Diagnostic significance of TCR gene clonal rearrangement analysis in early mycosis fungoides.

0.82 MB·English

by  Xu, Chen

Chinese Journal of Cancer
Original Article
Chen Xu 1 , Chuan Wan 2, Lin Wang 1, Han鄄Ju n Yang 1 , Yuan Tang 3 , and Wei鄄P ing Liu 3
吟
Abstract
Mycosis fungoides (MF), the most common type of cutaneous T鄄ce ll lymphoma, has various unspecific
clinical and histological characteristics. Its early diagnosis is challenging. The application of T鄄ce ll receptor
(TCR) gene clonal rearrangement to the diagnosis of MF has been widely studied. In this study, we used
polymerase chain reaction (PCR) to investigate the diagnostic significance of detecting TCR鄄酌 and 鄄茁
gene clonal rearrangement in the early diagnosis of mycosis fungoides. PCR for TCR鄄酌 and TCR鄄茁 gene
rearrangement was performed on 19 patients with suspected early MF, 6 with typical MF, and 6 with
chronic dermatitis. Of the 19 patients with suspected early MF, 13 had TCR鄄酌 gene clonal rearrangement,
whereas none had TCR鄄茁 gene clonal rearrangement. All patients with typical MF had TCR gene clonal
rearrangement, in which 4 showed TCR鄄酌 clonal rearrangement, 1 showed TCR鄄茁 gene clonal
rearrangements, and 1 showed both. No patients with chronic dermatitis had TCR gene clonal
rearrangement. These results indicate that TCR gene clonal rearrangement analysis is a useful tool in
diagnosing early MF. TCR鄄酌 gene is recommended to the routine analysis, whereas TCR鄄茁 gene has
potential in combination toward intractable cases.
Key words Lymphoma, T鄄ce ll, mycosis fungoides, immunophenotype, gene rearrangement, T鄄ce ll receptor
Mycosis fungoides (MF) is the most common type of  to a lack of specific clinicopathologic presentation, the 
primary cutaneous T­cell lymphoma. Most patients have  early diagnosis of MF is a greatchallenge. 
an infiltrationof small to medium size mature helper CD4  During the differentiation of stem cells to T cells, 
T cells with cerebriform nuclei; a few patients have an  non­transcriptional gene fragments are rearranged into a 
infiltration of cytotoxic CD8 T cells. MF often starts as an  complete gene in a process catalyzed by DNA 
unspecific erythema (patch phase) similar to many  recombinase. Thenewgene gains transcriptional activity, 
inflammatory skin diseases. Progression from patch  expressing antigen­specific receptor as T cells continue 
phase to plaque and tumor phase often takes many  differentiating. In contrast, if gene rearrangement fails, 
years. The diagnosis of MF depends on a combination of  cells undergo apoptosis. This process is termed T­cell 
clinical and pathologic examination. However, early MF  receptor (TCR) gene rearrangement. Polyclonal 
(patch phase) pathology is unspecific and may simulate  rearrangement happens during normal T­cell 
a variety of benign inflammatory skin diseases, such as  development. However, all hematopoietic and lymphoid 
chronic eczema, neurodermatitis, and lichen planus. Due  tumor cells share the same TCR gene rearrangement, 
namely monoclonal rearrangement. Accordingly, by 
using Southern blot and polymerase chain reaction 
(PCR), researchers analyze clonality by detecting TCR 
1D epartment of Dermatovenerology, West China
gene rearrangement of T cells in skin lesions. Previous 
Hospital, Sichuan University, Chengdu, Sichuan 610041, P. R. China
2D ermatology Hospital of Jiangxi Province, Nanchang, Jiangxi 330118, P. R曰. gene rearrangement studies have relied heavily on 
China 3D epartment of Pathology, West China Hospital, Sichuan Southern blot analysis. However, PCR has several 
曰
University, Chengdu, Sichuan 610041, P. R. China; 吟N ow work at advantages (simple technique, short cycle, no 
Department of Dermatology, Chongqing Ninth Hospital, Chongqing radioactive contamination, compatible with 
400700, P. R. China.
paraffin­embedded tissue samples, and high detection 
Lin Wang, Department of Dermatovenerology,
rate) and has been widely used in clinical and 
West China Hospital, Sichuan University, Chengdu, Sichuan 610041, P. R.
China. Tel: +86鄄01 8980601701; Email: [email protected]. experimental detection to replaceSouthern blot analysis. 
264 www.cjcsysu.com  CACA Chinese Anti鄄Ca ncer Association
Chen Xu et al.  TCR gene in early mycosis fungoides
As T cells (including 琢/茁  and 酌/啄  T cells) completely  sensation/itch.
keep the rearranged TCR­酌 gene with relatively simple 
structure, designing PCR primers to obtain a high  Histological examination and immunophenotyping
detection rate is easy. Therefore, most investigators 
choose TCR­酌 as a target gene for lymphoma clonal  HE staining was conducted on paraffin sections of 19 
gene rearrangement [1] . As a part of the European  early MF suspected cases and 6 typical MF cases to 
BIOMED­2 program, the emergence of specific primers 
observe morphologic characteristics, such as 
for TCR­茁 gene has greatly enhanced the detection rate  epidermotrophism, Pautrier microabscess, involvement 
of clonalTCR­茁generearrangement [2,3] .  of dermis and subcutaneous tissue, and heteromorphism 
In recent years, investigators have gradually applied  of infiltrated cells. Ten immunophenotypes (茁F 1, CD2, 
PCR to analyze TCR gene rearrangement in cutaneous  CD3着, CD4, CD7, CD8, CD20, CD45RO, CD68, and 
lymphoma [4­8] , primarily detecting the rearrangement of  CD79a) were examined. 茁F 1 and CD4 were examined 
TCR­茁  and TCR­酌.  Because of the complexity and  with Elivision and EnVision immunohistochemical 
diversity of TCR gene rearrangement and differences in 
staining; other immunophenotypes were stained with the 
primer design andnumbers, samples and PCR products,  SP method.
results of these studies have discrepancies, showing 
50% to 90% of T­cell lymphomas had TCR­酌  gene 
Detection of TCR gene rearrangement
rearrangement. When narrowed down to the MF stages, 
the detection rates were 52% to 75% for patch phase, 
DNA was phenol­chloroform extracted from paraffin 
73% to 83% for plaque phase, and 90% to 100% for the 
tissue with a concentration range of 0.5 to 2.6 滋g /滋L and 
tumor [4­8] . 
an  of 1.7 to 2.1. Three TCR­酌   gene 
In this study, we detected TCR­茁 and TCR­酌 gene  260/280 
rearrangement primer sets (T  /T  [11] , V  /V  /JGT [12] , 
rearrangement in paraffin­embedded tissue samples  VG  JX  2­5  8­12  1 
and the BIOMED­2­TCR­酌 multiplex PCR amplification 
from patients with early suspected MF to further explore 
system [13]) , two TCR­β  gene rearrangement primer sets 
the application of TCR gene rearrangement to early 
(D J  [14]  and the BIOMED­2­TCR­茁  multiplex PCR 
diagnosis of MF. 1 2 
amplification system [13] ), and the PCR targets are listed 
in Tables 1 and 2. PCR was conducted in a 25 滋L  
reaction volume containing 0.2 mmol/L dNTP, 1.5 
Materials and Methods
mmol/L MgCl, 1 滋m ol/L primers, 1.25 U Taq enzyme 
2
(Takara Bio Inc.), and 0.5 to 1 滋g  template DNA. 
Patients and samples Amplifications with primer T  /T  were done with 
VG  JX 
touchdown PCR. The amplifying conditions for 
The specimens were from 19 patients with suspected  V 2­5/ V 8­12/ JGT1 were as follows: denaturation at 95益 for 3 
early MF, 6 patients with typical MF (2 plaque phase and  min, 30 cycles of 94益 for 30 s, 60益 for 45 s, and 72益 
4 at tumor phase as positive control), and 6 patients with  for 45 s,followed by an extension at 72益 for10min;the 
chronic inflammatory skin disease (3 lichen planus, 2  amplifying condition for BIOMED­2­TCR­酌/茁   and D 1J  2 
psoriasis, and 1 neurodermatitis as negative control)  were as follows: denaturation at 95益  for 3 min, 35 
examined in the Pathology Division at the Department of  cycles of 95益 for 30 s, 60益 for 40 s, and 72益 for 1 
Dermatology and the Department of Pathology in the  min, followed by an extension at 72益 for 10 min. The 
WestChinaHospitalof Sichuan University between 2001  PTC­200 thermal cycler from MJ Research Inc. was 
and2007. Histological diagnosis of MF was confirmed by  used for all PCR reactions. 茁­g lobin was amplified as an 
a senior cutaneous pathologist and a lymphatic  internalcontrol. A DNA sample from Jurkatcells, a T­cell 
hematopoieticpathologistaccordingtoLever sdiagnostic  lymphoma line, was used as a positive control. PCR 
criteria [9]  and the 2005 WHO­EORTC clas爷sification of  products were electrophoresed on an 8% polyacrylamide 
cutaneous lymphoma [10] .  gel and analyzed after silver staining. For heteroduplex 
Early suspected patients were 9 men and 10 women,  analysis of gene rearrangement, 10 to 20 滋L of PCR 
aged 15 to 79 years with a mean age of 53 and median  product from BIOMED­2 system was denatured at 95益 
age of 59. All 19 patients had multiple skin lesions in the  for 5 min and then annealed at 4益 for 60 min. The 
upper limbs (16 cases), trunk (15 cases), and lower  resulting PCR products were electrophoresed on a 10% 
limbs (14 cases). Some patients had skin lesions in the  polyacrylamide gelandanalyzed aftersilverstaining.
face, neck, and head. The lesions mainly characterized 
as multiple erythema and papule covered with small  DNA sequencing
scales. A few patients had pigment changes, skin 
atrophy, and ichthyosis­like skin lesions. Most patients  The PCR product of a case of TCR gene 
had varying degrees of itching and 1 had a burning  rearrangement was randomly selected for DNA
www.cjcsysu.com Chin J Cancer; 2011; Vol. 30 Issue 4 265
Chen Xu et al. TCR gene in early mycosis fungoides
Name Sequence 5' 3' Product length
寅
鄄 gl obin PC03: ACACAACTGTGTTCACTAGC 110 bp
茁
PC04: CAACTTCATCCACGTTCACC
T /T T : AGGGTTGTGTTGGAATCAGG 160-190 bp
VG JX VG
T : CGTCGACAACAAGTGTTGTTCCAC
JX
V /V /JGT V: CTTCCTGCAGATGACTCCTACAACTCCAAGGTTG
2-5 8-12 1 2
V: CTTCCTGCAGATGACGTCTCCACCGCAAGGGATG 170-230 bp
3
V: CTTCCTGCAGATGACTCCTACACCTCCAGCGTTG
4
V: TTCCTGCAGATGACGTCTCCAACTCAAAGGATG
5
V: CTTCCTGCAGATGACTCCTACAACTCCAGGGTTG
8
V: GG(A/T/G/C)ACTGCAGGAAAGGAATCTGGCATTCCG
9
V : CTCTGCAGAATCCGCAGCTCGACGCAGCA
10
V : CACTGCAGGCTCAAGATTGCTCAGGTGGG
11
V : ACTCTGCAGCCTCTTGGGCACTGCTCTAAA
12
JGT: AAGTGTTGTTCCACTGCCAAA
1
BIOMED鄄2鄄 TC R鄄 V : GGAAGGCCCCACAGCRTCTT Tube A:
酌 r1f
V : CGGCACTGTCAGAAAGGAATC V 1 f+V 1 0+J 1 .1/2.1+ J 1 .3/2.3
r9 酌 酌 酌 酌
V : AGCATGGGTAAGACAAGCAA 145-255 bp
r10
V : CTTCCACTTCCACTTTGAAA
r11
J : TTACCAGGCGAAGTTACTATGAGC Tube B:
r1.1-1.2
J : GTGTTGTTCCACTGCCAAAGAG V 9 +V 1 1+J 1 .1/2.1+J 1 .3/2.3
r1.3-2.3 酌 酌 酌 酌
80-140 or 160-220 bp
Name Sequence 5' 3' Product length
寅
DJ D1: CAAAGCTGTAACATTGTGGGGAC 50-110 bp
1 2
J2: AGCACCGTGAGCCTGGTGCC
BIOMED鄄2鄄 TC R鄄 V 2 : AACTATGTTTTGGTATCGTCA Tube A:
茁 茁
V 4 : CACGATGTTCTGGTACCGTCAGCA V + J 1 .1-1.6, 2.2, 2.6, 2.7
茁 茁 茁
V 5 /1: CAGTGTGTCCTGGTACCAACAG 240-285 bp
茁
V 6 a/11: AACCCTTTATTGGTACCGACA
茁
V 6 b/25: ATCCCTTTTTTGGTACCAACAG
茁
V 6 c: AACCCTTTATTGGTATCAACAG Tube B:
茁
V 7 : CGCTATGTATTGGTACAAGCA V + J 2 .1, 2.3, 2.4, 2.5
茁 茁 茁
V 8 a: CTCCCGTTTTCTGGTACAGACAGAC 240-285 bp
茁
V 9 : CGCTATGTATTGGTATAAACAG
茁
V 1 0: TTATGTTTACTGGTATCGTAAGAAGC
茁
V 1 1: CAAAATGTACTGGTATCAACAA
茁
V 1 2a/3/13a/15:ATACATGTACTGGTATCGACAAGAC
茁
V 1 3b: GGCCATGTACTGGTATAGACAAG
茁
V 1 3c/12b/14: GTATATGTCCTGGTATCGACAAGA
茁
V 1 6: TAACCTTTATTGGTATCGACGTGT
茁
V 1 7: GGCCATGTACTGGTACCGACA
茁
V 1 8: TCATGTTTACTGGTATCGGCAG
茁
V 1 9: TTATGTTTATTGGTATCAACAGAATCA
茁
V 2 0: CAACCTATACTGGTACCGACA
茁
V 2 1: TACCCTTTACTGGTACCGGCAG
茁
V 2 2: ATACTTCTATTGGTACAGACAAATCT
茁
V 2 3/8b: CACGGTCTACTGGTACCAGCA
茁
V 2 4: CGTCATGTACTGGTACCAGCA
茁
J 1 .1: CTTACCTACAACTGTGAATCTGGTG
茁
J 1 .2: CTTACCTACAACGGTTAACCTGGTC
茁
J 1 .3: CTTACCTACAACAGTGAGCCAACTT
茁
J 1 .4: CATACCCAAGACAGAGAGCTGGGTTC
茁
J 1 .5: CTTACCTAGGATGGAGAGTCGAGTC
茁
J 1 .6: CATACCTGTCACAGTGAGCCTG
茁
J 2 .1: CCTTCTTACCTAGCACGGTGA
茁
J 2 .2: CTTACCCAGTACGGTCAGCCT
茁
J 2 .3: CCCGCTTACCGAGCACTGTCA
茁
J 2 .4: CCAGCTTACCCAGCACTGAGA
茁
J 2 .5: CGCGCACACCGAGCAC
茁
J 2 .6: CTCGCCCAGCACGGTCAGCCT
茁
J 2 .7: CTTACCTGTAACCGTGAGCCTG
茁
266 Chin J Cancer; 2011; Vol. 30 Issue 4 Chinese Journal of Cancer
Chen Xu et al. TCR gene in early mycosis fungoides
sequencing. The PCR product was purified with DNA  of lymphoid cells, including 4 had linear arrangement of 
Gel Extraction Kit (Axygen Biosciences Inc), cloned into  lymphoid cells in the basal layer (Figure 1A). Epidermal 
a T­vector (PMD 20­T Vector, Takara Bio Inc.) and  infiltrated cells had distorted or cerebriform nuclei and 
sequenced with an ABI 377 DNA sequencer. The  perinuclear halo which were deeply stained. All patients 
sequencing result was analyzed by the BLASTing gene  had lymphoid cell infiltration in superficial middle dermis: 
database in NCBIfor gene rearrangement. 9 had perivascular infiltration; 2 had striated infiltration in 
superficial dermal; 8 had scattered infiltration in the 
Statistical methods dermis. Dermal infiltrating cells were of small to medium 
size and individually were relatively large. In 2 cases, 
Statistical analysis was done with SPSS13.0  dermal infiltrating cells were smaller, had less cytoplasm 
software. The Fisher exact test was applied and the  and were stained deeper than those in the epidermis; 
difference was statistically significant when  < 0.05. some cells had distorted and heterogeneous nuclei 
(Figure 1B). Three cases had papillary dermal fibrosis. 
All suspected cases expressed two or more T cell 
markers (CD2, CD3着, CD7, and CD45RO) (Figure 2); 
Results
17 (89.5%) expressed 琢/茁  T­cell marker 茁F 1 (Figure 
2); 14 (73.7%)expressed CD4 (Figure 2); 13 (68.4%) 
Morphology and immunophenotype
expressed CD8 in some cells (10% to 80%) but not 
B­cell markers (CD20 and CD79a) or monocyte/ 
Nineteen patients with suspected early MF 
macrophage marker CD68.
predominantly had lesions within the epidermis and 
shallow middle dermis. Eleven patients had epidermal 
hyperkeratosis and/or parakeratosis, acanthosis, and  PCR amplification and DNA sequence
epidermal protuberance extension, including 5 had 
edema in the spinous layer and 3 had focal basal  PCR results are shown in Table 3. Of the 19 patients 
liquefaction degeneration. Fifteen had epidermotrophism  with suspected early MF, 13 (68.4%) had TCR­酌 gene
Figure 1. Histopathologic changes of a skin lesion of suspected early mycosis fungoides (MF)援T he skin lesion from a patient with suspected
early MF was stained with HE and observed under a microscope. A, medium鄄si zed epidermal lymphocytes with prominent cytoplasmic halos are
aligned within the basal layer (arrow). Lymphocytes with pleomorphic nuclei infiltrated in the dermis are smaller than those in the epidermis. B,
presence of haloed cells with round or hyperconvoluted cerebriform nuclei infiltrate in the epidermis; particularly, single cells are present at the
rete ridges with epidermotropism.
www.cjcsysu.com Chin J Cancer; 2011; Vol. 30 Issue 4 267
Chen Xu et al. TCR gene in early mycosis fungoides
Figure 2. Immunophenotype of lymphocytes in a skin lesion of suspected early MF. The skin lesion from a patient with suspected early MF was stained with
respective antibodies by the SP, Elivision, or EnVision method. The lymphocytes infiltrated in the epidermis express T鄄ce ll-associated antigens CD2, CD3 , CD7, and
着
CD45RO (SP method). Lymphocytes express F 1 (Elivision) and CD4 (EnVision) derived from cells with a helper T cell profile.
茁 琢茁
Case TCR鄄 TCR鄄
酌 茁
number T /T V /JGT BIOMED/ Total (TCR鄄 ) DJ BIOMED/ Total (TCR鄄 ) Total
VG JX 2-5,8-12 1 酌 酌 1 2 茁 茁
1 R R N R N N N R
2 R R N R N N N R
3 N N N N N N N N
4 N N N N N N N N
5 N R N R N N N R
6 N R N R N N N R
7 N R N R N N N R
8 N N N N N N N N
9 N N N N N N N N
10 N R N R N N N R
11 N R N R N N N R
12 R R N R N N N R
13 R R N R N N N R
14 N N N N N N N N
15 R R N R N N N R
16 N R N R N N N R
17 N R N R N N N R
18 N N N N N N N N
19 R R N R N N N R
Summary 6 13 0 13 0 0 0 13
Positive rate 31.6% 68.4% 0 68.4% 0 0 0 68.4%
R, rearrangement; N, no rearrangement.
268 Chin J Cancer; 2011; Vol. 30 Issue 4 Chinese Journal of Cancer
Chen Xu et al. TCR gene in early mycosis fungoides

clonal rearrangement (Figures 3, 4A and 4B), none had  rearrangement (Figure 4C), 2 had TCR­茁  gene 
TCR­茁 gene rearrangement. All patients with typical MF  rearrangement (Figures 4D and 4E), and 1 had both. No 
had TCR gene rearrangement: 4 had TCR­酌  gene  TCR gene rearrangement was detected in the 6 patients

Figure 3. Agarose electrophoresis
M P B 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 of 鄄 gl obin PCR products of 19
茁
patients with suspected early MF. 鄄
茁
globin was amplified using primer
PC03/PC04. Lane M, molecular
weight marker DL2000; lane P,
250 bp positive control; lane B, blank control;
lanes 1-19, PCR products of the 19
100 bp 110 bp patients. 茁鄄 gl obin gene, with a single
band of 110 bp, was detected in all
cases of suspected early MF.DNA
extraction with phenol鄄ch loroform
was qualified by the results.
M P B 1 2 12 13 15 19 Figure 4. Poly鄄ac rylamide gel
electrophoresis (PAGE) of PCR
250 bp
A products of TCR gene clonal
200 bp 190 bp rearrangement in the 19 patients with
150 bp suspected early MF and the 6
patients with typical MF. Lane M,
molecular weight marker DL50; lane
M P B 5 6 7 10 11 16 17
P, positive control; lane B, blank
control; lanes 1-19, PCR products of
250 bp
B 200 bp 200 bp the 19 patients. A, TVG/TJX, with a
single band of 190 bp, was detected
150 bp in the suspected early MF cases 1, 2,
12, 13, 15 and 19, confirming TCR鄄
酌
clonal rearrangement. B, V /V /JGT ,
M P B 3 5 6 2-5 8-12 1
with a single band of 200 bp, was
300 bp detected in the suspected early MF
C
250 bp cases 5, 6, 7, 10, 11, 16 and 17,
230 bp confirming TCR鄄 clonal
200 bp 酌
rearrangement. C, BIOMED鄄2鄄 TC R鄄 ,
酌
M P B 3 with a single band of 230 bp, was
detected in cases 3, 5 and 6 of 6
200 bp typical cases of MF, confirming TCR鄄

D 酌 clonal rearrangement. D, D1J2, with
150 bp a single band of 110 bp, was
110 bp detected in typical MF case 3,
confirming TCR鄄 clonal
100 bp 茁
rearrangement. E, BIOMED鄄2鄄 TC R鄄 ,
茁
M P B 2 with a single band of 250 bp, was
detected in typical MF case 2,
300 bp
confirming TCR鄄 clonal
E rearrangement. 茁
250 bp 250 bp
200 bp
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Chen Xu et al. TCR gene in early mycosis fungoides
with chronic inflammatory skin diseases. The PCR  and established a simple clinical scoring system to 
product from a case of TCR­酌  gene rearrangement  facilitate MF diagnosis. They found that the histological 
amplified with primers T /T was sequenced and the  diagnosis of MF and TCR gene rearrangement are 
VG  JX 
sequence was analyzed in NCBI BLAST gene database.  closely related (odds ratio = 14.4) and proposed a 
The results showed a 99.4% homology (170/171)  scoring program: TCR gene rearrangement positive 
between the PCR product and the chromosome 7  scores 2.5, classical morphology scores 2.0, and classic 
genomic contig, alternate assembly RA_TCAGchr7v2  clinical manifestation scores 1.5; a total score equal 
gene (GI: 079592) in the NCBI Genbank. Comparison of  to or greater than 3.5 indicates a high probability of 
TCR­酌  gene rearrangement in suspected cases and  MF (> 85%). In our study, the clinical manifestations, 
typical MF cases and 2 test showed that the positive  histopathologic and immunologic phenotypes suggest all 
字
rates of the two groups had no significant difference (  >  suspected cases were of possible MF. TCR­酌  gene 
0.05). rearrangement was detected in 13 patients (68.4%). 
Thus, we believe that these 13 cases can be diagnosed 
as early MF (patch phase). Four of the 13 patients were 
Discussion  followed up and diagnosed with MF according to disease 
developmentand treatment response.The diagnosis of 6 
Early MF has a variety of clinical manifestations and  patients was still unclear and further observation and 
unspecific pathologic characteristics. Typical  follow­up was needed. 
characteristics include focal perivascular dermal  This study showed that the detection rate of clonal 
infiltration of small to medium­sized lymphoid cells with  TCR­酌  gene rearrangement was higher than that of 
cerebriform nuclei, or arrangement along the junction of  TCR­茁.  No clonal TCR­茁  gene rearrangement was 
the epidermis and dermis and edema in the spinous  detected in the suspected cases. Currently, universal 
layer. Pautrier microabscesses and epidermotrophism,  PCR primers can be designed at the V and J regions of
which have important diagnostic value, are not obvious  琢,  茁,  啄 and 酌chains. However, all T cells (including 琢/茁   
or are missing in early MF [15] . MF may simulate chronic  and 酌/啄  T cells) keep the rearranged TCR­酌  gene. 
eczema, neurodermatitis, or other inflammatory skin  TCR­酌 gene structure is relatively simple and has no 
diseases and requires long­term follow­up and repeated  variable D region, which facilitates primer design and 
biopsy. As an important diagnostic method for MF,  PCR amplification. For this reason, researchers usually 
immunohistochemistry can determine the origin of tumor  choose TCR­酌 when analyzing TCR clonality. Therefore, 
cells but can not determine the clonality of proliferating  TCR­酌 gene is still a good target for the detection of 
cells. Moreover, as early MF is often accompanied by  clonalgene rearrangementin earlyMF. 
reactive lymphocytes, many cases do not show a  In addition, no TCR­茁  gene rearrangement was 
characteristic immunophenotype. For these cases,  detected in the suspected cases. But false negative 
immunohistochemistry has a limited diagnostic value.  could not be ruled out because the PCR may have been 
Thus, molecular diagnosis is needed.  affected by pre­treatment of the archived 
Analysis of clonal gene rearrangement plays an  paraffin­embedded tissue samples. Although 
important role in the diagnosis of lymphoproliferative  amplification of control 茁­g lobin (110 bp) was good, 
disease. Combining this approach with pathologic and  some target genes of greater or lesser size than 茁­g lobin 
immunohistochemical examinations may aid in the  (such as 250 to 300 bp or 80 to 100 bp fragments) may 
diagnosis of difficult cases. Currently, detecting TCR­酌   have been destroyed. In this study, we used primers to 
gene rearrangement by PCR has become an important  amplify 80 to 285 bp long fragments, so damaged DNA 
adjunctto the diagnosis of cutaneous T­cell lymphoma [16] .  fragments could not be amplified in our PCR reaction, 
In this study, TCR gene rearrangement was detected in  possibly causing false negative results. 
all typical MF cases but not detected in all chronic  False results are also inevitable in the interpretation 
inflammatory skin disease cases, indicating that  of PCR results. Through optimizing PCR primer design 
detecting TCR rearrangement by PCR is an effective  and selecting a higher resolution technique, one can 
adjunct to MF diagnosis.  reduce false results. In this study, we used the higher 
In addition, when PCR was used to analyze gene  resolution techniques of polyacrylamide gel 
rearrangement in tissues of atypical cutaneous T  electrophoresis and silver staining to display 
lymphocyte proliferative diseases, the sensitivity was  rearrangement bands and used heteroduplex analysis to 
83.5% and specificity was 97.7% [8] . The positive rate of  verify gene rearrangement, both of which could reduce 
TCR­酌gene rearrangement in earlyMF suspected cases  false positive results. In addition, the sensitivity of PCR 
was reported to be 40% to 71% [6,7] , which is useful for  has a certain range and false negatives can arise when 
early diagnosis. Stevens  . [17]  conducted logistic  the ratio of DNA from monoclonal proliferating 
regression analysis on some clinical and laboratory data  lymphocytes to that from reactive lymphocytes is less
270 Chin J Cancer; 2011; Vol. 30 Issue 4  Chinese Journal of Cancer
Chen Xu et al. TCR gene in early mycosis fungoides
than 1%. If the amount of template DNA is too small,  independence of each other, the occurrence of clonal 
DNA from a single or several lymphocytes is highly  gene rearrangement does not indicate a malignant 
amplified,similar bands like a monoclonal rearrangement  lesions, because some benign or reactive inflammatory 
may arise, namely pseudoclonality. Therefore, for  lesions may also have clonal rearrangement. For 
paraffin­embedded tissue samples, the initial amount of  example, clonal gene rearrangements could be detected 
template DNA should be no less than 50 ng, of which  in benign monoclonal 酌­g lobulin disease, acute 
DNA from reactive lymphocytes must be more than 5  smallpox­like moss­like rash, other skin inflammatory 
ng 18] . Under the premise of not affecting PCR, an  diseases, and immune deficiency disease with severe 
appropriate increase of the amount of template DNA can  Epstein­Barr virus infection [19] . Conversely, the lack of 
reduce pseudoclonality. In this study, template DNA was  monoclonal gene rearrangement is not equivalent to 
within a 200 to 500 ng range and the proportion of tumor  benign lesions. In many conditions, such as the low 
cells was higher than 1%, thereby reducing false positive  coverage of primers, the absence of a tested gene 
results.  rearrangement in the tumorgenome, DNA degradation in 
Based on this study, we believe that, when MF is  the sample, and the number of tumor cells below the 
suspected and diagnosis cannot be made on clinical  detection sensitivity threshold, clonal gene 
manifestations, regular histopathologic examination, and  rearrangement may not be detected. Therefore, the early 
immunohistochemistry, TCR gene rearrangement is a  diagnosis of MF must be based on pathologic changes, 
valuable diagnostic adjunct. The detection of clonal  with immunohistochemistry and gene rearrangement 
TCR­酌  gene rearrangement is recommended and the  analysis as important adjunct diagnostic tools. For some 
analysis of TCR­茁 rearrangement can be performedalong  suspected cases with no clonal gene rearrangement, 
with TCR­酌rearrangement for a few difficult cases.  long­term follow­up and multiple pathologic examinations 
However, it must be noted that gene rearrangement  should bedone to confirm the diagnosis [20] .
of antigen receptor is not involved in the malignant 
transformation of lymphocytes. These two processes 
occur independently. In general, gene rearrangement  Acknowledgments 
occurs before tumor transformation. On one side, 
clonally proliferating tumor cells harbor the same antigen  This work was supported by grant from Sichuan 
gene rearrangement (monoclonality), whereas each T or  Provincial Sci­Tech Research Foundation (No. 
B cell from benign lesions carries a unique type of gene  07SG004009). 
rearrangement (polyclonality). Therefore, in theory, the 
genetic rearrangement analysis of antigen receptor gene  Received: 2010­07­06; revised:2010­12­21; 
is helpful to distinguish proliferating lymphoma and  accepted: 2011­01­14
.
reactive lesions. On the other side, due to the 
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