Flow Cytometry: useful method to less invasive diagnosis in pediatric lymphomas

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1 Flow Cytometry: useful method to less invasive diagnosis in pediatric lymphomas Prof. Elaine Sobral da Costa Universidade Federal do Rio de Janeiro UFRJ Fac. Medicina - Departamento de Pediatria elainesc.ufrj@gmail.com

2 Declaração de Conflito de Interesse Declaro que possuo conflito de interesse na (s) categoria (s) abaixo: A palestrante é autora de patentes licenciadas a Cytognos S.L.

3 Casos novos em 2014 nos EUA Ward E et al. Cancer J Clin 2014; 64:83-103

4 Clinical Presentation Mass in different localizations Fast growth Extrinsic compression Dysfunction of the organs involved Wasting syndrome is uncommon

5 Risk of Invasive Procedures Extensive Para-aortic involvement

6 Diagnosis in pediatric tumor masses Histology: Neuroblastoma Primitive Neuroectodermic tumor - PNET Non-Hodgkin lymphoma Cytology: Neuroblastoma PNET Non-Hodgkin lymphoma

7 Histopathologic description of Neuroblastoma these cancer subtypes: Histology: IT IS A BLUE SMALL ROUND CELLS TUMOR PNET Non-Hodgkin lymphoma Origin in embryonic tissues Cytology: principally ectoderm mesenchyme Neuroblastoma PNET Linfoma não-hodgkin

8 Conventional diagnosis in pediatric solid tumors Immunohistochemistry:

9 Less Invasive Procedures Fine Needle Aspirate Dissociated cells in suspension Effusions Aspirate Bone Marrow CSF Vitreous puncture

10 Less Invasive Procedures Fine Needle Aspirate Effusions Aspirate Bezerra et al Bone Marrow CSF Vitreous puncture

11 Conventional diagnosis in acute leukemias Cytology: Flow Cytometry: (Since the 80) Acute Lymphoblastic Leukemia T Lymphoblasts T Lymphocytes NK cells B Lymphocytes T-cell precursors Acute Lymphoblastic Leukemia

12 Immunohistochemistry vs. Flow Cytometry

13 Why not flow Cytometry in pediatric solid tumors? Morphology & Immunohistochemistry VS. Flow Cytometry Advantages: tissue organization No need of tumor disaggregation or viability a lot of experience in this area Advantages: Fast execution samples with low cellularity Lower cost Simultaneously several proteins less represented tumor subclons Disadvantages: Diagnostic delay Background less represented tumor subclons 1 protein in each study Disadvantages: Loss of the tissue organization Need tumor disaggregation & viability Few appropriate antibodies to FC Lack of diagnostic criteria Few experience in the area

14 What exists using Flow Cytometry in lymphomas? Barrena et al. Histopathology, 2011

15 What exists using Flow Cytometry in lymphomas? Sensitivity 77% Specificity 97% VPP 77% VPN 100% Bezerra AMPS et al, Einstein 2011

16 What exists using Flow Cytometry in Pediatric lymphomas? Shen H et al, Leukemia & Lymphoma 2012

17 What exists using Flow Cytometry in Pediatric lymphomas?

18 What exists using Flow Cytometry in Pediatric lymphomas? Author Year n = FC Sample Country Patel et al (COG) % BM USA Canadá Singapura Bezerra et al. 2011? Bx/FNA Brasil (Einstein) Razack % FNA South Africa Ferreira-Facio et al. Shen et al % (L) 100%(R) Bx/Fluids/ BM/PB Brasil (UFRJ) Bhaker et al % FNA/Fluids India Mussolin et al. (AEIOP) % BM/ PB Italy Silowash et al % FNA USA

19 What have we done? Ferreira-Facio et al, 2013 Ferreira-Facio et al, unpublished

20 Standardization of procedures and panels Calibration and compensation with EuroFlow procedures (Kalina et al. 2012) Mechanical dissociation More than 50 antibodies tested in combinations Panels establishment and test according our previous tests Analysis strategy standardization On going - validation Ferreira-Facio et al, unpublished

21 What have we done? A Non-hematopoietic cells Endothelial cells Stromal cells T lymphocytes B lymphocytes Neutrophils B NEUROBLASTOMA E PNET C F D RHABDOMYOSARCOMA G Ferreira-Facio et al. PLOSone, (3):e55534

22 MFC vs. HP+IM Agreement degree in hematopoietic tumors: 41/53= 77.3%

23 MFC vs. HP+IM Agreement degree in reactive samples: 53/53= 100%

24 Reactive Sample Tube 1 T lymphocytes Neutrophils B lymphocytes T lymphocytes Tube 2 B lymphocytes

25 FSC CD10 Igλ Reactive vs. lymphoma samples CASE 1 reactive lymph node SSC CD20 SmIgκ CASE 2 abdominal mass Burkitt lymphoma CASE 3- pleural liquid Burkitt lymphoma

26 Immunophenotype of lymphomas B-cell lymphoblastic lymphoma CD19 CD22 CD19 CD19 NuTdT CD19 Cybcl2 CD10 EBV-linked T-cell lymphoma mcd3 mcd3 CD4 CD5 CyCD3 CD7 CD8 mcd3

27 Hodgkin or Anaplasic Lymphoma 1st panel was insufficient Tube1: Tube 2 1st change in routine Tube 1 CD15 CD30 CD5 CD56 CD10 HLADR CD45 CD20 Perfomed routinelly once tubes were 1&2 negatives Further, 100% (4/4) Anaplasic Lymphoma were correctly diagnosed

28 Hodgkin or Anaplasic Lymphoma 2nd change in routine Tube 1 CD15 CD30 CD5 CD56 CD10 CD45 CD20 Acquisition of 5 millons of events Further, 89% (8/9) Hodgkin Lymphoma were correctly diagnosed

29 Hodgkin Lymphoma by Flow Cytometry cervical lymph node from a 5y boy with Hodgkin lymphoma, mixed cellularity

30 Distribution of immune cells in pediatric solid tumors

31 Langerhans Cells Histiocytosis Abdominal mass

32 Conclusion Flow Cytometry is useful method for pediatric lymphomas diagnostic, especially for - Low infiltrated samples (tumor staging) - Minimally invasive diagnostics - Tumor hegerogeneity - Tumor immune infiltration But not only

33 Team of Flow Cytometry Lab IPPMG/UFRJ