5/16/2018 HEMATOPATHOLOGY FOR CYTOPATHOLOGISTS HEMATOPATHOLOGY FOR C CYTOPATHOLOGISTS I HAVE NOTHING TO C DISCLOSE

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1 HEMATOPATHOLOGY FOR C CYTOPATHOLOGISTS Kathryn Lindsey, MD McKee Cytology Symposium HEMATOPATHOLOGY FOR C CYTOPATHOLOGISTS Kathryn Lindsey, MD McKee Cytology Symposium I HAVE NOTHING TO C DISCLOSE Kathryn Lindsey, MD McKee Cytology Symposium 1

2 What I won t be discussing. CSFs nobody can save these Exhaustive review of CD markers 7,000+ Lymphoma Classifications by the WHO 7,000+ Benign Lymphadenopathy Classifications Bone marrow aspirates Coagulation Cascade Histopathology of esoteric lymphomas DSMVI Classifications of Hematopathologists Personalities T-Cells REASONABLE, NOT OBSESSIVE Overview Cases based that tricked Cytology discussion mesaved Do you hear what they re saying about us? It may surprise you. Case 1 Case 4 Flow Cytometry Primer or Review; Choose Your Own Adventure Monomorphic Populations of Lymphocytes The Precious, The Few, The Monotonous Lymphomas Case 2 Case 3 Benign Lymphadenopathy Simplified Reasonable, not obsessive, remember? When Flow Fails The Horror The Horror Case 5 Case 6 Other Hematolymphoid Neoplasms The Unusual Suspects Case 7 DO YOU HEAR WHAT C THEY RE SAYING ABOUT US? It may surprise you. 2

3 Do you hear what they re saying about us? 20% of primary or recurrent lymphoma will undergo FNA Obvious benefits of the FNA Prevailing view is that a primary diagnosis should be confirmed by biopsy FNA may be 1 st line diagnostic procedure for the pathologic diagnosis of lymphadenopathy It is accepted in HEMEPATH literature and textbooks that FNA for the diagnosis lymphadenopathy in adult and pediatric patients is 94% Sensitive and 99% Specific What is the accepted sensitivity and specificity of fine needle aspiration (FNA) for the diagnosis lymphadenopathy in adult and pediatric patients? A. 53% sensitivity; 54% specificity B. 27% sensitivity; 72% specificity C. 78% sensitivity; 79% specificity D. 94% sensitivity; 99% specificity 100% 0% 53% sensitivity; 54% specificity 27% sensitivity; 72% specificity 0% 0% 78% sensitivity; 79% specificity 94% sensitivity; 99% specificity Do you hear what they re saying about us? We can do this In fact, we do a great job of suspecting and diagnosing lymphomas Stay with me I ll show you 3

4 C CASE 1 Case 1 60 year old Neck mass History of Sjogren s Syndrome Case 1 60 year old Neck mass History of Sjogren s Syndrome 4

5 /16/2018 Case 1 60 year old Neck mass History of Sjogren s Syndrome CD3 CD20 Case 1 60 year old Neck mass History of Sjogren s Syndrome CD5 BCL CD19 PE Case 1 60 year old Neck mass History of Sjogren s Syndrome CD5 - FITC 5

6 What is the diagnosis? A.Benign Reactive Lymph Node B.Atypical Lymphocytes C.Small Lymphocytic Lymphoma D.Follicular Lymphoma E.Mantle Cell Lymphoma 4% 4% 76% 12% 4% Benign Reactive Lymph Node Atypical Lymphocytes Small Lymphocytic Lymphoma Follicular Lymphoma Mantle Cell Lymphoma Cytologic Diagnosis Small B-Cell Lymphoma Monomorphic population of lymphocytes are present with round regular nuclei with coarsely clumped chromatin. Flow cytometric analysis reveals a clonal B-cell population. Surgical Diagnosis Chronic Lymphocytic Leukemia / Small Lymphocytic Lymphoma Case 1 60 year old Neck mass History of Sjogren s Syndrome Clinical Follow-up This is unlikely to be a CLL/SLL in a patient with Sjogren s. Additionally, it is stage I. Are you sure that it is not a Marginal Zone Lymphoma? Cytologically, it is consistent with CLL. Flow, however, was negative for CD23 Case 1 60 year old Neck mass History of Sjogren s Syndrome 6

7 MONOMORPHIC C POPULATIONS OF LYMPHOCYTES There are precious few monotonous lymphomas. Monomorphic Populations of Lymphocytes Surrogate terminology to mean it looks monotypic or clonal Small-Cell Lymphomas or Low-Grade Lymphomas Small Cell Lymphomas Lymphoplasmacytic Lymphomas Small Cleaved-Cell Lymphomas Because Polymorphic Population of Lymphocytes means it looks polytypic or reactive Monomorphic Populations of Lymphocytes Small Mature B-Cell Lymphomas Small Cell Lymphomas CLL/SLL, perhaps Mantle Cell Lymphomas Lymphoplasmacytic Lymphomas Marginal Zone Lymphoma Small Cleaved-Cell Lymphomas Follicular Lymphoma 7

8 Monomorphic Populations of Lymphocytes CLL/SLL Composed of two cell populations Small round lymphocytes with coarsely clumped chromatin Prolymphocytes, fewer in number, larger, prominent cookie cutter nucleoli Mantle Cell Lymphoma Legitimately monotonous population- not large intermediate sized Delicate clefts- not specifically cleaved, dispersed chromatin Blastoid variant large cells with nucleoli Marginal Zone Lymphoma and Lymphoplasmacytic Lymphoma Composed of small lymphocytes, plasmacytoid lymphocytes, plasma cells, and immunoblasts Follicular Lymphoma Composed of centrocytes (small cleaved lymphocytes) and centroblasts in varying proportions Admixed are Follicutar Dendritic Cells and tangible body macrophages C CASE 2 Case 2 63 year old Lymphadenopathy Abdominal pain 8

9 Case 2 63 year old Lymphadenopathy Abdominal pain Case 2 63 year old Lymphadenopathy Abdominal pain Case 2 63 year old Lymphadenopathy Abdominal pain 9

10 What is the diagnosis? A.Benign Reactive Lymph Node B.Atypical Lymphocytes C.Small Lymphocytic Lymphoma D.Follicular Lymphoma E.Mantle Cell Lymphoma 89% 4% 0% 4% 4% Benign Reactive Lymph Node Atypical Lymphocytes Small Lymphocytic Lymphoma Follicular Lymphoma Mantle Cell Lymphoma Cytologic Diagnosis B-Cell Lymphoma Polymorphic population of lymphocytes showing tangles of smeared nuclei. The nuclei are enlarged and frequently cleaved. Flow cytometric analysis reveals a clonal B-cell population. Case 2 63 year old Lymphadenopathy Abdominal pain Surgical Diagnosis Follicular Lymphoma (Grade1-2) Follicular Lymphoma Small cleaved-cell lymphoma this is a beautiful descriptor Case year old Long-standing history of Follicular Lymphoma New onset ascites 10

11 C CASE 3 Case 3 72 year old History of Lymphoplasmacytic Lymphoma Diagnosis made on bone marrow biopsy not available for review New 18cm retroperitoneal mass Ascites fluid because that s a reasonable specimen Case 3 72 year old History of Lymphoplasmacytic Lymphoma Diagnosis made on bone marrow biopsy not available for review New 18cm retroperitoneal mass Ascites fluid because that s a reasonable specimen 11

12 Case 3 72 year old History of Lymphoplasmacytic Lymphoma Diagnosis made on bone marrow biopsy not available for review New 18cm retroperitoneal mass Ascites fluid because that s a reasonable specimen Case 3 72 year old History of Lymphoplasmacytic Lymphoma Diagnosis made on bone marrow biopsy not available for review New 18cm retroperitoneal mass Ascites fluid because that s a reasonable specimen Case 3 72 year old History of Lymphoplasmacytic Lymphoma Diagnosis made on bone marrow biopsy not available for review New 18cm retroperitoneal mass Ascites fluid because that s a reasonable specimen 12

13 What is the diagnosis? A.Positive for Lymphoma consistent with patient s diagnosis B.Positive for Malignant Cells C.Atypical Lymphocytes D.Reactive Mesothelial Cells E.Melanoma 53% 37% 5% 5% Positive for Malignant Cells Atypical Lymphocytes Reactive Mesothelial Cells Melanoma Cytologic Diagnosis Positive for Malignant Cells, Consistent with Plasma Cell Neoplasm Malignant plasmacytoid cells are present. These cells have voluminous cytoplasm and markedly irregular nuclei and nucleoli. Mitotic figures are readily identified. Surgical Diagnosis still waiting for that biopsy Now her diagnosis is listed as Rare Aggressive Lymphoplasmacytic Malignancy Case 3 72 year old History of Lymphoplasmacytic Lymphoma Diagnosis made on bone marrow biopsy not available for review New 18cm retroperitoneal mass Ascites fluid because that s a reasonable specimen Monomorphic Populations of Lymphocytes Very few lymphomas are actually monomorphic ALL CD5+ lymphomas should be interrogated for Mantle Cell characteristics ALL OF THEM! Cytologic atypia within a polymorphic population remains cytologic atypia Don t be unreasonable just because there was a weird heme diagnosis 13

14 BENIGN C LYMPHADENOPATHY SIMPLIFIED Reasonable, not obsessive, remember? Benign Lymphadenopathy Simplified Lymphadenitis infectious or inflammatory Acute Granulomatous Reactive Lymphoid Hyperplasia diverse and diagnostically challenging Involvement Patterns Paracortical Hyperplasia Follicular Hyperplasia Sinus Expansion The Lymph Node Flow will catch a small B-cell population Benign Lymphadenopathy Simplified Commonly Sited Features that Favor Reactive Lymphoid Hyperplasia Polymprphic population T-Cells > B-Cells Tingible Body Macrophages Frequent Mitoses The Features that I think Favor Reactive Lymphoid Hyperplasia No large atypical cell population Lymphocytes with HIGH N:C The radiologist doesn t know why it needs a biopsy Lymph node size < 2 cm 14

15 Which of the following is false regarding differentiating between neoplastic and non-neoplastic lymphoid hyperplasias? A. The smear pattern and lymphocyte population vary considerably based on the stage of the reactive process and compartment of the lymph node that the process primarily affects. B. A high mitotic rate can be seen in both neoplastic and nonneoplastic lymphoid hyperplasias C. A polymorphic population is the best indicator of a non-neoplastic process. D. B and T-cell lymphomas and Hodgkin Lymphomas are the most often encountered in clinical practice. The smear pattern and lymp... 0% 0% 0% 0% A high mitotic rate can be see... A polymorphic population is... B and T cell lymphomas and... C CASE 4 15

16 Case 4 50 year old Para-aortic lymphadenopathy History of Renal Cell Carcinoma Case 4 50 year old Para-aortic lymphadenopathy History of Renal Cell Carcinoma Case 4 50 year old Para-aortic lymphadenopathy History of Renal Cell Carcinoma 16

17 Case 4 50 year old Para-aortic lymphadenopathy History of Renal Cell Carcinoma What is the most appropriate response to you surgical pathologist friend? A.Let it go looks reactive B.There were some really bad cells cytologically. Let s order some more stains. 94% 6% Let it go looks reactive There were some really bad c... CD45 Case 4 50 year old Para-aortic lymphadenopathy History of Renal Cell Carcinoma 17

18 CD30 Case 4 50 year old Para-aortic lymphadenopathy History of Renal Cell Carcinoma Cytologic Diagnosis Suspicious for malignant cells Polymorphic population of lymphocytes showing very rare, markedly atypical cells. Surgical Diagnosis CD30+ Anaplastic Large Cell Lymphoma Case 4 50 year old Para-aortic lymphadenopathy History of Renal Cell Carcinoma Benign Lymphadenopathy If the lymph node is less than 2cm, let it go Small mature B-cell lymphomas can be missed and it s okay. P.S. Flow should catch those Large atypical cells should not be let go Or force heme to take the fall 18

19 C FLOW CYTOMETRY Primer or Review Choose your Own Adventure Flow Cytometry A word on Fluorescence Flow Cytometry A word on Fluorescence 19

20 Flow Cytometry Antibodies and Fluorochromes Flow Cytometry Antibodies and Fluorochromes Flow Cytometry Antibodies and Fluorochromes Each tube has only 4 different fluorochromes conjugated to 4 different antibodies Lambda LC Kappa LC CD19+20 CD45 CD5 CD19 CD10 CD45 CD7 CD2 CD3 CD45 CD8 CD38 CD4 CD45 Viability Sample Size History 20

21 Flow Cytometry Antibodies and Fluorochromes Tube FITC PE PC5 ECD Disease Screen 1 Lambda LC Kappa LC CD19+20 CD45 B-cell clonality 2 CD5 CD19 CD10 CD45 Types B-cell neoplasms 3 CD7 CD2 CD3 CD45 TCL & NKL 4 CD8 CD38 CD4 CD45 TCL & PC 5 CD11b CD123 CD56 CD45 AML & MDS 6 CD15 CD117 CD34 CD45 AML & MDS 7 CD16 CD13 HLA-DR CD45 AML & MDS 8 CD14 (Mo2) CD33 CD64 CD45 AML & MDS Flow Cytometry Antibodies and Fluorochromes B-cell CLL cell Fluorochrome tag CD20 antibody Flow Cytometry Antibodies and Fluorochromes B-cell Hairy Cell Fluorochrome tag CD22 antibody 21

22 Flow Cytometry - Sample Prep Flow Cytometry - Sample Prep Flow Cytometry Fluid Dynamics Flow Tube Detection 22

23 LASER Flow Cytometry Lasers Flow Tube Little Complexity Small Shadow Detection LASER Flow Cytometry Lasers Flow Tube Little Complexity Small Shadow Detection LASER Flow Cytometry Lasers Flow Tube Greater Complexity Bigger Shadow Detection 23

24 LASER Flow Cytometry Lasers Flow Tube Greatest Complexity Bigger Shadow Detection LASER LASER Flow Cytometry Lasers Flow Tube Side Scatter Forward Scatter Greater Complexity Bigger Shadow Little Complexity Small Shadow Detection LASER LASER Flow Cytometry Lasers B-cell T-cell CD5 CD19 CD10 CD45 B T Detection 24

25 LASER Flow Cytometry Lasers LASER T B Side PC5 FITC Scatter CD10 CD5 TB B-cell T-cell CD5 CD19 CD10 CD45 ECD PE EDC - CD19 CD45 Detection Flow Cytometry Data Output All the data for each cell collected as events Three pieces of information for each event Size Complexity Fluorescence User defined axes for each graph Isn t that nice? Flow Cytometry Examples CD CD Clonal B-cells Kappa Restricted Kappa Lambda 25

26 Flow Cytometry Examples CD Kappa CD5 + Clonal B-cells Lambda Restricted CD Lambda Flow cytometers give information about a cell s internal complexity and size. Which of the following is the correct pairing? A. Forward scatter is an indicator of size, and side scatter is an indicator of internal complexity. B. Forward scatter is an indicator of internal complexity, and side scatter is an indicator of size. C. The ratio of forward scatter to side scatter is an indicator of size, and side scatter is an indicator of internal complexity. D. Forward scatter and side scatter are used to determine a cells viability not to indicate size or internal complexity. 77% 8% 15% 0% Forward scatter is an indicat.. Forward scatter is an indicat.. The ratio of forward scatter t... Forward scatter and side scatt.. Flow Cytometry Flow is no longer ancillary just like immunostains or molecular it s expected We need to understand and use it Not that complicated The pre-analytical phase is still the most important If you don t enjoy getting blank requisitions, don t send the flow cytometry lab blank requisitions 26

27 C WHEN FLOW FAILS The Horror; The Horror When Flow Fails My Experience To flow is better than not to flow even when large cells are the question 16% failure to detect a clonal population by flow despite diagnostic material of both large and small cell lymphomas Size is often sited as a cause Large is bigger than an endothelial cell nucleus (17-20um) Most flow cell apertures are 150um or greater C CASE 5 27

28 Case 4 60 year old Previous lung nodule Suspicious for Focal Diffuse Large B-cell Lymphoma FNA of that very same lung nodule Case 4 60 year old Previous lung nodule Suspicious for Focal Diffuse Large B-cell Lymphoma FNA of that very same lung nodule Case 4 60 year old Previous lung nodule Suspicious for Focal Diffuse Large B-cell Lymphoma FNA of that very same lung nodule 28

29 Case 4 60 year old Previous lung nodule Suspicious for Focal Diffuse Large B-cell Lymphoma FNA of that very same lung nodule Case 4 60 year old Previous lung nodule Suspicious for Focal Diffuse Large B-cell Lymphoma FNA of that very same lung nodule What is the diagnosis? A.Atypical Lymphocytes B.Suspicious for Malignant Cells C.Positive for Malignant Cells D.Large B-cell Lymphoma 29% 12% 24% 35% Atypical Lymphocytes Suspicious for Malignant Cells Positive for Malignant Cells Large B cell Lymphoma 29

30 Cytologic Diagnosis Large B-Cell Lymphoma Polymorphic population of lymphocytes showing scattered large malignant lymphocytes. Immunohistochemical staining of smears reveals these cells to be B-cell lineage. Flow cytometric analysis shows no B-cells, likely due to sampling or poor viability. Surgical Diagnosis performed 11 months later, paraspinal mass Diffuse Large B-cell Lymphoma, Activated B-cell Subtype Case 4 60 year old Previous lung nodule Suspicious for Focal Diffuse Large B-cell Lymphoma FNA of that very same lung nodule C CASE 6 Case 5 22 year old Presents with shortness of breath Large anterior mediastinal mass 30

31 Case 5 22 year old Presents with shortness of breath Large anterior mediastinal mass What is the diagnosis? A.Positive for malignant cells B.Atypical Lymphocytes C.Hodgkin Lymphoma D.Thymoma 90% 10% Positive for malignant cells Atypical Lymphocytes 0% Hodgkin Lymphoma 0% Thymoma Cytologic Diagnosis Positive for Malignant Cells Polymorphic population of lymphocytes showing scattered large malignant lymphocytes in a background of small lymphocytes, plasma cells, and scattered eosinophils. The malignant lymphocytes show bizarre and anaplastic forms. There are frequently binucleate forms, consistent with Reid- Sternberg cells. Surgical Diagnosis Nodular Sclerosis Classical Hodgkin Lymphoma Case 5 22 year old Presents with shortness of breath Large anterior mediastinal mass 31

32 When Flow Fails If it s malignant, it s still malignant Even as a primary diagnosis Just Malignant is fine if it is not unequivocally lymphoma I support running B and T cell tubes on all cases There are characteristic findings in T-cells OTHER C HEMATOLYMPHOID NEOPLASMS The Unusual Suspects Other Hematolymphoid Neoplasms Trilineage hematopoiesis Leukocytes Erythrocytes Thrombocytes Leukemias Lymphocytic Myeloid 32

33 This image cannot currently be displayed. 5/16/2018 Other Hematolymphoid Neoplasms Why cytopathologists should care: CSFs FNAs of masses Just like squamous vs. adenocarcinoma the difference matters If we can t tell, I recommend the use of the term hematolymphoid C CASE 7 Case 6 50 year old History of Acute Myeloid Leukemia Bilateral Preauricular masses 33

34 This image cannot currently be displayed. 5/16/2018 Case 6 50 year old History of Acute Myeloid Leukemia Bilateral Preauricular masses What is the appropriate diagnosis? A. Positive for lymphoma; B-cell screen flow cytometry. B. Atypical lymphocytes; B-cell screen flow cytometry. C. Positive for malignant cells; Acute leukemia flow cytometry. D. Lymphocytes present; Attempt cell block for immunostains. 0% Positive for lymphoma; B cell... Atypical lymphocytes; B cell... 75% 13% 13% Positive for malignant cells;... Lymphocytes present; Attemp... Cytologic Diagnosis Positive for Malignant Cells Malignant cells showing marked nuclear pleomorphism, fine chromatin, nucleoli, and granulated cytoplasm are present. The findings are consistent with the patient s known Acute Myeloid Leukemia. In the appropriate clinical setting of a destructive mass lesion, the diagnosis is Myeloid Sarcoma. Surgical Diagnosis the patient died a week later Autopsy showed myeloid blast infiltrates and masses, everywhere Case 6 50 year old History of Acute Myeloid Leukemia Bilateral Preauricular masses 34

35 Reasonable Conclusions Focus on the cytologic features Smear description should match the actual smear Don t force square pegs in round holes Understand flow cytometry and USE it Lymphoid and myeloid lineages are different We re good at this; we are still expected to be. References Das, DK. Value and Limitations of Fine-Needle Aspiration Cytology in Diagnosis and Classification of Lymphomas: A Review. Diagnostic Cytopathology, Vol 21, No 4. Classification of Tumours of Haematopoietic and Lymphoid Tissues. World Health Organization. Swerdlow, et. al. Ed Non-Neoplastic Hematopathology and Infections. Cualing, HD, et.al. Ed Seidel TA, Garbes AD. Cellules grumelees. Old terminology revisited. Regarding the cytologic diagnosis of chronic lymphocytic leukemia and well differentiated lymphocytic lymphoma in pleural effusion. Acta Cytol 1985;29: Acknowledgements My Cytopathology and Hematopathology Mentors Drs. Jack Yang and John Krause two of the finest and most reasonable pathologists I know Our wonderful cytotechs very much my mentors as well Dr. Olga Chajewski Our Hematopathology and Cytopathology Fellows My Family they know that I am unreasonable and love me anyway 35