Pearls and pitfalls in interpretation of lymphoid lesions in needle biopsies

Pearls and pitfalls in interpretation of lymphoid lesions in needle biopsies Megan S. Lim MD PhD University of Pennsylvania October 8, 2018

Objectives To understand how the trend toward less invasive lymph node biopsies impacts the practice of hematopathology To learn how to triage small specimens to maximize diagnostic utility To understand the application of ancillary tests to lymph node core biopsies To raise awareness of common pitfalls in diagnosing lymphoma on core biopsies 2

Core Needle Lymph Node Biopsy https://www.cigna.com/individuals-families/health-wellness/hw/medical-tests/lymph-node-biopsy-hw232506 3

Evolution of the core needle biopsy University of Michigan Experience 2004 2012 Increase FNA 181 574 300% CNB 24 126 500% Excision 140 (40% of total LN Bx) 170 (20% of total LN Bx) Current state Dramatic increase in FNA and core needle biopsies for suspected lymphoma 4

Why? Ultrasound guided approaches for LN located in deep, inaccessible sites Better ancillary techniques Improved diagnostic criteria Better yield of diagnosis Increased opportunity for disease monitoring 5

The Problem Is The WHO classification system based on histologic findings from excisional biopsies. 6

Core needle biopsies (CNB)in lymphoma diagnosis Multi-institutional study Determine the diagnostic approach most likely to yield a definitive and accurate diagnosis Total 532 patients with CNB of LN evaluated Czader M et al., USCAP abstract 2014 7

Core needle biopsies (CNB)in lymphoma diagnosis Multi-institutional study RESULTS 23% no definitive diagnosis 83% specific WHO diagnosis made; longer CNB and larger needle gauge IHC performed on 72% FCM performed on 69% FNA yielded better material for FCM than tissue cores Czader M et al., USCAP abstract 2014 8

Core needle biopsies (CNB)in lymphoma diagnosis Multi-institutional study RESULTS 128 CNB diagnosis was confirmed in 40% of LN excision biopsies 50% cases showed minor changes in diagnosis 9% cases showed major changes in diagnosis 77% of diagnosed as lymphoma were treated based on CNB diagnosis Czader M et al., USCAP abstract 2014 9

Core needle biopsies (CNB)in lymphoma diagnosis Multi-institutional study Conclusions Definitive WHO diagnosis is rendered in the majority of CNB cases Significant number are treated based on CNB diagnosis 23% of CNB cases are non-diagnostic 10% with significant diagnostic discrepancies on LNEx Aggregate core lengths improves diagnostic accuracy Czader M et al., USCAP abstract 2014 10

Core needle biopsies are here to stay! 11

Pearls and pitfalls in the diagnostic workup of small lymph node biopsies I. Nodular lymphoid proliferations II. Assessment of transformation in CNB III. Polymorphous lymphoid proliferations IV. Lymphoid proliferations with Reed- Sternberg-like cells V. Lymphoid infiltrates with extensive sclerosis VI. Diffuse large cell proliferations 12

Nodular lymphoid proliferations Reactive Follicular hyperplasia PTGC Castleman disease B-cell Non-Hodgkin lymphoma Follicular lymphoma Chronic lymphocytic leukemia/ small lymphocytic lymphoma Mantle cell lymphoma Marginal zone lymphoma Mantle cell lymphoma Neoplastic Hodgkin lymphoma NLP Hodgkin lymphoma Classical Hodgkin lymphoma T-cell Follicular T-cell lymphoma Nodal PTL with TFH phenotype Angioimmunoblastic T cell lymphoma 14

CNB: left neck mass of 55 year old man 15

Cytologic features 16

Differential Diagnoses Reactive Small B cell lymphomas T cell lymphoma of small T cells Precursor lymphoid neoplasms 17

Proliferation centers in CLL/SLL 18

Immunophenotyping All suspected lymphoma samples should have basic immunohistochemistry and / or FCM Flow cytometry if there is viable tissue IHC if there is sufficient tissue even if necrotic 19

What will FCM tell us? Exclude lymphoblastic lymphoma/leukemia Determine if there is a clonal B cell population Determine the presence of CD5, CD10 + B cells or clonal plasma cells Ten-color flow cytometry can distinguish many subtypes of B-cell lymphoma Determine the presence of T cell antigen aberrancies 20

FCM is not very good at identifying: The presence of NLPHL or CHL Identifying and characterizing large B-cell neoplasms The presence of a clonal T cell population 21

Association between length of tissue core and cell number for FCM and diagnostic concordance Chi, P et al., Cytometry Part B 2018 22

Concordance between morphology and flow cytometry Chi, P et al., Cytometry Part B 2018 23

Back to our case 24

CD10 BCL6 25

CD5 26

Cyclin D1 Cyclin D1 27

Diagnosis Mantle cell lymphoma Other CD5+ B-cell lymphomas CLL/SLL (beware of BTKi that downregulate CD5,CD43) De novo CD5+ DLBCL Marginal zone lymphoma (rare) 28

Can we assess transformation in small biopsies? can be helpful in evaluating suspected transformation to higher grade disease but should not be definitive 29

CNB of inguinal node of 69 year old man with history of FCL 30

CD20 highlights nodules and diffuse areas 34

CD3 highlights nodules and diffuse areas 35

BCL-6 highlights nodules and diffuse areas 36

Ki67 demonstrates 2 different populations Ki67 5-10% Ki67 40-80% 37

Diagnosis B-cell lymphoma of germinal center origin Definitive subclassification cannot be made Raise concern for: FCL and possible DLBCL FCL grade 3 Low grade FCL with high proliferation index 38

Be aware of FCL with high proliferation index 39

FCL with high proliferation index CD20 CD3 Ki67 Flow cytometry: CD10+, CD19+, CD20+, FMC7+, lambda+, CD5-, CD23-40

60 year old woman with nasopharyngeal No previous medical history presented to the primary care physician lesion Diffuse, destructive submucosal infiltrate Significant necrosis of the superficial mucosa 42

Histologic features Large pleomorphic cells with variably prominent nucleoli Numerous mitotic figures and numerous apoptoses with occasional Reed- Sternberg-like cells 43

occasional Reed-Sternberg-like cells 44

Polymorphous lymphoid proliferations Reactive Paracortical hyperplasia Infectious mononucleosis Wegener s Granulomatous lymphadenitis Neoplastic Hodgkin lymphoma NLP Hodgkin lymphoma Classical Hodgkin lymphoma Non-Hodgkin lymphoma B-cell T cell rich histiocyte rich B cell lymphoma EBV+ polymorphic lymphoma FCL with RS-like cells Composite lymphomas T-cell Peripheral T cell lymphoma,nos Anaplastic large cell lymphoma NK/T cell lymphoma Angioimmunoblastic T cell lymphoma

CD20 Immunohistochemical studies CD30 46

Immunohistochemical studies Antibody Results Antibody Results CD15 Negative CD20 Var Positive EMA Negative CD19 Positive, weak CD10 Negative CD138 Positive, weak T cell antigens CD56 TIA1 Negative Negative Negative PAX5 CD45 CD30 Var Positive Var Positive Large cells HHV8 Negative 47

EBER-1 Diagnosis EBV-positive diffuse large B-cell lymphoma 48

Other EBV+ Large Cell Lymphoma Plasmablastic lymphoma Primary effusion lymphoma Lymphomatoid granulomatosis Pyothorax associated lymphoma T/NK cell lymphoma AITL Classical Hodgkin lymphoma 49

Pearls and pitfalls in the diagnostic workup of small lymph node biopsies I. Nodular lymphoid proliferations II. Assessment of transformation in CNB III. Polymorphous lymphoid proliferations IV. Lymphoid proliferations with Reed-Sternberg-like cells V. Lymphoid infiltrates with extensive sclerosis VI. Diffuse large cell proliferations 50

71-year-old woman with retroperitoneal mass 51

Differential Diagnosis Reactive Retroperitoneal fibrosis Chronic inflammation Neoplastic Classical Hodgkin lymphoma B-cell non-hodgkin lymphoma Peripheral T-cell lymphoma 53

CD20 CD3 54

CD30 CD15 FASCIN 55

LCA BCL6 56

Immunophenotype Immunostain Result CD20 + small and large cells CD30 + large cells CD15 - Fascin - BCL6 + small and large cells CD10 - LCA + small and large cells PAX-5 + small and large cells 57

Differential Diagnosis Reactive Peripheral T-cell lymphoma B-cell non-hodgkin lymphoma Classical Hodgkin lymphoma Composite lymphoma 58

Immunophenotype CHL B-cell NHL Morphology of large cells Sheets of small B-cells Sclerosis BCL6 + CD30 + LCA + 59

Reed-Sternberg cells--chl? No inflammatory background CD20 should not be stronger than CD30 on R-S cells in CHL R-S cells are negative for LCA & BCL6 Fascin should be positive on R-S cells Good evidence against CHL if negative Can be positive on other lymphomas (DLBCL) 60

Additional information about this case Small paratrabecular lymphoid aggregate in core biopsy Monoclonal CD10 positive B-cell population by FCM 61

Diagnosis Follicular lymphoma (grade 1-2) Focal transformation? R-S cells counted as centroblasts (area of grade 3 or DLBCL?) Very small area with >15 R-S cells/hpf 62

Features supporting FCL Sclerosis FCL in retroperitoneum OFTEN sclerotic Immunophenotype BCL6 + CD10 + in marrow 63

NHL with R-S-like cells Low grade B-NHL Follicular lymphoma CLL with HRS cells Marginal zone lymphoma Mantle cell lymphoma DLBCL Primary mediastinal B-cell lymphoma T-cell rich histiocyterich B-cell lymphoma 64

T-NHL with R-S-like cells Anaplastic large cell lymphoma Angioimmunoblastic T-cell lymphoma Peripheral T-cell lymphoma, NOS CD30+ CD15+ 65

Other CD30+ Large Cell Lymphoma Diffuse large B-cell lymphoma, anaplastic variant Classical Hodgkin lymphoma (MC, LD) Plasmablastic lymphoma Primary effusion lymphoma Pyothorax associated lymphoma NK/T cell lymphoma 66

FCL with extensive sclerosis 68

Diffuse large B-cell lymphoma with sclerosis are germinal center derived CD20 FCM: CD10 positive lamda restricted B cells without CD5, CD23 and CD38. 69

Primary mediastinal B-cell lymphoma CD20 70

ALK+ anaplastic large cell lymphoma presenting with retroperitoneal fibrosis ALK1 71

45 year old man with lesion in oral pharynx 73

Differential Diagnoses Reactive Infectious Viral Neoplastic Hematopoietic Non-Hodgkin lymphoma diffuse large B-cell lymphomas with anaplastic features, Anaplastic large cell lymphoma (ALK-positive and ALK-negative) Extramedullary myeloid tumor Hodgkin lymphoma Anaplastic myeloma Non-hematopoietic Melanoma Carcinoma (anaplastic variants) Inflammatory myofibroblastic tumor 74

Diffuse large B-cell lymphoma CD20 CD3 75

Subclassification of DLBCL is possible on small lymph node biopsies DLBCL GC type DLBCL Non-GC type Prognostic information Therapeutic implications 76

DLBCL, non-germinal center type CD20 CD3 CD10 MUM1 77

Synctitial variant of classical Hodgkin lymphoma CD30 78

Differential Diagnoses Diffuse large cell infiltrate DLBCL Double hit lymphoma B-cell lymphoma intermediate Blastoid mantle cell lymphoma Marginal zone lymphoma Anaplastic large cell lymphoma Histiocytic proliferations (Sinus histiocytosis, LCH, histiocytic sarcoma) Flow cytometry has low sensitivity in the diagnosis of Diffuse large B cell lymphoma 79

Take home points 80

Lymphoma work-up for small biopsies FNA: can be helpful and also submitted for FCM Tiny cores only: submit entire specimen for permanent sections Larger or multiple cores or fragments: attempt complete lymphoma work up, priority is permanent sections for histology 81

CNB in lymphoma diagnosis Definitive WHO diagnosis is rendered in the majority of CNB cases Significant number are treated based on CNB diagnosis 23% of CNB cases are non-diagnostic 10% with significant diagnostic discrepancies on LNEx Aggregate core lengths improves diagnostic accuracy Czader M et al., USCAP abstract 2014 82

Core biopsy is useful Inaccessible tumours such as those in the retroperitoneum Do allow a minimal assessment of architecture and immunophenotyping Allows definitive diagnosis and subclassification if IHC/FCM is incorporated appropriately Remember the utility and caveats about antibodies against tumor-specific proteins (ALK, IDH1, and BRAF VE1) 83

Core Biopsy Is Not useful Where much of the lymphoid infiltrate is reactive T-cell rich B-cell lymphoma MALT Angioimmunoblastic-type peripheral T-cell lymphoma Classical Hodgkin lymphoma Nodular lymphocyte predominant Hodgkin lymphoma Tumors where the architecture is important Low grade non-hodgkin lymphoma Nodular lymphocyte predominant Hodgkin lymphoma 84

When to ask for more tissue? Inadequate tissue: tiny, crushed, necrotic Flow is negative but there are lots of B cells or T cells by IHC Flow is negative but there is a polymorphous infiltrate DDX of CHL vs PTCL FCL with high proliferation index 85

Role of Cytogenetic and Molecular Genetic Analysis of SLN Biopsies FISH can be performed on touch preps Cytogenetic evidence is essential for the diagnosis of Burkitt lymphoma FISH should be used as adjuncts to diagnosis and not requested as a routine investigation on all samples PCR results should not form the sole basis for a diagnosis 86

Be cautious in interpretation of molecular data: unexpected findings Clonal populations or chromosomal translocations can be observed in non-neoplastic conditions Low level disease or pre-neoplastic conditions Multiple clonal populations can be present in one lesion Double hits in morphologically low grade B-cell lymphomas 87

Histopathology Thin sections, 4 um More Testing with Less Tissue Cell suspension Aspirate Cell Block Aspirate Smear Diff-Quik Stained Morphologic analysis Immunohistochemistry Fluorescent in situ hybridization Core needle biopsy Imprints for Wright stain Immunocytochemistry Fluorescent in situ hybridization 10-color flow cytometry Cytogenetics Cytogenomics NGS

Thank you for your attention! 89