1. ICCS e_Newsletter CSI Spring 2014 Suzanne Vercauteren MD, PhD, FRCPC Division of Hematopathology BC Children’s Hospital, Vancouver University of British Columbia

2. e-CSI - Clinical History 4 year old boy with a 4 week history of unexplained fevers, massive hepatosplenomegaly, diffuse lymphadenopathy and bicytopenia Differential Diagnosis: – Infection – Lymphoma – Primary/Secondary Hemophagocytic lympho-histiocytosis (HLH)

3. e-CSI – Peripheral Blood CBCReference Range WBC: 14.4 x 10 9 /L(5.3 - 16.0 x 10 9 /L) RBC: 3.51 x 10 12 /L(3.90 - 5.30 x 10 12 /L) Hgb: 89 g/dL(105 – 135 g/dL) MCV: 76.9 fl(75.0 – 87.0 fl) RDW-CV: 0.149(0.117 – 0.157) Plts: 46 x 10 9 /L(200 – 490 x 10 9 /L) Differential CountReference Range Neutrophils: 10.46 x 10 9 /L(1.50 – 8.50 x 10 9 /L) Lymphocytes: 3.29 x 10 9 /L(2.00 – 8.00 x 10 9 /L) Monocytes: 0.61 x 10 9 /L(0.00 – 0.90 x 10 9 /L) Eosinophils: 0.02 x 10 9 /L(0.00 – 0.50 x 10 9 /L) Basophils: 0.02 x 10 9 /L(0.00 – 0.20 x 10 9 /L)

4. e-CSI – Biochemistry ParameterValueNormal range ALT1.010 - 25 U/L AST34010 - 25 U/L G GT12710 - 25 U/L LDH1227470 - 900 U/L Ferritin11909 – 30 ug/L

5. Bone Marrow: – Normocellular marrow with trilineage hematopoiesis – Mild hemophagocytosis – Loose granulomas – CD20, CD3, CD4 and CD8 immunohistochemistry non contributory – Lymphoma screen panel ( see slide 18) by flow cytometry → normal Extensive infectious work up including TB as per patients recent travel to Cambodia e-CSI –Work-Up

6. Work up for HLH: – Fever present – Splenomegaly present – Cytopenias present – Ferritin ↑ ↑ – Triglycerides ↑ ↑ – Soluble IL-2 receptor ↑ ↑ – NK cell function → normal – Mild hemophagocytosis in BM → Criteria for HLH met (at least 5/8) e-CSI –Work-Up

7. Infectious work up continued: – Tuberculosis work up as granulomas seen in BM → pertinently negative – EBV and all other viruses → negative Work up for primary immune deficiency → Flow cytometric immunophenotyping on peripheral blood e-CSI –Work-Up

8. Acquisition: Beckman Coulter Navios flow cytometer Data analysis: Beckman Coulter Kaluza (software version 1.2) T+B cell panel on peripheral blood: e-CSI – Flowcytometric Studies FITCPC5RD1ECD Tube 1CD45CD3CD56CD19 Tube 2CD45CD3CD4CD8

9. e-CSI – Flowcytometric Studies Tube 1

10. e-CSI – Flowcytometric Studies Tube 2

11. This is strange!!!! (Repeat specimen showed same results) Aberrant CD3 dim-neg/CD4+/CD8+ population

12. Differential Diagnosis of aberrant T cell immunophenotype in this child: Hemophagocytic Lymphohistiocytosis (aberrant immunophenotype of CD8 + T cell (described) Pediatric T cell Lymphoma: T cell lymphoblastic Lymphoma Anaplastic Large Cell Lymphoma EBV positive T cell lymphoproliferative disorders of childhood e-CSI – Differential Diagnosis

13. Lymphoma panel on peripheral blood: e-CSI – Flowcytometric Studies PC5PEFITCECDPC7 Tube 1IgG1 Tube 2CD45CD4CD8CD20CD34 Tube 3CD45CD30CD25CD3CD5 Tube 4IgG1Cont PECont FITCIgG1 Tube 5CD45LambdaKappaCD10CD19 Tube 6IgG1cIgG1 IgG1 Tube 7CD45cCD79acTdTCD3CD19 cIgG1 = cytoplasmic IgG1

14. e-CSI – Flowcytometric Studies

15. Tube 2 Tube 3 ? some CD30, no CD25? Normal T cells CD4+/CD8+ e-CSI – Flowcytometric Studies

16. Tube 5 Tube 7 e-CSI – Flowcytometric Studies no TdT No B or T cells ? aberrant population

17. Blood specimen send for T and B cell clonality studies by PCR → CLONAL T cell population Lymph node biopsy suggested but surgeons wary about low platelet count (<20 x 10 9 /L at this time) e-CSI –Work-Up

18. Lymphoma screen panel on cells from supraclavicular cervical lymph node biopsy: e-CSI – Flowcytometric Studies PC5PEFITCECDPC7 Tube 1CD45IgG1 Tube 2CD45CD4CD8CD20CD2 Tube 3CD45LambdaKappaCD10CD19 Tube 4IgG1cIgG1 IgG1 Tube 5CD45cCD79acTdTCD3CD19

19. e-CSI – Flowcytometric Studies Lymph Node CD4+/CD8+ Bright CD2+

20. e-CSI – Flowcytometric Studies Lymph Node Dim CD3+

21. Sinus extended by large cells with convoluted nuclei Immunohistochemistry stains: – CD30 positive – ALK positive – CD3, CD4, CD8, CD68, CD1a and S100 negative – Brisk mitotic activity FISH shows NPM-ALK translocation e-CSI – Lymph Node Results

22. Aberrant T cell population detected in blood and lymph node but not bone marrow of this patient suggestive of mature T cell lymphoproliferative disorder CD3 expression on aberrant population in lymph node brighter compared to blood Immunohistochemistry studies differ from flow cytometry studies in that the aberrant cells are CD4 and CD8 negative by immunohistochemistry and CD30 positive in contrast to flow cytometry studies Circulating cells of this case of Anaplastic Large Cell Lymphoma were negative for CD30 and CD25 by flow cytometry (unfortunately these markers were not run on the lymph node) e-CSI – Case Conclusions

23. T cell lymphoblastic Lymphoma: 15% of childhood ALL BM always involved CD3+, CD2+, CD5+, CD7+, CD4 and CD8 often coexpressed BUT BONE MARROW NEGATIVE AND NO PERIPHERAL BLASTS SEEEN EBV positive T cell lymphoproliferative disorders of childhood: Mostly in Asia Sites of involvement: Liver, Spleen, LN, Bone marrow CD2+, CD3+, CD8+ (some CD4 and CD8 positive cases reported) BUT EBV serology NEGATIVE e-CSI – Pediatric T cell lymphomas

24. Anaplastic Large Cell Lymphoma 10-20% of childhood lymphomas Often advanced disease with B symptoms ALK+ in all pediatric cases CD3 often negative CD2, CD5 and CD4 often positive BUT almost always CD8 negative

25. Anaplastic Large Cell Lymphoma (ALCL) most common mature T-cell neoplasm in children and adolescents. approximately 15% of all non-Hodgkin lymphomas (NHL) in children and adolescents commonly present with advanced systemic disease nearly universally anaplastic large cell lymphoma kinase (ALK) positive event free survival (EFS) rates of 65-75% e-CSI – Pediatric ALCL

26. e-CSI – Flow Cytometric Findings in ALCL McCall CMMcCall CM et al. Am J Clin Pathol. 2012 May;137(5):786-94Am J Clin Pathol. 15 cases of pediatric ALCL High forward and side scatter (monocyte or granulocyte region) Frequently expressed antigens: CD4 (85%), CD2 (77%), CD7 (62%), CD3 (54%) and CD5 (38%) CD3 often dimly expressed No CD8 expression!!!!!!

27. HLH is a hyperinflammatory syndrome Impaired cytotoxic T cells and NK cells Rare Primary (familial, inherited) and secondary (infection, hematological malignancy, rheumatological diseases) Often fatal Aberrant immunophenotype of T cells reported e-CSI – Hemophagocytic LymphoHistiocytosis

28. e-CSI – Flow Cytometric Findings in HLH McCall CMMcCall CM et al. Am J Clin Pathol. 2012 May;137(5):786-94Am J Clin Pathol.

29. 13/15 patients (87%) expressed activation marker HLA-DR on cytotoxic T cells 6/9 (67%)EBV associated HLH had aberrant expansion of CD8 + T cells with variable loss of CD3, CD5 and CD7 5/8 (63%) of non EBV associated HLH had expansion of CD8 + T cells but only one (familial HLH) had aberrant loss of markers CD3 or CD5 or CD7 → Care must be taken with diagnosing T cell lymphomas based on aberrant CD8 + T cells in the setting of HLH e-CSI – Flow Cytometric Findings in HLH

30. Aberrant CD8 + T cells are a common finding in HLH Therefore HLH can mask an underlying CD8 + T cell lymphoma Although rare, CD8 expression can be seen on ALCL e-CSI – Conclusions

31. References Pediatr Blood Cancer. 2013 Nov;60(11):1916-7. doi: 10.1002/pbc.24638. Epub 2013 Jul 19. Pediatric subcutaneous panniculitis-like T-cell lymphoma with features of hemophagocytic syndrome.Merritt BY 1, Curry JL, Duvic M, Vega F, Sheehan AM, Curry CV. Pediatr Blood Cancer.Merritt BY Curry JLDuvic MVega FSheehan AMCurry CV Pediatr Hematol Oncol. 2013 Sep;30(6):509-19. doi: 10.3109/08880018.2013.805347. Epub 2013 Jun 12. Anaplastic large cell lymphoma in children and adolescents. Lowe EJ 1, Gross TG. Pediatr Hematol Oncol.Lowe EJGross TG Am J Clin Pathol. 2012 May;137(5):786-94. Flow cytometric findings in hemophagocytic lymphohistiocytosis.McCall CM 1, Mudali S, Arceci RJ, Small D, Fuller S, Gocke CD, Vuica-Ross M, Burns KH, Borowitz MJ, Duffield AS. Am J Clin Pathol.McCall CMMudali SArceci RJSmall DFuller SGocke CDVuica-Ross MBurns KH Borowitz MJDuffield AS