Structural variation of centromeric endogenous retroviruses in human populations and their impact on cutaneous T-cell lymphoma, Sézary syndrome, and HIV infection.

BACKGROUND: Human Endogenous Retroviruses type K HML-2 (HK2) are integrated into 117 or more areas of human chromosomal arms while two newly discovered HK2 proviruses, K111 and K222, spread extensively in pericentromeric regions, are the first retroviruses discovered in these areas of our genome. METHODS: We use PCR and sequencing analysis to characterize pericentromeric K111 proviruses in DNA from individuals of diverse ethnicities and patients with different diseases. RESULTS: We found that the 5' LTR-gag region of K111 proviruses is missing in certain individuals, creating pericentromeric instability. K111 deletion (-/- K111) is seen in about 15% of Caucasian, Asian, and Middle Eastern populations; it is missing in 2.36% of African individuals, suggesting that the -/- K111 genotype originated out of Africa. As we identified the -/-K111 genotype in Cutaneous T-cell lymphoma (CTCL) cell lines, we studied whether the -/-K111 genotype is associated with CTCL. We found a significant increase in the frequency of detection of the -/-K111 genotype in Caucasian patients with severe CTCL and/or Sézary syndrome (n = 35, 37.14%), compared to healthy controls (n = 160, 15.6%) [p = 0.011]. The -/-K111 genotype was also found to vary in HIV-1 infection. Although Caucasian healthy individuals have a similar frequency of detection of the -/- K111 genotype, Caucasian HIV Long-Term Non-Progressors (LTNPs) and/or elite controllers, have significantly higher detection of the -/-K111 genotype (30.55%; n = 36) than patients who rapidly progress to AIDS (8.5%; n = 47) [p = 0.0097]. CONCLUSION: Our data indicate that pericentromeric instability is associated with more severe CTCL and/or Sézary syndrome in Caucasians, and appears to allow T-cells to survive lysis by HIV infection. These findings also provide new understanding of human evolution, as the -/-K111 genotype appears to have arisen out of Africa and is distributed unevenly throughout the world, possibly affecting the severity of HIV in different geographic areas.

Cytomegalovirus: its potential role in the development of cutaneous T-cell lymphoma.

To investigate the potential role of CMV in cutaneous T-cell lymphoma (CTCL), we studied cytomegalovirus (CMV) seroprevalence in parapsoriasis (PP), mycosis fungoides (MF) and Sézary syndrome (SS) compared with healthy control patients. In cases where CMV seropositivity was observed, CMV PCR analyses were performed on skin biopsies. CMV seroprevalence was 37.1% in the control group, 50.68% in the PP + MF + SS group (P = 0.08), 56.2% in the MF + SS group (P = 0.07), 40% in the PP group (P = 0.9), 66.67% in the MF group (P = 0.009), 42.86% in the SS group (P = 0.9). CMV PCR in initial skin biopsies were all negative. However, PCR CMV was positive in two SS skin biopsies realized at an advanced stage. Our results show that latent CMV infection may play a role in the susceptibility of MF in predisposed subjects by inducing T-cell proliferation and resistance to apoptosis. Concerning SS, an immunosuppressive state may be responsible for CMV reactivation that in turn may interfere with evolution of the disease.

No evidence of HTLV-I infection in patients with mycosis fungoides and Sezary syndrome.

The involvement of human T-cell lymphotropic virus type I (HTLV-I) in the etiology of cutaneous T-cell lymphomas (CTCL) is still controversial. The aim of the study was to evaluate the role of HTLV-I in the pathogenesis of mycosis fungoides (MF) and Sezary syndrome (SS) in Polish patients. The studied group consisted of 42 patients with MF, 5 with SS and 25 with chronic dermatitis. DNA was extracted from snap-frozen and paraffin-embedded skin biopsies and from peripheral blood. Polymerase chain reaction (PCR or nested PCR) was carried out for amplification of different regions of HTLV-I genome. Primer sets flanking pX, p 19, U5, tax and pol genes were used in the investigation. The presence of HTLV-I antibody was examined in 46 sera samples with the use of anti-HTLV-I/II EIA test. HTLV-I antibodies were not detected in any collected sera samples. PCR with two primer sets homologous to the pX region of HTLV-I showed negative results in all samples investigated. To confirm these results two other primer pairs specific for U5 and gag regions were designed. With these primer pairs no PCR product, except that in positive control, was observed. For more sensitive amplification a nested-PCR with pol and tax specific primers was performed. HTLV-I probably does not play an important role in the pathogenesis of MF in Polish patients.

[A woman with Sezary syndrome developed HTLV-1-associated myeloneuropathy. The reported case supports the theory of etiological connection]. / Kvinna med Sézarys syndrom utvecklade HTLV-1-associerad myeloneuropati. Rapporterat fall ger stöd för teori om orsakssamband.

The patient was a 58-year-old woman with a 10-year history of severely itching erythroderma, as well as enlarged lymph glands and circulating Sézary cells (i.e. Sézary's syndrome). Histological analysis of a skin biopsy revealed Pautrier's microabcesses with atypical lymphocytes characteristic of T-cell lymphoma. Her lymph glands were also found to contain atypical lymphocytes. Parallel to the skin disorder, the patient developed paraparesis with fainting strength of the musculature of the extremities. Ultimately, she was unable to walk. She had no feeling of vibration and had difficulties emptying her bladder. Babinski's sign was positive, bilaterally. Ten years before the present admission the patient's skin and blood lymphocytes were positive for sequences of human T-cell lymphoma virus-1 (HTLV-1) in a polymerase chain reaction (PCR). At the present admission, the patient presented a high titre of HTLV-1 antibodies both in the blood and cerebrospinal fluid on ELISA and Wesern blot analysis. The patient had been married to a man from Bolivia for 30 years. He was also strongly positive for HTLV-1 antibodies in blood. Adult T-leukemia (ATL) is common in areas where HTLV-1 is found endemically. ATL has skin symptoms similar to mycosis fungoides. The clinical findings in this patient indicate an etiological connection between some cases of T-cell lymphoma in the skin (Sézary's syndrome) and HTLV-1.

Cytomegalovirus seropositivity is significantly associated with mycosis fungoides and Sézary syndrome.

Although mycosis fungoides (MF) may arise through persistent antigen stimulation, cytomegalovirus (CMV) is not a known risk factor. To study the incidence of seropositivity to viral infections, we compared MF and Sézary Syndrome (SS) patients to healthy bone marrow donors and other historical control groups. Baseline screening serologies at baseline were performed on 116 biopsy-proven MF/SS patients at MD Anderson Cancer Center from 1992 to 2001 and on healthy bone marrow donors evaluated by the transplant service from 1988 to 2001. Antibodies to HTLV-I/II, HIV-1, EBV, and CMV were measured using standard enzyme-linked immunosorbent (ELISA) and membrane enzyme immunoassay (MEIA) assays. One hundred thirteen (97.4%) of all MF/SS patients had positive CMV IgG serologies at initial presentation. Early- and late-stage patients' seropositivity rates were significantly higher than healthy bone marrow donor controls (chi(2).05(df=1) = 71.79). By stage, 98.1% of early-stage MF patients (IA, IB, IIA; 52/53) and 96.8% of late-stage MF and SS patients (IIB-IVB; 61/63) were seropositive compared with healthy bone marrow donors whose seropositivity rate was 57.3% (757/1322). Because the rate of CMV seropositivity increases with age, a subset of cutaneous T-cell lymphoma (CTCL) patients 55 years or younger were compared to age-matched healthy donor controls; their seropositivity rate for CMV was also significantly higher (chi(2).05 05(df=1) = 20.4). EBV titers were positive by serology in 13 patients who were examined prospectively. CMV seropositivity is highly associated with MF and SS, even in the earliest stages of the disease, and is significantly higher than that of healthy and immunocompromised controls.

Interferon resistance of cutaneous T-cell lymphoma-derived clonal T-helper 2 cells allows selective viral replication.

Cutaneous T-cell lymphomas (CTCL) comprise a heterogeneous group of lymphoproliferative disorders that are characterized by an accumulation of T-lymphocytes in the skin and occasionally in blood known as Sézary syndrome (SS). In most cases the dominant clone displays T-helper 2 cytokines. Because IFN-gamma is a natural inhibitor of T-helper 2 cells and IFN-alpha is frequently used in CTCL, the impact of IFNs on SS-derived purified clonal T-helper 2 cells was studied using anti-Vbeta antibodies. Moreover, IFNs are known to mediate virus resistance in normal cells. The isolated clonal CD4(+) cells, but not the nonclonal CD4(+) cells, appeared resistant to IFN-gamma and IFN-alpha stimulation in terms of human leukocyte antigen up-regulation and MxA induction caused in part by alterations in Stat-1 molecule mRNA and IFNgammaR1 mRNA transcription. The IFN resistance of the patient-derived clonal cells was then targeted by vesicular stomatitis virus infection after IFN-alpha priming, resulting in selective viral replication in clonal cells. In contrast, nonclonal cells of the same patient showed IFN-dependent MxA expression, which is a major mediator protein of viral protection. The IFN resistance of the dominant T-helper 2 cells might be important for lymphomagenesis. Interferon signaling deficiencies can be targeted for purging patients' cells in vitro. Furthermore, this approach may allow specific molecular interventions, resulting in the efficient treatment of CTCL and other IFN-resistant neoplasms such as lung cancer.

Characterization of a canine long-term T cell line (DLC 01) established from a dog with Sézary syndrome and producing retroviral particles.

The canine DLC 01 cell line derives from a lymph node of a dog with Sézary syndrome. The DLC 01 cell phenotype is CD4-, CD8+, CD45+, DQ+, similar to that of original cells after treatment with dimethylsulfoxide or phorbol myristate. Canine cutaneous T cell lymphoma are usually CD4-, CD8+ in contrast to their human counterparts which are CD4+, CD8-. Therefore, the DLC 01 cell line appears to be a unique model to study the mechanism of all surface molecule expression in vitro. Viral particles with retrovirus type-C morphology were found in ultrathin sections of DLC 01 cell pellets. Retroviral particles are spontaneously produced after the 50th cell passage or after induction with 0.5% dimethylsulfoxide. This is the first description of a dog lymphoid cell line spontaneously growing and producing a retrovirus. It was found to share several features in common with feline and murine leukemia viruses.

Evidence for the cofactor role of human T-cell lymphotropic virus type 1 in mycosis fungoides and Sézary syndrome.

The aetiology of mycosis fungoides (MF) and Sézary syndrome (SS) is unknown. A pathogenic role for the human T-cell lymphotropic virus type 1 (HTLV-1) has been suggested but remains controversial. We used an animal model to test the possibility that peripheral blood mononuclear cells (PBMC) obtained from MF patients harbour the HTLV-1 virus which may be infective. The polymerase chain reaction (PCR) was used to detect HTLV-1 proviral DNA sequences in PBMC of 27 MF patients and one SS patient of non-Iranian origin. Positive results were found in six of the patients. Twelve of the 28 patients tested by Western blot showed HTLV-1 antibodies. Twenty-eight immunosuppressed inbred Fisher F344 rats were inoculated intravenously with cultures of PBMC obtained from the 28 patients. Eight of these 28 rats showed antibodies to HTLV-1 while the proviral genome was demonstrated in the blood of only two of the rats. PBMC from two MF patients, in spite of showing negative results for the proviral genome by PCR, still induced HTLV-1 antibody formation in the F344 rat model. None of 10 control rats inoculated with normal donor PBMC showed antibodies to HTLV-1, nor the proviral genome. The present study suggests that HTLV-1 plays a cofactor role in MF/SS patients.

Cutaneous T cell lymphomas: mycosis fungoides, Sezary syndrome and HTLV-I-associated adult T cell leukemia (ATL) in Mali, West Africa: a clinical, pathological and immunovirological study of 14 cases and a review of the African ATL cases.

Cutaneous T cell lymphomas (CTCL) are rare lymphoproliferative diseases, which are frequently suspected to be of viral origin. As very few data were available concerning cutaneous T cell lymphomas in tropical Africa, we undertook a clinical, histopathological, immunological and viro-molecular study of patients with a clinical diagnosis of cutaneous lymphoma, in Bamako, Mali. While prior to this study, no case of CTCL had been reported in this country, 14 patients (five women, nine men; mean age 58 years) with a diagnosis of cutaneous lymphoma were seen over a period of 30 months (1992-1994) in the only dermatological department in Mali. Clinically, the most frequent pattern was an infiltrated erythrodermia similar to Sezary syndrome. Nodular lesions and/or plaques were rarely observed. All these cutaneous tumors were T cell lymphoproliferations, only one expressing the CD8+ antigen. A comprehensive analysis of all the available data permitted characterization of three cases of adult T cell leukemia/lymphoma (ATL) associated with HTLV-I (one definitive case, of leukemic type, with demonstration of clonal integration of HTLV-I proviral genome and two probable ATL cases), three cases of Sezary syndrome (SS), two cases of mycosis fungoides (MF) and five cases of pleomorphic cutaneous lymphoma. In one case, the differentiation between MF and pleomorphic cutaneous lymphoma could not be established. HTLV-I serological and/or molecular markers were restricted to the three ATL cases. From the unique definitive ATL case, a T cell line was established from culture of peripheral blood mononuclear cells and sequence analysis of the env gene and the U3-LTR region demonstrated that the virus present in this patient belonged to the cosmopolitan subtype A. Thus, we report here the first evidence of HTLV-I infection and associated ATL in Mali. This is the second ATL case described for the whole Sahelian region (one ATL of the lymphoma type was reported previously in a Mauritanian patient). Furthermore, we demonstrate that the main types of CTCL described in Europe and North America are also present in this African area and that the prevalence of these diseases is greatly underestimated in such regions. Furthermore, no association was observed between HTLV-I/II infection and SS, MF or pleomorphic cutaneous lymphoma in Mali in contrast to other studies.

Increased anti-Epstein-Barr virus antibodies in epidermotropic cutaneous T-cell lymphoma: a study of 64 patients.

Epstein-Barr virus (EBV) is often associated with non-Hodgkin's T-cell lymphomas and has recently been found in the lesions of mycosis fungoides and Sézary syndrome. We sought to determine whether the anti-EBV antibody profile was disturbed in mycosis fungoides and Sézary syndrome and whether there are particular profiles characteristic of disease stage. Anti-EBV antibodies (anti-VCA, -EA and -EBNA) were studied in the sera of 64 patients. An immunoenzymatic technique was used, and the results were compared with the same number of age- and sex-matched healthy controls. Patients with mycosis fungoides and Sézary syndrome developed higher anti-VCA antibody titres (median 1200) than controls (median 320). Thirty-seven patients had anti-VCA > or = 1200 vs. 19 controls (P < 0.01). These elevated anti-VCA antibody titres were associated with positive EA in 19 patients versus three controls. No differences were found between the illness stages. Anti-EBV antibodies were most often found in mycosis fungoides and Sézary syndrome when the serological profile was similar to that of cellular immune deficiencies and EBV-related non-Hodgkin's lymphoma. EBV could be involved, either directly on lymphocytes or, more likely, indirectly by chronic antigenic stimulation.

HTLV-associated diseases: human retroviral infection and cutaneous T-cell lymphomas.

An array of neurologic, oncologic, and autoimmune disorders are associated with infection with the human pathogenic retroviruses human T-cell leukemia virus types I and II (HTLV-I, II), as well as the human immunodeficiency viruses (HIV). The cutaneous T-cell lymphomas, mycosis fungoides (MF) and its hematogenous variant Sezary Syndrome (SS), share similar clinical and pathological features to HTLV-I-associated adult T-cell leukemia (ATL) and speculation of a retroviral link to MF and SS, especially in areas non-endemic for ATL, has lead to an intensified search for HTLV- and HIV-like agents in these diseases. To further explore a potential role for human retroviruses in MF and SS, skin biopsy-derived or peripheral blood mononuclear cell-derived DNA from 17 patients (MF, n = 7; erythrodermic MF (EMF), n = 5; SS, n = 5) from the North Eastern United States were screened using gene amplification by PCR and a liquid hybridization detection assay. Previously published primers and probes for HTLV-I (LTR, gag, pol, env, and pX), and our own primers and probes for HTLV-I (gag, pol, and env), HTLV-II (pol and env) and HIV-I (gag and pol) were employed. Serum antibodies to HTLV-I were negative in all but one EMF patient. The single HTLV-I seropositive patient carrying a diagnosis of EMF generated positive amplified signals for all of the eight HTLV-I regions tested. Ultimately, this individual evolved to exhibit clinical manifestations indistinguishable from ATL. The other 16 patients were negative for all 12 HTLV and HIV retroviral regions. Our findings suggest that none of the known prototypic human retroviruses are associated with seronegative MF and SS. The uniformly positive results for HTLV-I in the seropositive patient suggests that this patient initially presented with a smoldering form of ATL and illustrates the difficulty that sometimes may be encountered in the differential diagnosis of MF, SS, and ATL based solely on clinical and histopathological criteria.

Demonstration of antibodies to human T-cell lymphotropic virus-I tax in patients with the cutaneous T-cell lymphoma, mycosis fungoides, who are seronegative for antibodies to the structural proteins of the virus.

Although most patients with the cutaneous T-cell lymphoma, mycosis fungoides (MF), are seronegative for human T-cell lymphotropic virus-I or -II (HTLV-I/II) when tested by assays that measure only antibodies to the viral structural proteins, the majority of such patients harbor HTLV-I-related pol and tax proviral sequences that encode proteins not included in routinely used serologic tests. Tax mRNA has also been detected in their peripheral blood mononuclear cells (PBMC). Therefore, it seemed possible that these patients have antibodies to the tax protein. To investigate this, enzyme-linked immunosorbent assays (ELI-SAs) and Western blot assays were set up, using as antigens the full-length HTLV-I tax cloned from the prototypic HTLV-I-infected cell line, C91PL, and from PBMC of a MF patient, as well as a synthetic peptide made to the carboxy-terminal 20 amino acids of tax-I. Of 60 MF patients whose PBMC were shown to be positive for tax proviral DNA and mRNA, 50 (83%) were shown to have tax antibodies. The antigen derived from the MF patient was most useful in detecting such antibodies. These results demonstrate the need for including other HTLV-related antigens in addition to gag and env in serologic tests used to identify HTLV-infected individuals. The findings underscore the fact that individuals considered seronegative on the basis of currently used tests can be infected with HTLV.