1. Winter 2013 e-CSI Case
Julianne Qualtieri, M.D., Director of Flow Cytometry
Nupam Patel, M.D., PGY-2
Department of Pathology and Laboratory Medicine
University of Cincinnati Medical Center

2. Clinical Presentation
A 67 year old man presents with fatigue and shortness of breath to the VA and is referred to our ER due to concern for acute leukemia.
Physical Examination shows massive splenomegaly
Past Medical History
Chronic lymphocytic leukemia (CLL) diagnosed 3.5 years ago by peripheral smear morphology
Last chemotherapy (no rituximab) four months ago
Reportedly, WBC count last month was 5 K/uL

3. Laboratory Values Parameter Value Units Reference Range WBC 334.3
x109/L
( )
RBC
1.89
x1012/L
( )
HGB
6.1
g/dl
( )
HCT
17.9 %
( )
MCV
94.7 fl
( )
MCH
32.4 pg
( )
MCHC
34.2
gm/dl
( )
RDW
15.4
( )
PLT 43
(140 – 400)

4. Laboratory Values CBC Differential Percentage Reference Range (%)
Neutrophils 1%
( )
Lymphocytes
( )
Monocytes
( )
Others
97%
(0-0)

5. Flow Cytometric Analysis
Tube 1: CD19 FITC / CD5 PE / CD45 ECD / CD20 PC5
Tube 2: CD19 FITC / CD34 PE / CD10 ECD / CD45 PC5
Tube 3: CD23 FITC / CD38 PE / CD19 ECD / CD45 PC5
Tube 4: CD79b FITC / CD43 PE / CD19 ECD / CD45 PC5
Tube 5: kappa FITC / lamba PE / CD19 ECD / CD45 PC5

6. Gating Strategy
The specimen is essentially composed of all lymphocytes.
Within the lymphocyte population, a large spectrum of size is seen (arbitrarily separated into smaller and large cells).

7. Tube 1
Smaller lymphocytes express CD20+ of moderate intensity, and large cells show brighter intensity.
CD5 is essentially negative

8. Tube 2 Lymphocytes are negative for CD10.
CD19 expression is brighter on larger cells.

9. Tube 3 Lymphocytes are negative for CD38.
Lymphocytes show dim partial expression of CD23.

10. Tube 4
Lymphocytes show dim CD79b with larger cells displaying brighter intensity.
Lymphocytes are essentially negative for CD43.

11. Tube 5 Lymphocytes show surface kappa light chain restriction.
Light chain expression is of moderate intensity, with large cells showing brighter intensity.

12. Peripheral Blood Smear

13. Cytogenetics Conventional karyotype (of marrow): complex
45-47,Y,-X[3] ,?add(X)(q22), der(1)del(1)(p36.1) add(1)(q31),-2,-8,-9,add(11)(p11.2),add(12)(p13), der(12)add(12)(p11.2) add(12)(q24.1),add(13)(q22), del(13)(q13q22),-15[4],add(16)(p13.1),add(17)(q25), del(17)(p11.2),+mar1-6[cp20]
CLL FISH Panel
Loss of 13q14.3 in 100 % of nuclei
Loss of p53 in 100 % of nuclei

14. Differential Diagnosis
B cell prolymphocytic leukemia (B-PLL): de novo versus transformation of a small B cell lymphoma
CLL with increased prolymphocytes

15. Prolymphocytic Leukemia
Definition
Neoplastic proliferation of prolymphocytes which comprise greater than 55% of lymphoid cells.
Subdivided into B-PLL and T-PLL, two distinctly different diseases.

16. B-Cell Prolymphocytic Leukemia
Epidemiology
Rare: Comprises approximately 1% of lymphocytic leukemias.
Increasingly rare since diagnosis excludes cases of atypical CLL, CLL with increased prolymphocytes, and lymphoid proliferation with t(11;14)(q13;q32).
Median Age: years old with equal male:female distribution.

17. B-Cell Prolymphocytic Leukemia
Clinical Features
Systemic B symptoms (fevers, night sweats, and weight loss)
Rapidly rising white blood cell count (typically >100,000/µL)
Massive splenomegaly
Anemia and thrombocytopenia may be present
Lymphadenopathy is uncommon

18. Origin of B-cell Prolymphocytic Leukemia
De novo (common)
Prolymphocytic transformation of CLL (rare)
Prolymphocytic transformation of SMZL (rarest)

19. Classic Immunophenotype of B-PLL
Strong CD19+
Strong CD20+
Strong CD79b+
Strong CD22+
Strong Surface Ig
Strong FMC-7+
CD5 - (70-80% of cases)
CD23 - (80-90% of cases)
ZAP-70 +/--
CD38 +/--

20. Prolymphocytic Transformation of CLL
Immunophenotypic changes
Decreased CD5 expression
Increased pan-B cell marker (e.g. CD20) and surface Ig intensity
Acquisition of FMC-7
Some cases may retain same immunophenotype as CLL
Review of previous material, when available, is extremely helpful to verify the CLL component

21. Prolymphocytic Transformation of Splenic B cell Lymphomas
Recently described entity [Hoehn]
Case series describes 3 splenic marginal zone lymphomas (SMZL) and one splenic diffuse red pulp small B cell lymphoma
All showed strong p53 expression by immunohistochemistry
Morphologic clues
Cytoplasmic projections
Bone marrow involvement typical of SMZL
Del(7q) in SMZL cases

22. CLL with Increased Prolymphocytes
Prolymphocytes range from 10-54% in peripheral blood
Both p53 deletion/mutations and C-MYC translocations have been correlated with increased prolymphocytes in CLL [Bacher, Huh].
Associated with more aggressive disease
Distinction between B-PLL
Currently morphology-driven
Gene expression profiling works but is not routinely available [Del Giudice]

23. Our Final Diagnosis
“Atypical B cell lymphoma with increased prolymphocytes”
Favor an atypical CLL with increased prolymphocytes
By morphology, prolymphocytes ~25%
Case discussed at interdepartmental heme-onc conference
Marked change in tempo of disease requires aggressive treatment, as if a true prolymphocytic transformation
p53 deletion also necessitates aggressive treatment

24. Treatment Strategy for Lymphoid Malignancies with p53 Abnormalities
Resistant to conventional chemotherapy (purine analogs).
Use of anti-CD20 monoclonal antibodies have shown some improvement.
Deletions or mutations of p53 have shown good response to treatment with alemtuzumab [Deardon].
Hematopoietic stem cell transplant should be considered for suitable candidates.

25. References
Bacher, U., W. Kern, C. Schoch, W. Hiddemann, and T. Haferlach. "Discrimination of Chronic Lymphocytic Leukemia (CLL) and CLL/PL by Cytomorphology Can Clearly Be Correlated to Specific Genetic Markers as Investigated by Interphase Fluorescence in Situ Hybridization (FISH)." Annals of Hematology  (2004):
Dearden, Claire. "B- and T-cell Prolymphocytic Leukemia: Antibody Approaches." American Society of Hemotology Education Book .1 (2012):
Dearden, Claire. "How I Treat Prolymphocytic Leukemia." Blood .120 (2012):
Del Giudice, I., N. Osuji, T. Dexter, V. Brito-Babapulle, N. Parry-Jones, S. Chiaretti, M. Messina, G. Morgan, D. Catovsky, and E. Matutes. "B-cell Prolymphocytic Leukemia and Chronic Lymphocytic Leukemia Have Distinctive Gene Expression Signatures." Leukemia . (2009):
Hoehn D, Miranda RN, Kanagal-Shamanna R, Lin P, Medeiros LJ. Splenic B-cell lymphomas with more than 55% prolymphocytes in blood: evidence for prolymphocytoid transformation. Hum Pathol Nov;43(11):
Huh, Yang O., Katherine I-Chun Lin, Francisco Vega, Ellen Schlette, C. Cameron Yin, Michael J. Keating, R. Luthra, L. Jeffrey Medeiros, and Lynne V. Abruzzo. "MYC Translocation in Chronic Lymphocytic Leukaemia Is Associated with Increased Prolymphocytes and a Poor Prognosis." British Journal of Haematology  (2008):
Lens, Daniela, J.J.C. De Schouwer, Rifat A. Hamoudi, and et al. "P53 Abnormalities in B-Cell Prolymphocytic Leukemia." Blood . 89 (1997):