Transcript ICCS e-Newsletter CSI Winter 2014

ICCS e-Newsletter CSI
Fall 2014
UniPath - Denver, CO
Richard Quinones, MLS(ASCP)
Hong Lin, PhD
Cristina McLaughlin, MD
Presentation
Clinical History
• 67-year-old female with history of T-prolymphocytic leukemia (T-PLL).
• Originally presented in 2010 with leukocytosis, anemia, thrombocytopenia,
peripheral lymphadenopathy and massive splenomegaly.
• Currently status post second allogeneic stem cell transplant, after multiple rounds
of treatment with anti-CD52 antibody (Campath; alemtuzumab).
– First transplant with matched related donor in 2013 after first relapse.
– Second transplant with matched unrelated donor in 2014 after 2 extramedullary relapses.
Specimens Submitted for Analysis
• Bone marrow for 60-day evaluation post second transplant.
–
–
–
–
Peripheral blood received in EDTA for morphology.
Bone marrow aspirate received in EDTA for morphological and molecular testing.
Bone marrow aspirate received in NaHep for flow cytometry and cytogenetics.
Two trephine needle core biopsies of bone marrow (0.6cm and 0.8cm in length by 0.4cm each in
diameter) received in B+ for fixation and decalcification.
• Subsequent soft tissue biopsy of a right neck mass for flow cytometry.
• Follow-up peripheral blood for morphology and flow cytometry.
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Flow Cytometric Analysis
Instrumentation
• Acquired on Beckman Coulter Gallios 10 Color Flow Cytometer
• Analyzed using Beckman Coulter Kaluza Software, Version 1.2
Tubes Acquired (on all 3 specimens)
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
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60 days post second transplant
BONE MARROW FINDINGS
Peripheral Blood Count at time of BM
Parameter
Result
Unit
Normal Range
WBC
4.7
103/μL
4.5-11.0
RBC
3.09
106/μL
4.10-6.00
HGB
9.8
g/dL
14.0-18.0
HCT
28.4
%
40.0-54.0
MCV
91.7
fL
79-101
MCH
31.6
pg
28.0-34.0
MCHC
34.5
g/dL
32.0-36.0
RDW
17.5
%
11.5-14.7
PLT
111
103/μL
150-400
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WBC Automated Differential
Parameter
Granulocytes
Lymphocytes
Monocytes
Eosinophils
Basophils
Result
Unit
Normal Range
57.2
%
--
2.68
103/μL
1.50-7.50
23.3
%
--
1.09
103/μL
1.00-4.00
12.0
%
--
0.56
103/μL
0.00-1.00
6.6
%
--
0.31
103/μL
0.00-0.80
1.0
%
--
0.05
103/μL
0.00-0.30
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Bone Marrow Differential
Parameter
Result
Unit
Normal Range
Blast
1.50
%
0.1-1.7
Promyelocyte
1.50
%
1.9-4.7
Maturing Myeloid Cells
52.75
%
28.8-52.7
Lymphocytes
10.50
%
8.6-23.8
Monocytes
3.50
%
0.1-0.6
Eosinophils
7.50
%
0.4-7.4
Basophils
0.25
%
0.0-0.2
Plasma Cells
1.00
%
0.0-3.5
Erythroid Cells
21.50
%
13.9-37.3
M/E Ratio
3.1:1
--
2.1-4.1
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Morphology - Bone Marrow
Aspirate
• There is trilineage hematopoiesis with maturation. Myeloid and
erythroid lineages show no evidence of dysplasia. No increase in
blasts. No lymphoid or plasma cell aggregates.
• Iron staining demonstrated markedly increased storage iron with
decreased iron utilization. No ring sideroblasts were noted.
Particle Prep
• Rare interstitial and perivascular aggregates of small, mature
lymphocytes which show slight/moderate nuclear irregularities,
condensed chromatin, and scant cytoplasm.
Core Biopsy
• Patient exhibits normal cellularity of 30-40%. Bone trabeculae are
appropriate.
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Morphology - Bone Marrow
The particle preparation shows an interstitial aggregate of small, mature lymphocytes
with nuclear irregularities, condensed chromatin, and scant cytoplasm (blue arrows).
Immunostains show the lymphocytes are positive for CD3, CD2, CD4, with rare CD8
positive T cells and rare CD20 positive B cells.
Flow Data - Bone Marrow - Tube 1
CD45 vs Side Scatter plot shows normal distribution of lymphocytes,
monocytes, granulocytes, dim CD45 population and debris. The
Blymphocytes are clearly polyclonal with no expression of CD5.
Polyclonal B-lymphocytes
B-lymphocytes
Non-B-lymphocytes
Non-B-lymphocytes
B-lymphocytes
Bright light blue= B cells
Blue= T cells
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Flow Data - Bone Marrow - Tube 2
The abnormal T lymphocyte population, which represents 0.57% of total
events (shown in maroon), expresses partial CD2, CD5, CD7, CD4, bright
CD45; and is negative for CD3, CD8, CD16 and CD56.
Polytypic T cells
NK cells
Maroon=abnormal T cells
Blue= normal polytypic T cells
Gold= NK cells
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Cytogenetic Studies - Bone Marrow
Chromosome Analysis
• Patient shows a normal karyotype.
FISH
• A dual color assay using a break
apart translocation probe for the
human T-cell receptor alpha/delta
(TCRAD) was performed.
• 3.3% of cells analyzed post
transplant were positive for the
TCRAD rearrangement at the
14q11 locus.
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Patient developed an increasingly uncomfortable and rapidly growing
right neck mass. Needle core biopsy with flow cytometry analysis was
performed one week after the bone marrow biopsy.
RIGHT NECK MASS
Morphology - Right Neck Lymph Node
Needle core biopsy:
Sheets of small to
medium sized
mature
lymphocytes with
irregular nuclear
contours,
condensed
chromatin,
moderate
cytoplasm and
atypical mitoses
(blue arrow) and
apoptotic cells
(white arrow).
Flow Data - Right Neck Mass - Tube 1
The CD45 vs Side Scatter plot shows mostly degenerating cells with dim to
negative CD45, exhibiting the low viability (56%). There is a small
lymphocyte population with bright CD45. Very few B lymphocytes are
detected.
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Flow Data - Right Neck Mass - Tube 2
The lymphocytes show an abnormal population with a nearly identical
immunophenotype to the bone marrow aspirate: positive for CD2, CD4,
CD5, CD7, and bright CD45; negative for CD3,CD8, CD16, and CD56.
Polytypic T cells
cells
NK cells
Abnormal T cells
Maroon=abnormal T cells
Blue= polytypic T cells
Orange= NK cells
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The right neck mass was treated with palliative radiation. Approximately
one month later, the patient presented with a lower extremity deep
venous thrombosis and a rapidly increasing white blood cell count (up to
51,000, with 20% lymphocytes). Therefore, peripheral blood was
submitted for flow cytometry analysis.
PERIPHERAL BLOOD
Morphology- Peripheral Blood Smear
and Cytospin Preparation
Both the cytospin and the
peripheral smear show
numerous medium sized
atypical lymphocytes featuring
mildly irregular nuclear
contours and central,
prominent nucleoli (highlighted
by black arrow).
Flow Data - Peripheral Blood-Tube 1
In tube 1, very few B lymphocytes were detected with no aberrant
immunophenotype.
B lymphocytes
Polyclonal B cells
debris
Bright light blue= B cells
Blue= T cells
Grey=debris
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Flow Data - Peripheral Blood-Tube 2
There is a significantly abnormal population of T lymphocytes (41.9% of
total events) shown in maroon, which express bright CD45, partial CD3,
partial CD2, CD5, CD7, CD4; and are negative for CD8, CD56 and CD16.
Polytypic T cells
debris
Abnormal T cells
NK cells
Maroon=abnormal T cells
Blue= polytypic T cells
Orange= NK cells
Grey=debris
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Final Diagnosis
Recurrent T-Prolymphocytic Leukemia (T-PLL)
• Following a 2nd allogeneic, matched, unrelated bone marrow transplant, a
persistent small abnormal T-cell population was detected in marrow and
tissues (0.57% and 2.11% respectively).
• A peripheral blood drawn 90 days post transplant exhibited full relapse
with the aberrant T-cell population growing to 41.90% of total events.
• Key immunophenotypic findings across all specimens included negative to
partial/dim surface CD3; and CD2, CD4, CD5, CD7, bright CD45 positive;
CD8, CD16, CD56 negative.
• Morphology consistently showed medium-sized atypical lymphocytes with
slightly dispersed chromatin, 1-3 variably prominent nucleoli, irregular
nuclear contours, and moderate lightly basophilic cytoplasm.
• The patient previously showed a complex abnormal karyotype with a
derivative chromosome 14 from a t(14;14)(q11;q32), which is the second
most common cytogenetic abnormality seen in T-PLL (10% of patients).
FISH for TCRAD rearrangement was positive in the relapsed specimens,
c/w persistence of the rearrangement of the TCRAD locus at 14q11.2.
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T-Prolymphoctyic Leukemia (T-PLL)
WHO Classification/Definition
• An aggressive T-cell leukemia characterized by a proliferation of small to
medium-sized prolymphocytes.
Epidemiology
• Represents only 2% of mature lymphocytic leukemias in adults over 30.
• Median age of incidence is 65 years, with a range of 30-94 years.
Sites of Involvement
• Peripheral blood and bone marrow are the major sites of involvement.
• Infiltrates may also be seen in the spleen, liver, and skin.
Clinical Features
• Anemia, thrombocytopenia, and absolute lymphocytosis.
• Hepatosplenomegaly and generalized lymphadenopathy are common.
• Skin infiltration in 20% of cases, with serous effusions appearing in few cases.
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T-Prolymphoctyic Leukemia
Diagnostic Testing
• Peripheral Blood/Bone Marrow Morphology
– Abundant small to medium-sized lymphs.
– Non-granular basophilic cytoplasm.
– Round, oval, or markedly irregular nuclei and visible nucleolus.
• Immunophenotype is consistent with that of peripheral T-cells
– Negative for TdT, CD1a, CD16, CD56; Positive for CD2, CD3 (may be weak to negative
on membrane), CD5, CD7, bright CD45.
– CD4+8- (60% of cases), CD4+8+ (25% of cases), CD4-8+ (15% of cases).
– CD52 testing may be requested to determine treatment.
• Cytogenetic testing
– Rearrangement between T-cell receptor genes beta and gamma.
– Inv (14)(q11q32) in 80% of cases; t(14;14)(q11;q32) in 10% of cases
– Trisomy 8q, idic (8p11), and t(8;8)(p11-12;q12) in 70-80% of cases.
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T-Prolymphoctyic Leukemia
Prognosis
• Aggressive disease course with a median survival of less than 1 year.
• Chronic courses may accelerate after 2 to 3 years.
• Patient declined further therapy and opted for hospice care.
Treatment Options
• Monoclonal antibody therapy with anti-CD52 (alemtuzumab). Has a
median survival of 20 months.
• Autologous or allogeneic stem cell transplant following successful
immunotherapy and remission. Median survival of 48 months.
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References
• Dearden C. B- and T-cell prolymphocytic leukemia: antibody approaches.
Hematology Am Soc Hematol Educ Program. 2012;2012:645-51. doi:
10.1182/asheducation-2012.1.645. Review. PubMed PMID: 23233647.
• Dearden C. How I treat prolymphocytic leukemia. Blood. 2012 Jul
19;120(3):538-51. doi: 10.1182/blood-2012-01-380139. Epub 2012
May 30. PubMed PMID: 22649104.
• Foucar K. Mature T-cell leukemias including T-prolymphocytic leukemia,
adult T-cell leukemia/lymphoma, and Sézary syndrome. Am J Clin
Pathol. 2007 Apr;127(4):496-510. PubMed PMID: 17369126.
• Graham RL, Cooper B, Krause JR. T-cell prolymphocytic leukemia. Proc
(Bayl Univ Med Cent). 2013 Jan;26(1):19-21. PubMed PMID:
23382603; PubMed Central PMCID: PMC3523759
• Swerdlow, Steven H. “T-cell prolymphocytic leukemia." WHO classification
of tumours of haematopoietic and lymphoid tissues. 4th ed. Lyon,
France: International Agency for Research on Cancer, 2008. 270-271.
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