NORTH CAROLINA SOCIETY OF PATHOLOGISTS

Transcription

1 NORTH CAROLINA SOCIETY OF PATHOLOGISTS 2018 Annual Meeting and Shelley Lecture April 13-14, 2018 The Ballantyne Hotel Charlotte, NC FOCUS ON HEMATOPATHOLOGY SATURDAY HANDOUTS This continuing medical education activity is jointly provided by the North Carolina Society of Pathologists and Southern Regional Area Health Education Center.

2 A Practical Approach to the Diagnosis and Classification of Low Grade B-cell Lymphoproliferative Disorders Nathan D. Montgomery, MD, PhD Assistant Professor University of North Carolina School of Medicine Department of Pathology & Laboratory Medicine Divisions of Hematopathology & Molecular Genetic Pathology Speaker Disclosure The speaker has recent financial relationships with the following commercial interests: Ventana Biomedical Systems (prior funding) ArcherDx (reagents for pilot studies) The content of this presentation is not related to either relationship. 2 Low grade Lymphoproliferative Disorders Non-Hodgkin lymphomas were separated into low, intermediate, and high grade categories in some older classification schemes (e.g. The Working Formulation) In contrast, the current World Health Organization (WHO) Classification does not separate lymphomas into overarching categories based on histologic grade In practice, however, we regularly build a differential diagnosis based on whether lymphoproliferative disorders (LPDs) are predominantly small or large B-cells Here, we will focus on the former group of B-cell lymphomas (i.e. morphologically low grade) 3 1

3 Outline and Objectives Objective 1: Review common entities in the differential diagnosis of small/low grade B-cell lymphomas and leukemias Follicular lymphoma Small lymphocytic lymphoma/chronic lymphocytic leukemia Mantle cell lymphoma Nodal and extranodal marginal zone lymphomas Lymphoplasmacytic lymphoma Objective 2: To provide a broad diagnostic approach to diagnosis and classification of low grade B-cell lymphomas and leukemias Objective 3: To review less common morphologic and immunophenotypic variants that may cause diagnostic uncertainty Objective 4: To highlight the growing appreciation of clonal but not malignant lymphoid proliferations 4 Classification of morphologically low grade B-cell LPDs: 1994 vs REAL Classification B-cell chronic lymphocytic leukemia / Prolymphocytic leukemia / Small lymphocytic lymphoma Lymphoplasmacytic lymphoma Mantle cell lymphoma Follicle center lymphoma Marginal zone lymphoma - MALT type - Nodal type Splenic marginal zone lymphoma Hairy cell leukemia 5 Blood : Classification of morphologically low grade B-cell LPDs: 1994 vs REAL Classification 2017 WHO Classification B-cell chronic lymphocytic leukemia / Prolymphocytic leukemia / Small lymphocytic lymphoma Lymphoplasmacytic lymphoma Mantle cell lymphoma Follicle center lymphoma Marginal zone lymphoma - MALT type - Nodal type Splenic marginal zone lymphoma Chronic lymphocytic leukemia / Small lymphocytic lymphoma B-cell prolymphocytic leukemia Lymphoplasmacytic lymphoma Mantle cell lymphoma Follicular lymphoma Pediatric follicular lymphoma Extranodal marginal zone lymphoma Nodal marginal zone lymphoma Splenic marginal zone lymphoma Splenic B-cell lymphoma/leukemia, unclassifiable Hairy cell leukemia Hairy cell leukemia 6 Harris et al., Blood : ; Swerdlow et al., IARC

4 Classification of morphologically aggressive mature B-cell lymphomas: 1994 vs WHO Classification 1994 REAL Classification Diffuse large B-cell lymphoma Burkitt s lymphoma High grade B-cell lymphoma, Burkitt-like Diffuse large B-cell lymphoma, NOS T-cell/histiocyte-rich large B-cell lymphoma Primary DLBCL of the CNS Primary cutaneous DLBCL, leg type EBV-positive DLBCL, NOS Primary mediastinal large B-cell lymphoma Intravascular large B-cell lymphoma ALK-positive large B-cell lymphoma DLBCL associated with chronic inflammation Fibrin-associated DLBCL Large B-cell lymphoma associated with IRF4 rearrangement Plamablastic lymphoma Primary effusion lymphoma HHV8-pos large B-cell lymphoma, NOS Burkitt lymphoma Burkitt-like lymphoma with 11q aberration High grade B-cell lymphoma associated with MYC and BCL2 rearrangement High grade B-cell lymphoma, NOS B-cell lymphoma, unclassifiable, with features intermediate between DLBCL and CHL 7 Harris et al., Blood : ; Swerdlow et al., IARC Approach to diagnosis (and then classification) of morphologically low grade B-cell LPDs Clinical information Morphology Immunophenotype Light chain restriction (flow, stains) Immunophenotypic aberrancy Genetics Cytogenetics Clonality studies Sequencing data 8 Classification of morphologically low grade B-cell LPDs: immunophenotype Low grade B-cell LPD CD10 + CD5 + CD5 - / CD10-9 3

5 Classification of morphologically low grade B-cell LPDs: immunophenotype Low grade B-cell LPD CD10 + CD5 + CD5 - / CD10 - Follicular lymphoma, grade Follicular Lymphoma Clinical and epidemiological features ~20% of all lymphomas Less common in eastern Europe, Asia, and resource limited settings Nearly always has extensive nodal involvement at presentation Exceptionally rare in pediatric patients (and should always raise the possibility of an alternative diagnosis) 11 Follicular Lymphoma: Morphology Reactive lymphoid hyperplasia Follicular lymphoma 12 4

6 Follicular Lymphoma: Morphology Morphologic features Classically back-to-back follicles with attenuated mantle zones Follicular lymphoma Lack of polarization in the follicle centers Can be highlighted by Ki Follicular Lymphoma: Ki-67 Reactive lymphoid hyperplasia Follicular lymphoma 14 Follicular Lymphoma: Immunophenotype Immunophenotype Germinal center origin CD10+ BCL6+ RLH Immunophenotypic aberrancy: Expression of BCL2 in > 90% of cases Rare cases don t read the book (e.g. < 5% CD5 pos) FL CD10 BCL2 15 5

7 Follicular Lymphoma: Genetics Genetics t(14;18)(q32;q21) identified in 90% of grade 1-2 FL Less commonly, BCL2 translocations to light chain loci (2p12 and 22q11) Individual gene mutations increasingly appreciated and impact outcomes but not yet part of routine practice in most centers (Pastore et al., Lancet Oncol : ). 16 Follicular Lymphoma: Grading Cytologic features Mixture of small cleaved centrocytes and larger centroblasts Grading defined by centroblast count in the follicles Grade Definition centroblasts per HPF centroblasts per HPF 3a > 15 centroblasts per HPF centrocytes present 3b > 15 centroblasts per HPF No centrocytes present Standard high power field = mm 2 Image courtesy of Y. Fedoriw 17 Cells of the follicle center Follicular dendritic cell Centrocyte Centroblast Image courtesy of Y. Fedoriw 18 6

8 Grade and Ki-67 Ki-67 correlates with histologic grade but imperfectly Mixed data in the literature regarding the clinical significance of grade 1-2 FL with higher proliferation index by Ki Wang SA et al. Am J Surg Pathol : Grade and Ki-67 Diagnosis: Follicular lymphoma, grade 1-2, with elevated proliferation index by Ki-67 staining (See Comment) Comment: The significance of the high Ki-67 staining in this grade 1-2 follicular lymphoma is uncertain. While cases of low grade follicular lymphoma with a high proliferation index have been associated with more aggressive behavior in some cohorts, the significance of this finding has not been consistent in studies published to date. 20 Wang SA et al. Am J Surg Pathol : Follicular lymphoma: Variants Diffuse variant of FL Variant of grade 1-2 follicular lymphoma with diffuse architecture Most commonly in the inguinal region Expresses germinal center markers CD10, BCL6, LMO2 Variable BCL2 staining Generally lacks a BCL2 translocation Diffuse defined by lack follicular dendritic cell meshworks by CD21/CD23/CD35 IHC Most cases have deletions or mutations of TNFRSF14 (chromosome 1p36), which can also be seen in conventional FL 21 Katzenberger et al., Blood : ; Siddiqi IN et al. Mod Pathol :

9 Duodenal type FL Follicular lymphoma: Variants Multiple small polyps in the the duodenum Immunophenotypic & genetic characteristics are consistent with typical cases of grade 1-2 FL CD10, BCL2 positive by IHC Diagnosis: t(14;18)(q32;q21) Follicular lymphoma, by FISH grade 1-2, involving the duodenum (See Comment) However, often IgA given mucosal site Very good prognosis = watch and wait approach may be reasonable Must be distinguished clinically from more typical cases of follicular lymphoma with bowel involvement Shia J et al. Am J Surg Pathol : FL: Alternative diagnostic considerations Primary cutaneous follicle center lymphoma Cutaneous lesion, typically on head or trunk Consistently BCL6 pos BCL2 and CD10 are variable but strong expression points to systemic FL BCL2 translocations in 10-40% Pediatric follicular lymphoma Should be definitively excluded whenever considering a diagnosis of FL in a younger person Typically grade 3 but excellent prognosis BCL6 and CD10 positive BCL2 negative or weak BCL2 translocation negative 23 In situ follicular neoplasia Formerly called follicular lymphoma in situ Colonization of follicle centers by cells harboring BCL2 translocation (and aberrantly expressing BCL2 by IHC) Generally, the involved follicles are indistinguishable from adjacent reactive germinal centers by H&E Most important to appreciate that this finding is not equivalent to follicular lymphoma < 5% progress to overt follicular lymphoma BCL2 IHC 24 Jegalian AG et al. Blood :

10 Age-related clonal populations harboring BCL2/IGH translocations in peripheral blood The BCL2/IGH translocation arises in early B-cell precursors in the bone marrow This driver mutation is not sufficient for lymphomagenesis BCL2/IGH translocations identified in over 50% of patients over age 50 (at a very low level) 4% of patients have > 1 in 25,000 cells positive In situ follicular neoplasia likely represents the nodal equivalent of this phenomenon 25 Dolken G, Dolken L, Hirt C, et al. J Natl Cancer Inst Monogr : Karyotypically abnormal clones are relatively common in benign lymphoid tissues 497 consecutive specimens submitted for routine karyotype, in which the final pathologic diagnosis was non-neoplastic 5% had a clonal karyotypic abnormality Abnormal clones tended to be small (median = 2 metaphases, 16/19 < 5 of 20 metaphases) Clonality, though often used as a proxy for malignancy, is not synonomous with malignancy 26 Montgomery ND, et al. Cancer Genet : Classification of morphologically low grade B-cell LPDs: immunophenotype Low grade B-cell LPD CD10 + CD5 + CD5 - / CD10 - CLL/SLL Mantle cell Rarely others 27 9

11 Chronic Lymphocytic Leukemia / Small Lymphocytic Lymphoma Clinical and epidemiological features ~7% of non-hodgkin lymphomas Leukemic form (CLL) is the most common adult leukemia in western countries Median age of onset is 70 Risk of transformation (most often to DLBCL) related to a variety of cytogenetic and molecular features 28 Chronic Lymphocytic Leukemia: Cytology Peripheral blood Typically small lymphocytes with soccer ball chromatin May be larger and more atypical in cases with trisomy 12 Numerous smudge cells Variable number of prolymphocytes (definitionally < 55% at diagnosis) 29 Montgomery ND, Mathews SP. Surg Pathol Clin : Small Lymphocytic Lymphoma: Morphology Lymph node Architectural effacement by a proliferation of predominantly small lymphocytes Often a vaguely/pseudonodular appearance due to the presence of proliferation centers 30 10

12 Small Lymphocytic Lymphoma: Morphology Lymph node Architectural effacement by a proliferation of predominantly small lymphocytes Often a vaguely/pseudonodular appearance due to the presence of proliferation centers Proliferation centers contain numerous prolymphocytes and paraimmunoblasts High Ki-67, MYC 31 Montgomery ND, Mathews SP. Surg Pathol Clin : CLL/SLL: Immunophenotype Marker CD5 CD10 CD20 CD23 FMC7 Surface light chains Cyclin D1 Expression POS NEG DIM* POS NEG/DIM* NEG/DIM* NEG * Often brighter/stronger in atypical forms of CLL 32 CLL/SLL: Immunophenotype Marker CD5 CD10 CD20 CD23 FMC7 Surface light chains Cyclin D1 LEF1 Expression POS NEG DIM* POS NEG/DIM* NEG/DIM* NEG POS * Often brighter/stronger in atypical forms of CLL 33 11

13 CLL/SLL: Utility of LEF1 Lymphoid enhancer binding factor 1 Normally expressed in T-cells and immature B-cells Amongst small B-cell LPDs, very specific to CLL/SLL Tandon et al., Mod Pathol /92 SLL 0/53 mantle cell lymphoma 0/31 grade 1-2 follicular lymphoma 0/31 marginal zone lymphoma LEF1 is also expressed in subset of large B-cell lymphomas 34 Monoclonal B-cell Lymphocytosis and its Nodal Equivalent Small monoclonal B-cell population (< 5 x 10 9 / L) in blood without nodal or other extramedullary involvement Three types recognized in the 2017 WHO Classification CLL type Atypical CLL type (unusually bright CD20, sig, and/or negative CD23) Non-CLL type Nodal equivalents of CLL-type MBCL exist and have very favorable prognosis 35 Monoclonal B-cell Lymphocytosis and its Nodal Equivalent Features of cases likely to represent the nodal equivalent of MBL: Peripheral blood involvement < 5 x 10 9 /L Diagnosis: Absence Involvement of proliferation by centers CD5/CD23-positive B-cell population (see No comment) nodes > 1.5 cm Comment: While the immunophenotype is compatible with small lymphocytic lymphoma (SLL), correlation with peripheral blood flow cytometry and radiologic findings is necessary to determine whether this finding represents true SLL or a nodal equivalent of monoclonal B-cell lymphocytosis. 36 Gibson SE et al. Haematologica :

14 CLL/SLL: Progression and Transformation A subset of CLL/SLL cases will transform to a higher grade lymphoma, most commonly DLBCL (Richter s transformation) Clonal Richter s transformations to DLBCL have an abysmal prognosis (< 1 year) Risk of transformation is related to variety of morphologic and molecular features Accelerated phase morphology Cytogenetics Somatic hypermutation status Somatic mutations 37 Montgomery ND, Mathews SP. Surg Pathol Clin : CLL/SLL: Progression and Transformation The presence of expanded proliferation centers (> 20x field) may predict transformation and shortened survival Gine et al. Haematologica : CLL/SLL: Progression and Transformation The presence of expanded proliferation centers (> 20x field) may predict transformation and shortened survival Gine et al. Haematologica : Caution is appropriate in diagnosing Richter s 39 transformation on small biopsies 13

15 CLL/SLL: Cytogenetics and Survival Complex karyotype also associated with inferior outcomes 17p del 11q del 13q del +12q Standard evaluation requires karyotype and CLL FISH panel Donner H, et al. N Engl J Med : CLL/SLL: Molecular Features and Survival A number of molecular features are increasingly appreciated as strong predictors of outcome and have become part of routine clinical practice Somatic IGHV hypermutation status Positive (> 2% different from germline sequence) = good prognosis Negative ( 2%) = poor prognosis CD38 and ZAP70 expression are proxy markers Individual gene mutations most notably TP53 Sterotyped B-cell receptors using IGHV Mantle Cell Lymphoma Clinical and epidemiological features 5-10% of non-hodgkin lymphomas Despite small-to-intermediate cell size, typically associated with aggressive clinical behavior Middle aged to older individuals Male predominance (> 2:1) Lymph nodes commonly involved, as well as extranodal sites (e.g. GI tract) 42 14

16 Mantle cell lymphoma: Morphology Architecture is variable (diffuse, nodular, etc.) but proliferation centers should be absent 43 Mantle cell lymphoma: Morphology Architecture is variable (diffuse, nodular, etc.) but proliferation centers should be absent Classic cases are composed of monomorphic small-to-intermediate lymphocytes with irregular nuclear contours The presence of epithelioid histiocytes is not specific but is often seen Aggressive variants with blastoid or pleomorphic features are well described 44 Mantle cell lymphoma: Genetics t(11;14)(q13;q32) CCND1/IGH found in > 95% of cases, leading to cyclin D1 overexpression Most cases of mantle cell lymphoma have a complex karyotype with multiple additional abnormalities Cyclin D1 IHC 45 15

17 Mantle cell lymphoma: Immunophenotype Marker Mantle cell CLL/SLL CD5 > 90% POS POS CD10 NEG* NEG CD20 POS DIM FMC7 POS NEG/DIM Surface light chains BRIGHT NEG/DIM Cyclin D1 >95% POS NEG SOX11 > 90% POS NEG LEF1 NEG POS Cyclin D1 IHC * CD10 may be gained in aggressive variants (e.g. blastoid) 46 Mantle cell lymphoma: Cyclin D1-negative Variants < 5% of lymphomas with a gene expression profile of mantle cell lymphoma lack t(11;14)(q13;q32) and cyclin D1 overexpression > 50% of these cases will instead have CCND2 translocations (Salaverria et al., Blood : ) How are these to be recognized in practice? 47 Mantle cell lymphoma: utility of SOX11 SOX11 is a nuclear transcription factor with diagnostic value in mantle cell lymphoma Expressed in > 90% of mantle cell lymphomas, including cyclin D1 negative cases H&E Cyclin D1 SOX11 Prognostic significance is controversial, but diagnostic value is clear 48 Mozos A et al. Haematologica :

18 Mantle cell lymphoma: utility of SOX11 49 Nakashima MO et al. Appl Immunohistochem Mol Morphol : In situ mantle cell neoplasia Conceptually similar to in situ follicular neoplasia However, likely even rarer Defined by scattered cells with cyclin D1 expression and CCND1/IGH translocations limited to the intact mantle zones Should not expand and replace the native mantle zone, in which case a diagnosis of bona fide mantle cell lymphoma is appropriate Carvajal-Cuenca A, et al. Haematologica : An illustrative case 56-year old man with a 12-year history of chronic lymphocytic leukemia, which was previously treated at an outside institution Cytogenetics for prognostic stratification were not previously performed Now with a rising lymphocyte count at the high end of the reference range (4.9 x 10 9 / L) Peripheral blood flow cytometry was requested 51 17

19 52 Variable CD23 CD5+ B-cells Bright CD20 Moderate kappa 53 46,XY,t(11;14)(q13;q32) 54 18

20 Normal pattern CCND1 and IGH separate Fusion pattern CCND1 + IGH overlapping 55 Mantle cell lymphoma: Leukemic non-nodal mantle cell lymphoma Characterized by blood, bone marrow, and often splenic involvement but without adenopathy (< 1-2 cm) Cyclin D1 testing is important in the evaluation of all Simple karyotype with CD5+ t(11;14)(q13;q32); small B-cell neoplasms (either by FISH or IHC) CCND1/IGH Often SOX11 negative More indolent clinical course as a group, though some cases may behave more aggressively 56 Orchard J et al. Blood : Classification of morphologically low grade B-cell LPDs: immunophenotype Low grade B-cell LPD CD10 + CD5 + CD5 - / CD10 - LEF1+ t(11;14) Cyclin D1+ SOX11+ CLL/SLL Mantle cell Rarely others 57 19

21 Classification of morphologically low grade B-cell LPDs: immunophenotype Low grade B-cell LPD CD10 + CD5 + CD5 - / CD10 - LEF1+ t(11;14) Cyclin D1+ SOX11+ CLL/SLL Mantle cell Rarely others Includes CD5+ marginal zone and lymphoplasmacytic lymphomas 58 Classification of morphologically low grade B-cell LPDs: immunophenotype Low grade B-cell LPD CD10 + CD5 + CD5 - / CD10 - Extranodal MZL Lymphoplasmacytic lymphoma Nodal MZL 59 Classification of morphologically low grade B-cell LPDs: immunophenotype Low grade B-cell LPD CD10 + CD5 + CD5 - / CD10 - Extranodal MZL Splenic MZL Rarely others Lymphoplasmacytic lymphoma Nodal MZL Hairy cell leukemia 60 20

22 The diagnostic challenge of a lymphoma with a non-specific immunophenotype Unlike the entities discussed to this point, these lymphomas have an immunophenotype similar to normal B- cells (CD5 neg, CD10 neg) Direct or proxy methods of clonality can be very helpful in diagnosis Monotypic expression of light chains by flow cytometry Monotypic expression of light chains by ISH/IHC Molecular clonality studies Lambda ISH Kappa ISH Lambda ISH 61 RNAScope Lambda ISH Weaker monotypic expression in B cells Strong monotypic expression in plasma cells 62 Classification of morphologically low grade B-cell LPDs: immunophenotype Low grade B-cell LPD CD10 + CD5 + CD5 - / CD10 - Extranodal MZL Splenic MZL Rarely others Lymphoplasmacytic lymphoma Nodal MZL Hairy cell leukemia 63 21

23 MALT lymphoma Clinical and epidemiologic features 7-8% of B-cell lymphomas Occurs at native sites of MALT E.g. 50% of gastric lymphomas Also occurs at sites of acquired MALT due to chronic inflammation Thyroid (Hashimoto) Ocular MALT lymphoma 64 MALT lymphoma Clinical and epidemiologic features Many common sites reported to have infectious associations Gastric = H. pylori Ocular = C. psittaci Cutaneous = B. burgdorferi (controversial) Disseminated nodal involvement not typical at diagnosis Ocular MALT lymphoma 65 Nodal marginal zone lymphoma Clinical and epidemiologic features < 2% of all lymphomas Typically present with generalized lymphadenopathy Critical to exclude nodal dissemination of a MALT lymphoma Clinical information is essential for definitive classification 66 22

24 Lymphoplasmacytic lymphoma Clinical and epidemiological features Rare compared to other non-hodgkin lymphomas (annual incidence 3-4 cases / 1 million people) Bone marrow involvement is more common (~100%) than nodal or splenic involvement (15-30%) Can also involve mucosa associated lymphoid tissue Waldenstrom s macroglobulinemia: Lymphoplasmacytic lymphoma with bone marrow involvement + IgM monoclonal protein 67 MALT lymphoma: Pathologic features Morphology Monocytoid appearance Often plasmacytic differentiation Lymphoepithelial lesions May colonize germinal centers Ocular MALT lymphoma 68 Nodal marginal zone lymphoma Morphology Many monocytoid B-cells May have prominent plasmacytic differentiation Can have a variable number large cells, particularly in the colonized germinal centers 69 23

25 Lymphoplasmacytic lymphoma: Pathology Bone marrow morphology Variable patterns of involvement (nodular, diffuse, interstitial) Combination of lymphocytes (often Flow cytometry may be valuable to exclude an unappreciated plasmacytoid) and plasma cells clonal B-cell population in plasma cell neoplasms Clonal with the same light chain restriction Can be virtually all plasma cells, particularly after treatment 70 Lymphoplasmacytic lymphoma: Morphology Lymph node morphology Variable architecture Widely patent sinuses Few/absent follicles Abundant plasma cells 71 Pathologic features of MALT, MZL, LPL Immunophenotype Utility of immunophenotype to distinguish these entities is limited Immunophenotypic aberrancies do occur in a subset of cases, which may assist diagnosis (but less so classificatino) CD5 in < 20% of MALTs, MZLs, and LPLs CD43 in a subset of marginal zone lymphomas Cytogenetics Some recurrent, site-specific abnormalities do occur in MALT lymphomas [e.g. t(11;18)(q21;21) in gastric MALT lymphoma] 72 24

26 LPL: Molecular features Greater than 90% of cases of lymphoplasmacytic lymphoma (LPL) have a MYD88 p.leu265pro (L265P) mutation Mutations in MYD88 lead to activation of the NF-κB pathway, a common target of activation in B-cell lymphomas. MYD88 L265P is seen less commonly in some other B-cell lymphomas 20% of splenic marginal zone lymphomas 20% of nodal marginal zone lymphomas 5-10% of extranodal marginal zone lymphomas A subset of DLBCLs (non-gc type) Treon SP, et al. N Engl J Med : ; Jiminez C, et al. Leukemia : Varettoni M, et al. Blood : ; Xu L, et al. Blood : Classification of morphologically low grade B-cell LPDs: immunophenotype Low grade B-cell LPD CD10 + CD5 + CD5 - / CD10 - IgM monoclonal protein MYD88 L265P in > 90% Extranodal MZL Splenic MZL Rarely others Lymphoplasmacytic lymphoma Nodal MZL Hairy cell leukemia 74 My typical approach Flow cytometry whenever possible CD3, CD20 (or PAX5), CD5, CD10, Cyclin D1, and Ki-67 Evaluated in essentially all cases in which a low grade B-cell lymphoma is in the differential Add BCL2 IHC and BCL2 FISH in CD10 positive cases SOX11 and LEF1 in challenging CD5-positive cases Add CD138 + Kappa/Lambda ISH in cases CD5-negative/CD10-negative cases (or in cases with a prominent plasmacytic component) MYD88 testing in cases that I believe are or may be lymphoplasmacytic lymphoma (tend not to do it with marginal zone lymphomas when a positive result would not change my diagnosis) 75 25

27 Summary and Conclusions In many respects, the classification of low grade B-cell lymphomas and leukemias has proven the test of time Updates primarily reflect rare variants of common entities and highlight the utility of new immunohistochemical and molecular tools Increasingly, we appreciate the prevalence of clonal-but-not-malignant lymphoid lesions (as already appreciated in myeloid neoplasia) A systematic approach layering morphology, immunophenotype, and genetic features will lead to accurate diagnosis and classification of most low grade B-cell lymphomas and leukemias 76 References Carvajal-Cuenca A, et al. In situ mantle cell lymphoma: clinical implications of an incidental finding with indolent clinical behavior. Haematologica : Damle RN, et al. IgV gene mutation status and CD38 expression as novel prognostic indicators in chronic lymphocytic leukemia. Blood : Dolken G, et al. Age-dependent prevalence and frequency of circulating t(14;18)-positive cells in the peripheral blood of healthy individuals. J Natl Cancer Inst Monogr : Donner H, et al. Genomic aberrations and survival in chronic lymphocytic leukemia. N Engl J Med : Gibson SE, et al. Reassessment of small lymphocytic lymphoma in the era of monoclonal B-cell lymphocytosis. Haematologica : Hamblin TJ, et al. Unmutated Ig V(H) genes are associated with a more aggressive form of chronic lymphocytic leukemia. Blood : Harris NL, et al. A Revised European-American Classification of Lymphoid Neoplasms: A Proposal from the International Lymphoma Study Group. Blood : Jegalian AG, et al. Follicular lymphoma in situ: clinical implications and comparisons with partial involvement by follicular lymphoma. Blood : Jiminez C, et al. MYD88 L265P is a marker highly characteristic of, but not restricted to, Waldenstrom s macroglobulinemia. Leukemia : Katzenberger T, et al. A distinctive subtype of t(14;18)-negative nodal follicular non-hodgkin lymphoma characterized by a predominantly diffuse growth pattern and deletions in the chromosomal region 1p36. Blood : Montgomery ND, et al. Clonal karyotypic abnormalities associated with reactive lymphoid hyperplasia. Cancer Genet : Montgomery ND, Mathews SP. Transformation in low grade B-cell neoplasms. Surg Pathol Clin : Mozos A, et al. SOX11 expression is highly specific for mantle cell lymphoma and identifies the cyclin D1-negative subtype. Haematologica : Orchard J, et al. A subset of t(11;14) lymphoma with mantle cell features displays mutated IgVH genes and includes patients with good prognosis, nonnodal disease. Blood : Puente XS, et al. Non-coding recurrent mutations in chronic lymphocytic leukemia. Nature : Salaverria I, et al. CCND2 rearrangements are the most frequent genetic events in cyclin D1(-) mantle cell lymphoma. Blood : Shia J, et al. Primary follicular lymphoma of the gastrointestinal tract: a clinical and pathologic study of 26 cases. Am J Surg Pathol : Siddiqi INet al. Characterization of a variant of t(14;18) negative nodal diffuse follicular lymphoma with CD23 expression, 1p36/TNFRSF14 abnormalities, and STAT6 mutations. Mod Pathol : Swerdlow SH, et al. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues IARC. Revised 4 th edition, Volume 2. Tandon B, et al. Nuclear overexpression of lymphoid-enhancer binding factor 1 identifies chronic lymphocytic leukemia/small lymphocytic lymphoma in small B-cell lymphomas. Mod Pathol : Treon SP, et al. MYD88 L265P somatic mutation in Waldenstrom s macroglobulinemia. N Engl J Med : Varettoni M, et al. Prevalence and clinical significance of the MYD88 (L265P) somatic mutation in Waldenstrom s macroglobulinemia and related lymphoid neoplasms. Blood : Wang SA, et al. Low histologic grade follicular lymphoma with high proliferation index: morphologic and clinical features. Am J Surg Pathol : Xu L, et al. MYD88 L265P in Waldenstrom macroglobulinemia, immunoglobulin M monoclonal gammopathy, and other B-cell lymphoproliferative disorders using conventional and quantitative 77 allele-specific polymerase chain reaction. Blood :

28 How far is too far? Working up incidental laboratory findings in hematopathology Chad M. McCall, MD, PhD Assistant Professor of Pathology Duke University School of Medicine Disclosures I have no financial or other disclosures relevant to the content of this presentation. Objectives 1. To understand guidelines for evaluating monoclonal gammopathies of undetermined significance (MGUS). 2. To assess the significance of monoclonal B lymphocytosis. 3. To appreciate the prognostic significance of clonal hematopoiesis. 1

29 A day in the life of an electrophoresis lab director 63 year old male with a past medical history of chronic hepatitis C. Annual physical examination, no new complaints. PCP orders a comprehensive chemistry panel. Elevated total protein: what to do? Differential diagnosis: Polyclonal hypergammaglobulinemia (common in inflammatory conditions and liver disease) Monoclonal gammopathy Plasma cell myeloma Waldenstrom s macroglobulinemia Monoclonal gammopathy of undetermined significance The PCP rightly ordered a serum protein electrophoresis. Serum protein electrophoresis endosmotic flow of buffer cathode anode gamma beta alpha albumin pre alb 2

30 Serum protein electrophoresis McPherson, R.A. Ch. 19 in Henry s, 22 nd ed. Back to our patient Monoclonal spike of 1.2 g/dl Identifying an M spike: immunofixation electrophoresis IgG lambda monoclonal gammopathy 3

31 Monoclonal gammopathy of undetermined significance (MGUS) Non IgM MGUS IgM MGUS M spike level <3.0 g/dl <3.0 g/dl Bone marrow involvement Urine light chain excretion End organ damage (CRAB) Other clinical features <10% clonal plasma cells <10% involvement by clonal lymphoplasmacytic infiltrate <0.5 g per 24 hours N/A No evidence of amyloidosis No evidence No evidence of hyperviscosity, hepatosplenomegaly or lymphadenopathy WHO 2017 Monoclonal gammopathy of undetermined significance (MGUS) Epidemiology Very uncommon before age % of patients over age 50 >5% of patients over age 70 2X as frequent in African Americans 80 85% non IgM, 15 20% IgM Risk of progression: Non IgM (to myeloma): 1% per year Light chain only (to myeloma): 0.3% per year IgM (to lymphoplasmacytic lymphoma/other lymphomas): 1.5% per year WHO 2017 Can non IgM MGUS be risk stratified? Low risk High risk M spike level <1.5 g/dl >= 1.5 g/dl Isotype IgG IgA Serum free light chain ratio Risk factors above 0 (low risk) year risk of progression (%) Normal Abnormal Low risk patients without other clinical findings have a 7% chance of bone marrow involvement over MGUS level (10%), so bone marrow biopsy should NOT be recommended. Bone scan is also not needed (<2% of low risk patients had lesions) Mangiacavalli S et al, Eur J Haematol 2013;91(4):

32 If an M spike is found, now what? Lab studies to recommend: CBC and complete chemistry panel (to include calcium and creatinine) Serum free light chain assay Urine protein electrophoresis and immunofixation Quantitative immunoglobulins (sometimes SPEP doesn t quantify well) Bone marrow biopsy and bone survey only if high risk (>= 1.5 g/dl M spike, IgA, abnormal FLC ratio) Plasma cell myeloma (WHO 2017) Bone marrow plasma cells FLC ratio (involved to uninvolved) Smoldering myeloma 10 60% >60% <100 >=100 Plasma cell myeloma Bone lesions <=1 *on MRI* >=1 lytic lesion on bone scan, CT, PET/CT End organ damage None Present (hypercalcemia, renal insufficiency, anemia) M spike Urine M spike >= 3.0 g/dl >0.5 g/24 hours WHO 2017 Don t forget about comorbidities You can still diagnose MGUS if end organ damage is NOT from the plasma cell neoplasm: Anemia: many other causes Renal insufficiency: history of chronic kidney disease? Hypertension? Diabetes? Hypercalcemia: hyperparathyroidism? Bone lesions: metastatic carcinoma? 5

33 Before you diagnose IgM MGUS Recommend thorough clinical examination and cross sectional imaging: IgM paraproteins found in: Lymphoplasmacytic lymphoma Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) Marginal zone lymphoma (especially splenic) Follicular lymphoma Other non Hodgkin lymphomas Consider recommending a bone marrow biopsy to exclude Waldenstrom s macroglobulinemia (>10% involvement by lymphoplasmacytic lymphoma). Follow up to recommend Bone marrow biopsy and bone scan if not low risk. Repeat exam and labs in 6 months. After that: If Low risk (<1.5 g/dl M spike, IgG type, normal FLC ratio): yearly history and physical alone, no need to repeat SPEP/FLC unless new symptoms. All others: Annual SPEP/UPEP, CBC, creatinine, calcium. Transformation can be abrupt so patient education for signs/symptoms is crucial. Rajkumar SV, UpToDate v31.0 MGUS Summary Very common over age 50 (3%). Must NOT have clinical signs/symptoms associated with plasma cell neoplasms (CRAB criteria). Can be risk stratified by M spike level and free light chain ratio. Low risk patients may not even need follow up labs. Other patients need bone marrow biopsies, bone scans, yearly follow up with repeat labs. 6

34 A day in the life of the Duke hematopathology service 70 year old male, on chemotherapy for glioblastoma. CBC and differential normal with the exception of low platelets (attributed to therapy). No obvious abnormal lymphocytes detected. Quantitative lymphocyte subset testing was ordered to assess immune function, which found a small subset of B cells with abnormally dim CD19 expression. Flow cytometry results 5% CD5 positive, CD23 positive, kappa restricted monoclonal B cells. Absolute monoclonal B cell count of 300/mm 3 Monoclonal B lymphocytosis (MBL) WHO 2017 definition: monoclonal B cell count of <5,000/mm 3 (5 x 10 9 /L) in the peripheral blood in patients without: Lymphadenopathy Organomegaly Other extramedullary disease Other evidence of B cell lymphoproliferative disorders % of healthy individuals have a detectable MBL clone depending upon the sensitivity of the assay. Rare <40 years of age % of >90 year olds. WHO

35 Types of MBL CLL like Atypical CLLlike Frequency 75% 25% CD5 + + Non CLL like CD20 Dim Bright Variable CD23 + Variable Variable Surface light chain Dim to negative Moderate to bright Moderate to bright WHO 2017 CLL like MBL Low count MBL = <500/mm 3 Minimal risk of progression No need for further follow up High count MBL = >=500/mm 3 1 2% risk of progression to CLL per year. Higher count = higher risk My recommendation: yearly CBC with differential; repeat flow cytometry if lymphocyte count increases or new clinical signs/symptoms. WHO 2017 MBL other than CLL like Consider calling the ordering physician to emphasize that this needs working up: Atypical CLL like: need to rule out mantle cell lymphoma. Non CLL like: can be transient or indolent, but up to 17% develop splenomegaly and are related to splenic marginal zone lymphoma. WHO

36 Another case 77 year old female, no significant PMH Referred to outpatient hematology after a routine CBC with differential showed a low absolute neutrophil count (1200/mm 3 ). The CBC had normalized by the time she could get a hematology appointment, but Flow cytometry data 2% CD5 negative, CD23 dim monoclonal B cells Non CLL like MBL? Further workup No evidence of splenomegaly or enlarged lymph nodes Incidental 2.2 cm left renal mass. 9

37 Subsequent history Partial nephrectomy: clear cell renal carcinoma, Fuhrman grade 2 1 year follow up: normal CBC, no new symptoms. SPEP/immunofixation: now has a faint IgM lambda M spike, not quantifiable Normal FLC ratio IgM MGUS + non CLL MBL! Planned follow up every 4 months. Troubling cytopenias but no diagnosis 87 year old man with slowly progressive cytopenias: 2011: began having mild thrombocytopenia 2014: anemia starts, then worsens 2016: pancytopenia seen during pre op workup for spinal fusion surgery: WBC 2.1 Hgb 8.9 with an MCV of 119 Plt 95 Nutritional deficiencies have been ruled out. Bone marrow biopsies 2014: Normocellular marrow with mild erythroid atypia but not diagnostic. 2017: Hypercellular marrow with mild erythroid and myeloid atypia, but not diagnostic. Normal cytogenetics. 10

38 Diagnostic features of myelodysplastic syndrome Erythroid Megakaryocytic Myeloid Jaffe ES, ed., Hematopathology, 2 nd ed. What if morphologic features are not enough? In order to diagnose morphologic dysplasia, changes must: Be in >10% of the cell lineage. Not be explained by other disease processes (i.e., B12/folate/copper deficiencies, stress erythropoiesis) Cytogenetics can help: Many, but not all, cytogenetic abnormalities are diagnostic of MDS in and of themselves. MDS/AML is a multi step process (like many other cancers) Progenitor Cell TET2 DNMT3A IDH1/2 Epigenetic mutation(s) Clonal hematopoiesis SF3B1 ASXL1 Chromatin/ spliceosome mutation(s) MDS FLT3 PTPN11 NRAS Signaling/ proliferation mutation(s) AML Transcription factor mutation or fusion/translocation CEBPA RUNX1 inv(16) Preleukemic precursor Signaling/ proliferation mutation(s) AML After Viny AD, Levine RL. N Engl J Med 2016;374:

39 Genetic mutations are common in MDS 78% of MDS patients have at least one driver mutation Papaemmanueil E et al, Blood 122(22), 2013 Prognosis correlates with the number of mutations Papaemmanueil E et al, Blood 122(22), 2013 Hematopoietic driver mutations occur with increasing frequency with age in normal people Jaiswal et al, NEJM 2014;371(26) 12

40 Mutations occur mostly in known AML/MDS drivers Jaiswal et al, NEJM 2014;371(26) Mutations confer greatly increased risk of developing hematologic malignancies Jaiswal et al, NEJM 2014;371(26) How can we diagnose pre MDS states? Idiopathic Cytopenias of Undetermined Significance (ICUS) Clonal Hematopoiesis of Indeterminate Potential (CHIP) Clonal Cytopenias of Undetermined Significance (CCUS) Clonality No Yes Yes Cytopenias Yes No Yes Dysplasia No No No Risk of progression Low 0.5 1% per year Higher than CHIP Modified from Steensma DP et al, Blood 2015;126:

41 How frequent are clonal mutations in cytopenias and MDS? 22 33% of patients with unexplained cytopenias not meeting MDS criteria had at least one clonal mutation (two different studies). >85% of MDS patients have at least one detectable mutation in common genes. Steensma DP et al, Blood 2015;126:9 16 Hall J et al, Blood 2014;124(21) Kwok B et al, Blood 2014;124(21) Papaemmanuil E et al, Blood 2013;122(22):3616 Next generation (massively parallel) sequencing Millions of sequencing reactions performed and read simultaneously PCR amplification of target panel Deep Broad Sequencing of the whole exome or genome Thousands of reads of each target Fewer reads but many more targets Detection of very small mutant populations Detection of a broad range of mutations Illumina platform flow cell flow cell 14

42 Illumina platform Millions of patient DNA molecules flow cell Illumina platform Illumina platform Target DNA strand Sequencing primer 15

43 Illumina platform Target DNA strand Sequencing primer Fluorescentlytagged dntp Illumina platform Read (x millions) Illumina platform Remove fluor 16

44 Illumina platform + dntps Illumina platform Read (x millions) Illumina MiSeq Around 5 million short ( base) reads per flow cell. Relatively simple sample preparation. Most commonly used nextgeneration sequencer in the clinical (and research) laboratory. Other Illumina sequencers (NextSeq 500, HiSeq) have greater read depth potential. 17

45 Clinical next generation sequencing panels Panel Type Examples Pros Cons Hotspot panel Mayo OncoHeme Genoptix Panel UNC (Illumina TruSight) Duke Myeloid Panel Broad targeted panel Target known actionable and clinically relevant mutations. Good sensitivity for low level mutations. Relatively inexpensive. FoundationOne Heme Cover a wide array of targets, including fusions and large genomic deletions/insertions. Good sensitivity. Clinical exome GeneDx Very broad coverage Limited sensitivity for lowlevel mutations. Limited number of targets. Do not reliably detect fusions and large genomic deletions/insertions. Questionable utility of broader targeting. Relatively expensive. More applicable for constitutional genetic testing. Expensive. Back to our patient We performed a 55 gene NGS panel on his peripheral blood: A single mutation associated with clonal hematopoiesis was found at 55% variant allele fraction: A diagnosis of CCUS (clonal cytopenias of undetermined significance) was made. Cardiovascular mortality is increased with mutations Jaiswal et al, NEJM 2014;371(26) 18

46 Cardiovascular disease is definitely increased in patients with CHIP Jaiswal et al, NEJM 2017;377: TET2 loss of function leads to atherosclerosis Jaiswal et al, NEJM 2017;377: In summary Pre malignant, often incidental findings are becoming more frequent in hematopathology. MGUS multiple myeloma. MBL chronic lymphocytic leukemia and other lymphomas. CCUS/CHIP MDS AML Knowing the diagnostic methodologies for detecting these conditions (SPEP/IFE, flow cytometry, NGS) and their risk of progression will allow us to better advise clinicians of how to test and monitor their patients. 19

47 Case: 57 year old male with FNA of left cervical LN Diagnosis? 1 North Carolina Society of Pathologists Charlotte, North Carolina 14 April 2018 How to Diagnose Lymphoproliferative Disorders Utilizing FNA in 4 Easy Steps Simon Bergman, M.D. Michael W. Beaty, M.D. 2 Disclosures We disclose that we have no relevant financial relationships with commercial interests (Who would pay us to do anything, anyway?) 3 1

48 Lecture Goals Integrated diagnostic approach using FNA cytomorphologic features of primary nodal disorders: Benign, reactive lymph nodes Malignant lymphoproliferative disorders Hodgkin s lymphoma and non-hodgkin s lymphomas From the perspective of the 2016 WHO Classification Combined phenotypic and genotypic analyses of aspirated material Flow cytometry, immunohistochemistry Molecular analyses 4 A Reasonable Approach to FNAs of LPDs STEP 1. CLINICAL SUSPICION Patient age Immune status Known exposure Prior neoplasms Systemic manifestations Location of node(s) Localized vs generalized Physical characteristics of node(s) 5 A Reasonable Approach to FNAs of LPDs STEP 1. CLINICAL SUSPICION: FNA procurement STEP 2. MORPHOLOGY Systematic evaluation is essential Cellularity Major cell type Predominantly small cells Mixed intermediate cells Predominantly large cells Background 6 2

49 Predominance of Small Lymphocytes in the Aspiration Benign Reactive follicular hyperplasia Malignant Small lymphocytic lymphoma Follicular lymphoma Mantle cell lymphoma Burkitt lymphoma Lymphoplasmacytic lymphoma Small blue cell tumors of childhood 7 Mixture of Large and Small Lymphocytes in the Aspiration Benign Reactive hyperplasia Malignant Follicular lymphoma Large B-cell lymphoma T-cell lymphomas Marginal zone lymphoma Partial involvement by Hodgkin lymphoma 8 Predominance of Large Lymphocytes in the Aspiration Benign Florid hyperplasia, follicular or paracortical Various infections Mononucleosis Malignant Large B-cell lymphoma T-cell lymphomas Plasma cell neoplasm Hodgkin lymphoma Carcinoma Melanoma Other poorly-undifferentiated neoplasms, e.g. sarcoma 9 3

50 Reactive Lymphoid Hyperplasias 10 Reactive Hyperplasia Clinical Associations Infections Autoimmune diseases Draining primary cancers Idiopathic Differential Diagnosis Specific benign lymphoid proliferations, e.g. toxoplasmosis Hodgkin lymphoma Follicular lymphomas Partial involvement by neoplasms 11 Reactive Hyperplasia Cytomorphology Highly cellular with dispersed polymorphic lymphoid populations Small mature lymphocytes predominate Complete spectrum with transitional forms Tingible body macrophages Lymphohistiocytic aggregates Phenotype/Genotype - polyclonal 12 4

51 Reactive Lymphoid Hyperplasia 13 Reactive Lymphoid Hyperplasia 14 Reactive Lymphoid Hyperplasia 15 5

52 Reactive Lymphoid Hyperplasia 16 Reactive Lymphoid Hyperplasia (histology) 17 Infectious mononucleosis Clinical features Self-limited EBV infection Adolescents & young adults Fever, pharyngitis, splenomegaly, atypical lymphocytosis, lymphadenopathy (bilateral) Serologic confirmation 18 6

53 Infectious mononucleosis Cytomorphology Highly cellular Polymorphous population of lymphoid cells with a large proportion of immunoblasts and plasmacytoid cells, Reed-Sternberg-like cells Tingible body macrophages 19 Infectious Mononucleosis 20 Infectious Mononucleosis 21 7

54 Infectious Mononucleosis (histology) 22 Clonal Lymphoproliferative Disorders 23 B-cell Lymphomas: WHO Classification Precursor neoplasms Lymphoblastic lymphoma Mature neoplasms Diffuse large cell lymphoma Follicular lymphoma Small lymphocytic lymphoma Mantle cell lymphoma Nodal marginal zone lymphoma Burkitt lymphoma 24 8

55 B-cell NHL: Small Cell Patterns NEOPLASTIC CLL/SLL NON-NEOPLASTIC Follicular hyperplasia Mantle Cell Follicular, low grade (1 of 3) Lymphoplasmacytic 25 Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma (CLL/SLL) Clinical Features Most common leukemia of adults in Western countries Mean age 65 Males predominate Disseminated disease +/- peripheral blood Good prognosis 2-8% develop Richters transformation to large B-cell lymphoma 26 Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma (CLL/SLL) Cytomorphology Hypercellular Monotonous small lymphocytes without cohesion Round, smooth to slightly angulated nuclei Coarse clumped, dark chromatin Very high N/Cs Some cells may have plasmacytoid appearance Occasional prolymphocytes Phenotype/Genotype CD19/20 CD5 CD10 CD23 CD43 sig Other /- Tri 12, del(13), del(17p) 27 9

56 CLL/SLL 28 Mantle Cell Lymphoma Clinical Features Adults, median age 7 th decade Males predominate Disseminated disease GIT & Waldeyer s ring 10-20% Poor response Median survival 3-4 years Increased proliferation/blastoid variant 29 Mantle Cell Lymphoma Cytomorphology Hypercellular Dispersed monomorphic small lymphocytes with high N/C Prominent nuclear membrane irregularities - centrocyte-like Finely clumped to smooth chromatin Mitotic figures may be numerous (in blastic variant) Phenotype/Genotype CD19/CD20 CD5 CD10 CD23 CD43 sig Other t(11;14), cyclin D

57 Mantle Cell Lymphoma 31 Mantle Cell Lymphoma Cyclin D1 32 B-cell NHL: Mixed Cell Patterns NEOPLASTIC Follicular Lymphoma, grade 2, 3 NON-NEOPLASTIC Moderate follicular hyperplasia Marginal zone lymphoma Hodgkin Lymphoma Partial nodal involvement by lymphoma 33 11

58 Follicular Lymphoma Clinical Features 20% NHLs Mostly asymptomatic adults with generalized lymphadenopathy Indolent 34 Follicular Lymphoma Cytomorphology Moderate to high cellularity with aggregated & dispersed lymphoid cells Mixture of two cell types Centrocytes: buttock cells small/medium cells with irregular, angulated hyperchromatic nuclei with cleaves, notches & projections, inconspicuous nucleoli & scanty cytoplasm Centroblasts: large cells with round nuclei with fine chromatin & 1 to 3 distinct peripheral nucleoli Grading increased proportion of large cells higher grade Phenotype/Genotype CD20 CD5 CD10 CD23 CD43 sig Other 35 Follicular Lymphoma 36 12

59 Follicular Lymphoma 37 Follicular Lymphoma 38 Follicular Lymphoma Buttock Cells 39 13

60 Follicular Lymphoma (histology) 40 Follicular Lymphoma Grading 53 histologically proven FLs (low grade: 20 grade 1, 17 grade 2; high grade: 16 grade 3) In smears, counted # blasts per 300 lymphocytes In ThPr, counted # blasts in 10 HPFs Smears ThPr Grade # Centroblasts (mean) (5) (16) (151) (3) (17) 3 42 > 100 (79) 41 Brandao, et al. Cancer. 2006; 108(5): Follicular Lymphoma Grade 1 Grade 2 Grade

61 Nodal Marginal Zone Lymphoma Clinical Features Neoplasm of post-germinal marginal zone lymphocytes Mostly adults with median age in 7 th decade May be asymptomatic Localized or generalized lymphadenopathy Indolent with 75% 5 year survival 43 Nodal Marginal Zone Lymphoma Cytomorphology Hypercellular Polymorphic small to large lymphocytes Smooth nuclear outlines Moderately clumped chromatin Highly variable nucleoli Immunoblasts, plasmacytoid cells Phenotype nonspecific CD20+; CD5-, CD23-, CD10-44 Marginal Zone Lymphoma 45 15

62 B/T-cell NHL: Large/Intermediate Cell Patterns NEOPLASTIC Large-Cell Lymphoblastic NON-NEOPLASTIC Florid follicular hyperplasia Burkitt Anaplastic large cell 46 Diffuse Large B-cell Lymphoma Cytomorphology Hypercellular Non-cohesive uniform to variably sized large lymphocytes Variable nuclear contours Prominent nucleoli Variable N:C ratios 47 Diffuse Large B-cell Lymphoma 48 16

63 Diffuse Large B-cell Lymphoma 49 Diffuse Large B-cell Lymphoma 50 Burkitt Lymphoma Clinical Features Three clinical forms Endemic Sporadic Immunodeficiency-associated Very aggressive; potentially curable Limited Stage: 90% survival Advanced stage: 60-80% survival 17

64 Burkitt Lymphoma Cytomorphology Highly cellular with noncohesive uniform cells Nuclei: round with variable clumped chromatin & multiple nucleoli Cytoplasm: moderate, basophilic & vacuolated Numerous mitoses & TBMs Phenotype/Genotype CD20 +, CD10 +, PAX5 +, BCL2 MIB 100% + MYC translocation: t(8;14)(q24;q32) MYC/IGH Occasional cases may overlap DLBCL and BL Burkitt Lymphoma 53 Burkitt Lymphoma 54 18

65 Burkitt Lymphoma 55 Lymphoblastic Lymphoma Clinical Features 85-90% precursor T cell; 10-15% precursor B cell T cell LBL Adolescent & young adult males Mediastinal mass Low BM blast counts B cell LBL Young children & adolescents Cervical adenopathy Variable BM blasts counts 56 Lymphoblastic Lymphoma Cytomorphology Highly cellular with noncohesive uniform cells twice the twice the size of small lymphocytes Nuclei: variable shapes, often convoluted, fine chromatin, imperceptible nucleoli Very high N/Cs Numerous mitoses Phenotype T cell LBL: TdT, CD3, CD7, CD4, CD8 B cell LBL: CD19, CD79a, CD22, CD10, PAX5, TdT 57 19

66 Lymphoblastic Lymphoma 58 Lymphoblastic Burkitt Lymphoma 59 Lymphoblastic Burkitt Lymphoma TDT 60 20

67 T-cell Lymphomas: WHO Classification Precursor neoplasms Lymphoblastic lymphoma Mature neoplasms Peripheral T-cell Lymphoma, NOS Anaplastic Large Cell Lymphoma Adult T Cell Leukemia/Lymphoma Angioimmunoblastic Lymphoma 61 Peripheral T-cell Lymphoma, NOS Clinical Features Less than 15% of all NHL western world Over 50% of all PTCLs Most patients are adults Generalized disease involving nodes, skin, liver, spleen Aggressive, relapse frequent 62 Peripheral T-cell Lymphoma, NOS Cytomorphology Difficult diagnosis based on FNA morphology Variable cellular Heterogeneous mixture of small, medium sized, and large tumor cells with variability in predominance among patients. Small cells have marked nuclear irregularities, small nucleoli & high N/Cs Large cells have huge round to very irregular nuclei (multilobated), multi nucleation, massive nucleoli 63 21

68 Peripheral T-cell Lymphoma, NOS 64 Peripheral T-cell Lymphoma, NOS 65 Anaplastic Large Cell Lymphoma Clinical Features ALK positive ALCL First 3 decades Often extranodal CD30, EMA positive t(2,5) NPM/ALK, or variant Good prognosis ALK negative ALCL Patients tend to be older More nodal restricted CD30 positive, most EMA negative Lack t(2;5) NPM/ALK 66 22

69 Anaplastic Large Cell Lymphoma Cytomorphology Highly cellular Mostly dispersed but some cohesion Striking pleomorphism Large bizarre cells with one more massive irregular nuclei Multiple nucleoli & moderate to abundant cytoplasm Hallmark cells, wreath cells, donut cells Reed-Sternberg-like cells may be present Inflammatory background may be seen, including PMNs 67 Anaplastic Large Cell Lymphoma 68 Anaplastic Large Cell Lymphoma 69 23

70 Anaplastic Large Cell Lymphoma CD30 70 Hodgkin Lymphoma: WHO Classification Classical HL Four tissues morphologic variants (not FNA) Nodular sclerosis, Mixed cellularity, Lymphocyte depletion, Lymphocyte rich CD15 & CD30+; PAX5 (dim), CD20-, CD45-, EMA FNA DIAGNOSIS POSSIBLE Nodular lymphocyte predominant HL Background of small lymphocytes No classic R-S cells Popcorn cells with large polylobated vesicular nuclei, small nucleoli PAX5 (strong), CD20+, CD45+, EMA+; CD30 & CD15 NOT AN FNA DIAGNOSIS 71 Classical Hodgkin Lymphoma Cytomorphology Variable, but usually low to moderate cellularity Polymorphous lymphoid cells Small lymphocytes predominate Reed-Sternberg cells and variants Eosinophils 72 24

71 CHL (RS cell) 73 Classical Hodgkin Lymphoma 74 Classical Hodgkin Lymphoma 75 25

72 Classical Hodgkin Lymphoma CD30 76 Classical Hodgkin Lymphoma (histology) 77 Classical Hodgkin Lymphoma (histology) CD30 PAX

73 79 Case: 57 year old male with FNA of left cervical LN Diagnosis? 80 Entities should not be multiplied unnecessarily. - William of Ockham 81 27

74 Lymphoproliferative Neoplasms B-cell Lymphomas Account for > 90% of all NHL Common in developed countries, and increasing in incidence 83 T-cell Lymphomas Accounts for < 10% of all NHL Incidence varies widely on geographical regions/ethnic populations 84 28

75 FNA Materials for Ancillary Studies Cell Blocks Immunohistochemical staining In situ hybridization FISH PCR Smears Immunohistochemical staining FISH Dedicated pass for Flow Cytometry 85 A Reasonable Approach to FNAs of LPDs STEP 1. CLINICAL SUSPICION: FNA procurement STEP 2. MORPHOLOGY: Cell Size, background, etc STEP 3. PHENOTYPE: Antigen Expressions/Clonality Establishment of cell lineage Assessment of cell differentiation stage Assessment of clonality Differential diagnosis Assessment of prognostic/therapeutic factors 86 Immunophenotypic Methods Immunohistochemistry Flow Cytometry Less rapid (1-2 days) Rapid (within hours) Subjective Quantitative Architectural/cytologic Limited morphologic correlation correlation (cell size) Background staining Ability to wash cell surface Usually limited to single Multiple fluorochromes for Antigen antigen labeling (up to 10) Can be sent out to reference Difficult to defer/send out laboratory (loss of viability) 87 29

76 Principles of Immunophenotyping No single antibody diagnostic of disease entity ALK-1 protein: ALCL, DLBCL CD30: Classical Hodgkin lymphoma, ALCL, DLBCL ALK CD30 88 Principles of Immunophenotyping Antibodies should always be used in panels CD20 with CD3 kappa with lambda BCL-2 protein with CD20 & CD3 BCL2 appa 89 Principles of Immunophenotyping Stain interpretation made within context of histology Which cells are staining: tumor vs. reactive Normal cellular components positive internal controls EBV Staining patterns: membrane CD20 vs. cytoplasmic vs. nuclear CD

77 Case: 57 year old male with FNA of left cervical LN Interpretation? CD20 CD3 BCL2 CD5 91 FNA: Achieving Optimal Results In Flow Cytometry Requires cells in suspension Immediate preliminary interpretation made Cytochex reagent vial to receive the FNA sample Sample sent to Flow Lab held until notified Sample evaluation Sample type, clarity, appearance, presence/absence of clots The less manipulation of the sample the better Cell counts: 3 10 x 10 3 /ml is optimal amount for complete panel x 10 3 /ml amount needed for a modified panel < 0.5 x 10 3 /ml amount needed for a single tube assay 92 Phenotypic Assessment of B-cell Clonality B-cell Immunoglobulin (sig) light chains Critical in assessment of B-cell clonality ambda FSC-Height Count LAMBDA FITC/BD appa: ambda CD19 FITC CD19 Count appa Normal 2-4:1 Monoclonal >8:1 or < KAPPA FITC/BD 93 31

78 Phenotypic Differential Diagnosis Antigen Expression Profiles Expression Intensity Variability in the expression of an antigen Dim versus strong, e.g. CLL, MCL Aberrant phenotypes Loss of lineage specific/associated antigen T-cell lymphomas 94 Co-expression of non-lineage antigens Lineage Infidelity CLL Antigen Intensity and Expression Fluorescent intensity binding sites Negative Dim + Strong + Argon laser = 488 nm Emitted fluorescent light = 580 nm Emitted fluorescent light λ = 525 nm 95 Antigen Intensity and Expression CD5 CD

79 Flow Issues: False Negatives Minor malignant cell component May not show evidence of a clonal cell population <5% of cellular composition Minimal Residual Disease Detection Hodgkin Lymphoma (CHL and LPHL) T-cell rich large B-cell lymphomas Always correlate flow findings within context of pathologic and clinical features 97 Flow Issues: False Negatives Interfering stroma Dense or abundant stroma may interfere with results Cutaneous lymphoma Diffuse large B-cell lymphoma Always correlate flow findings within context of pathologic and clinical features 98 Flow Issues: Viability Cells may be fragile, apoptotic, or cycling May not show evidence of clonality Sample needs to be processed immediately Diffuse large cell lymphomas (B/T) Burkitt s lymphoma Lymphoblastic lymphomas (B/T) 7-AAD Dead Live Always correlate flow findings within context of pathologic and clinical features 99 33

80 Differential Diagnosis: Small Cell B-cell Lymphomas FL CLL MCL MZL LPL CD20 BASIC PANEL CD20 CD3 CD10 CD5 (CD23, Cyclin D1) CD43 BCL2 100 CD79a / CD10 CD23 FMC7 CD5 CD43 Cyclin BCL2 Differential Diagnosis: Aggressive B-cell Lymphomas DLBCL BL LBL Myeloid BASIC PANEL T-cell B-cell CD20 CD79a CD20 CD10 PAX5 CD3 CD43 CD10 BCL2 CD3 MIB-1 <95% 100% 100% 100% 100% TdT BCL2 CD34 CD45 MIB-1 Differential Diagnosis: Hodgkin Lymphomas DLBCL CHL ALCL BASIC PANEL CD30 CD15 CD20 CD3 PAX5 EBV CD CD30 -/ CD15 - +/- - CD20/79a + -/+ (weak) - PAX (weak) - CD /+ ALK - - +/- Other CD10, BCL2 EMA Clonality IGH IGH+/- TCR EBV -/+ -/

81 A Reasonable Approach to FNAs of LPDs STEP 1. CLINICAL SUSPICION: FNA procurement STEP 2. MORPHOLOGY: Cell Size, background, etc STEP 3. PHENOTYPE: Antigen Expressions/Clonality STEP 4. GENOTYPE: Clonality/Chromosomal Abnormalities Establishment of cell lineage Assessment of clonality Detection of chromosomal abnormalities Detection of minimal residual disease 103 PCR: Antigen Receptor Gene Rearrangements Amplification of specific DNA region Bioengineered primers flank region of interest Superior sensitivity: detect single cell in 10 6 cells Specimens Whole blood, bone marrow, body fluids, aspirates Fresh/frozen tissue Formalin fixed, paraffin-embedded tissue, cell blocks Interpretation Establish cell lineage: B cell ~ IG; T-cell ~ TCR Establish clonality Neoplasms have identical rearrangements Monoclonal lesions: 1/2 predominant bands Polyclonal lesions: ladder patterns/smear 104 PCR: IGH gene clonality Polyclonal sample Monoclonal sample Note the normal distribution of polymerase chain reaction products spanning 190 nucleotides. Mantle cell lymphoma. The ratio of the clonal peak to the background exceeds 2.0. Two alleles are rearranged

82 PCR Issues: Antigen Receptor Gene Rearrangements False negatives Inadequate DNA: poor extraction, poor fixation Imperfect consensus primers of complex genes: TCR, IgH Dependent on type of lymphoma Ig somatic hypermutations prevent primer annealing, e.g. FL Does NOT rule out lymphoma False Positives Pseudoclonality ~ few cells present 106 Detection of chromosomal abnormalities Do not occur in normal cells Juxtaposition of oncogene with ARG Lymphomagenesis Activated proteins involve proliferation/differentiation Transcription factors (c-myc) Signal transducers (abl) Cell cycling (cyclin D1) Apoptosis inhibition (bcl-2) Translocation splits 2 different gene loci Leukemogenesis 107 Recurring chromosomal abnormalities Abnormality Genes Follicular lymphoma t(14;18)(q32;q21) BCL2/IgH Mantle cell lymphoma t(11;14)(q13;q32) BCL1/IgH Burkitt lymphoma t(8;14)(q24;q32) & var MYC/IgH MALT lymphoma t(v;18)(v;q21) var/mlt t(1;14)(p22;q32) BCL10/IgH DLBCL t(3;v)(q27;v) BCL6/variable t(14;18)(q32;q21) BCL2/IgH ALCL t(2;5)(p23;q35) NPM/ALK Myeloma t(11;14)(q13;q32), 14q32 BCL1/IgH CLL 13q14, 12, 11q22-23, 17p

83 Chromosomal Translocation Detection: Fluorescence in situ Hybridization (FISH) Fluorescence in situ Hybridization - FISH Improved sensitivity 10 3 cells (upper limit of normal <1%) Specimens: Fresh/frozen tissues/fluids, touch preps, aspirate smears Formalin fixed, paraffin-embedded tissue, cell blocks Warning: You need to know what you re looking for. 109 FISH Strategies FUSION (specific) FL t(14;18)(q32;q21) BCL2/IGH MCL t(11;14)(q13;q32) CCND1/IGH BL t(8;14)(q24;q32) MYC/IGH DLBCL t(3;14)(q27;q32) BCL6/IGH ALCL t(2;5)(p23;q35) NPM/ALK MALT t(11;18)(q21;q21) API1/MLT B-LBL t(9;22)(q34;q11.2) BCR/ABL1 t(11;14)(q13;q32) CCND1/IGH 110 FISH Strategies BREAK APART (non-specific) FL BL DLBCL MALT B-NHL MCL, PCM ALCL 14q32 IGH or 18q21 BCL2 8q24 MYC 14q32 IGH, 3q27 BCL6 18q21 MLT 14q32 IGH 11q13 CCND1 2p23 ALK IGH rearrangement, partner gene unknown

84 FISH Strategies PANELS CLL del(13q14), +12, del(11q22-23), 17p- PCM B-LBL 11q13, 14q32 t(9;22), 11q23, t(12;21), t(1;19) 112 Summary and Conclusions Multiparameter approach: Systematic evaluation of cytomorphology Cellularity, homogeneity, background, etc. Pattern of arrangement: small, mixed, large cells Ancillary phenotypic/genotypic studies essential Appropriate procurement of material Cell blocks, aspirate smears (stained/unstained), dedicated flow Improved interobserver reproducibility Aids in differential diagnoses, e.g. FL vs. CLL vs. MCL Integration with clinical features Consultation with Hematopathology, Molecular Pathology, Genetics Consultation with Clinical Hematology colleagues 113 A Reasonable Approach to FNAs of LPDs STEP 1. CLINICAL SUSPICION: FNA procurement [Smears: cytology, FISH] [Cell suspensions: flow, PCR] [Cell block: immuno, PCR/FISH] STEP 2. MORPHOLOGY: Cell Size, background, etc STEP 3. PHENOTYPE: Antigen Expressions/Clonality STEP 4. GENOTYPE: Clonality/Chromosomal Abnormalities DIAGNOSIS

85 When in doubt, get the node out. - S. Bergman 115 Case: 33 year old male with disseminated lymphadenopathy underwent FNA of inguinal LN 116 Case: 33 year old male with disseminated lymphadenopathy underwent FNA of inguinal LN

86 Case: 33 year old male with disseminated lymphadenopathy underwent FNA of inguinal LN 118 Case: 33 year old male with disseminated lymphadenopathy underwent FNA of inguinal LN Flow: SS CD4 CD45 CD30 CD30 Cell block: 119 Case: 33 year old male with disseminated lymphadenopathy underwent FNA of inguinal LN FISH break-apart for 2p23 ALK

87 Case: 33 year old male with disseminated lymphadenopathy underwent FNA of inguinal LN Diagnosis ALK-positive Anaplastic Large Cell Lymphoma 121 Case: 61 yo female with FNA of periaortic lymph node and peripheral blood lymphocytosis discovered later 122 Case: 61 yo female with FNA of periaortic lymph node and peripheral blood lymphocytosis discovered later

88 Case: 61 yo female with FNA of periaortic lymph node and peripheral blood lymphocytosis discovered later Phenotyping Flow: CD19, CD20 (dim), lambda (dim) positive CD5, CD23 positive CD10, FMC7 negative Genotyping Karyotype of staging marrow 13q- 124 Case: 61 yo female with FNA of periaortic lymph node and peripheral blood lymphocytosis discovered later Diagnosis Small Lymphocytic Lymphoma/Chronic Lymphocytic Leukemia 125 Case: 85 yo female with FNA of peripancreatic lymph node

89 Case: 85 yo female with FNA of peripancreatic lymph node 127 Case: 85 yo female with FNA of peripancreatic lymph node Phenotyping Flow: not obtained (darn!) Cell block: CD10 CD20 BCL2 Genotyping FISH on cell block t(14;18) IGH/BCL2 positive 128 Case: 85 yo female with FNA of peripancreatic lymph node Diagnosis Follicular Lymphoma, low grade

90 Case: 57 yo male with FNA of inguinal lymph node 130 Case: 57 yo male with FNA of inguinal lymph node Phenotyping No cell block (darn!) Flow on aspirate: CD5 CD20 Genotyping FISH on aspirate smear t(11;14) CCND/IGH positive 131 Case: 57 yo male with FNA of inguinal lymph node Diagnosis Mantle Cell Lymphoma

91 Case: 46 yo male with FNA of retroperitoneal mass 133 Case: 46 yo male with FNA of retroperitoneal mass 134 Case: 46 yo male with FNA of retroperitoneal mass Phenotyping Flow: CD19, CD20, lambda positive CD10 positive CD5, TdT, CD34 negative Cell block: BCL2 MIB

92 Case: 46 yo male with FNA of retroperitoneal mass Genotyping FISH: t(8;14) MYC/IGH positive t(14;18) IGH/BCL2 positive 136 Case: 46 yo male with FNA of retroperitoneal mass Diagnosis Aggressive B-cell Lymphoma, Double Hit with both MYC and BCL2 rearrangements 137 Case: 32 yo female with FNA of axillary lymph node [No cell block or flow obtained]

93 Case: 32 yo female with FNA of axillary lymph node [No cell block or flow obtained] Diagnosis Large Cell Lymphoma, most consistent with Classical Hodgkin Lymphoma

94 Helping Pathologists Navigate in an Era of Evolving Health Care North Carolina Society of Pathology 2018 Annual Meeting Patrick E. Godbey, MD, FCAP April 14, 2018 College of American Pathologists. Thank you for inviting me to speak Southeast Georgia Health System Brunswick, Georgia College of American Pathologists. 2 Today s CAP Update Helping Pathologists Navigate in an Era of Evolving Health Care Mobilize Pathologists: Health care regulatory policy and advocacy Advance Quality: Laboratory quality solutions, partnerships, and collaborations Maintain Strength: Membership resources to stay on the forefront of pathology College of American Pathologists. 3 1

95 Mobilize Pathologists Preparing pathologists to meet the opportunities and challenges as health care evolves College of American Pathologists. College of American Pathologists. 4 Protecting the value of pathology services is a never-ending process. Since 2006, 47% of pathology CPT codes have been targeted for revaluation by the Centers for Medicare & Medicaid Services. College of American Pathologists. 5 A $15 million Difference: Advocacy on the Medicare Fee Schedule in 2017 and 2018 Medicare Payments ($Billions) $1.16 $1.15 $1.14 $1.13 $1.12 $1.127 Medicare Payments to Pathologists by Yearly CMS Regulatory Cycle Impact of CAP Advocacy 2018: +$7 million $1.154 Impact of CAP Advocacy 2017: +$8 million $1.135 $1.147 $1.11 Proposed Rule Final Rule Proposed Rule Final Rule CY2017 CY2018 College of American Pathologists. 6 2

96 PAMA and CLFS The CAP advocated on reforms mandated by the Protecting Access to Medicare Act (PAMA) to the clinical laboratory fee schedule (CLFS): o Direct meetings with CMS o grassroots campaign o Comment letters The CAP urged for delay in implementation to allow CMS to improve and redo data collection Final 2018 Medicare CLFS: o $670 million decrease in payments in first year o Top 100 codes: 84 decreased by 10% (max for year); only 4 increased College of American Pathologists. 7 PAMA and CLFS ACLA filed suit against HHS asking for summary judgement to set aside method used by CMS to collect market data The CAP filed amicus brief supporting ACLA suit College of American Pathologists. 8 Pathologists Quality Registry Launched at CAP17 o Offered free 2017 MIPS Reporting Solution o Register for 2018 Pathologists Quality Registry o In 2018, manual data entry via web portal QCDR Status o Earned Qualified Clinical Data Registry (QCDR) status from CMS in 2017 and 2018 o Allows CAP to offer one-stop-shopping for reporting Quality measures and Improvement Activities o 17 CAP-developed pathology measures for measures not available anywhere else College of American Pathologists. 9 3

97 2017 MIPS Reporting Solution: Helped Increase Medicare Bonus Potential Helped small practices: 67% of practices had 5 or fewer pathologists 100% attested to Improvement Activities o Most billing companies cannot do this for pathologists About 30% used the Solution for reporting QPP measures o About 8,000 cases were entered manually o Barrett s esophagus and melanoma reporting most frequently used measures College of American Pathologists. 10 Local Coverage Determination (LCD) Clarification Act Would result in... Upfront description of rationale and evidence for proposed LCD Open, public meetings of Contractor Advisory Committees with public posting of minutes Meaningful appeal process, with ombudsman assistance LCDs not becoming back door National Coverage Determinations College of American Pathologists. 11 LCD Clarification Act of 2017 LCD Clarification Act 15 Senate cosponsors 49 House cosponsors 7 states with at least a senator AND a representative sponsoring the bill. 21 states with either a senator OR a representative sponsoring the bill. 22 states with no sponsorship. College of American Pathologists. 12 4

98 Register Today: 2018 Policy Meeting College of American Pathologists. Washington Marriott, Washington, DC April 30 May 2 Fighting at the Local Level: North Carolina The CAP is continuing to work with NCSP to monitor the implementation of the Avalon Laboratory Benefit Management Program The CAP has issued state model legislation to limit the authority of laboratory benefit management programs, including curbs on prior authorization This spring, the CAP will be urging the AMA HOD to adopt new policy on laboratory benefit management in order to prevent insurance company abuses in administration College of American Pathologists. 14 Advance Quality Advancing quality of care for patients by ensuring the best practices in pathology and laboratory medicine through our laboratory quality solutions College of American Pathologists. College of American Pathologists. 15 5

99 Our Commitment to Laboratory Quality The CAP s laboratory quality solutions, initiated in 1947, currently have customers in more than 100 countries, accrediting more than 8,000 laboratories and providing proficiency testing to more than ~700 22,000 laboratory worldwide. Proficiency testing programs across 16 disciplines College of American Pathologists. 16 Evidence-Based Clinical Practice Guidelines The CAP has published 14 guidelines. Recently published: HPV Testing in Head and Neck Squamous Cell Carcinomas ASCP/CAP/AMP/ASCO: Molecular Markers for Evaluation of Colorectal Cancer CAP/ASH Algorithm for Initial Workup of Acute Leukemia Pathologists can take the lead by implementing CAP guidelines at their institutions and by participating in open comment periods. Visit cap.org > Protocols and Guidelines Coming soon Collaborations with ASCO on: Molecular and Cellular Diagnostics in Localized Prostate Cancer Workup of Initial Diagnosis of Diffuse Gliomas Checkpoint Inhibitor Testing College of American Pathologists. 17 Partnering to Advance the Specialty and Improve Patient Care College of American Pathologists. 18 6

100 Cancer Reporting Tools Help Pathologists Stay Current and Relevant CAP Cancer Protocols, CAP electronic Cancer Checklists (CAP ecc), and CAP electronic Forms and Modules (CAP efrm) Ease burden of staying current with the latest cancer protocol updates Help to ensure compliance to accreditation standards Provide information for big data Deliver confidence that your practice remains compliant with accreditation standards College of American Pathologists. 19 Test Ordering Program Carves out Value for Pathologists on the Health Care Team Test Ordering Program complimentary, members-only resource Offers modules that provide information on test selection, ordering, and interpretation to effect change in evidence-based manner. Topics include: BNP or NT-proBNP Cardiac Marker Testing Practices Hep C Virus Infection Testing Red Blood Cell Folate Testing College of American Pathologists. 20 Maintain Strength Providing membership resources to stay on the forefront of pathology College of American Pathologists. College of American Pathologists. 21 7

101 CAP Membership Reap Value at Every Stage of Your Career Become a member of world s largest organization of board-certified pathologists Join other residents 85% of residents are CAP Junior Members membership is free for residents Get resources for new-in-practice pathologists Access resources by and for practicing, seasoned pathologists o To remain current and relevant o To keep on the forefront of emerging areas of medicine o To stay engaged College of American Pathologists. 22 CAP s New-in-Practice Resources Prepare Members Online resources for new-in- practice CAP members address: Transition from fellowship and residency Job relocation Practice life adjustment Financial management Interpersonal skill enhancement Negotiation through conflice Performance assessment College of American Pathologists. 23 Education and Skills-Development Resources Prepare Members The CAP offers more than 350 educational offerings for pathologists across 50 specialty and professional topic areas to help members: Stay up to date Learn new skills Meet MOC requirements Manage their business College of American Pathologists. 24 8

102 Resources in Emerging Areas of Medicine Keep Members on the Forefront Genomics/Molecular Medicine, Personalized Medicine, and Clinical Informatics Resources Free webinars Pathology resource guides in precision medicine, digital pathology, in vivo microscopy, and clinical informatics SPECs (Short Presentations in Emerging Concepts) focused on molecular and IVM topics Explore a variety of resources on cap.org College of American Pathologists. 25 Resources in Emerging Areas of Medicine Introduction to In Vivo Microscopy (IVM) Interpretation Saturday, June 2, 2018 The Ritz-Carlton, Washington, DC Learn how new and emerging techniques of IVM can enhance your pathology practice. lvminfo@cap.org for more information. College of American Pathologists. 26 People are healthier because of excellence in the practice of pathology and laboratory medicine. College of American Pathologists. 27 9