Chronic lymphoproliferative disorders

1. CHRONIC LYMPHOPROLIFERATIVE DISORDERS PRESENTER- DR. ANKITA BAGHEL MODERATOR- DR. MANJULA K.

2. CONTENTS NORMAL HISTOLOGY WHAT IS CLPD CELL DIFFERENTIATION CLASSIFICATION INDIVIDUAL DISORDERS APPROACH TO DIAGNOSIS CYTOCHEMISTRY IMMUNOPHENOTYPING CYTOGENETICS REFERENCES

3. B CELL DIFFERENTIATION

4. T CELL DIFFERENTIATION

5. CHRONIC LYMPHOPROLIFERATIVE DISORDERS(CLPDS) are a heterogeneous group of malignancies characterised by the proliferation of mature B and rarely T lymphoid cells in the peripheral blood, bone marrow and/or lymph nodes/spleen and other lymphoid tissues. Classification- FAB- MORPHOLOGY+CYTOCHEMISTRY+IMMUNOHISTOCHEMISTRY WHO- Morphology Cytochemistry Immunophenotypic analysis Cytogenetic and molecular alterations.

6. PRECURSOR LYMPHOID NEOPLASMS Precursor-B lymphoblastic leukemia/lymphoma Precursor-T lymphoblastic leukemia/lymphoma

7. MATURE B-CELL NEOPLASMS CHRONIC LYMPHOCYTIC LEUKEMIA/ SMALL LYMPHOCYTIC LYMPHOMA B- CELL PROLYMPHOCYTIC LEUKEMIA LYMPHOPLASMACYTIC LYMPHOMA SPLENIC MARGINAL ZONE LYMPHOMAS EXTRANODAL MARGINAL ZONE LYMPHOMA MANTLE CELL LYMPHOMA FOLLICULAR LYMPHOMA NODAL MARGINAL ZONE LYMPHOMA HAIRY CELL LEUKEMIA PLASMACYTOMA/PLASMA CELL MYELOMA DIFFUSE LARGE B-CELL LYMPHOMA BURKITT’S LYMPHOMA PLASMABLASTIC LYMPHOMA WALDENSTROM MACROGLOBULINEMIA

8. MATURE T-CELL & NK-CELL NEOPLASMS T-CELL PROLYMPHOCYTIC LEUKEMIA LARGE GRANULAR LYMPHOCYTIC LEUKEMIA PERIPHERAL T-CELL LYMPHOMA UNSPECIFIED ANAPLASTIC LARGE CELL LYMPHOMA ANGIOIMMUNOBLASTIC T-CELL LYMPHOMA ENTEROPATHY-ASSOCIATED T-CELL LYMPHOMA PANNICULITIS-LIKE-T-CELL LYMPHOMA HEPATOSPLENIC T-CELL LYMPHOMA ADULT T-CELL LEUKEMIA/LYMPHOMA AGGRESSIVE NK/T-CELL LYMPHOMA NK-CELL LEUKEMIA SEZARY SYNDROME MYCOSIS FUNGOIDES

9. HODGKIN’S LYMPHOMA 1. CLASSICAL SUBTYPES- NODULAR SCLEOSIS MIXED CELLULARITY LYMPHOCYTE-RICH LYMPHOCYTE DEPLETION 2. NODULAR LYMPHOCYTE PREDOMINANT

10. CHRONIC LYMPHOID LEUKEMIA- SUBTYPES B- CELL TYPE 1. B-CELL CLL CLL(PROLYMPHOCYTES <10%) CLL/PLL(PROLYMPHOCYTES 11-55%) RICHTER SYNDROME 2. PROLYMPHOCYTIC LEUKEMIA(PROLYMPHOCYTES >55%) 3. HAIRY CELL LEUKEMIA 4. PLASMA CELL LEUKEMIA, WALDENSTROM MACROGLOBULINEMIA 5. LYMPHOMA SPILL OVER- SPLENIC LYMPHOMA WITH VILLOUS LYMPHOCYTES(SLVL) MANTLE CELL LYMPHOMA OTHER NON-HODGKIN’S LYMPHOMAS

11. T-CELL TYPE T-CELL PROLYMPHOCYTIC LEUKEMIA LARGE GRANULAR LYMPHOCYTIC LEUKEMIA AGGRESSIVE NK-CELL LEUKEMIA ADULT T-CELL LEUKEMIA/LYMPHOMA MYCOSIS FUNGOIDES/SEZARY SYNDROME PERIPHERAL T-CELL LYMPHOMA ANGIOIMMUNOBLASTIC T-CELL LYMPHOMA ANAPLASTIC LARGE CELL LYMPHOMA ALK + ve ANAPLASTIC LARGE CELL LYMPHOMA ALK - ve

12. B CELL CHRONIC LYMPHOCYTIC LEUKEMIA Median age at diagnosis- 70 years Incidence- Males= 2x Women Symptoms related to- Anemia Thrombocytopenia Neutropenia Neoplastic cells- Small and mature appearing Scant cytoplasm Nuclei usually round Chromatin regularly clumped(block-type chromatin) Nucleoli inconspicuous

13. Diagnosis- Lymphocytosis >5 x 10 9 /L Prolymphocytes <10% S mudge cells Cytogenetic abnormalities- Trisomy 12 Del 11q22.3-23.1 Del 6q21-23 Deletions at 17p13.1(p53 aberrations) 14q abnormalities and complex chromosomal abnormalities

14. PROLYMPHOCYTIC LEUKEMIA Aggressive leukemic disorder . Often does not respond to treatment. Incidence- 10% that of CLL. Phenotypes- Mature B cell Mature T cell

15. B CELL PLL 70% cases of PLL. Disease of adults Male:Female-4:1 Lymphocytes- Large cell Moderate amount of pale basophilic cytoplasm Moderately condensed chromatin Single prominent nucleolus

16. T CELL PLL Rare disorder of adults Lymphocyte- Medium size Convoluted nuclear outlines Prominent nucleolus

17. HAIRY CELL LEUKEMIA Uncommon malignancy of middle age. Male:female-7:1 Presentation- Massive Splenomegaly Extensive bone marrow involvement Pancytopenia WBC count low- Neutropenia And Monocytopenia

18. Neoplastic cell- Abundant pale staining cytoplasm Circumferential cytoplasmic projections(hairs) Oval or reniform nuclei Fine chromatin Markers- CD19 CD20(strong intensity) CD22 CD25 CD103 CD11c TRAP

19. Bone marrow aspiration- DRY TAP Bone marrow biopsy- monotonous infiltrate of abnormal lymphocytes with small nuclei and abundant pale-staining cytoplasm- FRIED EGG APPEARANCE

20. LARGE GRANULAR LYMPHOCYTIC LEUKEMIA LYMPHOCYTOSIS- Abundant pale staining cytoplasm Azurophilic cytoplasmic granules Nuclei with mature clumped chromatin Phenotypes- T LYMPHOCYTE TYPE NK CELL TYPE

21. T-LGL LEUKEMIA 80% of LGLL Age- 55years PRESENTATION- Anemia Neutropenia Thrombocytopenia Splenomegaly

22. NK CELL TYPE Median age-39 years Acute presentation and aggressive course with death within 2 months

23. CUTANEOUS T CELL LYMPHOMA Primary to skin MYCOSIS FUNGOIDES -most common variant of CTCL SEZARY SYNDROME- E rythroderma + Sezary cells Sezary cell- M ature memory helper T cell Irregular, convoluted( cerebriform ) nuclear outline Finely distributed chromatin CD3+, CD4+, CD7-

24. SUBGROUP OF CHRONIC LEUKEMIC LYMPHOID MALIGNANCIES KEY FEATURES MARKERS 1. B CELL CLL Lymphocytosis Smudge cells Prolymphocytes <10% CD19+,CD5+,CD20+ weak intensity, CD23+, FMC- Surface Ig weak intensity 2. B CELL PLL Splenomegaly Marked lymphocytosis Prolymphocytes >55% CD19+,CD20+,CD22+,CD5-/+,FMC-7+ Surface Ig strong intensity 3.T CELL PLL Splenomegaly Skin lesions Marked lymphocytosis Varied morphologic appearance 4. HAIRY CELL LEUKEMIA Pancytopenia Circulating hairy cells Bone marrow dry tap Bone marrow fried egg appearance TRAP+ CD19+,CD5-,CD20+ strong intensity,CD22+,CD103+,CD11c+,CD25+ Surface Ig strong intensity

25. 5. T CELL LGLL Lymphocytosis Anemia Neutopenia Thrombocytopenia Rheumatoid factor often present Indolent course CD2+,CD3+,CD4-,CD5+,CD7+,CD8+,CD16+,CD56-/+,CD57+/- T Cell receptor clonally rearranged 6. SEZARY SYNDROME Erythroderma (red skin) Cutaneous T cell lymphoma Circulating malignant cells CD2+,CD3+,CD4+,CD5+,CD7-,CD8-

26. PARAMETER HODGKIN LYMPHOMA NON-HODGKIN LYMPHOMA STAGE Usually localized Usually widespread DISTRIBUTION Usually central nodes Usually involves peripheral nodes MODE OF SPREAD Contiguous Non-contiguous EXTRANODAL DISEASE Uncommon Common PERIPHERAL BLOOD Never involved Can be involved CELL TYPE Abnormal bizarre cells Resembles normal lymphoid cells TREATMENT REGIMEN Often ABVD Often CHOP

27. SMALL LYMPHOCYTIC LYMPHOMA Tissue equivalent of CLL .

28. FOLLICULAR LYMPHOMA Neoplasm composed of cell originating from the germinal center. Median age-50-60years Presentation-generalized painless lymphadenopathy peripheral blood involvement bone marrow involvement Lymph node-infiltrate of lymphoid cells forming poorly circumscribed nodules Neoplastic follicles diifer from reactive follicle in lacking apoptosis of lymphocytes Tingible body macrophages Small cleaved cells(CENTROCYTES) and large cells(CENTROBLASTS)

29. GRADING OF FOLLICULAR LYMPHOMA GRADE DEFINITION 1 0-5 centroblasts per defined HPF 2 6-15 centroblasts per defined HPF 3 >15 centroblasts per defined HPF

30. MANTLE CELL LYMPHOMA Median age 60 years Male predominance Neoplastic cells-small to intermediate in size with round to slightly irregular nuclear outlines. Markers- CD19+ CD5+ CD23- FMC7+ sIg +(strong intensity) Cyclin D1

31. MALT LYMPHOMA Prsentation -localized extranodal disease A preceding chronic inflammatory disorder(chronic gastritis,sjogrens,hashimotos ) Neoplastic cells- Small lymphocytes Round to slightly cleaved nuclei

32. BURKITT LYMPHOMA High grade Non- hodgkin lymphoma High incidence in Africa(ENDEMIC SUBTYPE) 1/3 rd of pediatric lymphomas outside africa (SPORADIC SUBTYPE) Extranodal involvement Endemic-involves facial bones and jaw Sporadic- intestine,ovaries or kidney EBV associated

33. Biopsy- STARRY SKY APPEARANCE- The SKY represents the blue nuclei of the neoplastic lymphocytes The STARS formed by scattered pale staining tingible body macrophages. Infiltrating lymphoid cells are intermediate in size with nuclei approximately the same size as the nuclei of the tingible body macrophages. Multiple small nucleoli,mitotic figures and apoptotic bodies. Markers- CD19+ sIg + CD10+ CD5- t(8;14) , t(2;8), t8;22)

34. HODGKINS LYMPHOMA- CLASSIFICATION SUBTYPE SCLEROSIS LYMPHOCYTES TUMOUR CELLS VARIANTS VARIANTS CELL TYPE LYMPHOCYTIC PREDOMINANCE - ++++ + L AND H B-CELL NODULAR SCLEROSIS PRESENT ++ ++ LACUNAR UNCERTAIN CELL OF ORIGIN MIXED CELLULARITY - ++ ++ - UNCERTAIN CELL OF ORIGIN LYMPHOCYTIC RICH - ++++ + - UNCERTAIN CELL OF ORIGIN LYMPHOCYTIC DEPLETION -/+ + ++++ - UNCERTAIN CELL OF ORIGIN

35. ANN ARBOR STAGING OF HODGKIN’S STAGE FEATURE STAGE I Involvement of single lymph node region(I) or a single extra lymphatic organ/site(IE) STAGE II Involvement of two more lymph node regions on the same side of diaphragm(II) or Localized involvement of an extra lymphatic organ and one more more lymph node regions on the same side of the diaphragm(IIE) STAGE III Involvement of lymph node regions on both sides of diaphragm(III), which may also be accompanied by localized involvement of an extra lymphatic organ(IIIE) or involvement of the spleen(IIIS) or both(IIIES) STAGE IV Diffuse or disseminated involvement of one or more extra lymphatic organs or tissue s with or without associated lymph node enlargement.

36. SUBCLASSIFICATION A- without symptoms B- Systemic symptoms: Unexplained fever 38 degree Celsius. Unexplained weight loss 10% body weight in preceding 6 months. Night sweats.

37. MALIGNANT LYMPHOMA KEY FEATURE 1. FOLLICULAR LYMPHOMA Nodular growth pattern Lack of tangible body macrophages CD19+,CD20+,CD5-,CD10+ Surface Ig strong intensity t(14;18) BCL-2 protein overexpression BCL-2 gene rearrangement 2. MANTLE CELL LYMPHOMA Lack of large cells CD19+,CD20+,CD5+,CD23-,FMC-7+ Surface Ig + strong intensity Cyclin-D1 overexpression t(11;14) BCL-1 rearrangement 3. MALT LYMPHOMA Accompanied by infectious or autoimmune disease Often localized Extranodal Lymphoepithelial lesions Benign follicles Heterogeneous neoplastic infiltrate Phenotype and genotype not specific

38. 4. WALDENSTROM MACROGLOBULINEMIA Lymphoid malignancy with plasmacytic differentiation IgM Hypervicosity syndrome 5. BURKITT LYMPHOMA Can be associated with EBV Starry sky growth pattern CD19+,CD20+,CD5-,CD10+ Strong surface Ig t(8;14) C-MYC gene rearrangement 6. ANAPLASTIC LARGE CELL LYMPHOMA Bizarre,anaplastic cells can resemble HD T cell or Null phenotype LCA+/-,CD30+,CD15-,EMA+/-,EBV-,ALK-1+/- t(2;5) 7. CLASSIC HODGKIN LYMPHOMA Reed-Sternberg cells LCA-,CD15+,CD30+,ALK-1- Often EBV + 8. LYMPHOCYTIC PREDOMINANT HL Growth pattern frequently nodular L and H cells LCA+,CD20+,CD15-,CD30-,EBV-

39. PLASMA CELL DISORDERS PLASMA CELL MYELOMA (MULTIPLE MYELOMA) PLASMACYTOMA PRIMARY AMYLOIDOSIS HEAVY CHAIN DISEASE MONOCLONAL GAMMOPATHY OF UNDETERMINED SIGNIFICANCE (MGUS)

40. KEY FEATURES OF PLASMA CELL DISORDERS NEOPLASM FEATURES 1. MULTIPLE (PLASMA CELL) MYELOMA LYTIC BONE LESIONS “M” SPIKE ON SERUM/URINE ELECTROPHORESIS ROULEAUX ON BLOOD SMEAR >30% PLASMA CELLS IN BONE MARROW 2. PLAMACYTOMA LOCALIZED MASS MONOCLONAL PLASMA CELLS 3. MONOCLONAL GAMMOPATHY OF UNDETERMINED SIGNIFICANCE MONOCLONAL SERUM PROTEIN MONOCLONAL PROTEIN <3gm/dl LYTIC BONE LESIONS ABSENT BONE MARROW PLASMA CELLS <10%

41. CRITERIA FOR DIAGNOSIS OF MULTIPLE MYELOMA(SALMON AND DURIE) MAJOR CRITERIA- Plasmacytoma on biopsy Bone marrow plasmacytosis >30% plasma cells M band in serum IgG >3.5 g/dl OR IgA >2 g/dl Light chain excretion in urine >/= 1 g/24 HOURS MINOR CRITERIA- Bone marrow plasmacytosis 10-30% plasma cells Monoclonal globulin spike IgG <3.5 g/DL, or IgA <2g/DL Lytic bone lesions Normal IgM <50 mg/dl, IgA <0.1 g/dl OR IgG <0.6 g/dl CRITERIA FOR MM Diagnossi of myeloma is confirmed when at least- One major + one minor criteria OR 3 minor criteria, that must include 1 and 2 of minor criteria.

42. APPROACH TO DIAGNOSIS OF LYMPHOMA??

43. CELL SIZE DETERMINATION Neoplastic cells are compared with the Reactive Histiocytes interspersed among the lymphoma cells. SMALL- Nuclei smaller than that of reactive histiocytes . MEDIUM- Nuclei approx. same as that of histiocytes . LARGE- Nuclei larger than those of histiocytes .

44. MEDIUM SIZED FOLLICLES-APPROACH TO DIAGNOSIS MAJOR DIFFERENTIAL DIAGNOSIS Reactive follicular hyperplasia Follicular lymphoma Nodular mantle cell lymphoma

45. How to distinguish between reactive follicular hyperplasia and follicular lymphoma? FEATURE REACTIVE FOLLICULAR HYPERPLASIA FOLLICULAR LYMPHOMA FOLLICLES Discrete and separeated by interfollicular lymphoid tissue A pattern of back to back follicles with little interfollicular tissue TINGIBLE BODY MACROPHAGES Present Lack CELLULAR POLARIZATION Into light and dark zones present Absent POPULATION Heterogeneous population of follicular cells Predominance of centrocytes in the follicles

46. LARGE LYMPHOID NODULES: APPROACH TO DIAGNOSIS MAJOR DIFFERENTIAL DIAGNOSIS NLPHL N-LRCHL NSHL PTGC

47. DIFFUSE SMALL B-CELL LYMPHOMAS- APPROACH TO CLASSIFICATION MAJOR DIFFERENTIAL DIAGNOSIS CLL/SLL Lymphoplasmacytic lymphoma Mantle cell lymphoma Extranodal marginal cell lymphoma

48. LARGE CELL SCATTERED IN A BACKGROUND OF SMALL LYMPHOCYTES MAJOR DIFFERENTIAL DIAGNOSIS Reactive lymphoid hyperplasia T-cell/ histiocytic rich large B-cell lymphoma Hodgkin lymphoma

49. Possible diagnosis All large cells show morphologic features acceptable for immunoblasts or centroblasts ( nuclear size <x2.5 small lymphocyte nucleus: nuclear contour round to oval) Large cells are atypical(with irregular nuclear folding or granular chromatin),but most nuclei are <x2.5 small lymphocyte nucleus Most large cells have nuclei >x3 small lymphocyte nucleus Reactive lymphoid hyperplasia + - - TCRBCL + + + Hodgkin’s lymphoma - - +

50. CELL IDENTIFICATION BY CYTOCHEMISTRY CYTOCHEMICAL REACTION CELL TYPES 1. NON SPECIFIC ESTERASE MYELOID CELLS MONOCYTES MEGAKARYOCYTES T LYMPHOCYTES(dot like staining) 2. CHLOROACETATE ESTERASE MYELOID CELLS MAST CELLS 3. PEROXIDASE MYELOID CELLS EOSINOPHILS MONOCYTES 4. SUDAN BLACK B MYELOID CELLS MONOCYTES 5. METHYL GREEN PYRONINE PLASMA CELLS IMMUNOBLASTS 6.PERIODIC ACID-SCHIFF WITH DIASTASE PLASMA CELLS 7. TARTRATE-RESISTANT ACID PHOSPHATASE HAIRY CELL LEUKEMIA

51. PROBLEM TO BE ASSESSED FIRST-LINE ANTIBODIES B LINEAGE ? CD20( or CD79a, PAX5) T LINEAGE ? CD3( OR CD2) NK LINEAGE ? CD56, SURFACE CD3, CYTOPLASMIC CD3, TCR FOLLICULAR CENTER CELL ? CD10( OR BCL6, HGAL) FOLLICULAR LYMPHOMA OR HYPERPLASIA ? BCL2, CD10( interfollicular invasion) CLL ? CD5, CD23 NORMAL MANTLE ZONE CELLS ? IgD MANTLE CELL LYMPHOMA ? CYCLIN D1, CD5 BURKITT LYMPHOMA ? Ki67, CD10(+/-BCL6), BCL2, MYC IMMATURE(PRECURSOR LYMPHOBLASTIC)CELL ? TdT ANAPLASTIC LARGE CELL LYMPHOMA ? CD30, ALK PLASMA CELL ? CD20- , CD138+ HISTIOCYTE ? CD163( or CD68) INTERDIGITATIONG DENDRITIC OR LANGERHANS CELL ? S100( also LANGERIN/ CD207 for the latter cell type) FOLLICULAR DENDRITIC CELL ? CD21 or CD35 HODGKIN LYMPHOMA ? CD30, CD15, PAX5 MOST USEFUL ANTIBODIES FOR ASSESSMENT OF LYMPHOID PROLIFERATIONS

52. CHARACTERISTIC CYTOGENETIC FINDINGS IN SPECIFIC LYMPHOMA TYPES LYMPHOMA TYPE SPECIFIC CHROMOSOMAL TRANSLOCATION ONCOGENE OR TUMOUR SUPPRESSOR GEENE IMPLICATED Follicular lymphoma t(14;18)(q32;q21) BCL2 Mantle cell lymphoma t(11;14)(q13;q32) CCND1 Extranodal marginal zone lymphoma of MALT type t(11;18)(q21;q21) t(1;14)(p22;q32) t(14;18)(q32;q21) t(3;14)(p14.1;q32) API2, MALT1 BCL10 MALT1 FOXP1 Burkitt lymphoma t(8;14)(q24;q32) t(8;22)(q24;q11) t(2;8)(p12;q24) MYC T-lymphoblastic lymphoma/leukemia t(1;14)

53. RELATIVE FREQUENCIES OF B-CELL LYMPHOMA SUBTYPES IN ADULTS

54. RELATIVE FREQUENCIES OF MATURE T-CELL LYMPHOMA SUBTYPES IN ADULTS

55. REACTIVE LYMPADENOPATHIES AND THEIR DISTICTION FROM LYMPHOMA INFECTIOUS MONONUCLEOSIS AND OTHER VIRAL INFECTIONS HYPERSENSITIVITY REACTIONS(PHENYTOIN) KIKUCHI LYMPHADENITIS

56. REFERENCES MCKENZIE-2 ND EDITION,VOLUME 2 WHO CLASSIFICATION OF TUMOURS OF HAEMATOPOIETIC AND LYMPHOID MALIGNANCIES-4 TH EDITION WINTROBES-11 TH EDITION ROBBINS- 18 TH EDITION DIAGNOSTIC HISTOPATHOLOGY OF TUMOURS-FLETCHER 4 TH EDITION,VOLUME 2 IOACHIM’S LYMPH NODE PATHOLOGY INTERNET

57. THANK YOU