Hodgkin Lymphoma –

Pathological features and differential diagnosis

Steve Hamilton-Dutoit Institute of Pathology Aarhus University Hospital

Outline

Hodgkin lymphoma

• Background • Classification • Morphology & immunophenotype • Differential diagnosis

• Other malignancies • Benign lesions

1 Thomas Hodgkin 17981798----18661866

Thomas Hodgkin – dissection (Prof. Robert Carswell)

Thomas Hodgkin 17981798----18661866

Hodgkin’s original case: abdominal nodes

Gordon Museum, King’s College London

2 Thomas Hodgkin 17981798----18661866

Hodgkin’s original case: CD15 (1991)

Gordon Museum, King’s College London

Hodgkin Lymphoma --- clinical

The clinical picture is characteristic & often diagnostic

• nodal +/or mediastinal presentation • milt / liver / bone marrow • painless lymphadenopathy • contiguous spread • often toxic B symptoms • previously fatal • nownownow,now , highhighhighcurecurecure rates with chemotherapy +/+/+/-+/ ---radiotherapyradiotherapy; ; +/; +/-+/ ---targeted biological TxTxTx. Tx ...

3 Hodgkin Lymphoma

The histopathology is often characteristic & usually diagnostic

• an often scanty population of neoplastic cellscellscells • a diagnostic (host(host- ---derivedderivedderived)) stromal & cellular background • the HL ””microenvironmentmicroenvironmentmicroenvironment””

RS cell

Classical Hodgkin Lymphoma: WHO Classification Subclassification of HL is based on the Lymphocyte predominancebackground Hodgkin's stroma ca. 10% & cells (the HL microenvironment)

4 Dorothy Reed & the Reed-Sternberg cell

Age & sex: Hodgkin lymphoma (UK)

Cases per yearyearyear Age specific incidence

5 Hodgkin Lymphoma --- epidemiology

• USA: ca. 9000 annual cases EBV EBER • bimodal age distribution • incidence peaks in young adults & elderly • epidemiology suggests HL is a rare consequence of a common infection • HL maymaymaybebebe associated with EBV

• HL is commoner in industrialized countries EBV LMP1 • HL in developing countries is ––– • lesslesslesscommon • seenseenseenin children • highly associated with EBV • HL is onlyonlyonlymildly associated withwithwith immunodeficiency

Hodgkin Lymphoma Classification

6 WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues, 2008 – 2016/7

• WHO (2008…..2016/7) Defines entities based on: • Clinical features • Morphology • Immunophenotype • Molecular genetics

Hodgkin Lymphoma Classification

Relatively stable over the last 20 years

7 Lymphoma

Hodgkins lymphoma Non-Hodgkins lymphoma

Nodular LP HL B-cell T/NK-cell • precursor • precursor Classical HL • mature • mature HL, NS HL, LR HL, MC HL, LD

Lymphoma diagnosis

• Clinical features • Morphology • Immunophenotype • Molecular diagnosis

8 Lymphoma diagnosis

• Clinical features • Morphology • Immunophenotype • Molecular diagnosis

Hodgkin lymphoma: Two biological entities

Lymphocyte predominance ca. 10 % • a clonal B-cell lymphoma with functional lineage gene expression

Classical Hodgkin ca. 90% • a monoclonal neoplasm of germinal centre-derived B-cells • crippled, ineffective B-cell programme • the cHL microenvironment is crucial for: • diagnosis & subclassification • biological understanding • determining prognosis • as an emerging target for new biological/immunological treatments

9 ReedReed----SternbergSternberg cells • required for diagnosis • not specific to HL • large cells (>45um in diameter) • classically binucleate • most are actually mononuclear/multilobated • large prominent acidophilic nucleoli in a halo • ”owl’s eye” appearance

ReedReed----SternbergSternberg cells • required for diagnosis • not specific to HL • large cells (>45um in diameter) • classically binucleate • most are actually mononuclear/multilobated • large prominent acidophilic nucleoli in a halo • ”owl’s eye” appearance • variants: • mononuclear (Hodgkin cell) • mummified cell • lacunar cell • LP (L&H) cell in NLPHL

10 ReedReed----SternbergSternberg cells --- immunohistochemistry

Hodgkin lymphoma: CD30

Reed-Sternberg cells

HRS cells are required, but are not sufficient to diagnose Hodgkin lymphoma

11 Diagnosis: Infectious Mononucleosis

Infectious Mononucleosis: HRS-like cells

12 cHL vs. virus infection (IM)

cHL vs. virus infection (IM)

13 CD30

14 cHL vs. viral infction

• HRS-like cells commonly present in infections • CD30+ CD15+ • May be EBV+ • Check EBV latency (IHC for LMP1 & EBNA2)

Latency forms (latent antigen expression pattern) in EBV infections

Latency EBERs EBNA-1 EBNA-2 LMP-1 LMP-2

I + + - - -

II + + - + +

III + + + + +

Healthy carrier + +/- - - +

15 Latency form in EBV infections

Latency Disease EBERs EBNA-1 EBNA-2 LMP-1 LMP-2

I Burkitt lymphoma + + - - -

II NPC + + - + + Hodgkin lymphoma III I.M. + + + + + PTLD Healthy carrier + +/- - - +

cHL vs. infectious mononucleosis

• Age – IM usually in the young - but it can occur at any age • HRS-like cells commonly present in IM • CD30+ (CD15+) • Check EBV latency (IHC for LMP1 & EBNA2) • CD20 pos blasts in IM • HL: EBV Latency II LMP-1pos EBNA2 neg • IM: EBV Latency III LMP-1pos EBNA2 pos

16 EBV - immunology

Infectious Mononucleosis: EBV

EBER-ISH

EBV-RNA in situ hybridization (EBER)

17 Infectious Mononucleosis: EBV latency III

EBV-EBNA2 EBV-LMP1

Classical Hodgkin Lymphoma: WHO Classification Subclassification of HL is based on the Lymphocyte predominancebackground Hodgkin's stroma ca. 10% & cells (the HL microenvironment)

• Classical Hodgkin Lymphoma ca. 90% • Nodular sclerosis • Lymphocyte-rich • Mixed cellularity • Lymphocyte depletion

18 Nodular sclerosis classical Hodgkin lllymphomalymphoma (((NScHL (NScHLNScHL))))

• NScHL • Nodular pattern • Hyaline sclerosis • Lacunar type HRS cells • Commonest HL subtype

NScHL

19 NScHL: Lacunar cells

NScHL – cellular

20 NScHL – cellular

No fibrosis = HLNS, cellular phase

Diff. DX: TT----ALCALC

21 Grading HLNS

NS grade I NS grade II

• NSHL can be graded according to

• Number of RS cells

• Atypia of RS cells • Recommended for multicentre studies, not for routine

Classical Hodgkin Lymphoma: WHO Classification Subclassification of HL is based on the Lymphocyte predominancebackground Hodgkin's stroma ca. 10% & cells (the HL microenvironment)

• Classical Hodgkin Lymphoma ca. 90% • Nodular sclerosis • Lymphocyte-rich • Mixed cellularity • Lymphocyte depletion

22 Mixed cellularity cHL

Mixed cellularity cHL

• Diagnostic HRS cells • No hyaline fibrosis • Polymorphic infiltrate • Lymphocytes • Eosinophils • Histiocytes • Plasma cells • Often EBV positive

23 Mixed cellularity cHL

• Diagnostic HRS cells • No hyaline fibrosis • Polymorphic infiltrate • Lymphocytes • Eosinophils • Histiocytes • Plasma cells • Often EBV positive

Lymphocyte depleted cHL • Numerous atypical HRS cells • Densely fibrotic stroma • Rare < 1% cHL • Most diagnosed now as other NHLs

24 cHL vs. granulomatous inflammation

• Epithelioid clusters and larger sarcoid granulomas may dominate in cHL

• Remember to look behind the granulomas to diagnose HL

Hodgkin Lymphoma: SarcoidSarcoid----likelikelikelikegranulomas

25 Hodgkin Lymphoma: Epitheliod cell Clusters

Hodgkin Lymphoma: Epitheliod cell Clusters

CD30 EBER

26 Lymphoma diagnosis

• Clinical features • Morphology • Immunophenotype • Molecular diagnosis

Useful antigens in haematopathology

• CD45 • Other • Other • CD30 • EBV • B-cell ‘specific’ • CD10 • LMP1 • CD19 • Bcl-2 • EBNA2 • CD20 • Bcl-6 • (EBER) • CD56 • CD79 ααα • ALK • CD57 • Pax-5 • c-myc • EMA • CD21 • OCT-2 / BOB1 • S100 • CD23 • Ig • CD68 • CD15 • CD163 • T-cell ‘specific’ • TdT • CD3 • Cyclin-D1 • CD5 • SOX-11 • CD2 • CD56 • CD7 • TIA-1, granzyme, • CD1a perforin • CD4 • CD8 • PD-1/CXCL-13 (TFH)

CD20 CD5 CD30 PD1 EBV LMP1

27 IHC panel for lymphoma diagnosis • CD45 • CD20 • CD79 ααα • (PAX-5) • kappa/lambda • CD3 • CD5 • CD30 • CD43 • CD163 (CD68) • Bcl-2 • Bcl-6 • CD23 (CD21) • Cyclin-D1 • Ki-67

cHL: the classical immunophenotype

• always perform IHC

• typical immunophenotype in most cases facilitates diagnosis

• aberrant cases can be difficult

28 Classical Hodgkin Lymphoma: IHC

CD15 CD30

CD45 PAX5

Classical Hodgkin Lymphoma: IHC

EBER ISH EBV LMP1

DK: ca. 25% EBV positive

29 IHC panel for cHL

• CD3, CD20, CD45 • CD30 (CD15) • PAX-5 • MUM1, BCL-6, BOB-1, OCT-2, CD57 (NLPHL?) • ALK (ALCL?) • EBV

• HRS cells in cHL are: - CD30+ (>95%) - CD15+ (~70%) - CD20−/+ (~20%) PAX5 dim+ (> 95%) - CD45− (> 95%) - MUM1 (> 95%) - OCT2−/+, BOB1−/+ (~30%) & BCL6−

cHL: CD30

• TNF-R family • ’Ki-1 antigen’ • Activation antigen • Normal expression: • activated parafollicular immunoblasts • virally infected cells (EBV) Reactive LN: activated B-cells • some clones stain plasma cells (Ber-H2)

30 cHL: CD30

• Essential, characteristic marker • 100% positivity in technically adequate cases • membranous & Golgi positivity in all HRS cells • IHC can be challenging • Far from unique to HL

Hodgkins lymphoma: CD30

cHL panel: Pax-5 (BSAP)

• B-cell nuclear transcription factor

• Normal – many B cells Reactive

• cHL: • Essential marker - > 95% of cases of CHL • Correlates with B-cell origin of HRS cells • Characteristic expression is nuclear and weak HL, LP • Strong expression / loss of expression - reconsider!

HL, NS

31 cHL panel: CD45

• B-cell nuclear transcription factor

• Normal – many B cells

• cHL • HRS cells in classical HL are negative • (LP: Popcorn cells positive)

HL, NS

HL, MC: CD45

Hodgkin lymphoma: Two biological entities

Lymphocyte predominance ca. 10 % • a clonal B-cell lymphoma with functional lineage gene expression

32 Blood 96:188996:1889----1899,1899, 2000

cHL vs. nodular lymphocyte predominant HL • NLPHL is in reality a BCL with intact B-cell gene expression • definition: • (neoplastic) LP cells in a nodular/diffuse/mixed nodular/diffuse background of small lymphocytes • accompanied by epithelioid histiocytes & (in the nodular areas) by networks of follicular dendritic cells •NLPHL is a neoplasm of the germinal center and LP cells express GC-B-cell markers and occupy a GC microenvironment

33 cHL vs. nodular lymphocyte predominant HL LP cells: • Positive for: • B-cell antigens - CD20 (strong, diffuse), CD45 (> 90% cases), CD79A, OCT2, BOB1, BCL6, Ki67, EMA (~50%) & IgD (ca. 25% cases) • Negative for: • T-cell antigens • CD10, CD15, CD30 (weak in some cases) • EBV •Background small lymphocytes are mixed B- & T-cells •B-cells have a MZ immunophenotype •T-cells in NLPHL often form rosettes around LP cells and have a T-follicular helper immunophenotype (e.g. positive for PD1) •In nodular areas, many CD21/CD23/CD35+ follicular dendritic cells are seen

Hodgkin Lymphoma, LP

Nodular pattern

34 Case 2-3

Case 2-4

35 Case 2-5

Nodular Lymphocyte Predominant Hodgkin Lymphoma: LP cell (a.k.a. Popcorn Cell / L & H cell)

36 LP cell (popcorn cell)

CD20

Case 2-6

37 CD20

CD57

38 Nodular Lymphocyte Predominance Hodgkin Lymphoma

Popcorn cell CD20CD20CD20 CD57CD57CD57

EMAEMAEMA PD1 CD3CD3CD3

Expression of transcription factors PaxPax----5,5, OctOct----1,1, OctOct----22 and BOB.1 in Hodgkin Lymphoma

McClune et. Al. Modern Pathology (2006) 19, 10101010––––1018101810181018

39 Nodular Lymphocyte Predominance Hodgkin Lymphoma

OCT2OCT2OCT2

BOB1BOB1BOB1

Differential Diagnosis of HL

• NLPHL vs progressive transformation of germinal centres

• cHL, lymphocyte-rich vs B-NHL

• Classical HL vs viral infection

• LPHL vs T-cell / Histiocyte Rich B-cell Lymphoma • Classical HL vs immunodeficiency-associated NHL • Classical HL vs T-cell lymphoma • Classical HL vs anaplastic large cell lymphoma • Classical HL vs anaplastc malignant tumour

40 Progressive Transformation of Germinal Centres

• Common & distinctive form of follicular hyperplasia

• PTGC seen in 20 % of NLPHL • precede • accompany • follow

• resembles NLPHL morphologically

• also seen together with cHL

Progressive Transformation of Germinal Centres

41 Progressive Transformation of Germinal Centres

Progressive Transformation of Germinal Centres

42 NLPHL vs PTGC

NLPHL vs. LRcHL

43 NLPHL vs. Lymphocyte rich cHL

HL, nod LP

CHL, LR

LPHL vs LRCHL: Architecture

LPHL LRCHL

• B cell nodules • B cell nodules • FDC meshwork • atrophic germinal centres • L&H (popcorn) cell • RS cells in mantle • CD57 rosettes

Foss et al. Der Pathologe 21:113 ---123, 2000

44 HL Immunohistochemistry: Dealbreakers • CD30 • CD45 • PAX5 • BOB1/OCT2 co-expression • MUM1 • ALK • (CD15)

cHL vs. non-Hodgkin lymphomas

Diffuse large B cell lymphoma (DLBCL) & subtypes • DLBCL-NOS • Primary mediastinal large B-cell lymphoma • T-cell/histiocyte-rich large B-cell lymphoma • EBV-positive DLBCL • B-cell lymphoma, unclassifiable, with features intermediate between DLBCL and cHL, (grey zone lymphoma)

T-cell lymphoma • Anaplastic large cell lymphoma • Peripheral T-cell lymphomas

45 cHL vs. DLBCL & variants

IHC panel – ADD: • CD79a, MUM1/IRF4, OCT2, BOB1 and BCL6 • DLBCL: - CD20+ CD79a+ PAX5+ OCT2+ BOB1+ (90% - 95%) - CD30−/+ (20%) - CD45+ (> 95%) - MUM1 +/− (~60%), OCT2+ (> 95%), BOB1+ - CD15−

cHL vs. primary mediastinal large B cell lymphoma

• challenging, but important (different treatments) • major morphological similarities • large neoplastic cells in a fibrotic/sclerotic stroma • overlapling immunophenotypes • difficult if the atypical large cells are rare • inadequate (small/core) biopsy material - ASK FOR A NEW BIOPSY

46 cHL vs. primary mediastinal large B cell lymphoma

• ADD to panel: CD23, CD79A, CD45, p63 • PMLBCL: • positive: CD30; CD20, CD23, CD45 and CD79A • CD30 usually weaker/more variable than in cHL • CD20 & CD79A positive in cHL, but usually in fewer numbers & fewer cases • CD23/p63 rare in cHL - expression strongly favours PMLBCL • CD15 is rare in PMBCL • EBV expression (EBER/LMP1) rare in PMLBCL

CD20 CD30 Ki-67↑↑ MUM-1

Gray zone lymphomas – (mediastinal & non-mediastinal)

BBB-B---cellcell lymphoma, unclassifiable –––intermediate between DLBCL and cHL

47 WHO: Aggressive B-cell Lymphomas 20016/17 • Precursor B-lymphoblastic leukemia/lymphoma (precursor B-ALL) • 8 genetic subtypes • Mature B-cell neoplasms • Diffuse large B-cell lymphoma, NOS • Germinal centreB-cell type • Activated B-cell type • T-cell / histiocyte-rich • Primary DLBCL of the CNS • Primary cutaneous DLBCL, leg type • EBV+ DLBCL, NOS • EBV+ mucocutaneous ulcer • Diffuse large B-cell lymphoma with chronic inflammation • Lymphomatoid granulomatosis • Primary mediastinal large B-cell lymphoma • Intravascular large B-cell lymphoma • ALK+ large B-cell lymphoma • Plasmablastic lymphoma • Primary effusion lymphoma • HHV8+ DLBCL, NOS • Burkitt lymphoma • Burkitt-like lymphoma with 11q aberration • High grade B cell lymphoma, with MYC , BCL2 &/or BCL6 rearrangements • High grade B cell lymphoma, NOS • B cell lymphoma, unclassifiable, indeterminant between DLBCL and classical Hodgkin lymphoma • Mantle cell lymphoma • Immunodeficiency associated lymphoma • Post transplantation • AIDS-associated

cHL vs. non-Hodgkin lymphomas

T-cell lymphoma • Anaplastic large cell lymphoma • Peripheral T-cell lymphomas

48 cHL vs. non-Hodgkin lymphomas

T-cell lymphoma • Anaplastic large cell lymphoma • Peripheral T-cell lymphomas

T-cell / Histiocyte Rich B-cell Lymphoma vs LPHL

49 TTT-T---cellcell Rich Large BB----cellcell Lymphoma

CD20

T-cell / Histiocyte Rich B-cell Lymphoma vs LPHL

• Differential diagnosis with diffuse LPHL • Difficult, sometimes impossible to distinguish • Difference is clinically important • Favours TCRBCL: • predominance of T-cells • scattered large neoplastic B-cells • no FDC aggregates • CD20+ CD79+ Ig ± EMA ± • CD15- CD30- • few CD57+ T-cells • Ig rearranged

50 cHL vs. non-Hodgkin lymphomas

T-cell lymphoma • Anaplastic large cell lymphoma • Peripheral T-cell lymphomas

cHL vs. anaplastic large cell lymphoma

• Previously a challenging differential diagnosis, now made easier in typical cases by immunophenotyping • Hallmark cells of ALCL may mimic Hodgkin cells • cHL with syncytial growth of many HRS cells mimics ALCL

ALCL – sinus pattern CD30 ALCL

51 cHL vs. anaplastic large cell lymphoma

• IHC: - Both strongly express CD30 - Often lack pan B-cell (cHL) and T-cell (ALCL) markers

• Expression of PAX5 strongly supports cHL • Expression of ALK oncogene is diagnostic of ALCL • EBV expression strongly favors cHL • CD15 favours cHL • Expression of T-cell associated markers or cytotoxic markers strongly favour ALCL. • THERE ARE EXCEPTIONS TO MOST RULES!

HL vs ALC: Immunophenotype & Genotype

cccHLcHLHLHLALK ---pospospos ALK ---negnegneg T/T/T/nullT/ null ---ALCALCALC T/T/T/nullT/ null ---ALCALCALC

ALKALKALK ---+++ --- EBVEBVEBV > 25 % ------CD30 ++++++ +++ CD15 ca. 90 % < 5 %< %--- / +/ + EMAEMAEMA ---ca. 50 % ca. 50 % PAX5 > 80 % ------CD20 ca. 25 % ------CD3CD3CD3 ca. 2 % + / ---+ / --- CD45 ---ca. 50 % ca. 50 % CD43 ---most + most + Granzyme/ 10 –––20 %20 %ca. 90 % ca. 70 % perforin TCR genes GGGRG RR RRR Ig genes R (single cell)cell)cell) GG G GGG

52 HL vs ALC: Immunophenotype & Genotype

cccHLcHLHLHLALK ---pospospos ALK ---negnegneg T/T/T/nullT/ null ---ALCALCALC T/T/T/nullT/ null ---ALCALCALC

cHL vs. peripheral TCL +/- follicular helper (TFH) phenotype (incl. AITL)

• Often challenging due to morphological and immunophenotypical overlap • HRS-like cells are common in peripheral T-cell lymphomas (PTCL), e.g. angioimmunoblastic T-cell lymphoma (AITL) • In AITL, the HRS-like cells are often EBV-positive associated with neoplastic TFH cells, accompanied by a cellular milieu reminiscent of MC-cHL or LR-cHL

53 cHL vs. peripheral TCL +/- follicular helper (TFH) phenotype (incl. AITL)

ADD to primary panel: • pan-T-cell markers (CD2, CD5, CD7 & CD43) • CD4/CD8; TFH markers (CD10/BCL6/PD1/CKCL13) • FDC stains (CD21/CD23)

• Expansions of the FDC meshwork is characteristic in AITL, but not in cHL • T-cell marker expression is rare in cHL • Loss of/aberrant T-cell antigen expression can identify PTCL

cHL in bone marrow and extra-nodal sites

• Often challenging due to uncharacteristic morphology and scant biopsy material • De novo extra-nodal cHL is rare and should only be diagnosed after satisfying stringent morphological and immunophenotypical criteria • Beware of mimics, particularly in the setting of immunodeficiency and iatrogenic induced lymphoproliferations

54 cHL in an immunosuppressive setting • Weak increased incidence of cHL in several immunosuppressive diseases (e.g. HIV-infection) • True cHL in this setting is very often EBV-positive, increasing the differential diagnostic complexity • Neoplastic EBV-positive (cHL & Hodgkin-like) lymphoproliferations increasingly identified in the context of immunosuppression • PTLD • Treatment-related, e.g methotrexate, Remicade (& other anti-TNFs) • Cancer • Some respond to removal of immunosuppression, some don’t • Diagnose with care :)

cHL vs. reactive paracortical hyperplasia

• Most attention paid to diagnosing neoplasia

• Often the most difficult problems are distinguishing reactive and neoplastic lymphoproliferations

• Especially in the ”early” biopsy

55 Reactive paracortical hyperplasia

Reactive paracortical hyperplasia

56 Reactive paracortical hyperplasia – CD30

Reactive paracortical hyperplasia – PAX5

57 Reactive paracortical hyperplasia – CD15