ARTICLE
Recently, human T cell lymphotropic virus type 1 (HTLV-1) and Epstein-Barr
virus (EBV) infection have been implicated in the pathogenesis of mycosis
fungoides (MF) and Sézary syndrome (SS). HTLV-1 was isolated from
neoplastic cells derived from patients with adult T cell leukemia/lymphoma
(ATLL), an endemic disease in the south western part of Japan, the Caribbean
basin and south eastern United States, south America, central Africa,
and north eastern Iran [1, 2]. Subsequent studies have shown that HTLV-1-like
provirus and related genes can also be detected in patients with MF or
SS, suggesting that MF and SS may share the same etiology with ATLL [3-6].
However, this association has not been corroborated by other investigators
[7-10]. EBV, an ubiquitous human Herpes virus, has been recognized
as the cause of African Burkitt's disease, infectious mononucleosis, and
nasopharyngeal carcinoma. Furthermore, EBV has been demonstrated as being
associated with certain types of T cell lymphomas [11-15], and also with
MF or SS [16, 17].
This study was designed to evaluate the existence of the virus infection
in MF and SS in Japan, one endemic area for HTLV-1 and EBV infection.
We examined the HTLV-1 and EBV genes in a group of patients with cutaneous
lymphoproliferative disorders, including 18 patients with MF and SS.
Materials and methods
Patients
The diagnoses of patients included in this study have been made from
clinical features, histopathological findings, and immunohistochemical
study. All tissue and blood samples were obtained from various parts of
Japan, including HTLV-1 endemic areas. No patient diagnosed as having
MF or SS had antibodies to HTLV-1. Nine samples from healthy people were
used as negative controls. An ATLL cell line from one patient (KS-2),
which had been previously confirmed to be positive for HTLV-1 related
genes, was applied as the positive control in the present experiment.
Frozen biopsy specimens or blood samples were used for DNA extraction.
DNA extraction
Genomic DNA was extracted by proteinase K digestion, phenol/chloroform
extraction and ethanol precipitation, and subsequently resuspended in
TE (1 mmol/l EDTA and 10 mmol/l Tris-HCL, pH 7.5) buffer. One microgram
of genomic DNA was subjected to the PCR reaction.
Primers and PCR amplification
Primer sets used for the present study were designed to amplify the
gag, pol and pX genes of HTLV-1, and BamHI, W regions
of EBV. The sequences of primer sets are shown in Table
I.
Target genes were amplified in a 50 µl reaction mixture containing
10 mmol/l Tris-HCL (pH 8.0), 1.5 mmol/l MgCl2, 50 mmol/l KCl,
0.1 mg/ml gelatin, 200 µmol/l each of dATP, dGTP, dCTP, dTTP, 50
pmol/l of each primer, 1.25 units Taq polymerase (Takara, Japan). The
DNA was first pre-denatured at 94° C for 5 min, and was then subjected
to 35 cycles. Each cycle included denaturation at 94° C for 45 s,
annealing at 55° C for 45 s and extension at 72° C for 90 s.
For MF/SS samples, amplification of HTLV-1-related genes was repeated
by decreasing the annealing temperatures to 50° C, or increasing
PCR cycles up to 60. Taq DNA polymerase was added to the reaction mixture
after the first 30 cycles. Alternately, after the first 30 amplification
cycles, the PCR products were purified from agarose gel using a QIAquick
gel extraction kit (Qiagen, Germany) and were then processed for a second
amplification. The amplification of EBV consisted of 40 cycles of denaturation
at 94° C for 1 min, annealing at 52° C for 2 min, and extension
at 72° C for 2 min. PCR products were analyzed on 2% agarose gel
and visualized by staining with ethidium bromide.
Gene sequence analysis of PCR products
To specify the sequence of PCR products, sequence analysis was performed.
In brief, 4 ng PCR products were subcloned to 25 ng pGEM-T vector, then
transformed into competent cells (DH5alpha) for amplification. Plasmid
DNA was extracted and purified from DH5alpha cells by a NucleoSpin kit
(Bex Company), and then subjected to cycle sequencing using the single
primer extension method. The sequence was analyzed by a DNA sequence Model
4000L.
Results
Of 57 patients studied, gag, pol, and pX genes of HTLV-1
proviral DNA were positive in all 6 patients with ATLL and an ATLL cell
line (KS-2) (Fig. 1),
but none of them was detected in 14 patients with classic MF or 31 patients
with other cutaneous lymphoproliferative disorders (Table II).
Only HTLV-1 pol genes were detected by PCR in the skin lesions
of one patient with SS and two cases of T cell lymphomas (Fig.
2). The sequence of the pol gene product demonstrated by
PCR was identical to the registered sequence of the HTLV-1 pol
gene. Although PCR tests were repeated in different amplification conditions,
neither HTLV-1 gag nor pX sequence was detected in the 3
positive samples for the pol gene.
In contrast, a BamHI, W region of the EBV gene was demonstrated
in 13 cases, including 4 out of 4 angiocentric lymphomas, 4 out of 5 lymphomas
with hemophagocytosis, 4 out of 5 patients with hydroa vacciniforme-like
eruptions, and one with plasmacytoid lymphoma. EBV DNA sequences were
negative in all 17 patients with MF and SS (Fig.
3).
To confirm the presence of DNA in the PCR reaction, a human house-keeping
gene, ß-globin was amplified, and was found to be positive in all
patient samples. Both HTLV-1 DNA and EBV DNA were negative in 9 healthy
controls.
Discussion
HTLV-1 was one of the first viruses to be confirmed as an etiologic
agent for ATLL [1, 2]. However, previous reports have also demonstrated
the existence of HTLV-1 infection markers in MF, SS, and in other types
of T cell lymphoma [3, 4, 18, 19]. In 1991, deleted HTLV-1 provirus and
HTLV-1-like particles were demonstrated in MF patients by independent
investigators [3, 4]. Recently, the HTLV-1 pol or pX gene
was found in 46 out of 50 (92%) patients with MF or SS, either in peripheral
mononuclear cells or tumor lesions [18]. Furthermore, the same group demonstrated
the HTLV-1 tax proviral sequence in skin biopsies from 11 out of 12 patients
with MF using in situ PCR [20]. By contrast, no HTLV-1 DNA sequence
was detected in patients with MF and SS in at least four different laboratories
[7-10]. The conflicting results among the different authors might be due
to geographic differences of patients studied or mistaken diagnosis of
MF for chronic ATLL, etc. [10]. Also, a racial predominance of HTLV-1
infection can be considered. The present study was carried out in one
of the endemic area for HTLV-1 and EBV infection. The results showed that
no HTLV-1-related gag, pol, and pX genes were detected in
a group of Japanese patients with classic MF, although all patients with
ATLL were positive for HTLV-1 DNA. Our results suggest that HTLV-1 is
not involved in the pathogenesis of MF and SS in Japan. Because the HTLV-1
pol gene, but not gag and pX genes, was detected
in a few samples, we cannot exclude the possibility that the integration
of a largely deleted retrovirus genome might be responsible for the pathogenesis
of MF and SS, or be a cofactor for the illness [8, 21].
In addition to HTLV-1, EBV is another virus that has recently been suspected
of being involved in the pathogenesis of MF from evidence from immunologic
and molecular biology research [22]. Lee et al. [16] reported that
EBNA antibodies were detected in serum samples from all 21 patients with
cutaneous T cell lymphomas, by immunoblotting. With the in situ
hybridization method, EBV-encoded RNAs (EBER) were demonstrated in 8 out
25 (32%) patients with MF and SS [17]. However, other groups could not
confirm these findings [12, 23, 24].
In the present study, no patient with MF or SS demonstrated the EBV
DNA sequence by PCR analysis, in contrast to 13 positive cases in other
lymphoproliferative disorders (Tables
II and III).
These results do not support a pathogenic role for EBV infection in MF
and SS. Further studies to confirm latent EBV infection and clinicopathologic
findings in the EBV-associated disorders are currently underway.
In conclusion, HTLV-1 related genes are seldom detected in the majority
of classic MF or SS patients. EBV infection may be associated with certain
sets of cutaneous lymphoproliferative disorders, but is probably not involved
in the pathogenesis of MF and SS.
CONCLUSION
Acknowledgements
This publication was made possible by the generous donation of tissue
sections and medical notes from the following institutes: Departments
of Dermatology, Tohoku University (Drs. H. Tagami, N. Tabata, and M. Tanaka),
Sapporo Medical College (Drs. M. Kato, and S. Sugiyama), Aichi Medical
College (Drs. T. Ikeya, and Y. Nitta), Teikyou University (Dr. I. Ando),
Ohita Medical College (Drs. H. Terashi, and S. Takayasu), Yamaguchi Red-Cross
Hospital (Drs. K. Nishioka), Fujinomiya General Hospital (Drs. H. Igarashi,
and A. Aranami), and Tokyo Red-Cross Hospital (Dr. K. Shishiba), Nihonkai
Hospital (Dr. S. Anzai), and Department of Medicine, Tokyo Medical College
(Dr. Y. Katsura). This work was supported by a Grant-in-Aid for Scientific
Research from the Ministry of Education, Science and Culture of Japan
(KI, 08670979).
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