Reac%ve and Benign Flow Cytometry findings

Transcription

1 Reac%ve and Benign Flow Cytometry findings Lymph nodes and other /ssues Sindhu Cherian, MD University of Washington, Sea<le

2 Flow cytometry Well established role in the diagnosis of hematopoie/c neoplasms characterized by expansion of a uniform popula/on of cells with a typical aberrant immunophenotype Lymphoma Acute leukemia

3 In some samples, the neoplas%c cell dominates Consider this biopsy of a colonic mass in a 70 year old man

4 Easy to make a diagnosis of B cell lymphoma: Mantle cell lymphoma But what do we make of the other B cells in this sample?

5 What about the T cells in this sample?

6 What about the T cells in this sample? CD4:CD8 ra%o of 1:2 Double nega%ve T cells with bright CD3 CD4+ T cells that express decreased CD7 CD8+ T cells with low to absent CD5

7 GOAL: By the end of this hour you will be able to recognize the significance of each of the highlighted lymphoid subsets

8 Objec%ves Characterize reac/ve popula/ons one may see in a variety of segngs Lymph nodes, /ssues Differen/ate normal reac/ve changes from neoplasms that may be present in these same sites Avoid missing a small abnormal popula/on Avoid calling a reac/ve finding neoplas/c Determine how recogni/on of reac/ve popula/ons in some cases may inform the diagnosis

9 Lymph node Harbor ~40% of all lymphocytes in humans ~2% in PB by comparison A reac/ve lymph node has many compartments that can be recognized by morphology Many of these popula/ons can also be recognized by flow cytometry Laane et al Clinical Cytometry 64B; Lazuardi et al Immunology 114 (37-43)

10 Reac%ve Lymph Node

11 Reac%ve Lymph node 25-50% B cells 50-75% T cells 5% NK cells

12 B cells (20-50%) CD5+ B cells Plasmacytoid B cells/plasma cells/plasmablasts Follicular hyperplasia Kappa:Lambda ra/o of 1.5-2:1

13 CD5+ B cells CD5 is expressed on pre naive B cells (CD10 and CD38 posi/ve) and a subset of naïve B cells (CD10 and CD38 nega/ve) Predominate in fetal circula/onà less frequent in adult circula/on ~10-15% of B cells in adult circula/on Greater numbers in spleen and circula/on than lymph node Increased in circula/on in some condi/ons: Autoimmune disease, marrow regenera/on, post transplant Polyclonal with normal expression of CD19 and CD20 allowing dis/nc/on from CD5+ B cell lymphomas (CLL, mantle cell) Perez-Andres et al. Cytometry part B 78B;S47-S60. Gupta et al. Am J Clin Pathol. 2004;121(3):

14 Plasmacyotoid B cells/plasma cells

15 Plasma cells/plasmacytoid B cells/ Plasmablasts Paracor/cal hyperplasia, non-specific IgG4 related lymphadenopathy Dis/nguish from: Plasmacytoid component of a B cell neoplasm (cytoplasmic light chain) Plasma cell neoplasm (cytoplasmic light chain, normal CD19 and CD45 without aberrant CD56)

16 Follicular hyperplasia

17 Follicular hyperplasia Non-specific (infec/on, inflammatory process) Dis/nguish from follicular lymphoma

18 Reac%ve Follicular Hyperplasia versus Follicular Lymphoma Usually reac/ve follicular hyperplasia is polyclonal allowing easy dis/nc/on of the two In some cases dis/nguishing between reac/ve florid follicular hyperplasia and follicular lymphoma can be a challenge Poten/al pihalls: Reac/ve follicular hyperplasia can down-regulate light chains Nega/ve light chain expression has been reported Zhao et al. AJCP 2005:123: Reac/ve follicular hyperplasia can be clonal ~1.1% of excisional biopsy specimens over a 5 year period Kussick et al, AJCP 2004;121:

19 Reac%ve follicular Follicular lymphoma hyperplasia CD10 Increased Increased CD20 Increased Increased CD19 Normal Decreased CD38 Increased Normal/Low BCL-2 Decreased Increased * Intensi/es are reported as compared to normal CD10 nega/ve B cells in a lymph node References: Mantei and Wood. Cytometry Part B. 2009;76B: Ray et al. AJCP. 2005;124: Cornfield at al. AJCP 2000;114:

20 Reac%ve follicular hyperplasia Follicular lymphoma Follicular lymphoma may have decreased CD19 expression CD38 is usually low in follicular lymphoma as compared to reac%ve follicular hyperplasia BCL-2 is over expressed in most cases of follicular lymphoma but nega%ve (below background B cells and T cells in reac%ve follicular hyperplasia.

21 T cells (50-75%) Express pan-t cell an/gens CD2 CD3 CD5 CD7 CD4:CD8 ra/o 2-5, fluctuates with different states Reac/ve changes are onen accompanied by an/genic shins Notable T cell subsets will be described in the next few slides T cell LGLs, γδ T cells, Memory T cells

22 Large Granular T Lymphocytes CD3+ T cells expressing CD8 with decreased CD5 CD57+, CD56+/-

23 Large Granular T Lymphocytes CD3+ T cells expressing CD8 with decreased CD5 CD57+, CD56+/- Reac/ve LGLs may in increased in lymph nodes infiltrated by tumor

24 Gamma Delta T cells Bright CD3+ T cells that are double nega/ve for CD4/CD8 or express low CD8

25 Gamma Delta T cells Bright CD3 + T cells that are double nega/ve for CD4/CD8 or express low CD8 More frequent in the spleen and intes/ne than in lymph nodes

26 Memory T cells CD4+ T cell subset with decreased CD7 Frequent in the skin, increased in inflammatory segngs May show increased CD2 expression Sanders et al. The Journal of Immunology (5):1401-7

27 CD56 expression on T cell subsets CD56 may be seen on a subset of CD4, CD8, dual posi/ve or dual nega/ve T cells CD56 expression is associated with enhanced cytotoxicity and cytokine secre/on (CD8+, DN) CD4+/CD8+/CD56+ clonal T cells may be increased in the blood in pa/ents with CMV CD4+/CD56+ T cells may be increased in some tumors Glioblastoma mul/forme Kelly-Rogers et al. Human Immunology. 2006;67: Waziri et al. The Journal of Immunology 2008;180: Rodriguez-Caballero et al. Blood. 2008;

28 Skewed CD4:CD8 ra%o HIV 0.7+/-0.1 Mantle cell lymphoma (<2) Higher CD4:CD8 ra/o be<er overall survival Reac/ve lymphoid hyperplasia (2-5) IgG4 associated lymph adenopathy ( ) Occasionally CD4/CD8 double posi/ve popula/ons are seen 1-6%) Dermatopathic lymphadenopathy (increased) Age related changes Increase in the CD4:CD8 ra/o due to decrease in CD8+ T cells Nygren et al. Clinical Cancer Research 20(23) Grimm et al. Modern Pathology ; Laane et al. Clinical Cytometry 2005;64B; Lazuardi et al. Immunology 2005;114: Tedla et al. Clinical experimental immunology 1999;117:92-99.

29 NK cells ( 5%) NK cells Typically express CD2 and CD7 without surface CD3 or CD5 Localize to the parafollicular region of the LN A subset may lack CD2 NK cells express CD56 and CD16 Intensity of these an/gens is typically reciprocal NK cells highlighted in blue, 1.2% of all lymphocytes

30 CD16 and CD56 on NK cells CD56 bright NK cells have low CD16 (bright CD7) Predominate in reac/ve lymph nodes (~90%) Minority of PB NK cells (~10%) CD16 bright T cells have lower CD56 Majority in PB (90-95%) and spleen (~85%) Fehniger et al. Blood. 2003;101(8): Ferlazzo et al. The Journal of Immunology. 2004;172:

31 Reac%ve changes can give rise to immunophenotypic changes resul%ng in a variety of T and NK cell subsets These reac%ve changes may overlap with the immunophenotype of some lymphomas Knowledge of the varia%on in normal is required to avoid over interpre%ng a reac%ve T or NK cell subset

32 The following features should raise your suspicion for T cell lymphoma Loss of or markedly decreased expression of CD45 Complete loss of one or more pan-t cell an/gens Decreased expression of more than 2 pan T cell an/gens in conjunc/on with altered light sca<er proper/es CD4/CD8 dual posi/ve or dual nega/ve popula/ons Exclusions Thymus Autoimmunelymphoprolifera/ve syndrome-alps Some reac/ve T cell subsets (either dual posi/ve or dual nega/ve) Gorczyca et al Clinical Cytometry ;

33 T cell lymphoma-ptcl No one change is completely specific for T cell lymphoma Abnormal T cell popula%ons iden%fied by flow cytometry must be interpreted in the appropriate clinical and morphologic context Increased sca<er proper/es Loss of more than one an/gen CD7 Surface CD3 Aberrant expression of other an/gens Decreased CD5 Increased CD4

34 The distribu%on of lymphoid cells may vary based on the anatomic site Other lymphoid organs: Spleen Thymus Non-lymphoid /ssues where lymphoid cells may reside Small intes/ne Skin

35 Spleen Largest lymphoid organ in the human (25% of total lymphocytes) B cells 40-50% Plasma cell/plasmacytoid lymphocyte 3-6% 8-11% CD5+ (decreased with age) T cells 30-50% CD4:8 ra/o 1.2 +/-3 Alpha beta 30-40% Gamma delta 2-5% ~15% NK cells Wide range More than blood or marrow Colovia et al. Modern pathology ;

36 Thymus Site of T cell development Full T cell matura/onal spectrum is present With thymic hyperplasia, thymoma, the dual posi/ve popula/on onen predominates, though single posi/ve and double nega/ve popula/ons are seen. Occasionally immature T cell popula/ons can be seen in reac/ve lymph nodes Castleman s disease

37 Skin Benign dermatoses can be associated with: Increase memory T cells CD3+ T cells that express CD4 without CD7 Increased CD4:CD8 ra/o Clonal T cell popula/ons

38 Small intes%ne Intes/nal lymphocytes typically express CD103 CD103 is expressed by 80-85% of lymphocytes in the intes/ne CD8+ T cells outnumber CD4+ T cells in this anatomic site: CD4:CD8 ra/o of ~1:2 May include a subset of CD5 nega/ve T cells May include a subset of CD56+ T cells Gamma delta T cells account for 5-10% of lymphocytes Rela/vely higher in pa/ents with small intes/nal bacterial overgrowth and celiac disease than in irritable bowel syndrome Included in the CD5- T cell subset Remes-Troche et al. Journal of Clinical Gastroenterology. 2007;41(7)

39 Jejunum-reac%ve lymphocytes 4:8 ra/o of 0.09 Gamma delta T cells are 7.5% of T cells CD8 low+ CD3 bright CD5 absent CD56 subset

40 With an understanding of the normal and reac%ve findings in various %ssues, you can understand and explain normal findings and differen%ate that from neoplasm

41 In addi%on, in some cases, recogni%on of reac%ve T cell popula%ons may inform the diagnosis In par%cular in situa%ons where the neoplas%c cell is a minor component of the infiltrate and an inflammatory background predominates.

42 Classical Hodgkin lymphoma Nodular lymphocyte predominant Hodgkin lymphoma T cell rich large B cell lymphoma As compared to non-specific reac%ve hyperplasia Increased T cells Decreased B cells Increased CD4:CD8 ra/o Wu et al Clinical Cytometry 2015 e-published ahead of press

43 Classical Hodgkin lymphoma Most studies show Increased propor/on of T cells Decreased propor/on of B cells Increased CD4:CD8 ra/o as compared to reac/ve hyperplasia Increased T regulatory cells (CD4 bright, CD25+) Surrogates CD3+/CD4+ T cells with bright CD45 and CD7 CD3+/CD4+ with CD38 without CD26 (seen in PB too) Hudnall et al. Clinical Cytometry B;1-8. Gaetano et al. Annals of Hematology ; Seegmiller et al. Clinical Cytometry B Wu et al Clinical Cytometry 2015 e-published ahead of press

44 Classical Hodgkin lymphoma Normal CHL Key plot: T cells

45 Nodular lymphocyte predominant Hodgkin lymphoma Several studies have demonstrated the presence of CD4+/CD8+ mature T cells in NLPHL Double posi/ve T cells accounted for 10-38% of all T cells in NLPHL (seen in ~60% of cases) 38% in progressively transformed germinal centers 4% reac/ve lymphoid hyperplasia 6% in classical Hodgkin lymphoma 10% or more double posi/ve T cells Sensi/vity 58.3% Specificity 96.2% Thought to represent an ac/vated or reac/ve T cell subset Didn t seem to be correlated with # of CD57+ cells by IHC; however, reac/ve double posi/ve T cell popula/ons are seen in inflammatory segngs and known to express CD57. Rahemtulla et al. AJCP 2006;126; Rahemtulla et al. Leukemia and Lymphoma (10)

46 Nodular lymphocyte predominant Hodgkin Lymphoma Key plot: T cells Normal NLPHL

47 T cell rich large B cell lymphoma High CD4:CD8 ra%o (15.2) Mature B cells are decreased (<5%) Increased CD45 and CD7 on CD4+ T cells Normal for comparison

48 Classical Hodgkin lymphoma CD4+ T cells with bright CD7 and CD45 (76.5%) Nodular lymphocyte predominant Hodgkin lymphoma Increased double CD4+/CD8+ cells 11.7% Compare to 2.18%; 3.70% T cell rich large B cell lymphoma CD4+ T cells with bright CD7 and CD45 (92.3%) Highest CD4:CD8 ra/o (~10) Compare to 4.18; 5.44 B cells are rare (~5%) Compare to 27.4%; 29.6% Wu et al Clinical Cytometry 2015 e-published ahead of press

49 What are the popula%ons highlighted in the case we started with? Plasmacytoid lymphocytes/ Plasma cells/plasmablasts Reac%ve follicular hyperplasia

50 What about the T cells in this sample? CD4:CD8 ra%o of 1:2 Double nega%ve T cells with bright CD3 Gamma delta T cells CD4+ T cells that express decreased CD7 Memory T cells CD8+ T cells with low to absent CD5 Large granular lymphocytes

51 Conclusions Understanding the immunophenotype of normal and of common reac/ve pa<erns will help Recognize abnormal popula/ons Prevent calling a reac/ve finding neoplas/c Furthermore, understanding reac/ve popula/ons may provide a clue to the true underlying diagnosis

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