Immunopathology of Lymphoma

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1 Immunopathology of Lymphoma Noraidah Masir MBBCh, M.Med (Pathology), D.Phil. Department of Pathology Faculty of Medicine Universiti Kebangsaan Malaysia

2 Lymphoma classification has been challenging to pathologists. Prior to mid 70s, pathologists had great difficulty in defining lymphoma categories There were many classification schemes Earlier classifications depend largely on morphology

3 Previous Lymphoma Classification Schemes Rappaport classification (1966) Lukes and Collins (1974) Kiel classification /Lennert Group (1975) Cell morphology and growth pattern Based on immunological concepts. But little correlation with each other. Kiel also correctly identified morphologic types International Working Formulation (1982) Clinical prognostic groups. Does not include T cell lymphomas

4 International Lymphoma Study Group Adopted a new approach to lymphoma classification Revised European-American Classification of Lymphoid Neoplasms (REAL) Availability of immunohistological markers 2000 WHO Classification of Haematopoetic & Lymphoid Neoplasms (Revised 2008)

5 WHO Classification of Lymphoid Neoplasms Every lymphoma subtype is defined by integrating Tumour morphology, Immunophenotype, Genetic Clinical features. More than 40 subtypes are recognized Each is a distinct disease entity and has corresponding counterpart in normal lymphoid tissue.

6 Lymphomas are broadly categorized into 1. Precursor B and T lymphoid neoplasms 2. Mature B cell neoplasms 3. Mature T- and NK-neoplasms 4. Hodgkin lymphoma 5. Immunodeficiency-associated lymphoproliferative disorders

7 Lymphoma Immunophenotyping Specific immunophenotypic features essential for diagnosis of lymphoma entities. Two major methods: 1. Immunohistochemistry 2. Flow cytometry

8 B CELL NEOPLASMS B cell neoplasms most common lymphomas Tend to mimic stages of normal B cell differentiation. Their resemblance to normal cell differentiation is the basis for their classification. Knowledge of cellular composition and events in the differentiation of normal B cells is a prerequisite of understanding B cell lymphoma. It has led to the insight of tumor genesis in the lymphoid organ

9 Events in the development of B cells in the bone marrow and secondary lymphoid tissues and their characteristic phenotype at different stages of maturation.

10 B cell differentiation in peripheral lymphoid tissue Naïve mature B cell from bone marrow Mature B cell Mature B cell Antigen encounter MANTLE ZONE Centrocyte Memory B cell B blast GERMINAL CENTRE Centroblast Short-living plasma cell (Sagaert et al 2003) Long-living plasma cell

11 Cellular origin of B cell lymphomas Marginal zone Naïve B cell cells Mantle zone Each subtype of B cell lymphoma has its corresponding counterpart in normal lymphoid tissue. Germinal Centre B cells Plasmablastic cell Memory B cell plasma cell

12 Cellular origin of B cell lymphomas Naïve B cell cells Mantle cell lymphoma B CLL (unmutated V gene) Marginal zone Mantle zone MZL CLL (Mutated gene) HCL, DLBCL (ABC-type) Primary mediastinal B cell lymphoma CLL (unmutated V gene) Germinal Centre B cells Memory B cell Follicular lymphoma Burkitt lymphoma DLBCL (GC-type) Lymphocyte predominant Hodgkin lymphoma Plasmablastic cell Plasma cell neoplasm PTLD Classical Hodgkin lymphoma LPL PEL plasma cell

13 B Cell Lymphoma B Lineage-Specific Markers Detectable in Paraffin Embedded Tissues Molecule Classical pan-b cell markers Newer CD markers Signalling molecules Transcription factors Marker CD20, CD79a CD19, CD22, CD37 BLNK, PLCgamma2 PAX5, OCT2

14 B Cell Lymphoma CD20- For regulation of B cell proliferation and differentiation CD20 CD20 DLBCL CD20 Reactive follicle Homogeneous labelling for CD20 in DLBCL TCRBCL

15 B Cell Lymphoma CD20 CD20 Homogeneous labelling for CD20 in follicular lymphoma. Label tumour cells in interfollicular regions Follicular lymphoma CD20 Follicular lymphoma

16 CD20 B lymphoblastic lymphoma CD79a B lymphoblastic lymphoma B lymphoblastic lymphoma In B lymphoblastic lymphoma, most tumour cells express CD79a and not CD20

17 CD20 Multiple Myeloma CD79a Myeloma cells do not express CD20. Occasional CD20-positive lymphocytes as internal positive control Many tumour cells express CD79a

18 B Lineage-Specific Markers Progenitor B cell Pre B cell Immature B cell Mature naïve B cell Germinal centre B cell Memory B cell Plasma cell CD20 CD79a CD19 (B lymphoblastic lymphoma) (Mature B cell neoplasms) (Myeloma)

19 Using rabbit polyclonal anti CD19 antibody that is reactive on paraffin sections. Previous studies of CD19 on B cell neoplasia limited to flow cytometric analysis. Its expression in B cell neoplasm on routine tissue biopsy specimens has not been previously investigated.

20 CD19 Lymph node Tonsil CD19 labels the GC and mantle zone cells of a B lymphoid follicle in a comparable fashion to CD20 CD19 also labels reactive plasma cells found in tonsils

21 TCRBCL CD19 Double immunofluorescent labelling TCRBCL Weak CD19 expression CD19 absent Myeloma LPHD CD19 expression is absent in myeloma and LPHD

22 CONCLUSIONS CD19 was expressed in most B cell neoplasms In contrast to CD20, CD19 was absent/weak in FL, DLBCL, TCRBCL, myeloma, PTLD, chodgkin lymphoma and lymphocyte predominant Hodgkin lymphoma Significance of CD19 loss in B cell neoplasms?

23 Immunophenotypic profile of B cell lymphomas ALL CLL MCL MZL FL HCL LPL Burkitt CD / CD CD CD20 -/ CD / CD CD Cyclin D BCL / BCL TdT

24 Immunophenotypic profile of B cell lymphomas ALL CLL MCL MZL FL HCL LPL Burkitt CD / CD CD CD20 -/ CD / CD CD Cyclin D BCL / BCL TdT

25 Immunophenotypic profile of B cell lymphomas ALL CLL MCL MZL FL HCL LPL Burkitt CD / CD CD CD20 -/ CD / CD CD Cyclin D BCL / BCL TdT

26 Immunophenotypic profile of B cell lymphomas ALL CLL MCL MZL FL HCL LPL Burkitt CD / CD CD CD20 -/ CD / CD CD Cyclin D BCL / BCL TdT

27 Immunophenotypic profile of B cell lymphomas ALL CLL MCL MZL FL HCL LPL Burkitt CD / CD CD CD20 -/ CD / CD CD Cyclin D BCL / BCL TdT

28 Immunophenotypic profile of B cell lymphomas ALL CLL MCL MZL FL HCL LPL Burkitt CD / CD CD CD20 -/ CD / CD CD Cyclin D BCL / BCL TdT

29 Immunophenotypic profile of B cell lymphomas ALL CLL MCL MZL FL HCL LPL Burkitt CD / CD CD CD20 -/ CD / CD CD Cyclin D BCL / BCL TdT

30 New Immunostain Algorithm for DLBCL subtypes Subtype Germinal centre B cell like (GCB) type Activated B cell like (ABC) type Markers BCL6, CD10, GCET1 MUM1, FOXP1 (Chow et a.l Clin Cancer Res. 2009) Immunostains can be used to determine GCB and ABC subtype Closely approximated with the gene expression profiling results More accurate than the Hans algorithm

31 Identifying genetic abnormalities by immunohistochemistry Many types of B cell lymphoma carry reciprocal chromosomal translocation involving immunoglobulin gene and a proto-oncogene. This cause expression of the translocated gene product which can be detected by immunohistology

32 Identifying genetic abnormalities by immunohistochemistry Mantle cell lymphoma CCND1-IgH translocation t(11;14)(q13;q32) Cyclin-D1 protein

33 CD5 Mantle cell lymphoma Tumour cells Reactive T cells Cyclin D1 Homogeneous population of small to medium-sized cells

34 Identifying genetic abnormalities by immunohistochemistry Follicular lymphoma IgH BCL2 translocation t(14;18)(q32;q21) BCL2 protein

35 BCL2 expression in follicular lymphoma Classical Pattern: BCL2-positive follicular lymphoma with t(14;18) A case with a typical phenotype : CD20+, CD10+, BCL2+ and low proliferation fraction CD20 CD10 Ki67 BCL2 BCL2 is positive in essentially all neoplastic cells. BCL2 The labelling is seen in the neoplastic follicles and in the interfollicular cells

36 BCL2 expression in follicular lymphoma Case 2: t(14;18)-positive FL where BCL2 expression is heterogeneous. The tumour is heterogeneously positive for BCL2 and cell proliferation (Ki67) is moderate. FISH shows presence of BCL2 translocation (BCL2 split-apart probes) Double immunofluorescence BCL2(green)/Ki67(red: Lack of BCL2 expression in the proliferating neoplastic cells

37 BCL2 expression in follicular lymphoma Case 3: t(14;18)+ follicular lymphoma but BCL2 protein negative (7/33 cases showed this pattern) Stained with #124 BCL2 #124 The neoplastic follicles are negative for BCL2 Stained with #E17 BCL2 #E17 An adjacent section stained with #E17 shows positivity for BCL2

38 BCL2 expression in follicular lymphoma Case 3: t(14;18)+ follicular lymphoma but BCL2 protein negative #124 #E17 2 mutations that result in amino acid substitutions were found The first of these lies in the region recognized by the antibody #124 Sequencing of PCR products 1.CCG>GTG proline>valine 46 2.TAC>TTC tyrosine>phenylalanine 28. (A total of 11 mutations were found in 4 cases)

39 Conclusions BCL2 protein expression in follicular lymphomas with t(14;18) chromosomal translocations. This study has shed light on two areas of interest. Firstly, proliferating neoplastic cells commonly down-regulate BCL2, and it would be of interest to see if the minority of cases that fail to follow this rule have a more aggressive course. Secondly, mutations in the BCL2 gene in follicular lymphoma are common, and may give rise to false-negative immunostaining and possibly to altered protein function. (Masir et al Br J Haematol)

40 T- AND NK-CELL NEOPLASMS T/NK-cell Neoplasms Thymus or bone marrow precursor cells Mature T/NK cells Precursor lymphoblastic lymphoma /leukemia Nodal lymphoma Extra nodal Leukemia

41 T cell lymphomas that frequently involve lymph node 1. Peripheral T cell lymphoma, unspecified 2. Angioimmunoblastic T cell lymphoma 3. ALCL, ALK-positive, 4. ALCL, ALK-negative 5. Adult T cell leukemia/lymphoma

42 Peripheral T cell lymphoma, unspecified Immunophenotypic features: CD2+, CD3+, CD4>CD8, Frequent antigent loss (CD5, CD7, CD4/CD8, CD52) CD30-/+, CD10-, BCL6-

43 Peripheral T cell lymphoma, unspecified CD3 CD5 CD3 CD5

44 Peripheral T cell lymphoma, unspecified CD3 CD5 Loss of CD5 expression in many cells CD3 CD5

45 CD7 Peripheral T cell lymphoma, unspecified Tumour shows high proliferation rate Ki67 Loss of CD7 expression in many cells

46 Angioimmunoblastic T cell lymphoma CD3 Immunophenotypic features: CD2+, CD3+, CD4 or mixed CD4/CD8, CD10+/-, BCL6+/- PD1+. FDC hyperplasia, EBV+ CD20 B blasts

47 Angioimmunoblastic T cell lymphoma CD21 CD10 Follicular dendritic cell hyperplasia Tumour cells express CD10

48 Angioimmunoblastic T cell lymphoma CD79a LMP1

49 Anaplastic Large Cell Lymphoma, ALK-positive Translocation of the ALK gene Expression of the ALK protein and CD30

50 Anaplastic Large Cell Lymphoma, ALK-positive Chromosomal anomaly ALK partner ALK staining pattern % of cases T(2;5)(p23;q35) NPM Nuclear, diffuse cytoplasmic 84% T(1;2)(q25;p23) TPM3 Diffuse cytoplasmic with peripheral intensification 13%

51 Anaplastic Large Cell Lymphoma, ALK-positive A case of ALK+ ALCL that carry t(2;5) NPM/ALK translocation. ALK immunostain shows nuclear and cytoplasmic labelling

52 Anaplastic Large Cell Lymphoma, ALK-positive CD30 Ki67

53 Anaplastic Large Cell Lymphoma, ALK-positive CD3 EMA

54 Hodgkin Lymphoma Classical Hodgkin lymphoma Lymphocyte predominant Hodgkin lymphoma Subtype Nodular sclerosing Mixed cellularity Lymphocyte rich Lymphocyte depleted none Malignant cells Hodgkin cells, Reed Sternberg cells LP cells (L&H cells) Immunophenotype CD30+, CD15+/-, PAX5+, CD45- CD20-/+, CD79a- /+, OCT2-, BOB1-, EMA- CD30-, CD15-, PAX5+, CD45+ CD20+, CD79a+, OCT2+, BOB1+, EMA+/-

55 Hodgkin Lymphoma Classical Hodgkin lymphoma Lymphocyte predominant Hodgkin lymphoma Subtype Nodular sclerosing Mixed cellularity Lymphocyte rich Lymphocyte depleted none Malignant cells Hodgkin cells, Reed Sternberg cells LP cells (L&H cells) Immunophenotype CD30+, CD15+/-, PAX5+, CD45- CD20-/+, CD79a- /+, OCT2-, BOB1-, EMA- CD30-, CD15-, PAX5+, CD45+ CD20+, CD79a+, OCT2+, BOB1+, EMA+/-

56 Hodgkin Lymphoma Classical Hodgkin lymphoma Lymphocyte predominant Hodgkin lymphoma Subtype Nodular sclerosing Mixed cellularity Lymphocyte rich Lymphocyte depleted none Malignant cells Hodgkin cells, Reed Sternberg cells LP cells (L&H cells) Immunophenotype CD30+, CD15+/-, PAX5+, CD45- CD20-/+, CD79a-/+, OCT2-, BOB1-, EMA- CD30-, CD15-, PAX5+, CD45+ CD20+, CD79a+, OCT2+, BOB1+, EMA+/-

57 Classical Hodgkin lymphoma NSHL CD3 CD20 CD3 CD20

58 Classical Hodgkin lymphoma CD30 RS and Hodgkin cells are CD30+ and CD15+ CD15 CD15 labelling may be weaker. Golgi region only. Less number of CD15+ cells than CD30+ cell

59 Classical Hodgkin lymphoma CD30 only CD30/CD15 Double immunofluorescent studies

60 Classical Hodgkin lymphoma CD15 only CD30/CD15 Double immunofluorescent studies

61 Classical Hodgkin lymphoma CD30/OCT2 Double immunofluorescent OCT2 CD30 OCT2-positive B cells around CD30+ HRS cells

62 Lymphocyte predominant Hodgkin lymphoma

63 Lymphocyte predominant Hodgkin lymphoma CD20 LP cell CD20 CD20

64 CD79a Lymphocyte predominant Hodgkin lymphoma CD3 LP cell rimmed by T cells

65 Lymphocyte predominant Hodgkin lymphoma OCT2 LP cells express OCT2 and BOB1 BOB1

66 Lymphocyte predominant Hodgkin lymphoma EMA EMA is expressed in some LP cells

67 IMMUNOPATHOLOGY 1. Categorization of lymphoma according to lineage e.g., B, T, NK cell. Helpful in the differential of diagnosis of morphologically similar entities. 2. Gives information on degree of tumour differentiation / maturation/micro-anatomic localisation. 3. Identifies associated genetic abnormalities 4. Identifies prognostic markers

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