Prashant R. Tembhare, MD Flow Cytometry Unit Laboratory of Pathology NCI/NIH, USA

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

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

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

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

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

T Cell Receptor complex

T cell membrane

Science. 1996. 274: 209-219.

T cell Receptor (TCR) complex

SCIENCE. 272, 21 ;1996: 1755-62.

Thymus

α β polypeptides formation

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

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

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

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

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%

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

Human Immunology. 64, 689–695 (2003).

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

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

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

TCR Vβ Analysis: A case of PTCL

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?

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 ?

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.

Gating System -1

(GS-1)

Gating System -2 (GS-

2)

Gating Strategy

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


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


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

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

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

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

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

Prashant R. Tembhare, MD Flow Cytometry Unit Laboratory of Pathology NCI/NIH, USA

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