Lab Diagnosis of Chronic lymphoproliferative disorders (CLPD);�Flowcytometric Evaluation

Lab Diagnosis of Chronic lymphoproliferative disorders (CLPD);�Flowcytometric Evaluation

Basavatarakam Indo-American Cancer Hospital and Research Institute

1. Lab Diagnosis of Chronic lymphoproliferative disorders (CLPD); Flowcytometric Evaluation Dr. Siddartha . K Moderator: Dr. Faiq Ahmed Dr. Vishal Rao Basavatarakam Indo-American Cancer Hospital and Research Institute

2. Framework Definition WHO Classification of hematolymphoid malignancy Lymphoproliferative disorders Lab diagnosis Flowcytometry Limitations and Take home message

3. Definition Are set of disorders characterized by abnormal proliferation of lymphocytes into monoclonal population ( lymphocytosis ).

4. WHO Classification Of Tumors Of Hematopoietic And Lymphoid Tissues

5. WHO Classification Of Tumors Of Hematopoietic And Lymphoid Tissues

6. WHO Classification Of Tumors Of Hematopoietic And Lymphoid Tissues

7. WHO Classification Of Tumors Of Hematopoietic And Lymphoid Tissues

8. WHO Classification Of Tumors Of Hematopoietic And Lymphoid Tissues

9. B Cell Lymphoproliferative Disorders Chronic lymphocytic leukemia/ small lymphocytic lymphoma, B-cell prolymphocytic leukemia, Follicular lymphoma, Mantle cell lymphoma, Marginal zone lymphoma, Diffuse large B cell lymphoma, Hairy cell leukemia and its variant, Lymphoplasmacytic lymphoma

10. T cell prolymphocytic leukemia, T cell large granular lymphocytic leukemia, Adult T cell leukemia/lymphoma, Hepatosplenic T cell lymphoma, Sezary syndrome. Angioimmunoblastic T cell lymphoma Anaplastic large cell lymphoma; ALK + ve and – ve . T Cell Lymphoproliferative Disorders

11. NK Cell Lymphoproliferative Disorders Chronic Iymphoproliferative disorder of NK cells Aggressive NK cell lymphoma

12. Laboratory diagnosis of CLPD S. Lactate dehydrogenase β 2 microglobulin Microscopic lymph node examination Immunphenotyping

13. Biochemical Parameters S.Lactate dehydrogenase levels are increased in patients with lymphoproliferative disorders. Serum β 2 microglobulin levels are increased in pts with lymphoproliferative disorders.

14. Histopathological Examination

15. Morphology Of Cells Small cells with scanty cytoplasm -- CLL, MCL, MZL, low grade FL Small cells with moderate cytoplasm – HCL, MZL Small cells with eccentric nucleus and lymphoplasmacytic cells – LPL, MZL Mixed small cells, cleaved cells and large blastic cells – FL, CLL, CLL/PLL, MCL Large cells with or with out vacuoles – high grade FL, DLBCL, BL, B-PLL, blastic variant of MCL.

16. Immunophenotyping Immunohistochemistry Flowcytometry

17. Immunohistochemistry CD20

18. Flowcytometry Flow cytometry (FCM) has been used in the analysis of human lymphomas since the late 1970s Flow cytometery is the methodology used to detect cell surface antigens using monoclonal antibodies conjugated with different fluorochromes .

19. The Common Elements In All Flow Cytometers A light source. Fluid lines and controls to direct a liquid stream containing particles through the focused light beam. An electronic network for detecting the light signals coming from the particles as they pass through the light beam and then converting the signals to numbers that are proportional to light intensity. A computer for recording the numbers derived from the electronic detectors and then analyzing them.

20. The specimen must be in a monodisperse suspension. In this, isotonic fluid is forced under pressure where a fluid column with laminar flow and a high flow rate is generated (so-called sheath fluid). The sample is introduced into the flow cell in the center of the sheath fluid, creating a coaxial stream so that they are presented to the light beam one at a time.

21. The emitted light is focused by a lens onto fiber optic cables and transmitted to octagonal detectors. The sensors convert the photons to electrical impulses that are proportional to the number of photons received and to the number of fluorochrome molecules bound to the cell.

22. Flow Cytometry Gating - isolate a specific group of cytometric events from a large set. Selectively visualize the cells of interest while eliminating results from unwanted particles e.g. dead cells and debris. To separate b/w Neoplastic and non neoplastic Lymphoid and non lymphoid populations

23. Role of Flow cytometry in CLPD To assess the clonality For diagnosis Staging of lymphomas Evaluation of Prognostic markers Detection of target molecules for therapies Detection of minimal residual disease

24. Samples for flowcytometry Peripheral blood Bone marrow Body cavity fluids Needle aspirates Biopsies of lymph node, spleen and skin ( Peritoneal,Pleural,CSF )

25. CD5 CD19 CD23 CD10 CD20 CD103 FMC 7 Kappa Lambda CD38 CD56 IgG CD4 CD8 IgG / IgG /45/19 10/23/45/19 22/5/-/19 κ/ λ /20/19 FMC7/103/-/19 HLADR/34/45/19 38/3/-/19 56/4/8/19 Panel Of Antibodies At Our Centre

26. Mature B Cell Lymphoproliferative Disorders Identification of abnormal mature B-lymphoid cells. Immunoglobulin light chain restriction Aberrant antigen expression

27. Immunoglobulin Light Chain Restriction

28. Aberrant B-cell Antigen Expression CD5 expression in B cells. Normally CD5 is seen in T cells but not in B cells. But CD5 expression over B cells is aberrantly expressed - neoplastic proliferation. Presence of antigens not normally expressed by B cells. Aberrant expression of myeloid antigens like CD13, CD33 is found( less frequently). Eg . Lymphoplasmacytic lymphoma

29. Abnormal expression of antigens not typically in a subset of B cells belonging to a distinct biologic compartment. Bcl-2 is absent in germinal center B cells, but increased expression is seen in follicular lymphoma and some DLBCL. Alteration in intensity of staining for B lineage – associated antigens. Dim – CD20, CD22 and CD79a – CLL Moderate – CD20, – MCL Bright – CD20, CD22 – HCL

30. Lymphoid cells Clonal lymphoid cells B- Cells T -Cells

31. Clonal B Lymphoid cells CD5 + CD10- CD5 + CD10+ CD5 - CD10+ CD5- CD10- CLL/SLL MCL LPL DLBCL PLL DLBCL FL MCL CLL/SLL BL FL DLBL BL HCL MZL/SMZL LPL HCL/Variant DLBL FL MCL

32. CD5+ CD10-

33. Chronic Lymphocytic Leukemia / Small Lymhphocytic Lymphoma (CLL/SLL) Neoplasm composed of monomorphic small, round to irregular B lymphocytes in the PB, bone marrow, spleen and lymph nodes, admixed with prolymphocytes and paraimmunoblasts forming proliferation centers in tissue infiltrates. Antigen experienced B-cell CD20,CD22,CD79b,surface IgM / IgD -- weak CD23 and CD5 – mod positive FMC7 Negative CD38 & ZAP 70 prognostic markers

34. Atypical CLL CD5 neg CD23 neg FMC7 Positive CD 11c Positive CD79b positive S Ig strongly positive

35. Mantle Cell Lymphoma(MCL) Monomorphic small to medium-sized lymphoid cells with irregular nuclear contours and CCND1 translocation. Peripheral B-cell of inner mantle zone. M/c site - LN; the spleen and BM with or without PB involvement. CD5 + ve , CD10 neg.( + ve CD10 in aberrant) CD19, CD20, CD22, CD79a + ve . Intense surface IgM / lgD ( λ > κ ). CD23 weak or neg FMC7 + ve and CD43 neg .

36. Cyclin D1 -- in all the cases Cyclin D1 negative MCL show positivity for Cyclin D2 or Cyclin D3. t(1 1.14)

37. B-cell Prolymphocytic Leukaemia (B-PLL) Neoplasm of B prolymphocytes affecting the PB, BM and spleen. CD5 -- 20-30% + ve . CD23 – 10-20% + ve . IgM / IgD strong + ve CD 19, 20, 22, 79a, 79b, FMC7 + ve . ZAP-70 and CD38 are expressed in 57% and 46% of the cases respectively.

38. CD5- CD10-

39. Lymphoplasmacytic Lymphoma(LPL) Neoplasm of small B lymphocytes, plasmacytoid lymphocytes, and plasma cells usually involving BM, lymph nodes and spleen. IgM , IgG + ve and rarely IgA . But IgD will be neg. CD138 + ve CD5 CD10 CD23 CD103 CD19 CD20 CD22 CD79a - ve + ve

40. Hairy Cell Leukaemia (HCL) Indolent neoplasm of small mature B lymphoid cells with oval nuclei and abundant cytoplasm with "hairy" projections involving PB and diffusely infiltrating the BM and splenic red pulp. Activated memory B-Cell . CD5 & CD10 – neg . Bright expression of CD20, CD22 and CD11c. CD103, CD25, CD123, T-bet, Annexin A1, DBA.44, FMC-7 will be + ve

41. CD5 & CD10 – neg Annexin 1 -- negative CD123, CD25 – negative DBA.44, Pan-B-cell antigens, CD11c, IgG , CD 103 and FMC7 -- pos Hairy Cell Leukaemia - variant(HCL-V)

42. Marzinal Zone Lymphoma Lymphoma cells may be found in the peripheral blood as villous lymphocytes. IgM / IgD + ve CD20, CD79a  + ve CD5, 10, 23, 43 are negative Annexin 1, cyclin D1 and CD103 are neg

43. CD5- CD10+

44. Follicular Lymphoma(FL) Germinal center B cell FL predominantly involves LN, spleen, BM, PB and Waldeyer ring. Usually CD5 neg and CD10 positive. B- cell Ag- CD 19,20,22, CD79a  + ve BCL-2 & BCL-6  + ve Grade 3B cases may lack CD 10, but retain BCL6 expression. CD10 expression is often stronger in the follicles than in interfollicular neoplastic cells . May be absent in the interfollicular component, as well as in areas of marginal zone differentiation,

45. IRF4/MUM1 is typically absent in FL. BCL2 protein is expressed by a variable proportion of the neoplastic cells in 85-90% of cases of grade 1 and grade 2 FL, but only 50% of grade 3 FL using standard antibodies.

46. Diffuse large B Cell lymphoma(DLBCL) Peripheral B-cells of either germinal centre or post germinal centre (activated B-cell ) origin. CD 19, 20, 22, CD79a-- + ve Surface and/or cytoplasmic immunoglobulin ( lgM > lgG > lgA ) can be demonstrated in 50-75% of cases. CD30 - especially in the anaplastic variant. CD5 + ve in 10% of cases. CD 10 + ve - (30-60)% BCL6 + ve – (60-90)% MUM1 + ve – (35-65)%

47. Subgrouping DLBCL, NOS by immunophenotyping

48. Clonal T Lymphoid cells CD4 + CD8- CD4 + CD8+ CD4 – CD8+ CD4- CD8- T-PLL ATLL SEZARY Syn ALCL AITCL PTCL-NOS T-PLL ATLL T-LGL PTCL-NOS T-LGL NK-LGL HSTCL SEZARY syn EATCL HSTCL Aggressive NK Cell Leukemia

49. CD4+ CD8-

50. T Cell Prolymphocytic Leukemia(T-PLL) Aggressive T-Cell leukemia – small to medium size prolymphocytes . Cytoplasmic protrusions or blebs. HTLV-1 negative. CD1a, TdT – neg , TIA -1 neg Mem CD3 – weak CD4 + CD8 - ve --- 60% CD4 + CD8 + ve --- 25% CD4 - CD8 - ve --- 15% CD7 positive CD25 variable CD52(CAMPATH-1 Ag) dense positive http://www.bloodjournal.org/content/bloodjournal/120/3/538/F2.large.jpg?sso-checked=true Protrusion

51. Adult T-cell Leukemia / Lymphoma(ATLL) CD4 + CD8 – ve ( rarely both positive) CD 25 Strong staining CD2, CD3, CD5 positive CD7 negative Large transformed cells + CD30 (ALK Neg ) HTLV-1 positive

52. Sezary Syndrome/ Cutaneous T Cell Lymphoma(CTCL) CD4 + CD8 – ve CD2+, CD3+ , TCR β + and CD5+. CD7 & CD 26 - negative, CD 25 variable staining Erythroderma , Generalized LAP Sezary cells

53. Mature T-Cell Leukemias Including T- Prolymphocytic Leukemia, Adult T Cell Leukemia/Lymphoma, and Sézary Syndrome Kathryn Foucar , MD

54. Anaplastic Large Cell Lymphoma(ALCL) PB involvement seen in small cell variant . CD4 + CD8 – ve . Pan T cell marker (CD3) – neg. CD2 frequently + ve but CD5 and CD7 are negative. CD 30 strong positive in large cells (weak in small and medium) CD13, CD15, CD33 (aberrant myeloid markers + ve ). t(2 ;5)/ NPM-ALK will be positive

55. Angio Immunoblastic T Cell Lymphoma(AITCL) CD4 Follicular Helper T Cell CD4 + CD8 – ve Pan T cell marker CD2, CD3, CD5 – positive. Decreased expression of CD3 and CD7 . CD10, BCL6, CXCL13, PD-1 + ve (Normal T helper cells) .

56. Peripheral T Cell Lymphoma, Not Otherwise Specified(PTCL-NOS) PB is sometimes involved, but leukemic phase is uncommon. CD4 + CD8 – ve ( uncommon both + ve and both neg ). Aberrant loss of CD7 & CD5. TCR β chain is expressed. CD 30 + ve . Campath 1 Ag + ve in 40%.

57. CD4- CD8+

58. T Cell Large Granular Cell Leukemia(T-LGL) Persistent increase in the no. of PB large granular lymphocytes (2-20 ×10 9 /L). Sustained immune stimulation. Interstitial or intrasinusoidal infiltrate is seen. CD4 neg , CD8 + ve . CD3, TCR αβ + ve . CD16 & CD57 + ve ( few are + ve for CD56). Abnormally diminished or lost expression of CD5 and / or CD7 is common. Uncommon Variants CD4 + ve TCR αβ + ve , CD4 + ve TCR γδ + ve .

59. Chronic Lymphoproliferative Disorders Of NK Cells Persistent increase in the no. of PB NK cells (≥ 2 ×10 9 /L) with out identified cause. CD4 neg , CD8 + ve . Surface CD3 is neg but Cyt CD3 is often + ve . Diminished or lost expression of CD2, CD7 and CD57. CD16 + ve , CD56 weak positive.

60. CD4- CD10-

61. Hepatosplenic T Cell Lymphoma(HSTCL) Cytotoxic T cells usually of γδ TCR type. 20% of HSTL arise in the setting of chr . Immuno supression . CD4 neg , CD8 neg ( rarely CD8 + ve ) CD3 + ve Complete absence of CD5 CD16, CD56 + ve , CD57 neg TIA-1 pos. γδ TCR + ve Isochromosome 7q is seen.

62. Aggressive NK-Cell Leukaemia A systemic neoplastic proliferation of NK-cells almost always associated with Epstein-Barr virus and an aggressive clinical course. The most commonly involved sites are PB, BM, liver and spleen. CD4 neg , CD8 neg CD2+, surface CD3-. CD3 ε +, CD56+ CD16 is frequently positive, but CD57 is negative CD11b may be expressed

63. Limitations of flow cytometry Main drawback is, it requires fresh and unfixed tissue for analysis. When reactive cells are more than the neoplastic cells . Dilution of sample. Cannot grade the Follicular lymphoma. Cost .

64. Take home message Though morphology is the gold standard for diagnosing lymphoproliferative disorder, however Immunophenotyping is essential for subtyping Targeted therapy is possible Genotypic studies when needed can be done

65. References WHO Classification of tumours of haematopoietic and lymphoid tissues , 4 th edition. Ioachims lymphnode pathology 4 th edition. Wintrobes text book of clinical hematology.