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5/23/2013 Acknowldegements Case examples from: Diagnosis of Lymphoma by University of California San Francisco Cytology Study Set Fine Needle Aspiration Biopsy Massachusetts General Hospital Cytology Study Set Ronald Balassanian, MD

  1. 5/23/2013 Acknowldegements Case examples from: Diagnosis of Lymphoma by University of California San Francisco Cytology Study Set Fine Needle Aspiration Biopsy Massachusetts General Hospital Cytology Study Set Ronald Balassanian, MD Assistant Clinical Professor University of California San Francisco I have nothing to disclose Patient Evaluation History Duration of lymphadenopathy Change in size, fluctuation History of cancer, lymphoma Recent travel Exposure to infectious agents 1

  2. 5/23/2013 Patient Evaluation Patient Evaluation Physical exam: Review of systems Size Recent viral illness, cold flu, sinusitis … think back Characteristics: Dental procedures, problems Soft, firm, mobile, fixed, matted adjacent B symptoms: Fever, night sweats, chills, lymph nodes weight loss, headache Location Rash, itching, skin changes Cough, back pain, Amenable to biopsy Smoking… time to quit palpation image guidance, US, CT Lymph node FNAB Lymph node FNAB Establish clinical index of suspicion Clinically benign/reactive Possibly infectious Possibly metastatic Lymphoma 1 st pass 2 nd pass and additional passes Reactive lymph node Reactive lymph node Other Acute or chronic lymphadenitis Acute or chronic lymphadenitis Infectious process Infectious process Surprise!!! and Lymphoma, non-Hodgkin 2

  3. 5/23/2013 Lymph node FNAB Lymph node FNAB MAXIMIZE the diagnostic yield Smears – save unstained for testing Flow cytometry Cell block, IHC, special stains, Molecular testing 2 nd and additional passes Sclerotic lesions FISH Nodular sclerosing Hodgkin Lymphoma Sclerosing large cell lymphoma PCR Metastatic lesions Sequencing Clinically benign/reactive Smears, flow cytometry Infectious Smears, culture, +/- cell block, +/- flow cytometry Lymphoma Smears, flow cytometry, unstained, cell block, pcr, karyotyping Possibly metastatic Smears, cell block, +/- flow cytometry Other/surprise Adjust as needed 3

  4. 5/23/2013 Rapid Interpretation Desired Test Processing Smears Diff-Quik, Pap, other Lymph node FNAB Benign/reactive Flow cytometry Saline or RPMI/media Smears Diff-Quik, Pap, other Lymphoma AFB, GMS, Gram stain Unstained smear air-dry Infectious Microbiology culture Saline or culture media Morphology Cell block Formalin needle rinse Monomorphic or heterogenous? +/- Flow cytometry Saline or RPMI/media Smears Diff-Quik, Pap, other or both? Flow cytometry Saline or RPMI/media Cell size- how to judge FISH Unstained smear air-dry Lymphoma Cell block for IPEX Formalin needle rinse Flow cytometry Karyotyping Saline or RPMI/media - assess flow results in the context of cell size PCR Saline Smears Diff-Quik, Pap, other - assess cell size in the context of flow results Metastasis Cell block for IPEX Formalin needle rinse +/- Flow cytometry Saline or RPMI/media Adjust as needed. Other/surprise Get extra samples to reserve for unanticipated tests. Lymph node FNAB FNAB approach String of pearls Assessing the first FNAB pass Smears Heterogeneous Reactive, Unstained Infectious, Flow cytometry Hodgkin’s Cell block monomorphic Smears Monomorphic B-cell Flow cytometry lymphoma Unstained +/- cell block T-cell Heterogeneous Smears Lymphoma, Malignant, Flow cytometry polymorphic DLBCL, lymphoid PCR, Metastasis cell block Other 4

  5. 5/23/2013 Lymph node FNAB Lymph node FNAB When to use molecular cytogenetics B- cell Lymphomas Monomorphic- string of pearls and other ancillary testing Flow confirmation Cell size Discordance: Resolve with: compared to macrophage nucleus • Clinical impression • FISH small • Morphology • PCR • Flow cytometry • Karyotyping intermediate • IPEX large B-cell lymphoma size B- cell Lymphomas of small cells Small Intermediate Large Follicular lymphoma Diffuse large B-cell Follicular lymphoma Burkitt lymphoma •CD20+, CD10+ • CD20+, CD10+ •CD20+, CD10+/- • t(14;18) • t(8;14), t(2;8), t(8;22) • t(14;_) or t(14;18) Mantle cell lymphoma • Ki67 • Karyotype Mantle cell lymphoma • CD20+, CD5+ • Karyotype • t(11;14) cyclin D1 Small lymphocytic/CLL Lymphoblastic lymphoma • CD20+, CD23+, CD5+ • Clinical presentation Small lymphocytic lymphoma/CLL • FISH panel • CD20+, CD10+, TdT + Marginal zone/MALT • t(9;22) •CD20+, CD5-, CD23- • Pax5 • MALT panel Marginal zone lymphoma t(11;18), t(14;18), t(3;14), t(1;14) 5

  6. 5/23/2013 Lymph node FNAB Lymph node FNAB Follicular lymphoma Follicular lymphoma immunophenotype 20% of all lymphomas kappa or lambda light chain restriction B cell markers: CD19+, CD20+, CD22+, CD79a Median age is in the sixth decade and CD10+ Morphology: also BCL2+, BCL6+ Monotonous population of small cells Centrocytes - small cells with cleaved irregular nuclear Negative: CD5, CD23 contours and inconspicuous nucleoli Centroblasts - larger cells with round to oval nuclear Grade 3 FL may lack CD10 contours and prominent nucleoli Lymph node FNAB Lymph node FNAB Follicular lymphoma grading Follicular lymphoma grading Based on the proportion of centroblasts Cytology Requires perfect smears and samples Number of centroblasts/10 hpf in follicles grade may be suggested: grade 1: 0-5 centroblasts/10 hpf Follicular lymphoma, favor grade 1-2 grade 2: 6-15 centroblasts/10 hpfs Follicular lymphoma, favor grade 3 grade 3: >15 centroblasts/10 hpfs Proliferation index on cell block MIB-1 to support grade interpretation WHO recommends: Grade 1-2 proliferation fraction <20% Grade 3 proliferation fraction >30% grade 1-2 or grade 3 6

  7. 5/23/2013 Lymph node FNAB Case 1 Follicular lymphoma genetics 45 year old woman who presents with t(14;18)(q32;q21) 90% of cases axillary lymphadenopathy FISH is sensitive and specific for detection ROS: She denies fever, chills, weight loss. No Immunoglobulin heavy and light chains are rearranged history of breast cancer. Nearly 100% can be detected by PCR 20 pack-year smoking history PE: 3 firm mobile lymph nodes in the axilla, largest measures 2.0 cm Follicular architecture UCSF Cytology Study Set MGG 2x 7

  8. 5/23/2013 Follicular architecture Follicles UCSF Cytology Study Set Pap stain 2x UCSF Cytology Study Set MGG 40x Monomorphic pattern Macrophage UCSF Cytology Study Set MGG 40x UCSF Cytology Study Set Pap stain 60x 8

  9. 5/23/2013 Case 1 Flow cytometry: Monoclonal population of lambda restricted B-cells expressing CD19+, CD20+, CD22+, CD10+ Negative for: CD5, CD23, IPEX: CD20+, CD10+, Ki-67 low proliferation, Bcl-2 + FISH t(14:18) Follicular lymphoma, favor grade 1-2. UCSF Cytology Study Set Surgical excision Follicular lymphoma, grade 1-2 UCSF Cytology Study Set Surgical H&E stain 2x 9

  10. 5/23/2013 Lymph node FNAB Lymph node FNAB Mantle cell lymphoma Mantle cell lymphoma 3-10% of all lymphomas Cytomorphology: Median age of 60 - small to medium size lymphoid cells with irregular nuclear contours, may resemble centrocytes Most patient present with stage III or IV - large cell transformation does not occur Lymphadenopathy, hepatosplenomegaly and bone - blastoid variant marrow involvement - pleomorphic variant Peripheral blood involvement by flow cytometry - monomorphic population of small lymphoid cells with mitoses helps identify mantle cell lymphoma Aggressive clinical course Lymph node FNAB Lymph node FNAB Mantle cell lymphoma immunophenotype Mantle cell lymphoma genetics t(11;14)(q13;q32) Ig expression Kappa > Lambda (intense) ~100% of cases (rare cases negative). B cell markers: CD19+, CD20+, CD22+, CD79a cyclin D1 gene (CCND1) and CD 5 Negative CD10, CD23 (or weak) rare variants t(2:12)(p12;p13) Blastoid variant may lack CD5 Proliferation fraction: MIB-1 stain 10

  11. 5/23/2013 Case 2 79 year old man presented with an inguinal mass. ROS: He reports increasing fatigue no other B-symptoms. PE: A 2.0 cm firm ovoid slightly mobile inguinal lymph node MGH Cytology Study Set Diff Quik stain 2x Mitotic figures Macrophage MGH Cytology Study Set Pap stain 40x MGH Cytology Study Set Diff Quik stain 40x 11

  12. 5/23/2013 Mitotic figures MGH Cytology Study Set Pap stain 40x MGH Cytology Study Set Cell block Case 9 Cell block Ki67 40x MGH Cytology Study Set Cyclin D1 MGH Cytology Study Set Mib-1 12

  13. 5/23/2013 Case 2 Flow cytometry: Monoclonal population of kappa-restricted B cells Positive for: CD19+, CD20+, CD5+ Negative for: CD10-, CD23-, FISH: CCND1 rearrangement. Lymph node FNAB Lymph node FNAB Small lymphocytic lymphoma/CLL Small lymphocytic lymphoma/CLL morphology - Small lymphocyte with clumped chromatin, round Most common adult leukemia in Western countries nucleus, rare small nucleolus 6.7% of non-Hodgkin lymphoma (slightly larger than a normal lymphocyte) - Larger forms: Clinical presentation - Prolymphocytes- small to medium sized with May be subtle clumped chromatin, small nucleoli Fatigue, anemia, infections, splenomegaly - Paraimmunoblasts- larger cells with round to oval Lymphadenopathy may be subtle nuclei, prominent nucleoli Asymptomatic - Richters transformation: large cell lymphoma, Hodgkin lymphoma 13

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