1. ICCS e-newsletter CSI Sean R McMaster and George Deeb
Department of Pathology & Laboratory Medicine
Emory University School of Medicine
Emory University Hospital
1364 Clifton Road NE
Atlanta, GA 30322

2. Clinical History-I
37 year old female with a history of human immunodeficiency virus (HIV) infection and sickle cell anemia
CD4 count of 30/µL at presentation
Presented to the emergency department with progressively worsening facial swelling and shortness of breath
The patient had generalized weakness and fatigue accompanied by weight loss over a month
The radiology work-up revealed diffuse bulky axillary, mediastinal, upper abdominal, and retroperitoneal lymphadenopathy compressing the superior vena cava and trachea above the carina

3. Complete blood counts
Results
Reference range
White Blood Cell Count
6.3 10^3/µL
Red Blood Cell Count
^3/µL
Hemoglobin
10.1 gm/dL
Hematocrit
32.2 %
MCV
75.8 fL
Red Cell Distribution Width-CV
18.4 %
Platelet Count
261 10^3/µL
A bone marrow biopsy and mediastinal mass fine needle aspiration and core needle biopsy were performed.

4. Flow cytometry analysis
Four-color multiparameter flow cytometry analysis were performed on submitted samples:
Samples were acquired as FCS3.0 files and analyzed using BD FACSDIVA software on a BD FACS Canto II cytometer
Selected relevant antibody combinations
Per-CP
FITC PE
APC
Tube 1
CD45
CD16
CD56
CD38
Tube 2
intracellular kappa light chain
-----
Tube 3
intracellular lambda light chain

5. Flow cytometry analysis-I Overview
The flow cytometric immunophenotyping of samples suspected of harboring hematological malignancies emphasizes the identification and characterization of both normal and abnormal cell populations
A “pattern recognition” approach is utilized whereby normal cells exhibit a predictable antigen expression pattern and abnormal populations are obviously deviated from such
Analysis requires familiarity with normal patterns in various sample types

6. Flow cytometry analysis-II Overview
14 (lymphoma/leukemia) and 16 (plasma cell myeloma) four-color tubes are usually performed on blood and bone marrow samples submitted for suspected hematolymphoid malignancies
The initial analysis begins with an overview of the principal cell populations expected to be seen in these samples (e.g. granulocytes, monocytes, and lymphocytes in a blood sample)
Thereafter, the assessment of each antibody combination focuses mainly on the mononuclear cells (mononuclear cell gate)
The cell population of interest will be gated on to be highlighted as needed
CD45 is a backbone marker in all tubes

7. Multiparameter flow cytometry analysis
Bone marrow
Multiparameter flow cytometry analysis
Per-CP
FITC PE
APC
Tube 1
CD45
CD16
CD56
CD38

8. Bone marrow sample analyzed using the four-color 14 tube panel for leukemia-lymphoma analysis
P1= mononuclear cell gate
P2= sub-gate encasing the cell population of interest, 0.7% of total acquired events (painted green, circled green)

9. Multiparameter flow cytometry analysis
Mediastinal mass
Multiparameter flow cytometry analysis
Per-CP
FITC PE
APC
Tube 1
CD45
CD16
CD56
CD38

10. Mediastinal mass sample analyzed using the four-color tube 16 panel for plasma cell myeloma
P1= mononuclear cell gate
P2= sub-gate encasing the cell population of interest, 8.9% of total acquired events (painted green, circled green)

11. Tube 2 Tube 3 Per-CP FITC PE APC CD45 intracellular kappa light chain
Per-CP
FITC PE
APC
Tube 2
CD45
intracellular kappa light chain
CD38
-----
Tube 3
intracellular lambda light chain

12. Flow cytometry analysis-I Interpretation
The analysis reveals a distinct neoplastic cell population that expresses CD38 (bright), CD56 (bright), and cytoplasmic lambda light chain, and lacks CD45 expression
The neoplastic population is negative for all other tested markers (including T-cell markers such as CD2, CD3, CD4, CD5, and CD8, B-cell markers such as CD19, CD20, CD22, and CD10, immature markers (CD34, CD117), and myeloid markers such as CD13 and CD33, among other tested markers)

13. Flow cytometry analysis-II Interpretation
The neoplastic population has light scatter and immunophenotypic characteristics commonly encountered in plasma cell neoplasm/plasma cell myeloma in the marrow sample
The neoplastic population has higher light scatter characteristics (obvious larger cell size) in the mediastinal mass sample
The mediastinal mass sample has variable blood contamination (as evident by prominent granulocytic population seen)

14. Morphologic assessment
Bone marrow
Morphologic assessment

15. Bone marrow aspirate smear, W-G, x400
Bone marrow biopsy, H-E, x400
Bone marrow biopsy, IHC, CD138, x200
Bone marrow biopsy, IHC, CD56, x200

16. The bone marrow aspirate smears show trilineage hematopoiesis and large neoplastic mononuclear cells with plasmablastic features
The core biopsy sections show 50% cellularity with trilineage hematopoiesis and involvement by neoplastic plasmablastic cells

17. Immunohistochemistry
Immunostains on core biopsy sections
The neoplastic cells are:
Positive for CD138, CD56, MUM1, and lambda
Negative for kappa, CD20, CD3, cyclin-D1, CD30, EMA, ALK1, HHV8, and EBV-LMP

18. Morphologic assessment
Mediastinal mass
Morphologic assessment

19. Mediastinal tissue, H-E, x200
Mediastinal tissue, IHC, CD138, x200
Mediastinal tissue, IHC, CD56, x200

20. Mediastinal tissue, IHC, Ki-67, x200
Mediastinal tissue, IHC, C-MYC, x200

21. The mediastinal biopsy tissue sections show extensive involvement by neoplastic large cells with plasmablastic and plasmacytic features

22. Immunohistochemistry
Immunostains on mediastinal tissue sections
The neoplastic cells are:
Positive for CD138, MUM1, lambda, CD56 (partial), C-MYC (90%), and Ki-67 (~ 100%)
Negative for CD20, PAX5, kappa, CD3, CD30, BCL6 and ALK1
In-situ hybridization for EBER is negative.

23. Cytogenetics Conventional karyotype on bone marrow sample
Abnormal complex karyotype including t(8;22)(q24.1;q11.2), among others
Fluorescence in-situ hybridization on bone marrow sample
Positive for translocation of MYC gene

24. Differential diagnosis
The major differential:
Plasmablastic plasma cell myeloma (PCM)
versus
Plasmablastic lymphoma (PBL)
Primary effusion lymphoma (extracavitary) is less likely based on the immunophenotypic features and the negative HHV8 status

25. The clinical (the presence of massive nodal involvement in an HIV-positive patient), morphologic (prominent plasmablastic morphology), immunophenotypic (mainly excluding B and T-cell neoplasms and acute leukemia by flow cytometry and high proliferation rate and C-MYC expression by immunohistochemistry), and cytogenetic (C-MYC translocation) findings are in support of plasmablastic lymphoma

26. Final diagnosis
Plasmablastic lymphoma involving bone marrow and extramedullary tissue (lymph node), EBV-negative, HHV8-negative, and C-MYC-positive with C-MYC gene rearrangement and t(8;22) [IGL/C-MYC] translocation, in HIV-positive patient

27. Conclusions-I
It is difficult without comprehensive clinicopathologic correlation to differentiate between plasmablastic plasma cell myeloma and plasmablastic lymphoma
The anatomic distribution of the disease is in support of the PBL
Mediastinal mass and generalized lymphadenopathy, bone marrow involvement, and absence of obvious PCM involvement pattern (e.g. lytic bone lesions)

28. Conclusions-II
In the bone marrow sample of this case, the neoplastic cells exhibit immunophenotypic features usually characteristic of plasma cell myeloma (CD38+ bright, CD56+ bright, and CD45-)
CD56 expression is encountered in about 25% of all PBL and in 27% of those occur in HIV+ patients
Blood. 2015; 125(15):

29. Conclusions-III
C-MYC gene rearrangement is seen in PBL and reported in those occuring in HIV+ patients
It could be also seen in aggressive PCM or with PCM with plasmablastic evolution
PBL is more commonly seen in HIV+ patients than PCM
The presence of Epstein-Barr virus (EBV) is in support of PBL over PCM
EBV status is negative in this case study and therefore of non-contributory diagnostic value

30. References
Castillo JJ, et al. The biology and treatment of plasmablastic lymphoma. Blood 2015, 125:
Lorsbach RB, et al. Plasma cell myeloma and related neoplasms. Am J Clin Pathol 2011, 136:
Valera A, et al. IG/MYC rearrangements are the main cytogenetic alteration in plasmablastic lymphomas. Am J Surg Pathol 2010, 34:
Taddesse-Heath L, et al. Plasmablastic lymphoma with MYC translocation: evidence for a common pathway in the generation of plasmablastic features. Mod Pathol 2010, 23:991-9.
Bogusz AM, et al. Plasmablastic lymphomas with MYC/IgH rearrangement: report of three cases and review of the literature. Am J Clin Pathol 2009, 132: