Transcript ICCS e-Newsletter CSI Spring 2014

ICCS e-Newsletter CSI
Spring 2014
Hong Lin, PhD and Cristina McLaughlin, MD
APP-Unipath
Denver, Colorado
History and Physical Examination
1. 28-year old African American female with painful
lymphadenopathy
2. Palpable lymph nodes in the right groin
Specimens Submitted for Analysis
• Peripheral blood received in EDTA.
• One tube of bone marrow aspirate received in NaHep for Flow
Cytometry testing
• One tube of bone marrow aspirate received in EDTA for morphologic
examination.
• Trephine needle core biopsies of bone marrow (2.4 cm in length by 0.2
cm in diameter) received in B+ for fixation and decalcification.
• Right groin lymph node received in RPMI without fixative for flow
cytometry testing, as well as portions in formalin for morphologic
examination.
Peripheral Blood Cell Counts (CBC)
Parameter
Result
Unit
Normal Range
WBC
4.4
103/μL
4.5-11.0
RBC
3.65
106/μL
4.10-6.00
HGB
11.5
g/dL
14.0-18.0
HCT
34.5
%
40.0-54.0
MCV
94.7
fL
79-101
MCH
36.7
pg
28.0-34.0
MCHC
33.4
g/dL
32.0-36.0
RDW
13.6
%
11.5-14.7
PLT
32
103/μL
150-400
MPV
9.9
fL
7.2-11.1
WBC Automatic Differential
Parameter
% of WBC
Absolute Count
(109/L)
Neutrophils
74.0
3.26
1.5-7.0
Lymphocytes
15.0
0.66
1.0-4.0
Monocytes
11.0
0.48
0.3-1.0
Eosinophils
0.0
0.0-0.5
Basophils
0.0
0.0-0.3
Normal Range
(absolute count 109/L)
No atypical cells observed on smear evaluation by hematopathologist.
Bone Marrow Differential
Parameter
Result
Unit
Normal Range
Blast
1.25
%
0.1-1.7
Promyelocyte
2.00
%
1.9-4.7
Maturing Myeloid Cells
53.25
%
28.8-52.7
Lymphocytes
13.50
%
8.6-23.8
Monocytes
3.00
%
0.1-0.6
Eosinophils
4.00
%
0.4-7.4
Basophils
1.00
%
0.0-0.2
Plasma Cells
3.75
%
0.0-3.5
Erythroid Cells
18.25
%
13.9-37.3
M/E Ratio
3.5:1
--
2.1-4.1
Flow Cytometric Studies
Flow cytometric immunophenotyping was performed on right groin lymph
node sample and bone marrow aspirate received in NaHep, to “rule out
lymphoma”. Data acquisition was performed on a 10-Color Gallios Flow
cytometer from Beckman Coulter.
Flow cytometric data was analyzed with Kaluza software from Beckman
Coulter, using two standard screening panels (Tube 1 and 2), with two addon custom tubes.
Panel
Markers (FITC/PE/ECD/PC5.5/PC7/APC/AF700/AF750/PB/KrO)
Tube 1
Kappa
Lambda
CD23
CD38
CD34
CD20
CD10
CD19
CD5
CD45
Tube 2
CD8
CD2
CD7
CD3
CD34
CD56
CD16
CD4
CD5
CD45
CD7
CD3
CD2
CD8
CD4
CD45
CD25
CD19
CD45
Tube 3
Tube 4
TCR γ/δ TCR α/β
CD52
CD30
CD3
CD20
Flow Cytometric Data Analysis – Tube 1
Specimen: right groin lymph node
Non-B lymphocytes
bleed into the monocytic
area, with high side
scatter and bright CD45
B lymphocytes
Non-B
lymphocytes
Maroon = abnormal lymphocytes
Orange = polyclonal B lymphocytes
Blue = polytypic T lymphocytes
Polyclonal B lymphocytes
B lymphocytes
Non-B
lymphocytes
The CD45/side scatter plot shows mostly lymphocytes, many which
show high side-scatter, causing bleed into the monocytic gate. The B
lymphocytes (orange) are clearly polyclonal, with a kappa:lambda
ratio = 1.8. The non-B lymphocytes do not exhibit CD10 expression.
Flow Cytometric Data Analysis – Tube 2
Specimen: right groin lymph node
Mixture of polytypic T
lymphocytes and
atypical lymphocytes
Maroon = abnormal lymphocytes
Orange = polyclonal B lymphocytes
Blue = polytypic T lymphocytes
Unremarkable T lymphocytes
Atypical T
lymphocytes
The remainder of lymphocytes consist of two populations of non-B
lymphocytes. There is an unremarkable mixture of polytypic T lymphocytes
(blue population), with a mixture of CD4 and CD8 positivity. There is also
an atypical cell lymphocytic population (maroon) that expresses higher side
scatter and CD4, but is negative for surface CD3 and CD8.
Flow Cytometric Data Analysis – Tube 2
Specimen: right groin lymph node
Maroon = abnormal lymphocytes
Orange = polyclonal B lymphocytes
Blue = polytypic T lymphocytes
The atypical lymphocytes (maroon) are negative for CD5, CD7, CD34, and
CD56, but brightly positive (as compared to normal T lymphocytes) for CD2.
Because of the atypical lymphocytic population, two additional add-on
tubes to analyze for T cell markers and for CD30 were added.
Flow Cytometric Data Analysis – Tube 3
Specimen: right groin lymph node
Maroon = abnormal lymphocytes
Orange = polyclonal B lymphocytes
Blue = polytypic T lymphocytes
The atypical lymphocytes (maroon) are negative for TCRαβ and TCRγδ, as
well as surface CD3, while showing bright CD2 as compared to the normal T
lymphocytes, which are predominantly TCRαβ positive.
Flow Cytometric Data Analysis – Tube 4
Specimen: right groin lymph node
Maroon = abnormal lymphocytes
Orange = polyclonal B lymphocytes
Blue = polytypic T lymphocytes
The atypical lymphocytes (maroon) show variable positivity for CD30, CD25
and CD52.
Key Immunophenotypic Findings
The atypical cells show the following findings:
•
Cells are negative for pan B cell markers.
•
Cells express CD4, CD2.
•
Cells are negative for all other T cell markers, such as
CD5, CD7 and surface CD3.
•
There is no expression of TCRα/β and TCRγ/δ
•
CD30, CD25, and CD52 expression are heterogeneous.
Lymph Node Morphology Findings
Microscopic Description:
•
•
•
•
•
Large, anaplastic lymphoid cells
Eosinophilic cytoplasm
Large irregular nuclei with single or multiple macronucleoli
Rare Hallmark cells
Multiple foci of necrosis
Immunohistochemical Stains:
• CD30 is diffusely positive in the large neoplastic lymphocytes
• ALK1 is diffusely positive in the large neoplastic lymphocytes
Right groin lymph node, H&E, 40X
ALK-1 immunostain
CD30 immunostain
Bone Marrow Findings
Bone Marrow Morphology:
•
•
•
•
•
Trilineage hematopoiesis with appropriate maturation.
Core biopsy with 50% cellularity; mildly hypercellular for age.
Myeloid to erythroid ratio is normal at 3.5:1.
Blasts are not increased; no lymphoid or plasma cell aggregates.
Megakaryocytes are normal in number and morphology.
Immunohistochemistry analysis:
• No significant CD30 or ALK-1 positivity observed.
Flow Cytometry studies on bone marrow aspirate:
•
No Flow Cytometric evidence of hematolymphoid neoplasm.
Green = granulocytes
Purple = monocytes
Blue = lymphocytes
Orange = hematogones
Red = blasts
Gray = debris
Diagnosis:
Anaplastic Large Cell Lymphoma
(ALCL), ALK positive
• In this particular case, though absolute final classification could not
be made on flow analysis, the identification of an abnormal T cell
population with CD30 expression helped narrow the immunostain
workup and accelerated diagnosis by greater than 24 hours.
Identification of circulating ALCL cells by flow analysis can also help in
identifying patients at high risk of relapse, similar to minimal residual
disease studies in leukemia patients, and can help triage patients
biopsied by fine needle aspiration.
• By flow analysis, if the cells aren’t too large to survive processing,
ALCL tumor cells will be positive for CD30 and bright CD45, with many
cells bleeding into the monocyte gate on the CD45/SS plot, as we see
in our analysis. Given this tendency, an analysis strategy that
examines ungated populations as well as gated may yield the greatest
sensitivity.
Anaplastic Large Cell Lymphoma
(ALCL), ALK positive
• WHO 2008 Classification Definition:
• T cell lymphoma
• Exhibits a translocation involving the ALK gene, with
expression of ALK protein and CD30.
• Epidemiology:
• 3% of adult non-Hodgkin lymphomas
• 10-20% of childhood lymphomas
• Most frequent in first three decades of life, with a
male predominance (M:F ratio 1.5:1)
Anaplastic Large Cell Lymphoma
(ALCL), ALK positive
• Clinical Features
• Patients typically present with advanced stage disease,
with peripheral and/or abdominal lymphadenopathy
• Often extranodal infiltrates, including skin, bone, soft
tissues, lung and liver
• High fever
• Bone marrow involvement ranges from 10-30%
• Prognosis:
• The specific translocation does not appear to yield
prognostic significance.
• ALCL, ALK+ has a favorable prognosis compared with
ALCL, ALK negative, with a 5 year survival approaching
80%.
Anaplastic Large Cell Lymphoma
(ALCL), ALK positive
• Morphology
•
•
•
•
•
•
Cell size varies from small to very large
Eccentric, horseshoe- or kidney-shaped nuclei
Eosinophilic region near the nucleus
Nuclear chromatin is clumped or dispersed with basophilic nucleoli.
Basophilic or eosinophilic cytoplasm
Tumor cells often grow within sinuses and can resemble metastatic
tumor.
• Small cell pattern also recognized (represents 5-10% of ALCL, ALK+)
• Molecular /Cytogenetic
• TCR gene rearrangement
• Chromosomal abnormalities:
• t(2;5)(p23;q35)—NPM-ALK (84%)(IHC shows nuclear and
cytoplasmic positivity)
• t(1;2)(q25;p23)---TPM3-ALK (13%)(IHC shows diffuse cytoplasmic
positivity with peripheral intensification)
• Other variant translocations
Anaplastic Large Cell Lymphoma
(ALCL), ALK positive
• Immunophenotype
•
•
•
•
•
•
•
•
CD3 negative in more than 75% of cases
CD2, CD5 and CD4 positive in 70% of cases; can have null cell
CD8 is usually negative
CD30 expression can be detected both by
immunohistochemistry (most positive in the larger cells) and
flow cytometry
CD25 variably positive
CD13/CD33 expression is a sensitive marker, but not specific
EBV negative
ALK is absent in all postnatal normal human tissues except
rare cells in the brain; therefore a positive result by IHC
supplants molecular testing and confirms diagnosis
References
• Damm-Welk, C et al. “Flow cytometric detection of circulating tumour
cells in nucleophosmin/anaplastic lymphoma kinase-positive anaplastic
large cell lymphoma: comparison with quantitative polymerase chain
reaction”, British Journal of Haematology, 138: 459-466; 2007.
• Kesler, M et al. “Anaplastic Large Cell Lymphoma: A Flow Cytometric
Analysis of 29 Cases”, Am J Clin Pathol, 128: 314-322; 2007.
• Muzzafar, T et al. “Flow Cytometric Immunophenotyping of Anaplastic
Large Cell Lymphoma”, Arch Pathol Lab Med, 133: 49-56; 2009.
• Popnikolov, N. et al. “CD13-Positive Anaplastic Large Cell Lymphoma of TCell Origin – A Diagnostic and Histiogenetic Problem: A Case Report and
Review of the Literature)”, Arch Pathol Lab Med, 124: 1804-1808; 2000.
• Swerdlow SH, Campo E, Harris NL, et al., editors. WHO Classification of
Tumours of Haematopoietic and Lymphoid Tissues. Lyon, France: IARC;
2008.