Immunopathology of Lymphoma Noraidah Masir MBBCh, M.Med (Pathology), - - PowerPoint PPT Presentation

Immunopathology

• f Lymphoma

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

Immunopathology of Lymphoma

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

Immunopathology of Lymphoma

Previous Lymphoma Classification Schemes

Rappaport classification (1966) Cell morphology and growth pattern Lukes and Collins (1974) Based on immunological concepts. But little correlation with each

• ther. Kiel also correctly

identified morphologic types Kiel classification /Lennert Group

(1975)

International Working Formulation (1982) Clinical prognostic groups. Does not include T cell lymphomas

Immunopathology of Lymphoma

1994 - International Lymphoma Study Group

Adopted a new approach to lymphoma classification Availability of immunohistological markers

Revised European-American Classification of Lymphoid Neoplasms (REAL) WHO Classification of Haematopoetic & Lymphoid Neoplasms 2000 (Revised 2008)

Immunopathology of Lymphoma

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.

Immunopathology of Lymphoma

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

Immunopathology of Lymphoma

Specific immunophenotypic features essential for diagnosis of lymphoma entities.

Lymphoma Immunophenotyping

Two major methods:

• 1. Immunohistochemistry
• 2. Flow cytometry

Immunopathology of Lymphoma

• 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 B CELL NEOPLASMS

Immunopathology of Lymphoma

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

Immunopathology of Lymphoma

Short-living plasma cell Centroblast Centrocyte Long-living plasma cell MANTLE ZONE GERMINAL CENTRE B blast

B cell differentiation in peripheral lymphoid tissue

Antigen encounter Mature B cell Mature B cell Memory B cell Naïve mature B cell from bone marrow (Sagaert et al 2003)

Immunopathology of Lymphoma

Cellular origin of B cell lymphomas

Naïve B cell cells Mantle zone Germinal Centre B cells Marginal zone Memory B cell plasma cell Plasmablastic cell

Each subtype of B cell lymphoma has its corresponding counterpart in normal lymphoid tissue.

Immunopathology of Lymphoma

Cellular origin of B cell lymphomas

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

Molecule Marker Classical pan-B cell markers CD20, CD79a “Newer” CD markers CD19, CD22, CD37 Signalling molecules BLNK, PLCgamma2 Transcription factors PAX5, OCT2

Immunopathology of Lymphoma

B Cell Lymphoma

B Lineage-Specific Markers Detectable in Paraffin Embedded Tissues

Immunopathology of Lymphoma B Cell Lymphoma

Reactive follicle CD20

CD20- For regulation

• f B cell proliferation

and differentiation

TCRBCL CD20

Homogeneous labelling for CD20 in DLBCL

DLBCL CD20

Immunopathology of Lymphoma

CD20

B Cell Lymphoma

Follicular lymphoma

CD20

Follicular lymphoma

CD20

Homogeneous labelling for CD20 in follicular lymphoma. Label tumour cells in interfollicular regions

Immunopathology of Lymphoma

CD20 B lymphoblastic lymphoma CD79a

B lymphoblastic lymphoma B lymphoblastic lymphoma

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

Immunopathology of Lymphoma

CD20 CD79a

Multiple Myeloma

Many tumour cells express CD79a Myeloma cells do not express

• CD20. Occasional CD20-positive

lymphocytes as internal positive control

Progenitor B cell Pre B cell Immature B cell Mature naïve B cell Germinal centre B cell Memory B cell Plasma cell

Immunopathology of Lymphoma

B Lineage-Specific Markers

CD20 CD79a

(B lymphoblastic lymphoma) (Mature B cell neoplasms) (Myeloma)

CD19

Immunopathology of Lymphoma

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.

Immunopathology of Lymphoma

CD19

CD19 labels the GC and mantle zone cells of a B lymphoid follicle in a comparable fashion to CD20

Lymph node Tonsil

CD19 also labels reactive plasma cells found in tonsils

LPHD Myeloma CD19 expression is absent in myeloma and LPHD Immunopathology of Lymphoma TCRBCL

Weak CD19 expression

TCRBCL

CD19 absent

CD19

Double immunofluorescent labelling

Immunopathology of Lymphoma

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?

Immunopathology of Lymphoma

Immunophenotypic profile of B cell lymphomas

ALL CLL MCL MZL FL HCL LPL Burkitt CD5

• +

+

• /+
• CD10

+

• +
• +

CD19

+ + + + + + + +

CD20

• /+

+ + + + + + +

CD23

• +
• /+
• CD25
• +
• CD103
• +
• Cyclin D1
• +
• BCL2

+ + + + +/- + +

• BCL6
• +
• +

TdT

+

Immunopathology of Lymphoma

Immunophenotypic profile of B cell lymphomas

ALL CLL MCL MZL FL HCL LPL Burkitt CD5

• +

+

• /+
• CD10

+

• +
• +

CD19

+ + + + + + + +

CD20

• /+

+ + + + + + +

CD23

• +
• /+
• CD25
• +
• CD103
• +
• Cyclin D1
• +
• BCL2

+ + + + +/- + +

• BCL6
• +
• +

TdT

+

Immunopathology of Lymphoma

Immunophenotypic profile of B cell lymphomas

ALL CLL MCL MZL FL HCL LPL Burkitt CD5

• +

+

• /+
• CD10

+

• +
• +

CD19

+ + + + + + + +

CD20

• /+

+ + + + + + +

CD23

• +
• /+
• CD25
• +
• CD103
• +
• Cyclin D1
• +
• BCL2

+ + + + +/- + +

• BCL6
• +
• +

TdT

+

Immunopathology of Lymphoma

Immunophenotypic profile of B cell lymphomas

ALL CLL MCL MZL FL HCL LPL Burkitt CD5

• +

+

• /+
• CD10

+

• +
• +

CD19

+ + + + + + + +

CD20

• /+

+ + + + + + +

CD23

• +
• /+
• CD25
• +
• CD103
• +
• Cyclin D1
• +
• BCL2

+ + + + +/- + +

• BCL6
• +
• +

TdT

+

Immunopathology of Lymphoma

Immunophenotypic profile of B cell lymphomas

ALL CLL MCL MZL FL HCL LPL Burkitt CD5

• +

+

• /+
• CD10

+

• +
• +

CD19

+ + + + + + + +

CD20

• /+

+ + + + + + +

CD23

• +
• /+
• CD25
• +
• CD103
• +
• Cyclin D1
• +
• BCL2

+ + + +

+/-

+ +

• BCL6
• +
• +

TdT

+

Immunopathology of Lymphoma

Immunophenotypic profile of B cell lymphomas

ALL CLL MCL MZL FL HCL LPL Burkitt CD5

• +

+

• /+
• CD10

+

• +
• +

CD19

+ + + + + + + +

CD20

• /+

+ + + + + + +

CD23

• +
• /+
• CD25
• +
• CD103
• +
• Cyclin D1
• +
• BCL2

+ + + + +/- + +

• BCL6
• +
• +

TdT

+

Immunopathology of Lymphoma

Immunophenotypic profile of B cell lymphomas

ALL CLL MCL MZL FL HCL LPL Burkitt CD5

• +

+

• /+
• CD10

+

• +
• +

CD19

+ + + + + + + +

CD20

• /+

+ + + + + + +

CD23

• +
• /+
• CD25
• +
• CD103
• +
• Cyclin D1
• +
• BCL2

+ + + + +/- + +

• BCL6
• +
• +

TdT

+

Immunopathology of Lymphoma

New Immunostain Algorithm for DLBCL subtypes

Subtype Markers Germinal centre B cell like (GCB) type BCL6, CD10, GCET1 Activated B cell like (ABC) type 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

Immunopathology of Lymphoma

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

Identifying genetic abnormalities by immunohistochemistry

Mantle cell lymphoma CCND1-IgH translocation t(11;14)(q13;q32) Cyclin-D1 protein

Immunopathology of Lymphoma

Identifying genetic abnormalities by immunohistochemistry

Immunopathology of Lymphoma

Mantle cell lymphoma

Homogeneous population

• f small to medium-sized

cells CD5

Reactive T cells

Tumour cells

Cyclin D1

Follicular lymphoma IgH –BCL2 translocation t(14;18)(q32;q21) BCL2 protein

Immunopathology of Lymphoma

Identifying genetic abnormalities by immunohistochemistry

BCL2 expression in follicular lymphoma

A case with a typical phenotype : CD20+, CD10+, BCL2+ and low proliferation fraction CD20 CD10 Ki67

Classical Pattern: BCL2-positive follicular lymphoma with t(14;18)

BCL2 is positive in essentially all neoplastic cells. BCL2 The labelling is seen in the neoplastic follicles and in the interfollicular cells BCL2

Double immunofluorescence BCL2(green)/Ki67(red: Lack of BCL2 expression in the proliferating neoplastic cells The tumour is heterogeneously positive for BCL2 and cell proliferation (Ki67) is moderate.

Case 2: t(14;18)-positive FL where BCL2 expression is heterogeneous. BCL2 expression in follicular lymphoma

FISH shows presence of BCL2 translocation (BCL2 split-apart probes)

BCL2 #E17

An adjacent section stained with #E17 shows positivity for BCL2

Stained with #E17

Case 3: t(14;18)+ follicular lymphoma but “BCL2 protein negative”

BCL2 #124

The neoplastic follicles are negative for BCL2

Stained with #124

BCL2 expression in follicular lymphoma

(7/33 cases showed this pattern)

Sequencing of PCR products 1.CCG>GTG proline>valine 46 2.TAC>TTC tyrosine>phenylalanine 28 . #124 #E17

BCL2 expression in follicular lymphoma

Case 3: t(14;18)+ follicular lymphoma but “BCL2 protein negative”

2 mutations that result in amino acid substitutions were found The first of these lies in the region recognized by the antibody #124

(A total of 11 mutations were found in 4 cases)

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. 2009. Br J Haematol) Conclusions

Immunopathology of Lymphoma

Immunopathology of Lymphoma

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

Immunopathology of Lymphoma

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

Immunopathology of Lymphoma

Peripheral T cell lymphoma, unspecified

Immunophenotypic features: CD2+, CD3+, CD4>CD8, Frequent antigent loss (CD5, CD7, CD4/CD8, CD52) CD30-/+, CD10-, BCL6-

Immunopathology of Lymphoma

Peripheral T cell lymphoma, unspecified CD3 CD3 CD5 CD5

Immunopathology of Lymphoma

Peripheral T cell lymphoma, unspecified CD3 CD3 CD5 CD5

Loss of CD5 expression in many cells

Immunopathology of Lymphoma

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

Immunopathology of Lymphoma

Angioimmunoblastic T cell lymphoma

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

CD3

Immunopathology of Lymphoma

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

Immunopathology of Lymphoma

Angioimmunoblastic T cell lymphoma CD79a LMP1

Immunopathology of Lymphoma

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

Immunopathology of Lymphoma

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%

Immunopathology of Lymphoma

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

Immunopathology of Lymphoma

Anaplastic Large Cell Lymphoma, ALK-positive CD30 Ki67

Immunopathology of Lymphoma

Anaplastic Large Cell Lymphoma, ALK-positive CD3 EMA

Immunopathology of Lymphoma

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+/-

Immunopathology of Lymphoma

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+/-

Immunopathology of Lymphoma

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+/-

Immunopathology of Lymphoma

Classical Hodgkin lymphoma

CD3 CD20 CD20 CD3 NSHL

Immunopathology of Lymphoma

Classical Hodgkin lymphoma

CD30 RS and Hodgkin cells are CD30+ and CD15+

CD15 labelling may be

• weaker. Golgi region only.

Less number of CD15+ cells than CD30+ cell

CD15

Immunopathology of Lymphoma

Classical Hodgkin lymphoma

CD30 only CD30/CD15 Double immunofluorescent studies

Immunopathology of Lymphoma

Classical Hodgkin lymphoma

CD15 only CD30/CD15 Double immunofluorescent studies

Immunopathology of Lymphoma

Classical Hodgkin lymphoma

CD30/OCT2

OCT2 CD30 Double immunofluorescent

OCT2-positive B cells around CD30+ HRS cells

Immunopathology of Lymphoma

Lymphocyte predominant Hodgkin lymphoma

Immunopathology of Lymphoma

Lymphocyte predominant Hodgkin lymphoma

LP cell

CD20 CD20 CD20

Immunopathology of Lymphoma

Lymphocyte predominant Hodgkin lymphoma

CD79a CD3

LP cell rimmed by T cells

Immunopathology of Lymphoma

OCT2 BOB1

Lymphocyte predominant Hodgkin lymphoma

LP cells express OCT2 and BOB1

Immunopathology of Lymphoma

Lymphocyte predominant Hodgkin lymphoma

EMA EMA is expressed in some LP cells

Immunopathology of Lymphoma

• 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

IMMUNOPATHOLOGY