ICCS e-Newsletter- CSI CaseSpring 2015

Amr Rajab and Anna PorwitFlow Cytometry Laboratory, Department of

Laboratory Medicine, University Health Network, Toronto General Hospital,

Toronto, ON, Canada

Clinical History and Specimens Submitted

79 year old male diagnosed with splenic marginal cell lymphoma 2010.

Bone marrow aspirate and biopsy were performed due to suspect progression (increasing splenomegaly and some lymphadenopathy).

Peripheral Blood Cell Counts (CBC)

Hb: 140 g/LMCV: 92WBC: 6.1 x10e9/LPlt: 108 x10e9/LNeutrophils: 2.1 x10e9/LEosinophils: 0.2 x10e9/LLymphocytes: 3.2 x10e9/LMonocytes: 0.6 x10e9/LBasophils: 0 x10e9/L

Abnormal cytoplasm-rich lymphocytes are present in blood, some have villous cytoplasm, no increase of plasma cells in blood

Bone Marrow Aspirate

Sample Quality: AdequateFragments: PresentCellularity: ModerateErythropoiesis: Increased Left shiftedErythroid Morphology: NormalGranulopoiesis: Adequate for ageGranulocyte Morphology: NormalMegakaryopoiesis: PresentMegakaryocyte Morphology: NormalLymphocytes: IncreasedPlasma Cells: Increased, some binucleated forms seenIron Status: NormalRinged Sideroblasts: AbsentClot Section: Confirms findings in smears: variable cellularity,

clusters of lymphocytes and plasma cells noted

Bone Marrow Differential

Number of cells counted: 200Blasts: 0.5 %Promyelocytes: 0.5 %Myelocytes: 7 %Metamyelocytes: 10 %Neutrophils: 14 %Eosinophils: 1 %Basophils: 0 %Monocytes: 2 %Lymphocytes: 26 %Plasma cells: 13 %Erythroblasts: 26 %

Plasma cells in BM smears

Flow Cytometric Analysis

B Tube Multiple Myeloma

KAPPA FITC cyt. KAPPA FITC

LAMBDA PE cyt. LAMBDA PE

CD19 ECD CD56 ECD

CD38 PC5.5 CD138 PC5.5

CD20 PC7 CD34 PC7

CD34 APC CD117 APC

CD23 APC-A700 CD19 APC-A700

CD10 APC-A750 CD38 APC-A750

CD5 PB CD20 PB

CD45 KO CD45 KO

Flow cytometric analysis was performed on bone marrow aspirate.

Data acquisition was performed on a 10-Color Navios Flow cytometer from Beckman Coulter.

Flow cytometric data was analyzed using Kaluza software from Beckman Coulter.

Flow Cytometric Analysis

Surface staining

Wash cells x 2 with warm PBS Resuspend cells in 1% BSA in PBS Adjust cells to 5 x109 /L Add Abs cocktail to 100ul (5 x105 cells) of sample Incubate for 15 minutes in dark at RT. Lyse with 1mL VersaLyse (BC ref. IM3648) plus 25 uL IOTest 3

Fixative (BC ref. IM3515) Wash and suspend in 1 mL PBS and 12.5 uL IOTest 3 Fixative (BC

ref. IM3515)

Flow Cytometric Analysis

Intracellular staining

Wash cells x 2 with warm PBS Resuspend cells in 1% BSA in PBS Adjust cells to 5 x109 /L Add surface Abs cocktail to 50ul of sample Incubate for 15 minutes in dark at RT. Add 100 mL of IntraPrep Reagent ‘1’ (BC ref. A07803) to each tube Vigorously vortex tubes. Incubate at room temperature for 15 minutes, in the dark. Wash and suspend in 50uL PBS Add 100 mL IntraPrep Reagent ‘2’ (BC ref. A07803) to each tube and

GENTLY mix. Incubate at room temperature, in the dark, for 5 minutes. Add cytoplasmic antibodies. Gently vortex tubes Incubate at room temperature, in the dark, for 15 minutes Wash and Re-suspend cells in ~500 uL of PBS

Flow Cytometric Data Analysis – B Tube

A

Non-viable cells are gated using forward (FSC) vs. side scatter (SSC) gating (gate Debris, plot A). All consequent dot plots are created of Boolean gate (Living cells) excluding Debris.

Various cell subpopulations are mapped on CD45/Side scatter (SSC) plot (B) Gray: Granulocytes Yellow: Monocytes Cyan: Blasts Blue: Plasma cells Red & Purple: Lymphocytes

B

Plasma cells

Flow Cytometric Data Analysis – B Tube

A B

B-cells are enumerated using CD19+/SSC dot plot (A) Kappa vs. Lambda plot shows Lambda light chain

restriction (Red) (B).

Flow Cytometric Data Analysis – B Tube

The B-cell population (Red dots, 93% of B-cells) is negative for CD10, CD5, CD23, and CD34 (not shown) but brightly positive CD20 and CD38 (compared to background normal B cells – Green dots, 3% of CD19+). A small population of CD10+ B-cell precursors was noted (Cyan dots, 4% of CD19+)

Flow Cytometric Data Analysis – B Tube

T-cells are enumerated using CD5 (A&B). CD34+ cells are enumerated out of the total

living cells (C). Plasma cells are enumerated using

CD38++/SSC dot plot (D). Increased plasma cells prompted adding of the multiple myeloma panel.

A B C

D

Flow Cytometric Data Analysis – MM Tube

Debris and doublets were removed using sequential gating (A &B).

PCs were gated on CD38/CD138 plot (C) and viable PCs on FSC/SSC plot (D). Note many PC in the area for apoptotic cells (Myeloma panel performed 24 hours after aspiration).

B C

D

A

Flow Cytometric Data Analysis – MM Tube

PCs are positive for cytoplasmic Kappa, CD56 and CD117, but negative with CD19 and CD20 (A, B & C)

Eight-parameter Radar plot (D): Kappa and lambda are displayed horizontally from East to West. Antigens expressed on normal PCs are displayed vertically towards the North and those expressed aberrantly on clonal PCs vertically towards the South.

A B C

D

Immunophenotypic Findings

BMA: 9% of B-cells positive for lambda, CD19, CD20, CD38, negative for

CD5, CD10, CD23, and CD11c (not shown, evaluated in T-cell tube) 4% kappa+ monotypic plasma cells population positive for CD138,

CD38++, CD56+, CD117+ and negative for CD19, CD20 and CD45.

PB (received separately): 21% B-cells positive for lambda, CD19, CD20, CD38, negative for CD5, CD10, CD23, and CD11c.

Morphology Findings

Bone marrow smears showed up to 13% plasma cells near fragments, less in other more diluted areas

Bone marrow biopsy confirmed presence of CD20+ B-cell infiltrates and an increase of intra sinusoidal B-cells.

Clusters of plasma cells with positivity for kappa, CD56 and CD117 were noted.

CD20+ infiltrates in BM biopsy

Clusters of CD138+ plasma cells

Diagnosis: Bone marrow involvement by splenic marginal zone lymphoma (lambda+) and plasma cell neoplasm (kappa+).

Discussion Approximately 70 patients with concomitant involvement of the

bone marrow by plasma cell myeloma and B-cell lymphoma (mainly of CLL type) have been described in the literature (1,2). In most cases B-cell lymphoma preceded plasma cell myeloma.

Two processes may arise independently from the same stem cell (3) or arise from different B cells coincidentally (4).

Splenic marginal cell lymphoma may show marked plasmacytic differentiation and molecular study may be necessary to identify the 2 monotypic populations if the light-chain restriction is the same for both monotypic processes (5). In our case this scenario is unlikely since the plasma cell neoplasm showed kappa light chain restriction. Previous studies have shown that lambda loci in kappa-producing neoplastic B-cells are largely unrearranged while kappa loci in lambda producers are often rearranged and inactivated by rearrangements of the kappa-deleting element (6,7). Thus a possibility of kappa+ plasma cell neoplasm developing as progression of lambda+ B-cell lymphoma is not likely.

References1. Alley CL, Wang E, Dunphy CH, Gong JZ, Lu CM, Boswell EL, Burchette J, Lagoo

AS. Diagnostic and clinical considerations in concomitant bone marrow involvement by plasma cell myeloma and chronic lymphocytic leukemia/monoclonal B-cell lymphocytosis: a series of 15 cases and review of literature. Arch Pathol Lab Med. 2013 Apr;137(4):503-17.

2. Hussein S, Gill K, Baer LN, Hoehn D, Mansukhani M, Jobanputra V, Bhagat G, Alobeid B. Practical diagnostic approaches to composite plasma cell neoplasm and low grade B-cell lymphoma/clonal infiltrates in the bone marrow. Hematol Oncol. 2014 Jan3 (E-Pub)

3. Saltman DL, Ross JA, Banks RE, Ross FM, Ford AM, Mackie MJ. Molecular evidence for a single clonal origin in biphenotypic concomitant chronic lymphocytic leukemia and multiple myeloma. Blood. 1989;74(6):2062–2065.

4. Pereira Wde O, Bacal NS, Correia RP, Kanayama RH, Veloso ED, Borri D, Hamerschlak N, Campregher PV. Development of plasma cell myeloma in a B-cell chronic lymphocytic leukemia patient with chromosome 12 trisomy. BMC Res Notes. 2013 Oct 29;6:433.

5. Molina TJ, Lin P, Swerdlow SH, Cook JR. Marginal zone lymphomas with plasmacytic differentiation and related disorders. Am J Clin Pathol. 2011; 136(2): 211–225.

6. Korsmeyer SJ, Hieter PA, Sharrow SO, Goldman CK, Leder P, Waldmann TA. Normal human B cells display ordered light chain gene rearrangements and deletions. J Exp Med. 1982 Oct 1;156(4):975-85.

7. Bräuninger A, Goossens T, Rajewsky K, Küppers R. Regulation of immunoglobulin light chain gene rearrangements during early B cell development in the human. Eur J Immunol. 2001 Dec;31(12):3631-7