77R (REPEAT) Flow Cytometry: Basic Principles and Case Analysis. Charles Goolsby PhD Kristy Wolniak MD, PhD

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1 77R (REPEAT) Flow Cytometry: Basic Principles and Case Analysis Charles Goolsby PhD Kristy Wolniak MD, PhD 011 Annual Meeting Las Vegas, NV AMERICAN SOCIETY FOR CLINICAL PATHOLOGY 33 W. Monroe, Ste Chicago, IL 60603

2 77R (REPEAT) Flow Cytometry: Basic Principles and Case Analysis This session will present basic principles of flow cytometry analysis of hematopoietic malignancies through short lecture and mutlitple case presentations including demonstrated listmode analysis of the cases. Although both acute and chronic processes will be presented, there will be an emphasis on chronic lymphoid and lymphoma cases. The case presentations will also stress the analysis of the flow cytometry results in the context of the morphology and patient presentation. Appreication of the variety of flow cytometry analysis approaches necessary for sensitive and specific analysis of hematopoietic malignancies. Understand basic technical aspects/prinicples including epitope deletion/masking and defining positive and negative staining including in cases of dim staining. Understand the goals and basic guidelines of flow cytometric analysis of hematopoietic malignancies. FACULTY: Charles Goolsby PhD Kristy Wolniak MD, PhD Practicing Pathologists Hematopathology Hematopathology.0 CME/CMLE Credits Accreditation Statement: The American Society for Clinical Pathology (ASCP) is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education (CME) for physicians. This activity has been planned and implemented in accordance with the Essential Areas and Policies of the Accreditation Council for Continuing Medical Education (ACCME). Credit Designation: The ASCP designates this enduring material for a maximum of AMA PRA Category 1 Credits. Physicians should only claim credit commensurate with the extent of their participation in the activity. ASCP continuing education activities are accepted by California, Florida, and many other states for relicensure of clinical laboratory personnel. ASCP designates these activities for the indicated number of Continuing Medical Laboratory Education (CMLE) credit hours. ASCP CMLE credit hours are acceptable to meet the continuing education requirements for the ASCP Board of Registry Certification Maintenance Program. All ASCP CMLE programs are conducted at intermediate to advanced levels of learning. Continuing medical education (CME) activities offered by ASCP are acceptable for the American Board of Pathology s Maintenance of Certification Program.

3 Flow cytometry: Basic analysis of hematopoietic malignancies Charles Goolsby Kristy Wolniak Northwestern University Medical School Flow cytometry: Immunphenotyping in diagnosis of hematopoietic malignancies Key, critical component of primary diagnostic workup Adjunctive: Critical is correlation with Morphology Other laboratory/pathology data Clinical data/presentation Cytogenetics/molecular History Flow cytometry: Lineage determination B versus T Lymphoid versus myeloid Establishing B cell clonality Benign (polyclonal) versus malignant (clonal) Clonal implies malignant??? Diagnostic classification CLL versus mantle cell lymphoma (MCL) Follicular lymphoma HCL Prognostic ZAP-70 8 CD3 Integral component WHO classification Detection of bone marrow involvement in lymphoma Sensitive methodology (< 1%) Detection of circulating lymphoma cells Residual disease detection 1

4 Flow cytometry: Flow cytometry: Potpourri of general analysis comments +/- Intensity Epitope deletion/masking Dim staining General analysis guidelines Example case analyses : What is + and -? Internal Cellular Controls 4 Intensity 3 Intensity 0 ed Light Intensity ed Light Intensity Intensity CD7 Intensity

5 : Staining Intensity CD0 DIM CD0 MODERATE CD0 BRIGHT Comparison to normal staining intensity Quantitative number of bound antibodies : Dim staining comments Dim staining = dim antigen? Establish staining level where confident Below that, what to do Competitive inhibition Epitope not practical, cost prohibitive Unlabeled antibody, expensive : Dim staining comments Dim staining = dim antigen? Establish staining level where confident Below that, what to do Competitive inhibition Epitope not practical, cost prohibitive Unlabeled antibody, expensive Independent antibodies, brighter fluorochrome No staining not necessarily informative Increase or change blocking reagents Routine 30% FBS 50% up to 100% NMS other 3

6 : Dim staining comments 30% FBS 80% NMS CD45 CD45 Surface Surface : Dim staining With 50% normal mouse serum With 100% normal mouse serum S/N ~ 1.3 S/N ~ 3.1 CD10 CD10 : Epitope deletion/masking No staining=no / antigen Kappa Lambda Kappa Lambda -Frequent with Kappa/lambda -Can happen with any antigen 4

7 : Epitope deletion/masking CD1 19 CD PE A CD3* 10 5 PE A CD3* *Independent anti-cd3 antibodies : Epitope deletion/masking In ntensity In ntensity CD7 Intensity* CD7 Intensity* * Independent anti-cd7 antibodies General guidelines Analysis Goals Pattern recognition abnormal patterns Subjective, experience needed Scatter characteristics of a subset Scatter/antigen ti pattern Multiple antigen pattern Are unusual /aberrant patterns abnormal or reactive change? Pattern of reactivity with a panel antigens Characteristic immunophenotypic signatures CLL vs mantle cell Follicular lymphoma/burkitt s 5

8 General guidelines Account for all cells Are they normal/abnormal? Positive identification of cells of interest Minimally two + antigens Minimally one antigen More the better Pan restricted vs associated antigens Boolean gating virtually all cases Scatter gate to remove debris/dead cells, etc Antigen sets to identify subsets Linked to assess other antigen expression Multiple strategies will be needed Always assess un-gated for all antigens FS vs SS, In way best attuned to note aberrant patterns Color eventing/gating Powerful if done to add information content General guidelines CD Forward Sc catter CD General guidelines atter Forward Sca CD5 CD5 6

9 General guidelines 4 Intensity 3 Side Kappa Scattered Intensity Light Intensity ed Light Forward Lambda Scattered Intensity Light CD0 Intensity Intensity Intensity Intensity CD5 Intensity CD103 Intensity CD11c Intensity General guidelines Intensity Kappa Intensity Kappa Intensity ed Light Lambda Intensity Lambda Intensity Kappa Intensity Intensity Intensity Lambda Intensity Kappa Intensity Lambda Intensity General guidelines Intensity Intensity Kappa Intensity CD10 Intensity CD5 Intensity Lambda Intensity Intensity Intensity CD0 Intensity CD79b Intensity 7

10 General guidelines Intensity Gate 5 Gate 3 Gate 4 Kappa Intensity CD10 Intensity Lambda Intensity Kappa Intensity Kappa Intensity Kappa Intensity Lambda Intensity Lambda Intensity Lambda Intensity Patient #1: Case History 78 year old female with a relative lymphocytosis Asymptomatic Laboratory values WBC 11,300/uL Hemoglobin 1.9 g/dl Platelets 195,000/uL Peripheral blood Patient #1 Forward Scatter CD45 - Debri 81% 1% 17% Lymphocytes +: 41% CD5 Lymphocytes Lymphocytes + cells +: 55% CD5 NK: 5% CD CD5 CD16/56/57 CD8 8

11 Patient #1 + cells Lymphocytes Kappa Lambda CD3 Lymphocytes Lymphocytes Lymphocytes FMC7 CD79b CD0 Peripheral blood smear Mantle cell lymphoma + Cells Kappa Lambda CD5 CD3 FMC7 CD79b CD0 9

12 Mature B cell malignancies-panel of antigens +/ CLL Mantle Follicular Marginal CD CD sig dim CD (+) - CD0 dim (bright) + FMC CD79b CD11c - (+) - (+) - (+) + -(dim) CD5 - (+) CD Distinctly increased scatter light intensity CLL variability in archetypical pattern ~40% of cases aytpical (ie moderate or bright) staining one or more of the pan B cell antigens* 36% CD0 19% CD79b 7% FMC7 No correlation atypical pan B cell antigen staining intensity and morphology (CLL/PL or transformed) or extent of BM involvement* CD3 generally moderate to bright Can be variable Subset mantle cell lymphoma CD3+ * Monaghan SA et al, Clinical Cytometry, 003 Mantle cell lymphoma Intensity Intensity CD5 Intensity CD3 Intensity Kappa Intensity Lambda Intensity Intensity FMC7 Intensity Cyclin D1 + t(11;14) + 10

13 100 Mantle cell lymphoma Event-free, CD3-negative Event-free CD3-positive Percent survival Percent survival Time in Months Overall, CD3-negative Overall, CD3-positive Time in Months 30-40% CD3 + 8% all sites 11% discordant between sites Pertinent clinical differences Bulky disease (13% vs 39%) Splenomegaly (73% vs 4%) 4 year EFS: 47% vs 18%, p=0.0 4 year OS: 76% vs 5%, p=0.05 Multivariate Cox regression CD3, LDH, and HSCT Hazards ratio (EFS) CD3: 0.31, p=0.06 LDH: 0.5, p=0.18 HSCT: 0.99, p=0.99 Keleman et al, AJCP, 008 Patient #: Case History 41 year old male with a rectal mass The patient has a history of HIV and recent left lower quadrant pain Laboratory values WBC 1,500/uL (low) Hemoglobin 8.7 g/dl (low) Platelets 3,000/uL (low) Bone marrow aspirate Patient # 04 + cells Forward Scatter Kappa Lambda Normal cells BM 10 #1 4 # CD10 # Gate 5 # PE C 7 A CD0 Kappa Lambda Kappa Lambda 11

14 Normal bone marrow Bone marrow Bone marrow 1

15 Burkitt Lymphoma Rapidly proliferating B cell lymphoma Frequently associated with translocation of MYC Three types of Burkitt lymphoma Endemic Children ages 4-7, predominantly males Equatorial Africa Most are associated with EBV Sporadic Mainly in children and young adults Low incidence world-wide Immunodeficiency-associated Particularly associated with HIV Often occurs before CD4+ counts are reduced Burkitt Lymphoma Diagnosis Clinical features, morphology, immunophenotype, and genetics Clinical features Extranodal sites often involved Endemic form jaw and orbital lesions Majority of patients present at an advanced stage Highly aggressive, but can be cured Characteristic morphology Medium-sized cells, basophilic cytoplasm, small round nuclei Starry sky pattern in solid tissue with admixed tingible body macrophages Frequent mitoses Genetics t(8;14)(q4;q3) found in a majority of cases (c-myc and IgH fusion) CD10+ differential CD10+, sig+, clonal/monotypic B cell population Follic lar Follicular Large cell lymphoma Burkitt s Rare immature acute B cell lymphoblastic leukemia 13

16 Patient #3: Case History 79 year old female with an incidental finding of lymphadenopathy on imaging In good health and asymptomatic Laboratory values WBC 7,000/uL Hemoglobin 13. g/dl Platelets 30,000/uL Lymph node Patient #3 Forward Scatter CD5 Kappa Kappa CD10 Lambda Lambda Patient #3 CD0 CD5 CD0 CD10 CD10 Kappa Side Lambda Scatter CD79b CD3 14

17 Patient #3 Two monotypic (surface Kappa) + populations CD5+, CD10- CD3+ FMC7-, dim CD79b+ Dimmer CD0+ CD10+, CD5- Brighter CD0+ Increased scattered light intensity FMC7+, CD79b+ Lymph node Patient #4: Case History 68 year old male with skin lesions Multiple erythematous plaque lesions on thighs and upper extremities. No other symptoms. Laboratory values WBC 6700/uL Hemoglobin 14.5 g/dl Platelets 0,000/uL Peripheral blood 15

18 0 0 10/8/011 Patient # CD4 CD8 CD CD5 CD CD6 Peripheral blood Peripheral blood 16

19 Mycosis Fungoides/Sezary Syndrome: Pan T cell antigen loss/alteration Mycosis Fungoides (MF) Most common CTCL (~50% of primary cutaneous lymphomas) Epidermotropic clonal T cell malignancy Skin lesions (patches, plaques, etc) Cells with characteristic cerebriform nuclei Primarily older adults Slight male predominance (1.5 to :1) Indolent 85-90% 5 year survival Sezary syndrome Many common pathology/immunophenotypic features with MF Disease of adults Skin lesions and generalized lymphadenopathy Malignant cells in skin, lymph node, and peripheral blood Refer to Blood 105:3768, 005 for diagnostic criteria Aggressive disease ~5% 5 year survival Mycosis Fungoides/Sezary Syndrome Immunophenotype Mature T cell malignancy (surface +) Most frequently, T helper immunophenotype +CD4+ Characteristically, CD7- May be partial Can be CD7+ Typically, CD+, CD5+ But deletion can be seen Altered expression of any of the pan T cell antigens can be seen Memory cell immunphenotype (CD45RO+, CD9+, etc) Most frequently, CD5- CD5+ in ~10-0% of cases CD7 Modulation in Reactive T cells Reactive T cells can modulate pan T cell antigen expression Most temporally short CD7 can be significant and persistent Dim CD7 to CD7- reactive T cells can be CD4+ CD5+, CD6+ (activated immunophenotype) Overlaps with MF/Sezary and ATLL immunophenotype Pattern of CD7 staining can be helpful 17

20 CD7 Modulation in Reactive T cells CD4 CD7 CD8 Mycosis Fungoides/Sezary Syndrome CD6 T cell activation antigen Membrane and secreted protein Modulates chemokine activity Co-stimulatory molecule (both and CD pathways) Reported to be useful differentiating Sezary cells from reactive T cells Jones et al, Am J Clin Path 115: 885, 001 Bernengo et al, Br J Dermatol 144(#1):15, 001 Circulating Sezary cells frequently CD6- Reactive Dim CD7 to CD7- cells which would be CD6+ Mycosis Fungoides/Sezary Syndrome CD7 CD6 CD7 CD6 18

21 Mycosis Fungoides/Sezary Syndrome How useful? % % 73% 69% 43% CD6 expression (%) CD4/CD8 >10 >5% convoluted lymphocytes >1000/mm 3 convoluted lymphocytes Positive TCR gene rearrangement Overall, ~64% of cases show CD4+CD6- Keleman et al, AJCP, in press, 007 Lymphoblastic Lymphoma/Leukemia 14 year old male Shortness of breath and cough Chest discomfort Chest x-ray revealed a large mediastinal mass and left pleural effusion CBC: normal Received mediastinal mass biopsy Lymphoblastic Lymphoma/Leukemia CD4 CD4 CD CD8 CD1 CD5 CD5 CD5 TdT s c 19

22 Acute Lymphoblastic Leukemia Bone marrow aspirate Bone marrow core biopsy Lymphoblastic Lymphoma/Leukemia Proliferation of malignant blast cells T-LBL and T-ALL same disease T-LBL ~80-85% of lymphoblastic lymphomas Primarily, young males Most frequently presents with mediastinal mass Can involve lymph node, spleen, skin, liver Pleural effusions common Peripheral blood/bone marrow (if extensive T- ALL) Lymphoblastic Lymphoma/Leukemia Immunophenotype Most frequent is common thymic T cell immunophenotype s-, c+, CD+, CD5+, CD7+ Less often dim s or s+ staining can be seen CD4+/CD8+, CD1a+ TdT+, 4- Frequently, CD10+ Loss or modulation of CD, CD5, or CD7 can be seen Aberrant expression of CD13 and/or 3 can be seen CD79a rarely Although rare, more immature immunphenotypes can be seen 0

23 Patient #5: Case History 60 year old male with incidental finding of a mediastinal mass on pre-op evaluation Asymptomatic Laboratory values WBC 3500/uL Hemoglobin 14. g/dl Platelets 9,000/uL Patient #5 Forward Scatter CD7 Gate 1 Surface Surface CD5 CD4 Cytoplasmic CD5 CD CD1a CD8 Mediastinal mass 1

24 Mediastinal mass Cytokeratin immunohistochemistry Lymphoblastic Lymphoma/Leukemia Frequent differential in patient with mediastinal mass Thymoma vs T-LBL/T-ALL Immunophenotype of T-LBL/T-ALL (s-, c+, CD+, CD5+, CD7+, CD4+/CD8+, CD1a+, TdT+) can overlap with normal immature thymic T cells T cells differentiate in the thymus Normal thymus mixture of T and non-hematopoietic cells Immature, common thymocyte T cells More mature, medullary thymocytes Mature T cells exiting to the periphery Lymphoblastic Lymphoma/Leukemia Immature Thymus Peripheral 4 CD CD CD CD5 TdT CD CD7 CD5 CD1 TdT CD7 CD5 CD7 CD5 CD7 CD4/CD8 CD4 or CD8 CD4 or CD8 Common Thymocyte Medullary Thymocyte Normal is a process of differentiation Common and medullary populations Transition immunphenotypes as well Pattern of staining can be helpful in differentiating normal vs abnormal

25 Pattern of expression s CD4 CD1a CD8 Pattern of expression Normal BM Archetypical B lymphoblastic leukemia CD10 CD10 CD0 CD0 Patient #6: Case History 7 year old female with diffuse lymphadenopathy and hepatosplenomegaly Anorexia, fatigue, dyspnea, and fevers Laboratory values WBC 13,100/uL (high) Hemoglobin 8.4 g/dl (low) Platelets 138,000/uL Lymph node 3

26 Patient #6 4 Forward Scatter 8 6 CD45 % 77% 0 CD T cells 81% T cells CD4 18% CD5 CD CD8 Patient # CD10 CD All T CD4 0 0 CD10 CD8 Lymph node 4

27 Lymph node Patient #7: Case History 81 y.o. female Found on floor at home, too weak to walk/stand (spot easy access to bathroom) Denies passing out H/O anemia (refused w/u, takes Fe) Fatigue -3 wks, Myalgias, chills, fever (100.4F), drenching night sweats No appetite or regular meals for ~10 days Patient #7: Case History cont. Treated with antibiotics, mild improvement Thought she had the flu Labs WBC: 33.1 (~11.0 a few months prior), HGB: 8., HCT: 5., MCV: 77, RDW: 19.7, PLT:117 BP 97/5, T 98.6, H 103, RR 16 PE: Alert, no sign of leukostasis Heme/Onc fellow evaluation Blood smear: mature appearing lymphocytes No hemolysis Peripheral blood sample received by flow lab 5

28 Patient #7 Forward Scatter F CD Forward Scatter Lymphoblast CD45 Myeloblast Immature Granulocyte

29 Patient #7 Forward Scatter CD CD CD CD CD7 Patient #7 5 5 CD CD CD CD CD CD13 3 Patient #7 CD45 MPO Cytoplasmic CD79a TdT Cytoplasmic CD CD7 CD5 CD64 7

30 Patient #7 ~97% dim CD45, low to intermediate SS intensity cells (<0.1% B cells) Immature antigens 4+, CD117+ TdT- Myeloid associated antigens Myeloperoxidase+, CD13+, 3+ CD64-, CD11b- T cell associated antigens Dim aberrant CD7 and CD5 - (cytoplasmic and surface), CD- B cell associated antigens -, CD0-, CD79a- Other HLADR+ Myeloperoxidase Patient #7: Follow-up Follow up Diagnosis AML Not treated per family decision Transferred to Hospice 8

31 AML Flow Cytometric Analysis AML Lineage Sub-classification No Uitility Correlations Aberrant antigen expression CD7: ~30 to 35% CD: ~0% CD5: rarer : ~0% TdT: ~30% Helpful in confirming abnormal Summary Analysis of a panel of antigens Patterns of staining with multiple antigens Recognition of normal and abnormal Account for all cells + and antigens for all populations Recognition of abnormal (normal) is subjective and pattern recognition driven Experience needed +/-, intensity, scattered light characteristics Multiple gating/data presentation approaches required Technical issues including Dim staining vs antigen expression Lack of staining vs lack of antigen (epitope alteration) Careful correlation with morphology, pathology, molecular, cytogenetics,. Flow cytometry: Northwestern Memorial Clinical Flow Cytometry Laboratory Laura Marzalek Adella Khong Janet McLaughlin Keisha Hughes Carolina Ostiguinin Maybelle Tiongson Hematopathology Team LoAnn Peterson Beverly Nelson Diana Variakojis Amy Chadburn Yihua Chen Bill Karpus 9

32 Flow cytometry: Basic analysis of hematopoietic malignancies Discussion i and Questions 30