A Practical Guide To Diagnose B-Cell Lymphomas on FNAs. Nancy P. Caraway, M.D.
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1 A Practical Guide To Diagnose B-Cell Lymphomas on FNAs Nancy P. Caraway, M.D.
2 Major Factors Impacting Dx Lymphomas on Small Bxs Classification systems Immunophenotyping by multiprobe flow cytometry and immunohistochemistry Molecular analysis High resolution imaging Image-guided biopsies
3 Classification System Impacts Use of FNABs to Diagnosis Lymphomas Incorporation of cytomorphology, immunophenotype, genetic features, & clinical findings: 1994 Revised European-American Classification of Lymphoid Neoplasms (REAL) 2001 WHO Classification Updated 3 rd ed WHO Classification Updated 4 th ed WHO Classification Updated Re 4 th ed.
4 2017 WHO Classification of Haematopoietic and Lymphoid Tissues 17 new entities Mature B-cell NHL: T-cell NHL Hodgkin variants 5 5 WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues Rev 4 th ed.
5 Small Needle Bxs in Dx Lymphomas Assess lymphadenopathy Primary diagnosis of lymphoma Diagnose recurrence of lymphoma Assess for transformation to higher grade lymphoma Diagnose secondary neoplasms Assess adequacy of lesion for additional sampling (i.e., necrosis, protocol studies) Assess adequacy of material for ancillary studies (FCM, molecular) AFIP Tumors of the Lymph Nodes and Spleen Series 4.
6 Advantages of Concurrent Core Needle Bx & FNA Both can be done in an outpatient setting Increase likelihood of Diff Dx with cyto & histo findings correlated Optimal for directing sampling (necrotic) Can assess the adequacy for ancillary studies In this era of small biopsies, optimal to obtain as much tissue as possible with one procedure
7 Only 12% of FNA had concordant with excisional bx (most common dx were atypical, non-dx and lymphoma without subtype); 29% given an acceptable classification (REAL/WHO) One of the most referred to papers on FNAs in the workup of lymphomas HOWEVER, No ROSE, limited FLOW results, limited material, no workflow for triaging FNAs 2004
8 Diagnosing Lymphomas on FNABs Requires knowledge of the most recent WHO classification; know the limitations of FNAB Multiparameter approach ROSE helpful in obtaining optimal material/ aspirator willing to aspirate for ancillary studies &/or core needle biopsy Workflow for proper handling of specimens Takes time & effort (can t sign out in 1 or 2 days) Collaboration -hematopathologist with expertise in flow cytometry
9 Triaging Small Needle Biopsies 1st 2nd
10 Challenges of Dx Lymphomas by FNABs Identifying cases with partial involvement Assessment of pattern in FL and MCL Grading follicular lymphoma; however, low vs. high is often achievable Diffuse large B-cell vs. Burkitt lymphoma CLL/SLL with prominent pseudofollicles vs. Richter syndrome Composite lymphomas AFIP Tumors of the Lymph Nodes and Spleen Series 4.
11 Challenges of Dx Lymphomas on FNABs AITL vs reactive lymphadenopathy AITL vs peripheral T-cell lymphoma (PTCL) Variants of ALK-positive ALCL ALK- ALCL vs classic Hodgkin lymphoma ALK-ALCL vs CD30+ PTDL Natural killer cell lymphoma Nodular predominant HL vs progressive transformation of germinal centers Nodular predominant HL vs T-cell rich LBCL Subclassification of HL AFIP Tumors of the Lymph Nodes and Spleen Series 4.
12 FREQUENCIES OF B-CELL LYMPHOMA SUBTYPES IN ADULTS MCL CLL/SLL MALT FOLLICULAR LYMPHOMA DLBCL Diffuse large B-cell Follicular lymphoma MALT lymphoma Mantle cell lyphoma CLL/SLL Primary med large B-cell High grade B, NOS Burkitt Splenic marginal zone Nodal marginal zone Lymphoplasmacytic WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. Ed. Swerdlow S.
13 ??? Future of FNABs FNABs are optimal for mutational analysis in era of small bx & personalized medicine Aid in the further subtyping of lymphomas Prognostic marker Therapeutic indicator Need to collaborate with Hematopathology Advocates for LN FNABs
14 Strategy For Obtaining Optimal Material for Ancillary Studies Immediate assessment with optimal preparations (Pap & DQ smears) Triage material for ancillary studies (optimal 5-10 million cells; 10 ml of RPMI+ media) Correlate cytology & ancillary studies Use WHO classification Coulter counter: evaluates # of mononuclear cells
15 Multiparameter Approach Clinical history - age, prior neoplasms, LN site & size, localized vs. general lymphadenopathy, systemic symptoms Cytomorphology - major cell type, cellularity, arrangement, background Ancillary studies
16 Ancillary Studies Flow Cytometry -Routinely send on all cases with clinical/radiographic suspicion of a lymphoma except for Hodgkin lymphoma** Gene Rearrangements Selected cases that have been difficult to classify by immunophenotyping or suspected T-cell lymphomas (rinse) Fluorescence in situ hybridization (FISH) Selected cases suspected lymphoma with a characteristic translocation that is difficult to classify (DQ slides, cytospin, cell block)
17
18 Lymph Node Cell Population Dendritic reticulum cells Cleave cells Tingible body macrophage Large non-cleaved cell
19 Differential Diagnosis of Monomorphous Lymphoid Population Based on Size Monomorphous Population of Lymphoid Cells Small Cells Reactive Lymph Node Small Lymphocytic Lymphoma Lymphoplasmacytic Lymphoma Follicular Lymphoma Mantle Cell Lymphoma Intermediate Cells Lymphoblastic Lymphoma Burkitt Lymphoma Large Cells Large Cell Lymphoma Follicular Lymphoma Grade 3 Blastoid variant mantle cell lymphoma
20 LYMPHOMAS COMPOSED OF SMALL CELLS?
21 Case 1: 81 y/o with right axillary mass Paraimmunoblast Paraimmunoblast
22 Case 1: Differential Diagnosis Malignant Small lymphocytic lymphoma Follicular lymphoma, lowgrade Mantle cell lymphoma Lymphoplasmacytic lymphoma Hodgkin lymphoma (nodular lymphocyte predominant) Benign Reactive lymph node
23 Differential Diagnosis of Small Cell B-Cell Lymphomas LYMPHOMA IMMUNOPHENOTYPES CYTOGENETICS CD5 CD23 CD10 CD20 Small Lymphocytic Trisomy 12, del 13q14 Lymphoplasmacytic No specific abnormality Mantle Cell t(11;14)(q13;32) Marginal Zone MALT Trisomy 3; t(11;18)(q21;q21) Follicular, low grade - -/+ + + t(14;18)(q32;q21)
24 Case 1: Ancillary Studies 10 4 Flow Cytometry: CD20+ (dim), CD19+, CD5+ CD23+, CD200+, CD10-, FMC CD CD19 KAPPA LAMBDA 10 3 CD CD19 CD5 (weak +) Ki-67
25 Case 1: Ancillary Studies 10 4 Flow Cytometry: CD20+ (dim), CD19+, CD5+ CD23+, CD200+, CD10-, FMC-7- CD FLOW CD19 KAPPA LAMBDA CYTOSPIN CD CD19 CD5 (weak +) Ki-67
26 Small Lymphocytic Lymphoma Accounts for 7% of non-hodgkin lymphomas Often associated with chronic lymphocytic leukemia Majority of patients are >50 years and have involvement of lymph nodes, liver and spleen Clinical course is indolent but not curable; may transform to large cell lymphoma & rarely HL
27 Things To Think About Typically SLL can be differentiated from other small lymphomas with immunophenotyping SLL with prominent pseudofollicles vs SLL accelerated phase /transformed LBCL may be challenging
28 Richter Syndrome 2-8% of pts with SLL/CLL transform 2-8% Diffuse large B-cell lymphoma 0.5% Hodgkin lymphoma Sudden onset of rapidly progressive lymphadenopathy & B symptoms: fever, night sweats, &/or weight loss Common sites: lymph nodes, BM, PB, spleen DLBCL: clonally related or unrelated to SLL/CLL immunophenotype Poor prognosis
29 Transformed Large B-cell Lymphoma in Patient with Hx of SLL/CLL TBM
30 FNA of Axillary LN with hx of CLL/SLL &
31 Metastatic Merkel to LN with SLL Squamous cell ca, Merkel cell ca, adenocarcinoma have been describes met to LN with SLL/CLL
32 Case 2: 70 y/o man with a 1.8 cm axillary nodule
33 Case 2: Differential Diagnosis Malignant Small lymphocytic lymphoma Follicular lymphoma, lowgrade Mantle cell lymphoma Lymphoplasmacytic lymphoma Hodgkin lymphoma (nodular lymphocyte predominant) Benign Reactive lymph node
34 Case 2: Ancillary Studies Immunophenotyping: Kappa light chain restricted, CD20+, CD19+, CD5+, CD10-, CD23-, CD200- H & E CB CyclinD1 Sox11
35 Mantle Cell Lymphoma Comprises about 4% of all non-hodgkin lymphomas Occurs primarily in men in 7 th decade and presents with generalized lymphadenopathy MCL should be distinguished from other small cell B-cell lymphomas, because clinically more aggressive Classic type composed of monomorphous small to medium-sized lymphocytes with irregular nuclei; other variants including blastoid, pleomorphic, MCL-HPI
36 Mantle Cell Lymphoma Characteristic t(11;14)(q13;q32) translocation Reciprocal translocation results in the juxtaposition of the transcriptional enhancer on IgH locus (14q32) next to the CCND1 gene (11q13) which is thought to lead to overexpression of cyclin D1 No Fusion Dual Fusion
37 Things to Think About MCL is more aggressive than other small cell lymphomas Immunophenotyping important Patterns cannot be determined on FNAs (MCLs with mantle-zone pattern are considered to be more indolent and maybe tx ed differently than those with nodular & diffuse patterns) Blastoid & pleomorphic variants are more difficult to diagnosis on FNABs
38 Things to Think About CyclinD1 staining on cytospins is not as reliable as on cell block preparations Immunstaining for Cyclin D1 has been reported in some cases of HCL& plasma cell myeloma as well as histiocytes & many types of ca (breast, thyroid, colorectal, urinary bladder) Immunostaining for SOX11 has been reported in some cases of HCL, Burkitt lymphoma, & plasma cell myeloma Need Ki-67 to detect MCL with HPI, can t tell on cytomorphology alone
39 Case 3: 68 y/o man with a 9 cm pelvic mass
40 Case 3: Differential Diagnosis Benign Reactive Hyperplasia Malignant Follicular lymphoma MCL, SLL, MZL Partial involvement of lymphoma
41 Case 3: Ancillary Studies Flow Cytometry: Positive: Kappa light chain restricted, CD20, CD19, CD10, CD23 Negative: CD5, CD200, CD30 Ki-67: Approximately 10% of cells staining CD20 ->
42 Follicular Lymphoma Accounts for 35% of non-hodgkin B-cell lymphomas in US and 22% worldwide Usually occurs in older adults and primarily involves lymph nodes Composed of follicle center B-cells (cleaved and non-cleaved cells) type cells vary with grade
43 Follicular Lymphoma Immunophenotype: CD20+, CD19+, CD10+, CD5-, CD23+ (BCL-2+ and BCL- 6+; both need architecture to evaluate ) Update WHO classification defines 3 histologic grades based on number of centroblasts/hpf in tissue On cytology Low-grade (grade 1 & 2) High-grade (grade 3/Large cell lymphoma of follicle center cell origin)
44 Follicular Lymphoma Characteristic t(14;18)(q32;q21) translocation Reciprocal translocation results in the fusion of the IgH gene and the BCL2 gene leading to overexpression of the BCL2 protein On FISH, fusion signal; can be done on cytospin Dual fusion indicative of t (14;18)
45 Sensitivity for dx FL was 89% & 66% of LG FL; later increase to 94% if performed Review of not-graded: 45% LG, 35 % indetermine due to polymorphous lymphoid cells with increased large cells, & 20% scant cellularity Part-time faculty did not grade 54% vs full-time cytopathologist 19%
46 FNA: Follicular lymphoma, low-grade Bx: Follicular lymphoma, grade 1
47 FNA: Follicular lymphoma, NOS Bx: Follicular lymphoma, grade 1-2
48 FNA: Follicular lymphoma, suggestive of gr. 2-3 Bx: Follicular lymphoma, grade 3A
49 Case 3: Things to Think About FNA cannot reliably determine the pattern in FLs Grading FL on FNAs Low grade (grades 1 & 2) High grade (grades 3) Distinguishing between grades 1 & 2 or 2 & 3A can be problematic Subset with polymorphous population with increased large cells may be difficult to grade Can t reliably differentiate FL HG from CD10+ diffuse large cell lymphoma
50 Case 4: 40 y/o man with abdominal lymphadenopathy
51 Case 4: Differential Diagnosis Burkitt lymphoma Diffuse Large B-cell lymphoma Morphologic variants Molecular variants Germinal center B-cell subtype Activated B-cell subtype High-grade B-cell Lymphoma High-grade B-cell lymphoma with MYC & BCL2 &/or BCL6 rearrangements High-grade B-cell lymphoma, NOS
52 Case 4: Ancillary Studies Flow Cytometry Positive for CD20, CD22, CD19, CD10, CD38 Negative for CD5, CD3 Kappa light chain restricted Immunocytochemistry Ki-67 > 95% cells staining
53 Burkitt Lymphoma B-cell Ag+, CD10+, EBER+ CD38+, (need architecture to assess BCL6+, BCL2-/+), TdT-, >90% Ki-67 Often associated with t(8;14)(q24;q32) results in MYC at 8q24 adjacent to heavy chain region on 14q32 and less frequently with t(2;8) or t(8;22) EBER Ki-67
54 Case 4: Things To Think About CD10+ BL can be misinterpreted as large B- cell lymphoma (LBCL) of germinal center origin Burkitt-like lymphoma with 11q aberration Some DLBCL can have round nuclei, cytoplasmic vacuoles, high mitotic index, and tingible body macrophages mimicking BL High-grade B-cell Lymphoma High-grade B-cell lymphoma with MYC & BCL2 &/or BCL6 rearrangements High-grade B-cell lymphoma, NOS
55 Diffuse Large B-cell Lymphoma with high-grade features
56 Case 5: 63 y/o woman had a hx of breast cancer 2 yr ago; now has a 2 cm axillary LN Ki-67
57 Case 5: Breast cancer 2 yrs earlier
58 Case 5: Rt. Breast FNA: Large B-cell Lymphoma (FCM: kappa restricted population positive for CD19, CD20, CD22, CD38, & CD44; negative for CD10, CD5, CD23, CD30; Ki67 >90%) Rt. Axilla FNA: Reactive polymorphous lymphoid tissue (FCM: Polytypic)
59 Large B-cell Lymphoma LBCL- no longer adequate dx for tx Morphologic variants: centroblastic, immunoblastic, anaplastic, other rare variants Molecular Subtypes Germinal center B-cell Better survival Activated B-cell Worse survival GCB ACB WHO Classification of Tumors of Haematopoietic and Lymphoid Tissues 2017
60 Case 5: Things to Think About Previous clinical hx is important Cancer doesn t necessarily mean carcinoma Compare with previous cytology/histology Ancillary studies may be helpful in selected cases (unexplained lymphadenopathy) Ki-67 is elevated in reactive LNs
61 Case 6: 22 y/o man with a hx of Hodgkin lymphoma and now has 2 cm neck node
62 Case 6: Differential Diagnosis Benign Reactive LN Lymphoma Hodgkin lymphoma B-cell lymphoma Follicular lymphoma Marginal zone lymphoma T-cell lymphoma
63 Case 6: Ancillary Studies Flow cytometry: polytypic B-cells, immunophenotypically unremarkable T-cells and NK-cells Note: Fine needle aspiration with flow cytometry is an insensitive means of detecting certain types of lymphoma (most notably, T- cell rich large B-cell lymphoma, lymphocyte predominant Hodgkin lymphoma and some subtypes of T-cell lymphoma).
64 Reactive Hyperplasia Infections Draining primary tumors Following biopsy Autoimmune Idiopathic
65 Case 6: Histology
66 Case 6: Cytology & Histology FNA Dx: Atypical lymphoid proliferation, favor reactive process Excisional Bx: Prominent follicular and interfollicular hyperplasia with monocytoid B- cells and multiple neutrophilic microabscesses Comment: Warthin-Starry stain shows clusters & single rod-like organisms (possible Bartonella) in some microabscesses, cultures had no growth
67 Cat Scatch Disease Necrotizing inflammation cause by Bartonella B. henselae causes CSD in mostly immunocompetent pts 90% of pts report contact with cats Bacilli transmitted to humans through bite or scratch of infected cat Granuloma formation results from activation and accumulation of histiocytes Bacilli identified by Warthin-Starry stain; also serology
68 Case 6: Things To Think About Immunophenotyping can be helpful in assessing clonality FNA cannot reliably assess reactive lymphadenopathies with architectural features (Castleman dz, Kimura dz, Kikuchi-Fujumoto dz, Toxoplasmosis, others) Aspirated material can be sent for cultures
69 Conclusions A multiparameter approach is imperative to diagnose lymphoproliferative disorders by FNAB ROSE for triaging material for ancillary studies is optimal
70 Conclusions Most B-cell lymphomas can be subclassified with the aid of immunophenotyping and proliferation indices When in DOUBT, get more tissue, core it or take it OUT
71 THANK YOU FOR ATTENTION!
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