1. ICCS e-Newsletter CSI Spring 2013 David A Westerman, MBBS FRACP FRCPA Department of Pathology Peter MacCallum Cancer Centre Melbourne, Australia

2. e-CSI - Clinical History: A 52 year Caucasian man presents with pancytopenia for investigation, and a previous history of “T cell lymphoma”.

3. e-CSI - Peripheral Blood: CBCNormal Range – WBC: 0.5 x 10 9 /l (4.0 – 11.0) – RBC:2.86 x 10 12 /l (4.5 – 6.5) – Hgb: 9.0 g/dl (13.0 – 18.0) – Hct: 27.0 % (40.0 – 54.0) – MCV:95.0 fl (80.0 – 96.0) – MCH: 32.0 pg (27.0 – 32.0) – MCHC: 32.9 g/dl (31.0 – 35.0) – RDW: 17.2% (11.7 – 15.7) – Plts: 25 x 10 9 /l (150 – 400)

4. e-CSI - Peripheral Blood: CBC Differential – Granulocytes:75% – Lymphocytes: 9% – Monocytes:11% – Eosinophils:2%

5. e-CSI - Clinical History: Bone marrow aspirate and trephine biopsy were performed. Flow cytometric immunophenotyping was performed on the bone marrow aspirate and the results from selected 8-color tubes are provided for review.

6. e-CSI - Flow Cytometric Studies: Acquired FACS Canto II, analyzed with Kaluza

7. e-CSI - Flow Cytometric Studies: Initial gating strategies excluded doublets and debris Further gating to include WBC’s and then a more “targeted” gated population based on an unusual CD45 vs SSC plot, in this case – “mononuclear gate”

8. e-CSI - Flow Cytometric Studies: 56% of mononuclear cells gated are CD2+, while T cells account for only 4% An expanded monocytic population is also identified with weak (but normal) CD4 expression (green)

9. e-CSI - Flow Cytometric Studies: CD2 vs CD3 allows separation of the NK cell population (black) and monocytes (green) in this case. Normal CD4+ and 8+ T cells can also be seen (mustard colour and blue)

10. e-CSI - Flow Cytometric Studies: CD2+ and 16/56+ NK cells (black), monocytes in green, residual T cells in mustard and light blue

11. e-CSI - Flow Cytometric Studies: NK cells are CD94+bright, 26+bright, 4-, 27+ Normal T cells 

12. e-CSI - Flow Cytometric Studies: Normal NK plot expressions Normal expression of pooled normal donors NK cells are in black, CD4+T cells mustard, CD8+ T cells in blue

13. e-CSI - Flow Cytometric Conclusion: 1.NK cells 56% of mononuclear gate expressing: CD2+, 3-, 4-, 5 partial, 7-, 8-, 16/56+, 26+ bright, 27-, 94+ bright, TCR αβ- & γδ- 2.Monocytes –normal expression compared to controls with this panel of markers but expanded in number

14. e-CSI – cont. Correlation with morphology and subsequently immunohistochemistry was performed

15. e-CSI – morphology The aspirate quality was poor. A trephine touch is depicted showing numerous large, immature haematopoietic precursors

16. e-CSI – morphology H & E section x 40 magnification

17. e-CSI – morphology H & E section x 200 magnification; Effacement by an undifferentiated population of large blast-like cells

18. e-CSI – morphology CD2 demonstrates a dominant positive population, including mitotic figures

19. CD3 shows cytoplasmic staining, in keeping with staining of CD3 epsilon chains of NK cells e-CSI – morphology

20. TIA shows a significant positive population, while some granzyme positivity was also seen (not shown), in keeping with the cytotoxic function of NK cells

21. e-CSI – morphology EBV ISH is not a validated in our mercuric chloride fixed trephines, so was not tested TP53 IHC (not shown) showed positivity in about 20% of cells Insufficient material was available for cytogenetics TCR gene rearrangement studies were polyclonal

22. e-CSI – Diagnosis Aggressive NK-cell leukaemia Reactive monocytosis

23. e-CSI – Aggressive NK-cell leukaemia This is a rare disorder with a preponderance in Asian and South American populations, and young to middle aged There is a strong association with EBV Patients present with fever, cytopenias, constitutional symptoms, leukaemic cells, DIC, or haemophagocytic syndrome. Skin involvement is uncommon

24. e-CSI – Aggressive NK-cell leukaemia Morphologically: NK cells in this disorder vary from typical large granular lymphocyte morphology to atypical forms with folded nuclei, and nucleoli to frank blasts The typical phenotype is CD2+ CD3- CD3ε+ CD56+ CD57- CD16+ CD11b+ FAS ligand (CD95) is found on neoplastic cells, and also in the serum T cell receptor genes are germline

25. e-CSI – Aggressive NK-cell leukaemia >90% cases are EBV positive Karyotypic anomalies vary and include 11q, 6q, 17p deletions The prognosis is poor and median survival measureable in just a few months Typically the disease is refractory to aggressive chemotherapy

26. e-CSI – References Chan JKC, Jaffe ES, Ralfkiaer E et al. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. 4TH edition, pp276-277. Dearden CE, Johnson R, Pettengell R et al. Guidelines for the management of mature T-cell and NK-cell neoplasms (excluding cutaneous T-cell lymphoma). Br J Haematol 2011; 153, 451–485. Kawa-Ha K, Ishihara S, Ninomiya T et al. CD3-negative lymphoproliferative disease of granular lymphocytes containing Epstein-Barr viral DNA. J Clin Inves 1989; 84: 51-55. Kwong YL, The Diagnosis & management of extranodal NK/T cell lymphoma, nasal-type and aggreesive NK-cell leukemia. J Clin Exp Hematopath. 2011; 51: 21-27. Suzuki R, Suzumiya J, Nakamura S et al. Aggressive natural killer-cell leukemia revisited: large granular lymphocyte leukemia of cytotoxic NK cells. Leukemia 2004; 18: 763–770.