prashant r. tembhare, md flow cytometry unit laboratory of pathology nci/nih, usa
DESCRIPTION
Flow Cytometric TCR V Beta Analysis for Determination of T cell Clonality and Its Clinical Application. Prashant R. Tembhare, MD Flow Cytometry Unit Laboratory of Pathology NCI/NIH, USA. Flow cytometric diagnosis of T-CLPD. Altered expression of pan T cell markers like CD7, CD5, CD3, CD2 - PowerPoint PPT PresentationTRANSCRIPT
Flow Cytometric TCR V Beta Analysis for Determination of T cell Clonality and Its Clinical
Application
Prashant R. Tembhare, MDFlow Cytometry Unit Laboratory of PathologyNCI/NIH, USA
Page 2
Altered expression of pan T cell markers like
CD7, CD5, CD3, CD2
Subset restriction CD4+ or CD8+
Increased expression of few markers like CD25,
Aberrant Expression of antigens like CD10, CD30, CD103
Helpful in diseases with high tumor cell count
Flow cytometric diagnosis of T-CLPD
Page 3
Altered expression of pan T-cell markers – Viral infections like IM,
CMV, others
Subset restriction CD4+ or CD8+ may be seen in Viral infections
like IM, HIV, Autoimmune diseases
Increased expression of few markers like CD25
- Increased T regulatory cells
Aberrant Expression of antigens like CD10, CD30 – limited to few
NHL only
- CD30+ positive T cells can be activated T cells
Problems in routine FCM
Page 4
Need of T cell Clonality
T cell clonality usually established by molecular techniques
Confirmation & isolation of clonal cell proliferation
Follow up of treatment – with low cell count / partially treated
Minimal Residual Disease
Low cellularity specimens like CSF, FNA deep LNs/organs
staging
PB - Sezary cells
Morphology and IHC is not typical and helpful
LGLL
Page 5
Mainly qualitative
Time Consuming
Needs Specific set up
Amplification of background T cells may interfere
Clonal peak can not define Cell type and does not help in
classification of CLPD
Highly Sensitive but less specific for malignancy
Limitations of Molecular Techniques
Page 6
T Cell Receptor complex
T cell membrane
Science. 1996. 274: 209-219.
T cell Receptor (TCR) complex
Page 7
SCIENCE. 272, 21 ;1996: 1755-62.
Thymus
α β polypeptides formation
Page 8
The a TCR – chromosome 7.
65 Vβ genes – 46 are functional
Vβ gene segments -26 subfamilies by cDNA
-75% sequence homology at the DNA level.
Palindromic & random additions and deletions
Unique V-D-J = 91 – Vβ segments
24 Vβ segments usage – 70% T cells
Each individual TCR-Vβ segment is expressed in only a small percentage of T cells.
SCIENCE. 272, 21 ;1996: 1755-62. Immunogenetics. 1995;42(6):451-3
TCR Vβ repertoire
Page 9
Clonal T-cell expansions are expected to express a single V domain
FCA demonstration of a V restricted population is evidence of a T cell clone
There are V -specific antibodies now that recognize 70% of all individual V domains –
- TCR V beta Repertoire
V restricted T cell populations can be quantitatively monitored during and after therapy
Expanded T Cell Population - Vβ Analysis
Page 10
SF10 1646 pb_21_V Beta mix A 3 ,2f, 4.fcs gate PerCP APC
V Beta 3
V B
eta
5poi
nt3
102
103
104
105
102
103
104
105 Vb 7.1
Vb 3
Vb 5.3
2.65%
95.45%
0.86%
1.05%
V 5.3b V 7.1b
V 3b
Anti-TCR Vβ Ab-fluorochrome for each TCR-Vβ segment -
identify a Vβ specific T cell subset
FITC
FITC+PE
FITC+PE
FITC
PE
PE
Page 11
The IOTest Beta Mark TCR-Vβ Repertoire kit – Beckman Coulter
24 Vβ-specific antibodies - recognize 70% of all individual Vβ domains
8 tube panel ( 8 X 3 = 24)
– 3 antibodies in
2 colors (FITC, PE and FITC+PE)
Cocktail Mix A Mix
B
Mix
C
Mix
D
Mix
E
Mix
F
Mix
G
Mix
H
FITC Vβ 3 Vβ 16 Vβ 20 Vβ 8 Vβ 12 Vβ 21.3 Vβ 14 Vβ 7.2
PE Vβ 5.3 Vβ 9 Vβ 18 Vβ 13.1 Vβ 5.2 Vβ 23 Vβ 11 Vβ 13.2
FITC+PE Vβ 7.1 Vβ 17 Vβ 5.1 Vβ 13.6 Vβ 2 Vβ 1 Vβ 22 Vβ 4
PerCP CD3 CD3 CD3 CD3 CD3 CD3 CD3 CD3
APC CD4 or
CD8*
CD4 or
CD8*
CD4 or
CD8*
CD4 or
CD8*
CD4 or
CD8*
CD4 or
CD8*
CD4 or
CD8*
CD4 or
CD8*
TCR Vβ Repertoire kit
Page 12
CD3 PerCP
CD
8 A
PC
100
101
102
103
104
100
101
102
103
104
CD3+CD8+
TCR Vβ Analysis – selection of T cells
SF10 1646 pb_21_V Beta mix A 3 ,2f, 4.fcs gate PerCP APC
V Beta 3
V B
eta
5poi
nt3
102
103
104
105
102
103
104
105 Vb 7.1
Vb 3
Vb 5.3
2.65%
95.45%
0.86%
1.05%
SF10 1646 pb_21_V Beta mix A 3 ,2f, 4.fcs Lymphs
CD3 PerCP
CD
4 A
PC
102
103
104
105
102
103
104
105
SF10 1646 pb_26_V Beta mix F 3 ,2f, 4.fcs
V Beta 21point3
V B
eta
23
102
103
104
105
102
103
104
105
Vb 1
Vb 21.3
Vb 23
0.91%95.75%
2.66%
0.67%
Page 13
0
3
6
9
12
15
CD4+ T CELLS
CD8+ T CELLS
TCR Vβ FamilyTCR Vβ Family
Vβ Repertoire usage in T cells from reactive lymphoid tissues%
V
bu
sag
e in
lym
pho
cyte
su
bse
ts M
ea
n +
2 S
D
Page 14 Human Immunology. 64, 689–695 (2003).
Page 15
ALL CD3+ T cells CD3+/CD4+ T cells CD3+/CD8+ T cells Mean Range Mean Range Mean RangeV beta 1 3.53 (Normal 1.89-11.7) 3.32 (Normal 1.62-14.2) 4.24 (Normal 1.4-8.21)V beta 2 8.3 (Normal 4.03-23.48) 9.36 (Normal 5.43-12.84) 5.43 (Normal 1.65-12.42)V beta 3 4.68 (Normal 0.52-15.7) 4.37 (Normal 0.66-10.04) 4.44 (Normal 0.32-13.8)V beta 4 1.91 (Normal 0.79-3.26) 2.03 (Normal 1.20-.2.83) 1.9 (Normal 0.61-4.34)V beta 5.1 5.45 (Normal 3.9-14.93) 6.71 (Normal 4.67-10.94) 3.22 (Normal 1.12-8.92)V beta 5.2 1.33 (Normal 0.49-4.98 ) 1.33 (Normal 0.5-2.87 ) 1.12 (Normal 0.18-3.53 )V beta 5.3 1.08 (Normal 0.37-2.98) 1.09 (Normal 0.36-2.1) 0.92 (Normal 0.32-2.64)V beta 7.1 2.56 (Normal 0.64-20.01) 1.93 (Normal 0.59-3.8) 3.39 (Normal 0.87-7.14)V beta 7.2 1.47 (Normal 0.05-5.45) 1.12 (Normal 0.00-3.10) 2.44 (Normal 0.01-12.10)V beta 8 4.68 (Normal 2.26-29.47) 4.81 (Normal 2.94-6.73) 4.06 (Normal 0.86-11.43 )V beta 9 3.13 (Normal 1.1-9.3) 4.07 (Normal 0.78-8.24) 3.47 (Normal 1.16-7.67)V beta 11 1.04 (Normal 0.25-5.11) 0.87 (Normal 0.3-1.9) 0.92 (Normal 0.14-2.25)V beta 12 1.66 (Normal 1-4.76) 1.82 (Normal 1.08-2.8) 1.29 (Normal 0.33-3.33)V beta 13.1 3.83 (Normal 1.62-8.16) 4.03 (Normal 1.93-7.7) 3.42 (Normal0.41-5.35)V beta 13.2 2.8 (Normal 0.8-5.28) 2.81 (Normal 0.72-7.27) 3.34 (Normal 0.96-9.62)V beta 13.6 1.86 (Normal 0.84-8.8) 1.86 (Normal 0.86-3.4) 1.6 (Normal 0.47-4.56)V beta 14 3.49 (Normal 1.33-8.03) 2.59 (Normal 1.57-4.68) 5.74 (Normal 1.5-14.3)V beta 16 0.92 (Normal 0.42-1.9) 0.95 (Normal 0.34-1.8) 0.8 (Normal 0.02-2.24)V beta 17 5.15 (Normal 2.28-12.61) 5.46 (Normal 3.12-8.32) 5.06 (Normal 1.83-11.18)V beta 18 1.49 (Normal 0.58-5.23) 1.92 (Normal 0.72-3.35) 0.57 (Normal 0.02-2.76)V beta 20 2.52 (Normal 0-9.73) 2.6 (Normal 0.04-5.3) 2.31 (Normal 0.08-5.61)V beta 21.3 2.38 (Normal 1.08-5.97) 2.46 (Normal 1.53-4.7) 2.39 (Normal 0.54-4.93)V beta 22 3.84 (Normal 1.99-9.89) 4.26 (Normal 1.98-8.48) 3.17 (Normal 0.54-6.47)V beta 23 0.85 (Normal 0.28-4.76) 0.48 (Normal 0.13-1.9) 1.34 (Normal 0.04-5.13) Total 69.95 (Normal 69.95) 72.25 (Normal 72.25) 66.58 (Normal 66.58)
TCR Vβ Repertoire usage in T cells : Normal Range
Page 16
SF08 62002.002
V beta 3 FITC
V b
eta
5.3
PE
100
101
102
103
104
100
101
102
103
104
0.18%0.18%
87.97%11.66%
Vb 5.3
Vb 3
Vb 7.1
SF08 62002.002
CD3 PerCP
CD
4 A
PC
100 101 102 103 104100
101
102
103
104
Dim CD3+ CD4+ T cells SF08 62002.003
V beta 16 FITC
V b
eta
9 P
E
100
101
102
103
104
100
101
102
103
104
0.56%26.73%
71.37%1.34%
Vb 9 Vb 17
Vb 16
SF08 62002.006
V beta 12 FITC
V b
eta
5.2
PE
100
101
102
103
104
100
101
102
103
104
3.84%
88.67%
6.33%1.16%Vb 5.2
Vb 12
Vb 2
SF08 62002.007
V beta 21.3 FITC
V b
eta
23 P
E
100
101
102
103
104
100
101
102
103
104
0.27%
2.21%
96.29% 1.24%
Vb 23
Vb 21.3
Vb 1
SF08 62002.004
V beta 20 FITC
V b
eta
18
PE
100
101
102
103
104
100
101
102
103
104
1.49%
94.41%
3.82%0.28%
Vb 18
Vb 20
Vb 5.1
SF08 62002.005
V beta 8 FITCV
bet
a 13
.1 P
E10
010
110
210
310
410
0
101
102
103
104
1.10%0.55%
96.88%
1.47%
Vb 13.1
Vb 8
Vb 13.6
SF08 62002.008
V beta 14 FITCV
bet
a 11
PE
100
101
102
103
104
100
101
102
103
104
0.72%96.56%
2.26%0.45%
Vb 11
Vb 14
Vb 22
SF08 62002.009
V beta 7.2 FITC
V b
eta
13.2
PE
100
101
102
103
104
100
101
102
103
104
0.69%0.49%
97.94% 0.88%
Vb 13.2
Vb 7.2
Vb 4
TCR Vβ Analysis: A case of ATLL
Page 17
Total V beta usage
Vbeta %5.3 1.057.1 0.863 2.659 3.4817 4.1616 0.6118 1.255.1 4.8620 2.48
13.1 5.4713.6 2.10
8 8.175.2 1.342 10.3812 1.3123 0.671 2.66
21.3 0.9111 0.8522 3.9514 1.43
13.2 4.354 2.35
7.2 0.91
TOTAL 68.25
Vbeta %5.3 0.18
7.1 0.18
3 11.66
9 1.34
17 71.37
16 0.56
18 0.28
5.1 3.82
20 1.49
13.1 1.10
13.6 0.55
8 1.47
5.2 1.16
2 6.33
12 3.84
23 0.27
1 2.21
21.3 1.24
11 0.45
22 2.26
14 0.72
13.2 0.69
4 0.49
7.2 0.88
TOTAL 114.54
Normal Direct Clonal Indirect Clonal
Page 18
TCR Vβ Analysis: A case of PTCL
Page 19
Suggestions in literature : Expression of a single TCR-Vβ
Lima M et al. Am J Pathol. 2001;159:1861-1868> 40% sensitivity 93% & specificity 80%> 60% sensitivity 81% & specificity 100%
Beck RC et al. Am J Clin Pathol. 2003;120:785-794.1.6 times of UNL
Morice WG et al. Am J Clin Pathol. 2004;121:373-383. 10-fold or > 50% of the T cells 40% to 49% suggestive > 70% failed to react with panel of Ab
B Feng,et al. J Clin Pathol 2010 63: 141-146.(1) > 50% of a gated T cell subset; or (2) >10 times above its normal ranges T cell subsets; or (3) >70% of gated T cells failed to react to any of the TCR-Vb antibodies
What levels of expanded T-cell subset as a monoclonal T-cell population?
Page 20
Questions for TCR - V beta analysis
1 – At what level or % – clonal ?
2 – Gating strategy ?
3 – Can we use it in samples like BM, CSF, FNA ?
4 – Can we use it for MRD ?
Page 21
Patients & Samples
Total number of patients analyzed for screening
41 Peripheral Blood - 37 +Bone Marrow Asp- 03 +
Total samples - 40
ATLL – 24CTCL – 07LGL - 06PTCL - 03T-PLL - 01
Total number of patients followed up to monitor MRD and response to treatment
14 Peripheral Blood - 48 +Bone Marrow Asp- 02 +Total samples - 50
NCI Study – 2007 to 2010
Tembhare P et al. Am J Clin Pathol 2011;135:890-900.
Concurrent molecular studies for TCRG gene rearrangement by PCR were performed in 36 cases.
Page 22
Gating System -1
(GS-1)
Gating System -2 (GS-
2)
Gating Strategy
Page 23
We found,
Using aberrant phenotype based gating strategy
i.e. GS-2
Demonstration of > 50% usage of single V beta domain
OR
Failure to demonstrate > 70% of immunoreactivity
Cut off levels for T cells Clonality
Tembhare P et al. Am J Clin Pathol 2011;135:890-900.
Page 24
MRD - 61(48 PB, 7 FNA, 4 CSF and 2 BM)
Sequential samples - 14 patients - 3 years
Short immunophenotypic panels and
Single clone specific TCR-Vβ antibody cocktails
Number of tumor cells per micro liter of blood
TCR-Vβ Monitoring of MRD T NHL
Tembhare P et al. Am J Clin Pathol 2011;135:890-900.
Page 25
0 1 2 3 4 5 6 7 80
1 0 0
2 0 0
3 0 0
4 0 0
5 0 0
6 0 0
7 0 0
8 0 0
9 0 0
1 0 0 0Cas e 10
Cas e 11
Cas e 14
Cas e 15
Cas e 16
Cas e 19
Cas e 21
Cas e 24
Cas e 26
Cas e 29
Cas e 30
Cas e 34
Cas e 35
Cas e 36
F O L L O W U P IN T E R V A L S
DA
YS
Follow up
Page 26
0 1 2 3 4 5 6 7 80
5 0 0 0
1 0 0 0 0
1 5 0 0 0
2 0 0 0 0
2 5 0 0 0
Cas e 10
Cas e 14
Cas e 15
Cas e 16
Cas e 19
Cas e 21
Cas e 24
Cas e 26
Cas e 29
Cas e 30
Cas e 34
Cas e 35
Cas e 36
Cas e 11
N u m b er of in terva ls
Tum
or ce
lls/u
L
Number of cells at each interval
Page 27
The minimum level of clonal T cells - 0.8 cells/ul
Ranged from 0.8 cells/ul - 306,603 cells/ul
Median value of 1132 cell/ul
MRD by TCR-Vβ repertoire
Page 28
TCR-Vβ Monitoring in Paucicellular specimens FNAs and CSF:
Case No Specimen
Immunophenotypic profile
TCR V β
Family
% of Aberrant T Cells Vβ
RestrictedCytology ResultsCD2 CD3 CD4 CD5 CD7 CD8 CD25 CD26 CD45 CD57
1 FNA + +/- + + - - ++ + - 1 100 NA
1 FNA +/- + - - +/- + - 1 94.8 Positive
2 FNA + ++ +*++
- 17 76.5 Suspicious
2 FNA + + +*++
- 17 53.4 Atypical cells
2 FNA + + +*-
- 17 76.1 Positive
2 FNA + ++ +* - - + - 17 60.0 Suspicious
2 FNA + + + + +* - - + - 17 60.4 Suspicious
3 FNA + +/- + + - - +/- - + - 13.1 90 Positive
4 FNA + +/- + ++ - - ++ + - 17 84 Positive
5 CSF +/- + - 17 72.4 Negative
7 CSF + + +/- + +* 4 86.5 Atypical cells
7 CSF + + +/- + - 4 93.3 Atypical cells
7 CSF + - +/- + - 4 91.9 Negative
7 CSF + + +/- + - 4 92.6 Negative
7 CSF + + +/- - 4 76.2 Positive
Patients: 6 ATLL & 2 PTCL-NOS
Tembhare P et al. Am J Clin Pathol 2012;137:220-226
Page 29
Summary
Sensitive method for detection of T cell clonality
Gating strategy – aberrant immunophenotypes based – superior than T cell subset based.
Clonality - > 50% TCR-Vβ subset restriction > 70% negative for all Vβ subsets TCR-Vβ Repertoire kit
Useful in low cellularity specimens such as FNA and CSF
Quantitative and highly sensitive MRD evaluation
Highly sensitive - treatment response & early relapse
Page 30