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
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
Laboratory Values
Parameter
Value
Units
Reference Range
WBC
334.3
x109/L
(3.8 - 10.8)
RBC
1.89
x1012/L
(4.20 - 5.80)
HGB
6.1
g/dl
(13.2 - 17.1)
HCT
17.9
%
(38.5 - 50.0)
MCV
94.7
fl
(80.0 - 100.0)
MCH
32.4
pg
(27.0 - 33.0)
MCHC
34.2
gm/dl
(32.0 - 36.0)
RDW
15.4
%
(11.0 - 15.0)
PLT
43
x109/L
(140 – 400)
Laboratory Values
CBC Differential
Percentage
Reference Range (%)
Neutrophils
1%
(40.0 - 80.0)
Lymphocytes
1%
(15.0 - 45.0)
Monocytes
1%
(0.0 - 12.0)
Others
97%
(0-0)
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
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).
Tube 1
• Smaller lymphocytes express
CD20+ of moderate intensity,
and large cells show brighter
intensity.
• CD5 is essentially negative
Tube 2
• Lymphocytes are negative
for CD10.
• CD19 expression is brighter
on larger cells.
Tube 3
• Lymphocytes are negative for CD38.
• Lymphocytes show dim partial expression
of CD23.
Tube 4
• Lymphocytes show dim
CD79b with larger cells
displaying brighter
intensity.
• Lymphocytes are
essentially negative for
CD43.
Tube 5
• Lymphocytes show surface
kappa light chain restriction.
• Light chain expression is of
moderate intensity, with
large cells showing brighter
intensity.
Peripheral Blood Smear
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
Differential Diagnosis
• B cell prolymphocytic leukemia (B-PLL): de novo versus
transformation of a small B cell lymphoma
• CLL with increased prolymphocytes
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.
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: 65-69 years old with equal male:female distribution.
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
Origin of B-cell Prolymphocytic Leukemia
• De novo (common)
• Prolymphocytic transformation of CLL (rare)
• Prolymphocytic transformation of SMZL (rarest)
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 +/--
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
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
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]
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
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.
References
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•
•
•
•
•
•
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 . 83.6 (2004):
349-55.
Dearden, Claire. "B- and T-cell Prolymphocytic Leukemia: Antibody Approaches." American Society of
Hemotology Education Book .1 (2012): 645-51.
Dearden, Claire. "How I Treat Prolymphocytic Leukemia." Blood .120 (2012): 538-51.
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): 2160-2167.
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. 2012 Nov;43(11):182838.
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 . 142.1
(2008): 36-44.
Lens, Daniela, J.J.C. De Schouwer, Rifat A. Hamoudi, and et al. "P53 Abnormalities in B-Cell Prolymphocytic
Leukemia." Blood . 89 (1997): 2015-023.