IGH/CMAF, D135319, LAMP1, p53 Plasma cell myeloma t(11;14)(q13;q32) CCND1 / IGH Mantle cell lymphoma t(11;18)(q21;q21) API2 / MALT1 Marginal zone B cell lymphoma t(14;18)(q32;q21) IGH / MALT1 Marginal zone B cell lymphoma t(8;14), t(2;8), t(8;22) c-myc / IGH, IgKappa, IgLambda Burkitt lymphoma, some DLBCL Deletions of 11q22, 13q14, 13q34, 17p13; Trisomy 12 ATM, D13S319, LAMP1, P53 CLL/SLL t(3;14)(q27;q32) bcl-6 Follicular lymphoma, DLBCL t(9;22)(q34;q11.2) BCR/ABL ph+ B-ALL MYELOID NEOPLASMS Chromosomal Abnormality Genes Involved Diagnosis t(9;22)(q34;q11.2) BCR/ABL CML t(8;21)(q22;q22) t(15;17)(q22;q12) 11q23 alterations: t(4;11)(q21;q23), t(9;11)(p22;q23), t(11;19)(q23;p13) AML1 / ETO (RUNX1 / RUNX1T1) PML / RARA MLL gene alterations Acute myeloid leukemia with recurrent genetic abnormalities (AML-M2 under FAB classification) Acute myeloid leukemia with recurrent genetic abnormalities (AML-M3 under FAB classification) AML and ALL Inv(16)/t(16;16)(p13;q22) inv16 / t(16;16)(p13;q22) / MYH11 AML, myelomonocytic with eosinophilia 5/del 5q, 7/del 7q, trisomy 8, deletion 20q Many genes involved Myelodysplastic and myeloproliferative neoplasms Table 3: PCR studies for hematopoetic neoplasms performed at PhenoPath Laboratories MYELOID MUTATIONAL/TRANSLOCATION ANALYSIS Mastocytosis Follicular dendritic cell sarcoma Myeloid-associated antigens, tryptase, c-kit/cd117, CD25, CD2 CD21, CD23, CD35, podoplanin (D2-40) JAK2 by Real Time PCR Detects the V617F mutation in the JAK2 gene, which is used in the diagnosis of polycythemia vera (PV), essential thrombocythemia (ET), and idiopathic myelofibrosis (MF). Post-transplant lymphoproliferative disorders/ HIV-associated lymphoma Pan-B cell antigens, EBER1 RNA, EBV LMP or HHV-8 (KSHV) in a minority of cases; kappa/ lambda light chains, CD138 Quantitative BCR-ABL1 Major & Minor Breakpoint Clusters by Real Time PCR Quantitative assay for the BCR-ABL1 major and minor breakpoint cluster region transcripts using international scale (IS) reporting. Classical Hodgkin lymphoma Nodular lymphocyte-predominant Hodgkin lymphoma Small blue round cell tumors (favor non-hematopoietic), by flow cytometry CD30, CD15, PAX-5, CD20, CD3, CD45, MUM1, Oct-2, Bob-1, fascin, EBER1 Pan B cell antigens, bcl-6, EMA, CD10, CD21, IgD CD45, EpCAM (epithelial cell adhesion marker), CD56, CD99, CD90 IGH Chain Gene Rearrangement Studies IGK (Kappa) Gene Rearrangement Studies B CELL (IGH, IGK) AND T CELL (TCR-y) B cell heavy chain clonality assessment B cell light chain clonality assessment (minimizes false-negatives compared to IGH assay alone) Potential therapeutic targets B cell (CD20, CD22, CD52); T cell (CD25, CD30, CD52); myeloid (CD33) TCR- Chain Gene Rearrangement Studies T cell clonality assessment 54 55