T-cell receptor gene in Sezary syndrome 
Clinical and laboratory study 
T-CELL RECEPTOR GENE 
REARRANGEMENT STUDIES 
IN SEZARY SYNDROME. 
C. Santangelo, R. Benucci, M. Ruffelli, M. Ribuffo, 
R. Simoni and O. De Pita. 
ABSTRACT 
Sezary syndrome (SS) is a cutaneous T-cell lymphoma characterized by pruritic erythroderma, generalized 
adenopathy and circulating Sezary cells in peripheral blood. The Sezary cells' percentage and morphology 
necessary for the diagnosis are still unknown; therefore, recently T-lymphocyte receptors gene rearrangement 
has been employed thus allowing to study the monoclonality of lymphoproliferative disorders. In order to 
verify the specificity and the diagnostic relevance of the above mentioned method, the DNA obtained from 
the peripheral blood of 17 subjects (10 affected by SS and 7 by inflammatory erythroderma) was analyzed 
by the Southern Blot Analysis (SBA). A monoclonal gene rearrangement of the TcR 13-chain was shown in 
6 subjects affected by SS. In 2 patients the analysis was repeated during the therapy with recombinant 
interferon u-2a (rIFNu-2a), in one patient the clonal population had disappeared. In 4 patients with CSCs 
and germline pattern in the peripheral blood ("false negatives") gene rearrangement bands were obtained in 
skin and lymphnodes except for in one patient. Seven controls with inflammatory erythroderma presented a 
germline pattern. 
These results suggest that the TcR gene rearrangement, although it is a useful diagnostic tool, must always 
be associated with clinical, histopathological and immunophenotypical data; Furthermore, this study highlights 
the need to make consecutive sampling, possibly with material from various organs (peripheral blood, 
lymphnodes, skin). 
KEY WORDS 
T-cell receptor, gene rea,rangement, Southem blat analysis, cutaneous T-cell lymphoma 
INTRODUCTION 
Sezary syndrome (SS) is a cutaneous T celi 
lymphoma (CTCL) characterized by pruritic erythro-
derma, generalized adenopathy and circulating atypical 
lymphoid cells in peripheral blood ( circulating Sezary 
cells, CSC) (1). There are different opinions regarding 
acta demiatovenerologica A.P.A. Vol 5, 96, No 2 
the percentage of CSC (2,3,4,5) and the morphological 
criteria (5,6) necessary to confirm the diagnosis: 
Cases of SS with moderately atypical nuclei of the 
circulating lymphocytes (7) and, on the contrary, 
reactive dermatitis or benign lymphoproliferative 
43 
T-cell receptor gene in Sezary syndrome 
Table I. Patients with Seza,y syndrome (SS) and the stage of their disease at the time of investigation. 
PATIENTS AGE / SEX CLINICAL FEATURE STAGE 
1 41/F 
2 62/F 
3 65/F 
4 /F 
5 72/M 
6 45/F 
7 61/M 
8 57/M 
9 63/M 
10 43/M 
disorders showing lymphoid Sezary-like cells in 
peripheral blood are known (2,5,8). 
The evaluation of the peripheral T-lymphocyte 
-J3-chain receptor (TcR J3) gene rearrangement 
allows to evaluate the monoclonality of a lympho-
proliferative infiltrate in skin (9,10,11), lymphnodes 
(12) and peripheral blood (6,9,13) . In the majority 
of peripheral T-lymphocytes, the TcR is made of 
two proteic a/J3 chains; only a small percentage 
expresses an alternative heterodimeric y/8 chain 
(13,14,15). All the four chains are coded by 
genes with a configuration similar to immu-
noglobulins (Ig); they are formed by the assembling 
of numerous VJC ( a and y) and VDJC (13 and 8) 
segments (Fig. 1). Such genic order defines the 
"germline" configuration. During the thymocyte 
differentiating process the VDJC segments are 
juxta-posed thus originating a "somatic-physiological 
rearrangement" specific for each T-cell (14). The 
clonal cells which originate from the T-lymphocyte 
proliferation have the same type of gene receptor 
rearrangement which is detected analyzing the 
DNA by Southern blot analysis (SBA). 
In order to verify the SBA sensibility and the 
specificity as well as the diagnostic and prognostic 
importance of this method, we analyzed the DNA 
obtained from peripheral blood of 17 subjects, 10 of 
whom were affected by SS and 7 by inflammatory 
erythroderma. 
In subjects affected by SS showing "false negative" 
results in molecular analysis of peripheral blood, in 
spite of the presence of a various percentage of 
CSC, the gene rearrangement was analyzed also in 
skin and lymphnodes. In two patients the band 
alterations of the TcR J3 gene rearrangement in 
peripheral blood were analyzed during the maintenance 
phase of the parenteral therapy with rIFNa-2a. 
44 
erythroderma T4NOMO 
erythroderma T4NOMI 
erythroderma T4NOMI 
plaques> 10% TZNOMO 
erythroderma T4NOM! 
erythroderma T4NOMI 
plaques> 10% TZNOMI 
plaques> 10% TZN0MO 
erythroderma TZNOMO 
erythroderma TZNIMO 
MATERIALS AND METHODS 
Subjects: We selected 10 subjects, 6 women and 4 
men 41 to 72 years old, affected by SS who showed 
typical clinical manifestations (7 patients with 
erythroderma, 3 with widespread patches > 10% ), 
with the Karnofsky index > 60% and life expectation 
> 12 months. The clinical diagnosis was confirmed 
by histopathological and immunophenotypical 
examination (Table II). The staging of the disease 
was defined according to the TNM classification of 
the T-cell lymphoma (Bunn et al, 1979) (Table I) 
(16). Two patients (case 1 and 2) had long-lasting 
inveterate mycosis fungoides (MF). The molecular 
investigations were made on all the subjects prior to 
the beginning of the therapy and were repeated on 
two patients ( case 3 and 10) during the maintenance 
phase of the parenteral therapy with an average 
dose of 9xl06 IU/day rIFNa--2a. Furthermore, in 4 
patients with SS the rnolecular investigation was 
made in the skin (case 5-7-9) and in the lymphnode 
( case 6). The molecular study on peripheral blood 
of 7 patients affected by inflammatory erythroderma 
was also made. 
Samples: Following centrifugation on Lymphoprep, 
the fraction of mononuclear cells of peripheral 
blood (in EDTA) was obtained. 
DNA extraction: The sarnples were DNA extracted 
and purified utilizing the ONCOR DNA Extraction 
Kit. 
The lymphnodes were incubated overnight at 60°C 
with the lysis buffer containing proteinase. Following 
incubation, the digested proteins were removed through 
double treatment with phenol and then with 
chloroform. The DNA precipitated, with cold ethanol 
and re-suspended in TRIS-EDTA (TE). 10 µg DNA 
of all the samples were digested with the restriction 
acta dermatovenerologica A.P.A. Vol 5, 96, No 2 
T-cell receptor gene in Sezary syndrome 
Table II. T/z e results of t/ze immunological and molecular investigations. 
PATIENTS LEUKOCYTES 
c/µl 
1 3840 
2 19100 
3 6430 
4 9040 
5 9000 
6 16400 
7 5580 
8 8430 
9 7540 
10 5700 
Legends: 
G.L. = germline pattern 
R= rearrangement 
LYMPHOCYTES SEZARY 
c/µl CELLS(%) / 
1100 10 
8500 15 
1800 10 
2010 6 
3100 10 
6230 5 
2050 5 
2400 6 
2140 o 
1800 5 
CD4/CDS 
RA.TIO 
1,4 G.L. 
14,6 G.L. 
5,2 R. 
5,1 R. 
9,9 G.L 
30,3 R. 
1,2 G.L. 
3 R. 
2,5 R. 
1,5 R. 
enzymes EcoRI, BamHI and Hind III. The DNA 
was submitted to electrophoresis on 0.7% agarose 
gel on Probe Tech 2 (ONCOR). At the same tirne, 
a sensitivity control (ONCOR), containing a mixture 
of 0.25 µg of EcoRI cut placenta DNA, 0.25 g of 
BamHI cut placenta DNA, and 0.25 µg of Hind III 
cut placenta DNA, was also submitted to electro-
phoresis. This DNA is mixed so that germline 
fragment bands from each enzyme are included. If 
these bands are visible, the test detects the equivalent 
of either: 1) a elana! population that contains one 
rearranged allele of - gene which is as small as 5% 
of the total sample population, or 2) a clonal 
population containing two rearranged alleles of the 
same gene as small as 2.5% of the total sample 
population. The darker the bands of the sensitivity 
control, the more sensitive the test. The lowest limit 
of detection represents a clonal population of 1 % 
when both alleles of 13-gene are rearranged or of 
2% when only one allele is rearranged. 
Following depurination and · denaturation, the 
separated fragments were vacuum transferred on 
nylon filters. After drying at 80°C in the oven, the 
filters were pre-hybridized. 
fcoR 1 · EcoR I fcr,R 1 fcoR 1 fcoR 1 
1 1 1 1 I 
i.---------11 Kb--------~1.8 Kb->l--4 .2 Kb-->1+---4.2 Kb~ro-&t 
1 ,' 1 1 1 
1 1 1 1 
1 D,,ll 1 J,,ll 1 C,,ll 1 1 Dr!l2 I J,,82 Cr,82 1 1 -,-----ili~~1,_1 .-1 t-+-ll .-1 -~[H{]~l - 1-oo 1111111 ~~---1 -
1 ( ::.· .. Kc .<•' ·> j D I h · : ., .c·n,·rx .... •;.c,: .. : 1 1 
1 i< •I ~ 1 1 1 . 1 Oncrll~robe 1 
1 
Onc~~,ll~obe 1 
I 1 1 l 1 1 
1 r--4,l Kb--,-wl<--3.7 Kb------7.2 Kb----~ 1 
/ Hind 111 Hind III Hind III Hind 111 --+j r- 1 Kb j 
k------------------24 Kb--------------------->l 
8omH 1 8omH 1 
Fig. l . Map of the gene codifying of the TcR -chain. 
acta dermatovenerologica A.P.A. Vol 5, 96, No 2 45 
T-cell receptor gene in Sezary syndrome 
HYBRIDIZATION 
The filters were hybridized with the biotynilated 
genomic probe (ONCOR JB/J!\), complementary to 
the J region of the TcR a/13 chain. Following 
hybridization (18 hours at 50°C), the filters were 
washed with SSC 0.1/SDS 0,1 %; then they were 
incubated with streptavidin and alkaline phosphatase, 
washed with SSC 1 and stained with a NBT 
chromogen and BCIP (Blue tetrazolium and 5-
bromo-4-chloro-3-indolphosphate) at 37°C far 4 to 
16 hrs. Following visualization of the bands, the 
filters were washed in distilled water, dried and 
kept in a dark place. 
RESULTS 
The results are summarized in Table I. The 
clonal rearrangement bands were observed in 
peripheral blood in 6 patients affected by SS ( cases 
3-4-6-8-9-10); in the other 4 patients ( cases 1-2-5-7) 
the pattern resulted to be germline; after further 
investigations ( cases 5-6-7-9), the TcR J3 gene re-
arrangement bands were observed in skin (cases 7-
9) and one lymphnode after histopathological 
examination revealed a pathologic result ( case 6). In 
two patients ( cases 3-7) the molecular investigation 
was repeated during the therapy with rIFNcx-2a: in 
one patient ( case 3) the CSC and the elana! 
rearrangement band of the TcR J3 in peripheral 
blood disappeared. Seven patients with inflammatory 
erythroderma (one showed 15% of Sezary-like lym-
phoid cells in circulation) showed a DNA germline 
pattern in peripheral blood. Sensitivity control was 
visible in all analyses of rearrangements performed, 
indicating that the tests were able to detect a clonal 
population containing one rearranged allele as small 
as at least 5% of the total sample population ar 
two rearranged alleles as small as 2.5% of the total 
sample population. 
In Fig. 2 the DNA rearrangement bands obtained 
by SBA of patients 3-4-10 are shown. The DNA, 
digested by the enzymes EcoRI, BamHI and Hind 
III, was hybridized with the JB/JJ3
2 
genomic probe. 
The rearrangement bands are marked with arrows. 
DISCUSSION 
The number and the morphology of CSC necessary 
far the diagnosis of SS are still unknown. 
Recent investigations of peripheral T-cell 13-chain 
46 
~ 
~ ~ ~ • 
Fig. 2. DNA Southem Blot Analysis of patients 3-4-10 
in peripheral blood. The digested DNA with the enzymes 
EcoRI, BamHI and Hind III was hybiidized with the 
genomic lf3/Jf32 probe. The bars indicate the germline 
bands; the arrows indicate the rearrangement bands. 
S. C. indicates the sensitivity control. 
receptor (TcRB) gene rearrangement, analyzed by 
the SBA, to evaluate the clonality of a lympho-
proliferative infiltrate in skin (9,10,11), lymphnodes 
(12) and peripheral blood (6,9,13) resulted in a 
high sensitivity (80-100%) and specificity (>90%) 
(6,15,17,18,19). 
The DNA obtained from peripheral blood of 17 
patients, 10 affected by SS and 7 by inflammatory 
erythroderma, was analyzed; the results of the 
molecular investigations were compared with the 
morphological and immunological data (Table II): 
the patients ( case 3-4-6-8-9-10) showed a TcR J3 
clonal rearrangement in peripheral blood samples, 
in absence of direct correlation with the number of 
CSC and the extension of the skin lesions. Weiss 
(6) studied the TcR clonal rearrangement in peripheral 
blood of 26 patients affected by MF/SS: 3 patients 
with SS presented TcR J3 gene rearrangement in 
correlation with the CSC presence; 2 patients showed 
a germline pattern with 3%-4% CSC. Witthaker et 
al. (11) showed in 8 patients out of 14 a clonal 
TcR rearrangement in peripheral blood samples, 
while the remaining 6 patients formed a germline 
pattern with a variable CSC percentage (0-5% ). 
According to these results, it is possible to find a 
TcRB germline pattern in peripheral blood in the 
presence of a small number of CSC. On the contrary, 
in one patient ( case 9) TcR J3 rearrangement bands 
without morphological CSC occurred. SS variants 
with a modest atypia of CSC nuclei (7) have also 
been published. 
Four patients ( case 1-2-5-7) presented a germline 
acta demwtovenerologica A.P.A. Vol 5, 96, No 2 
T-cell receptor gene in Sezary syndrome 
pattern, in peripheral blood ( case 1-2) and in the 
skin ( case 5), in spite of the high percentage of 
CSC. Bendelac (20) showed a case of acute non 
epidermotropic T-cell lymphoma with 67% CSC in 
the absence of the TcR J3 gene rearrangement but 
with rearrangement bands in the affected skin. The 
observation may be explained in different ways: the 
rearrangement band might have co-migrated with 
the germline band ar a deletion of chromosomic 
segments containing TcR genes occurred ar those 
atypical cells were activated polyelonal T-lymphocytes 
with a reactive value; furthermore, the use of the 
biotynilated genomic probe, complementary at the J 
region of the cx/13 TcR J3 chain, might not recognize 
the rearrangement of the y/8 chain (2,5,6,8,12,13). 
Case 6 shows similar monoclonal rearrangement 
bands in peripheral blood and in an affected 
lymphnode thus showing the monoelonality of the 
neoplastic infiltrate. In fact, the monoelonality of an 
infiltrate doesn't necessarily prave its malignancy 
but may indicate an abnormal cellular proliferation 
with possible progression to malignancy (9). The 
diseases with a benign course ( acute lichenoid pityriasis, 
lymphomatoid papulosis) present elana! proliferation 
of reactive T-lymphocytes (15). In our case, the 
presence of the same elana! population infiltrating 
the lymphonode and peripheral blood determined 
the diagnosis, although numerous reactive lymphocytes 
overlapped the infiltrate (12). 
In case 7 the rearrangement bands were shown in 
skin and not in peripheral blood, even if morpho-
logically 5% CSC were present. A similar observation 
has already been described in literature (11). The 
authors hypothesize that in CTCL the circulating 
polyclonal reactive lymphoid cells with a DNA gem11ine 
pattern are different from the elana! neoplastic cells 
which infiltrate lymphnodes and skin. A c!inical and 
biological explanation is at present not available (6). 
During the maintenance phase of the parenteral 
therapy with rIFNcx-2a at an average dose of 9xl06 
IU/day in 2 patients, one ( case 3) showed a partial 
elinical remission with the disappearance of CSC, 
the elonal TcR J3 rearrangement bands in peripheral 
blood and an improvement of the CD4/CD8 ratio, 
while in the other ( case 10), the immunological and 
molecular pattern was not changed. In literature 
one SS case with TcR gene rearrangement bands in 
peripheral blood has been reported, who after 
combined therapy with rIFNcx-2a and extracorporeal 
photochemotherapy displayed a germline pattern (21). 
These results indicate that the TcR J3 elana! rearrange-
ment studies by serial withdrawals of peripheral 
acta dermatovenerologica A.P.A. Vol 5, 96, No 2 
blood allow to monitor eventual molecular variations, 
possibly correlated with the elinical evolution of the 
disease. Ali the 7 patients with inflammatory 
erythrodermia showed a germline pattern, although 
one of them had 15% Sezary-like cells in circulation. 
Bakels et al. (19) showed an inflammatory erythro-
derma with elana! rearrangement of the TcR gene 
in peripheral blood. 
These data appear to be consistent with those of 
other authors (6,9,13,19) in which the results of the 
TcR J3 clonal rearrangement studies in peripheral 
blood were extremely sensitive. The use of the 
Polymerase Chain Reaction (PCR), the Denaturing 
Gradient Gel electrophoresis (DGGE) (22,23,24,25,26) 
and the withdrawals performed at different times 
(peripheral blood, skin, lymphnodes) may increase 
the sensitivity of the molecular investigations and 
thus strengthen the diagnosis. 
CONCLUSIONS 
Molecular biology makes possible a more accurate 
diagnostic evaluation, as well as the monitoring of 
the therapeutical effects and a better understanding 
of the etiopathological events of the lymphoproliferative 
diseases. The results have to be compared however 
with the clinical, histological and immunopathological 
investigations, therefore they represent a comple-
mentary diagnostic tool. 
ABBREVIATIONS' LIST 
ss = Sezary Syndrome 
MF Mycosis Fungoides 
TcR = T-cell Receptor 
rIFN- C( = recombinant Interferon alfa 
csc = Circulating Sezary Cells 
CTCL = Cutaneous T Celi Lymphoma 
DNA = Deoxyribonucleic Acid 
SBA = Southern Blat Analysis 
EDTA Ethylenediaminetetraacetic acid 
TE Tris-EDTA 
SSC/SDS = Saline-sodium Citrate/ Sodium 
Docecyl sulfate 
NBT = Nitroblue Tetrazolium 
BCIP = 5-bromo-4-chloro-3-indolphospha te 
PCR = Polymerase Chain Reaction 
DGGE = Denaturing Gradient Gel 
Electrophoresis 
47 
T-cell receptor gene in Sezary syndrome 
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AUTHORS' ADDRESSES 
Carmela Santangelo, PhD, Department of Immunodermatology, Istituto Dermopatico dell' Immacolata, 
Via Monti di Creta, 00167, Roma, Italia 
Roberto Benucci, MD, I Division of Dermatology, same address 
Marina Ruffelli, PhD - Immunodermatology, same address 
Marcella Ribuffo, MD - VI Division of Dermatology, same address 
Romeo Simoni, MD - III Division of Dermatology, same address 
Ornella di Pita, PhD - Immunodermatology, corresponding author, same address 
acta dermatovenerologica A.P.A. Vol 5, 96, No 2 49