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
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.
Casos novos em 2014 nos EUA Ward E et al. Cancer J Clin 2014; 64:83-103
Clinical Presentation Mass in different localizations Fast growth Extrinsic compression Dysfunction of the organs involved Wasting syndrome is uncommon
Risk of Invasive Procedures Extensive Para-aortic involvement
Diagnosis in pediatric tumor masses Histology: Neuroblastoma Primitive Neuroectodermic tumor - PNET Non-Hodgkin lymphoma Cytology: Neuroblastoma PNET Non-Hodgkin lymphoma
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
Conventional diagnosis in pediatric solid tumors Immunohistochemistry:
Less Invasive Procedures Fine Needle Aspirate Dissociated cells in suspension Effusions Aspirate Bone Marrow CSF Vitreous puncture
Less Invasive Procedures Fine Needle Aspirate Effusions Aspirate Bezerra et al. 2011 Bone Marrow CSF Vitreous puncture
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
Immunohistochemistry vs. Flow Cytometry
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
What exists using Flow Cytometry in lymphomas? Barrena et al. Histopathology, 2011
What exists using Flow Cytometry in lymphomas? Sensitivity 77% Specificity 97% VPP 77% VPN 100% Bezerra AMPS et al, Einstein 2011
What exists using Flow Cytometry in Pediatric lymphomas? Shen H et al, Leukemia & Lymphoma 2012
What exists using Flow Cytometry in Pediatric lymphomas?
What exists using Flow Cytometry in Pediatric lymphomas? Author Year n = FC Sample Country Patel et al (COG) 2012 59 77% BM USA Canadá Singapura Bezerra et al. 2011? Bx/FNA Brasil (Einstein) Razack 2012 290 96% FNA South Africa Ferreira-Facio et al. Shen et al 2013 2013 26 78% (L) 100%(R) Bx/Fluids/ BM/PB Brasil (UFRJ) Bhaker et al. 2015 15 100% FNA/Fluids India Mussolin et al. (AEIOP) 2015 65 49% BM/ PB Italy Silowash et al 2016 47 19% FNA USA
What have we done? Ferreira-Facio et al, 2013 Ferreira-Facio et al, unpublished
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
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, 2013 8(3):e55534
MFC vs. HP+IM Agreement degree in hematopoietic tumors: 41/53= 77.3%
MFC vs. HP+IM Agreement degree in reactive samples: 53/53= 100%
Reactive Sample Tube 1 T lymphocytes Neutrophils B lymphocytes T lymphocytes Tube 2 B lymphocytes
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
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
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
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
Hodgkin Lymphoma by Flow Cytometry cervical lymph node from a 5y boy with Hodgkin lymphoma, mixed cellularity
Distribution of immune cells in pediatric solid tumors
Langerhans Cells Histiocytosis Abdominal mass
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
Team of Flow Cytometry Lab IPPMG/UFRJ