ICCS e-Newsletter CSI Spring 2013

David A Westerman, MBBS FRACP FRCPA

Department of Pathology

Peter MacCallum Cancer Centre

Melbourne, Australia

e-CSI - Clinical History:

• A 52 year Caucasian man presents with pancytopenia for investigation, and a previous history of “T cell lymphoma”.

e-CSI - Peripheral Blood:

• CBCNormal Range– WBC: 0.5 x 109/l (4.0 – 11.0)– RBC: 2.86 x 1012/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 109/l (150 – 400)

e-CSI - Peripheral Blood:

• CBC Differential – Granulocytes: 75%– Lymphocytes: 9%– Monocytes: 11% – Eosinophils: 2%

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.

e-CSI - Flow Cytometric Studies:

• Acquired FACS Canto II, analyzed with Kaluza

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”

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)

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)

e-CSI - Flow Cytometric Studies:

CD2+ and 16/56+ NK cells (black), monocytes in green, residual T cells in mustard and light blue

e-CSI - Flow Cytometric Studies:

NK cells are CD94+bright, 26+bright, 4-, 27+

Normal T cells

e-CSI - Flow Cytometric Studies:• Normal NK plot expressions

Normal expression of pooled normal donorsNK cells are in black, CD4+T cells mustard, CD8+ T cells in blue

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

e-CSI – cont.

• Correlation with morphology and subsequently immunohistochemistry was performed

e-CSI – morphology

The aspirate quality was poor. A trephine touch is depicted showing numerous large, immature haematopoietic precursors

e-CSI – morphology

H & E section x 40 magnification

e-CSI – morphology

H & E section x 200 magnification; Effacement by an undifferentiated population of large blast-like cells

e-CSI – morphology

CD2 demonstrates a dominant positive population, including mitotic figures

CD3 shows cytoplasmic staining, in keeping with staining of CD3 epsilon chains of NK cells

e-CSI – morphology

e-CSI – morphology

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

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

e-CSI – Diagnosis

• Aggressive NK-cell leukaemia• Reactive monocytosis

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

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

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

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.