Non neoplasticneoplastic condictions. Dipartimento di Scienze Biomorfologiche e Funzionali, Università di Napoli, Federico II Naples, Italy

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1 European Federation of Cytology Societies 4tu Annual Tutorial in Cytopathology Trieste, June 6-10, 2011 Fine needle cytology of lymph nodes: Non neoplasticneoplastic condictions Pio Zeppa MD PhD Pio Zeppa MD, PhD Dipartimento di Scienze Biomorfologiche e Funzionali, Università di Napoli, Federico II Naples, Italy

2 Why cytology for lymph nodes? REACTIVE!!

3 Why cytology for lymph nodes? Peri carotideal lymph node, the surgeon refused to perform the excision, US guided FNC: NHL Deep cervical node: suspect NHL, FNC: metastasis t high h respiratory tract, nasopharyngeal carcinoma, RX therapy No surgery! Diffuse spleen enlargement patient t F. S.: fourth relapse from MCL! t(11;14)(q13;q32)

4 Why cytology for lymphoma? 1,5 million of new cases in the world Doubled incidence in the last 20 yrs patients and new cases yrs, in Italy Increased n. of complete remissions and prolonged survivals 30% of patients will develop recurrences 30% will develop non lymphomatous swellings Non invasive procedures do not produce definitive diagnoses

5 Why cytology for lymphoma?

6 Lymph nodal cytology and ancillary techniques Hodgkin s disease immunocytochemistry metastases large cell NHL Diff Quik flow cytometry reactive? small medium cell NHL? FISH PCR others

7 From Zajicek to flow cytometry Colorado M, et al: Simultaneous cytomorphologic and multiparametric flow cytometric analysis onlymph node samples is faster than and as valid as histopathologic study todiagnose most non Hodgkin lymphomas. Am J Clin Pathol. 2010; 133:81. Katz RL et al.: Fine needle aspiration cytology of peripheral T cell lymphoma. A cytologic, immunologic, and cytometric study. Am J Clin Pathol. 1989; 9:120. Dunphy CH, Katz RL et al: Leukemic lymphadenopathy: diagnosis by fine needle aspiration. Hematol Pathol. 1989; 3:35.

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9 GATING

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11 Normal lymph node histology

12 Reactive hyperplasia (BRH)

13 non-specific hyperplasia with follicular centre expansion Non-specific hyperplasia with follicular centre expansion: smears show numerous centrocytes and centroblasts with two or more eccentrical nucleoli intermingled with small mature lymphocytes with slightly lengthened nuclei and cytoplasmic tales, plasma cells and immunoblasts.

14 non-specific hyperplasia with follicular centre expansion Cy: numerous centrocytes and centroblasts intermingled with a variable number of small lymphocytes. Differential diagnosis with a NHL is often pointed out. FC :T and B-cell populations a follicular centre cell population which co-expresses CD19/CD10 and FC :T and B-cell populations, a follicular centre cell population which co-expresses CD19/CD10 and balanced expression of kappa and lambda light chains

15 non-specific hyperplasia with interfollicular expansion Non specific hyperplasia with interfollicular expansion: the smear shows a prevalence of mature Non specific hyperplasia with interfollicular expansion: the smear shows a prevalence of mature lymphocytes with large nucleolated immunoblasts and plasma cells.

16 non-specific hyperplasia with interfollicular expansion FC in non specific hyperplasia with interfollicular expansion may shows a prevalence of T-lymphocytes (CD5) with CD2/3/7 co-expression.

17 Viral and post-vaccinial FNC show small lymphocytes and numerous centrocytes t and centroblasts intermingled with small mature lymphocytes, plasma cells and immunoblasts. Capillary structures, phagocyting histiocytes and eosinophils may also be present. Differential diagnosis: follicular lymphoma (see NHL).

18 Mononucleosis FNC shows normal cell type constituents including centrocytes and centroblasts. Numerous immunoblasts with large nucleoli and a rim of blue cytoplasm are present. Macrophages and capillary structures may be present. Differential diagnosis: Hodgkin lymphoma (see HL).

19 Histiocytosis This is mainly observed in lymph nodes draining inflamed tissues or organs or cancer. Corresponding smears may show histiocytes, small lymphocytes and macrophages with engulfed cytoplasm. Differential diagnosis: Sinus histiocytosis with massive lymphadenopathy.

20 Suppurative lymphadenitis This condition can be observed in lymph nodes draining bacterial infections. Smears show of granulocytes and a variable amount of lymphocytes in a necrotic background. Attention should be paid because metastatic squamous cell carcinoma and rarely HL, may show a prevalent necrotico-suppurative background.

21 Granulomatous lymphadenitis Granulomatous lymphadenitis may be determined d by several infective agents or by different pathological processes, sarcoidosis and tuberculosis being the most frequent. Granulomatous lymphadenitis may also occur along hematological diseases or after chemotherapy or radiotherapy. Smears show epithelioid cells and/or multinucleated giant cells, either isolated or in a granulomatous arrangement in a lymphoid background.

22 Toxoplasmosis Reactive lymph node with toxoplasmosis:small groups of epitheliod cells intermingled with lymphocytes.

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28 FC sub classification of small/medium cell size NHL phenotype LpcL SLL MCL FCL MZL/ MALT SNCL PTCL NK k:λ>4:1 or <1: /- - - CD19/CD CD19/CD CD19/CD23+ +/ CD19/FMC7+ +/ / CD19/CD bl2+/cd10 bcl-2+/cd CD4+/CD8-, CD4-/CD CD3+, CD5+, CD CD2/CD3+CD7- or CD2/CD7+CD CD3+/- CD56+ CD

29 Cytology, phenotype (FC), IGH quantitative assessment (PCR), IGH integrity evaluation (FISH) of lymphoid cells IGH probe, split signal

30 IGH (14q32) is the most frequently involved in different translocations of B cell NHL t (14;18)(q32;q21) in FL and a subset of DLBCL t (11;14)(q13;q32) in MCL t (q14;q32) in 60 % in MM with different chromosomal partners t (9;14)(p13;q32) in 50% of LPCL and some cases of MZL t (8;14)(q24;q32) in 100% of BL The IGH FISH DNA probe, split signal, is a mixture of two fluorochrome: the green labelled DNA probe that binds to a 612 kb telomeric segment, and the red labelled that binds to a 460 kb centromeric segment, to the IGH breakpoint Therefore IGH FISH DNA probe, split signal should detect any breakage involving the IGH locus at chromosome 14q32.

31 Indirect FC markers of malignancy CD5/CD19 co expression CD19/CD10 co expression in >50% of the gated cells

32 Chronic lymphatic leukemia/small lymphocytic lymphoma (CLL/SLL)

33 Lymphoplasmacytoid Lymphoma (LPCL)

34 Mantle cell lymphoma (MCL)

35 Mantle cell lymphoma Cyclin D1/bcl-1 t(11;14)(q13;q32)

36 Follicular lymphoma (FL)

37 bcl 2 in follicular lymphoma Laane E et al.: Flow cytometric immunophenotyping including Bcl 2 detection on fine needle aspirates in the diagnosis of reactive lymphadenopathy and non Hodgkin's lymphoma. Cytometry B Clin Cytom. 2005;64:34. Bcl 2 quantification might be useful using antisense oligonucleotides

38 FL with dim or not expressed light chains t(14;18)(q32;q21) p T(

39 Marginalzone B cell lymphoma (MZL)

40 Extranodal B cell lymphoma of mucosa associated lymphoid tissue (MALT lymphoma)

41 Diffuse large B cell lymphoma

42 Burkitt lymphoma (BL)

43 Peripheral T cell lymphoma (PTL) CD45 Ro

44 Peripheral T cell lymphoma (PTL)

45 Peripheral T cell lymphoma (PTL) CD45Ro

46 NK cell lymphoma/leukemia

47 Pitfalls and shortcomings! Hodgkin Lymphoma anaplastic large cell lymphoma CD30

48 F, 57 yrs, FNC of 50 mm nodule in the left thyroidal area Cytological diagnosis: T cell lymphoproliferative process

49 Histological i l diagnosis: i benign type B1 ectopic thymoma CD45Ro CK-pan

50 Thymic lymphocytes: y mature polyclonal T cells profile: (CD2+,CD3+,CD5+,CD7+) CD10+ in an immature T cell subsets maturation: CD4 CD8 in the cortex, continues as CD4+ CD8+, reaches maturation as CD4+ CD8 or CD4 CD8+ in the medulla. CD4+/CD8+ is a specific feature of intrathymic T cells

51 40 yrs old female, HIV positive with right cervical swelling. The patient suffered from a FL, since 2 yrs, last chemotherapy 10 months before US: 2,5cm large lymph node, oval with hilus preservation FC: CD5:10%, CD19:49%, CD23: 10%, FMC7: 0%, CD10:40%, CD10/19:40%, lambda light chains 40%, kappa light chains 0%. Cytological FC diagnosis: FL

52 Final lhistological i l diagnosis: i reactive hyperplasia with florid follicular proliferation!

53

54 Incidence, phenotypical and molecular data of non-lymphomatous, clonal B-cell population: a review of the literature Author/year organ sample n. of cases clinical background light chain restriction FC ICC IHH ( pos./total) IgH clonality PCR (pos./total) Berthold F, 1989 lymph node histology 1 Herpes virus 6 NP + (1/1)* negative Matsubayashi S, 1990 thyroid FNA 15 Hashimoto thyroiditis FC/ + (7/15) NP NR Cartagena N, 1992 lymph node FNA 86 NP ICC/ + (1/86) NP NR Jordan RC, 1996 minor salivary histology 50 Sjogren s syndrome NP + (4/50) NR glands follow up Zhou XG, 1999 lymph node tonsils histology 9 NR NP + (9/9) negative Saxena A, 2000 stomach histology 20 Helicobacter pylori NP + (10/20) negative gastritis Kussik SJ, 2004 lymph node histology and FNA 6 5 negative,1 HIV FC/ + (6/6)* + (3/6) negative Chen H, 2006 thyroid histology 21 Hashimoto FC/ + (18/21) negative and FNA thyroiditis Venkatraman L, 2006 lymph node FNA 26 NHL, reactive, HIV (1case) ICC+ (1 HIV/26) + (1/26) negative Bangerter M, 2007 lymph node FNA 131 NR FC+ (3/131) NP negative Nam Cha SH, 2008 lymph node histology 8 Castleman s, LES, EBV, HIV (1 case) IHH/+ (8/8)* + (3/8) 6 negative, 2 developed NHL Bhargava P, 2008 lymph node FNA 1 HIV FC/+ negative Zeppa P, 2009 thyroid FNA 34 Hashimoto thyroiditis FC/+ (5/34) negative Fan HB, 2009 liver histology 40 HCV hepatitis + (4/40) negative

55 FUTURE PERSPECTIVES New antibodies for diagnosis Schniederjan SD et al.: A novel flow cytometric antibody panel for distinguishing Burkitt lymphoma fromcd10+ diffuse large B cell lymphoma. Am J Clin Pathol. 2010;133: expression of CD44 and CD54 differs significantly between BL and CD10+ DLBCL.. Leshchenko et al.: Genomewide DNA methylation analysis reveals novel targets for drug development in mantle cell lymphoma. Blood. 2010;116:1025..CD37 surface expression in 93% MCL.

56 FUTURE PERSPECTIVES

57 Non Hodgkin lymphoma, monoclonal lantibodies and Flow Cytometry Monoclonal antibodies have a direct action against IgG constant regions and a cross fire effect (radio conjugated) Flow cytometry may quantify the target cells by the evaluation of antigen coexpression and their numerical evaluation

58 FUTURE PERSPECTIVES Högerkorp CM et al.: Identification of uniquely expressed transcription factors in highly purified B cell lymphoma samples. Am J Hematol. 2010;85:418. AndréassonU et al.: Identification of molecular targets associated with transformed diffuse large B cell lymphoma using highly purified tumor cells. Am J Hematol. 2009;84:803.

59 acknowledgments Prof. L. Palombini Dr. A. Iaccarino Prof. A. Vetrani Prof. G. Troncone Dr. I. Cozzolino Dr. C. Frangella Dr. U. Malapelle Dr. F. Plaitano Dr. M. Russo Dr. M. Salatiello Dr. L.V. Sosa Fernandez

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61 mantle cell lymphoma and mtor gene activationation Farnesyltransferase (Rapamicyn): hampers the progression G1 S inhibiting the transduction of mtor, reduces cyclin D2 D3 and increases CDk and p27 p27 may be evaluated on FNC samples by FC or ICC P27/kip1

62 lymphoplasmacytic lymphoma

63 why cytology for lymphomas? Lymphomas are an increasing pathology: new cases per year in USA 30% of these patients will develop recurrences 30% develop non lymphomatous swellings Non invasive procedures do not produce definitive diagnoses* *Zinzani PL, et al.: Histological verification of positive positron emission tomography findings in the follow up of patients with mediastinal lymphoma. Haematologica. 2007; 9()77 92(6):

64 palpable lymph node 148 thyroid 13 parotid 15 breast 4 impalpable us/ct guided lymph node 98 spleen 20 liver 2 small bowel 1 FNC/FC inadequate 15 suspicious 10 BRH 135 NHL 70 soft tissue 5 soft tissue 1 NHLr 77 Total 185 Total 122 Total 307

65 Follicular lymphoma (FL)

66 new drugs for non Hodgkin lymphomas DRUG PATHOGENETIC TARGET MECHANISM TARGET Antisense oligonucleotides Bcl-2 over expression Inhibition of target gene Follicular lymphoma (Bcl-2 antisense) G3139 chemotherapy resistance Anti ubiquitin proteasome NF-kB over (Bortezomib) expression p21, p27 degradation Anti ubiquitin proteasome action Myeloma, p21, p27 restoration Mantle, Marginal, Small lymphocytic lymphoma Protease inhibitors Farnesyltransferase (Rapamicyn) mtor gene activation progression G1 - S Transduction inhibition of mtor cyclin D2, D3 reduction CDK, p27 increase Mantle cell lymphoma Monoclonal antibodies Clonal expansion of Direct action against IgG constant region, Follicular lymphoma (unconjugated and radioconjugated): anti CD20 (Rituximab) anti CD22(Epratuxumab) anti HLA-DR(Apolizumab) lymphocytes cross-fire effect (radioconjugated) Histone deacetylase inhibitors depsipepside, hydroxamic acids (TSA,SAHA) 3q27 translocation bcl-6 over expression Bcl-6 gene deacetylation Diffuse large B-cell lymphoma

67 new drugs for non Hodgkin lymphomas DRUG PATHOGENETIC TARGET MECHANISM TARGET Antisense oligonucleotides (Bcl-2 antisense) G3139 Bcl-2 over expression chemotherapy resistance Inhibition of target gene Follicular lymphoma Anti ubiquitin proteasome (Bortezomib) NF-kB over expression p21, p27 degradation Anti ubiquitin proteasome action p21, p27 restoration Myeloma, Mantle, Marginal, Small lymphocytic y lymphoma Farnesyltransferase, (Rapamicyn) mtor gene activation progression G1 - S Transduction Inhibition of mtor cyclin D2,D3 riduction CDK, P27 increase Mantle cell lymphoma Monoclonal antibodies (unconjugated and radioconjugated): anti CD20 (Rituximab) anti CD22 (Epratuxumab) anti HLA-DR (Apolizumab) Clonal expansion of lymphocytes Direct action against IgG constant region, cross-fire effect (radio conjugated) Follicular lymphoma Histone deacetylase inhibitors:depsipepside, hydroxamic acids (TSA, SAHA) 3q27 translocation bcl-6 over expression Bcl-6 gene deacetylation Diffuse large B-cell lymphoma

68 marginal zone B-cell lymphoma

69 Diffuse large B cell lymphoma (DLBCL)

70 Past Leukeran Present watch and wait (indolent) Chemotherapeutic agents: COP, Fludarabina, Antraciclina (low grade). CHOP (high grade) therapy of non Hodgkin lymphomas Future Protease inhibitors: Farnesyl transferase Antisense oligonucleotides: Bcl 2 antisense Monoclonal antibodies: Unconjugates: Rituximab (anti CD20), Epratuxumab (anti CD22), Apolizumab, (anti CD52) Radioconjugates: Iodine 131 anti CD20 CD20, 90 Yttrium anti CD20 Monoclonal antibody: Rituximab (anticd20) Bone marrow transplantation Histone deacetylase inhibitors: depsipepside, hydroxamic acids (TSA, SAHA) Vaccines

71 new drugs for non Hodgkin lymphomas Antifolate: Pralatrexate, PTL Heat Shock Protein Inhibitors DLBCL Antiangiogenetic Aggressive NHL 15th Congress of the European Hematology Association, Barcelona June 2010 Monoclonal antibodies bi-specific T-cell engager (BiTE) (Blimatumomab)

72 Lymphomas are the most complex and heterogeneous set of human malignancies Lymphomas comprise some of the fastest (Burkitt, lymphoblastic) and slowest growing (small lymphocytic, follicular low grade) malignancies

73 Anaplastic large cell lymphoma (ALCL)

74 NHL in pleural effusion

75 NHL in pleural leffusion

76 mantle cell lymphoma (MCL) MCL small boxes MCL centrocytic MCL n.o.s. MCL Ki67

77

78

79 Chronic lymphatic leukemia/small lllymphocytic lymphoma (CLL/SLL)

80 Chronic lymphatic leukemia/small lymphocytic lymphoma (CLL/SLL) lambda kappa ICC of SLL/CLL on cytospins showing negativity for lambda and positivity for kappa light chains (peroxidase/anti peroxidase immunostain)

81 LymphoplasmacytoidLymphoma Lymphoma (LPCL)

82 Diffuse large B cell lymphoma (DLBCL) GCB (germinal center B) ABC (peripheral B cell) 5 years overall survival GCB ABC 76% 24% Alizadeh AA et al: Distinct types of diffuse large B cell lymphoma identified by gene expression profiling. fl Nature 2000; 3;403:503.

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