From Morphology to Molecular Pathology: A Practical Approach for Cytopathologists Part 1-Cytomorphology. Songlin Zhang, MD, PhD LSUHSC-Shreveport

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1 From Morphology to Molecular Pathology: A Practical Approach for Cytopathologists Part 1-Cytomorphology Songlin Zhang, MD, PhD LSUHSC-Shreveport

2 I have no Conflict of Interest.

3 FNA on Lymphoproliferative Disorders

4 Why FNA for Lymphoproliferative Disorders? Initial diagnosis-less invasive than open biopsy. Confirming recurrent lymphomas. Unexpected lymphoma during routine work-up to rule-out metastatic lesions. New FNA techniques for deeply located lymph nodes, such as EUS and EBUS FNA. Available ancillary tests such as flow cytometry and immunocytochemistry for phenotyping and molecular testing.

5 Diagnosis of Lymphoproliferative Disorders Diagnosis: reactive vs lymphoma Classification: non-hodgkin B cell lymphoma, Hodgkin lymphoma, NK/T cell lymphoma and others. Subclassification: diffuse large B cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone lymphoma, Burkitt lymphoma, and different T-cell lymphomas. Grade: follicular lymphoma (low grade 1 and 2; high grade 3A and 3B).

6 Classic Flow Cytometry Phenotyping CD Lymphoma Small lymphocytic lymphoma Follicular lymphoma for Small B-cell Lymphoma CD19/ CD20 CD5 CD10 CD Mantle cell lymphoma Nodal marginal zone lymphoma

7 Case #1 25 year-old female with a 5.0 cm axillary lymph node and diffuse lymphadenopathy on CT.

8

9

10

11

12 Your Diagnosis?

13 Flow Cytometry Monoclonal B-cells positive for: CD19, 20, 10, 79b and lambda light chain restriction. Negative for: CD5 and 23.

14 Excisional biopsy, case #1

15 Burkitt Lymphoma Cytomorphology: monotonous intermediate rounded lymphocytes, nuclei with 2-5 distinct nucleoli, dense blue cytoplasm with lipid vacuoles, many tingible body macrophages. Immunophenotype: monoclonal B-cells with positive CD19, 20, 10, 22, BCL-6 and negative BCL-2. Cytogenetic abnormalities: most cases with MYC translocation [t(8;14); t(2;8); t(8;22)]; highly characteristic but not specific for Burkitt lymphoma.

16 Case #2 53 year-old male with diffuse 53 year-old male with diffuse lymphadenopathy and right pleural effusion. FNA of right cervical lymph node.

17

18

19

20 Your Diagnosis?

21 Flow Cytometry Monoclonal B-cells positive for: CD19, 20, 10, 79B, BCL-2 and kappa light chain. B-cells negative for: CD5 and 23.

22 Excisional biopsy case #2

23 Follicular Lymphoma Cytomorphology: heterogenous lymphocytes, small to medium centrocytes with irregular nuclei, large centroblasts with few nucleoli, visible cell aggregates (follicular centers), and often no tingible body macrophages. Immunophenotype: monoclonal B cells positive for CD19, 20, 22, 10, BCL-2 and BCL-6. Cytogenetic abnormalities: t(14;18) up to 90% grade 1-2 follicular lymphoma. Grading follicular lymphoma on cytology: using centroblast count.

24 Case #3 51 year-old female with right neck 51 year-old female with right neck mass for 8 months.

25

26

27

28

29 Your Diagnosis?

30 Flow Cytometry Mixed population of T and B cells. No evidence of light chain restriction (normal kappa/lambda ratio). Normal CD4/CD8 ratio. Small population of B cells with lambda predominance. Flow diagnosis-atypical but nondiagnostic.

31 Excisional biopsy, case #3

32 Diffuse Large B Cell Lymphoma, NOS Cytomorphology: diverse cytology with three common variants-centroblastic, immunoblastic and anaplastic. Immunophenotype: monoclonal B cells positive for CD19, 20, 22 and 79a; germinal center (GC)- like DLBCL with >30% cell CD10+ or CD10-, BCL-6+, IRF4/MUM1-; all others non-gc type. Cytogenetic abnormalities: 30% BCL6 translocation, 20-30% BCL2 translocation.

33 Smears from a recent case of DLBCL

34 Case #4 43 year-old female with left neck lymphadenopathy.

35

36

37

38 Your Diagnosis?

39 Flow Cytometry Mixed T and B cells. No light chain restriction on B cells. A population of CD45 (+) cells positive for CD2, 4, bright 25 and 52, but negative for CD3, 8, 5 and 7. Flow cytometry interpretation: atypical T lymphocytes, suspicious for T-cell lymphoma.

40 Excisional biopsy, case #4

41 Adult T-cell Leukemia/Lymphoma Cytomorphology: a broad spectrum of cytological features, typically medium-sized to large cells, pronounced nuclear pleomorphism, coarsely clumped chromatin, and sparse background inflammation. Immunophenotype: usually positive for CD2, 3, 5, but lack CD7; most CD4+; CD25 strongly positive in all cases. Cytogenetic: clonal rearrangement of T-cell receptor; monoclonal integration of HTLV-1.

42 Cytomorphologic Summary of Lymphoproliferative Disorders Monotonous lymphocytic population: Small: SLL, MCL, atrophic nodes Medium: Burkitt, lymphoblastic and MCL Large: DLBCL, T cell lymphoma Heterogenous: Mainly small: reactive, FL, marginal zone Large: reactive, FL, DLBCL, T cell and Hodgkin lymphoma Pleomorphic: ALCL, Hodgkin, and histiocytic sarcoma.

43 Flow Chart of Using FNA for Diagnosis of Lymphoproliferative Disorders Flow cytometry Cytology-reactive Flow cytometry-neg Cytology-positive or Atypical Flow cytometryinconclusive Cytology-positive or atypical Flow cytometry-positive for clonal Reactive LN Clinical correlation PCR or other molecular testing for clonality FISH or PCR for subclassification

44 Urine Cytology

45 Bladder Washing-Diagnosis?

46

47 Renal pelvic washing-diagnosis?

48

49 Case #1 67 year-old male with hematuria.

50 Bladder washing-diagnosis?

51 Case #1. Bladder biopsy

52 Case #2 63 year-old man with microscopic hematuria.

53 Bladder washing

54 Ureteral washing

55 Your Diagnosis?

56 Case #2. Ureteral biopsy.

57 Urine Cytology and UroVysion Urine cytology is quite sensitive for detecting high-grade urothelial carcinoma and carcinoma in-situ; The sensitivity for low-grade urothelial tumors is very poor. UroVysion is a FDA-approved FISH test performed on voided urine and targets chromosomes 3, 7, 17 and 9p21; Not all urothelial carcinomas are positive for UroVysion (especially low-grade tumors) and there is significant false positive rate in reactive urothelial cells.

58 FNA on Soft Tissue Tumors

59 Case #1 23 year-old male with back pain and a large soft tissue mass (12.0 cm) at T12 on CT scan.

60

61

62 Vimentin

63 Your Diagnosis?

64 Immunohistochemistry Results CD 99 (-) NSE and chromogranin (-) Neurofilament protein (-) S-100 (-) Pancytokeratin (-) Positive for Myo D1, myogenin and desmin.

65 Diagnosis Alveolar rhabdomyosarcoma. Which molecular test can be used to confirm this diagnosis?

66 Case #2 57 year-old female with left neck 2.0 cm nodule and history of resection clear cell sarcoma 6 months ago.

67

68

69

70 Your Diagnosis?

71 FNA Diagnosis High-grade malignant neoplasm with positive immunocytochemistry for S-100 and HMB-45. Metastatic clear cell sarcoma? Metastatic malignant melanoma?

72

73 Mart-1 S-100

74 Diagnosis What is your diagnosis? What molecular tests may help you to differentiate between clear cell sarcoma and melanoma?

75 FNA Cytology on Soft Tissue Tumors Cytomorphology: Myxoid: myxoma, myxoid liposarcoma, myxoid MFH Spindle: fibromatosis, Schwannoma, fibrosarcoma, leiomyosarcoma, GIST Pleomorphic: MFH, pleomorphic liposarcoma, pleomorphic leiomyosarcoma Round: rhabdomyosarcoma, Ewing s/pnet, DPSCL Polygonal (epithelioid): epithelioid sarcoma, alveolar soft part sarcoma, granular cell tumor Chromosomal translocations: Ewing s/pnet: t(11;22)(q24;q12), t(21;22), t(7;22) DPSCL: t(11;22)(q13;q12) Clear cell sarcoma: t(12;22) Alveolar rhabdomyosarcoma: t(2;13), t(1;13)

76 Thank You!

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