Long-term implications of T-cell receptor gene rearrangement analysis by Southern blot in patients with cutaneous T-cell lymphoma

BACKGROUND: T-cell clonality analysis by Southern blot (TSB) in skin biopsy specimens suggestive of mycosis fungoides may be helpful in confirming the diagnosis of a cutaneous lymphoma. However, there are no data available regarding the long-term prognostic implication of such results. OBJECTIVES: We sought to determine the long-term prognostic significance of TSB results from skin biopsy specimens of patients with mycosis fungoides. METHODS: We reviewed the records from the Cleveland Clinic Foundation and Northwestern University Medical Center for cases of biopsy-proven mycosis fungoides with results available for skin biopsy TSB from 1987 to 1990. RESULTS: The detection of clonality by TSB correlates with a higher TNM stage (median stage for positive TSB, IIb vs negative TSB, Ib; P <.05), but not with age at presentation (62 vs 59 years) or duration of disease before presentation (6.2 vs 5.9 years). Although the long-term survival was not significantly different between the 2 groups, there was a trend for patients with positive TSB to die earlier (5-year survival of 67% vs 87%). Disease progression did not correlate with TSB results. Higher clonality rates were noted among patients with biopsy specimens showing a denser lymphoid infiltrate and a higher grade of cytologic atypia. CONCLUSIONS: Detection of clonality with TSB requires a significant clonal burden. Although clonality can be detected in patients with patches and plaques (T1 and T2) most cases with positive results were obtained from patients with advanced disease (T3 and T4). In our experience, detection of clonality by TSB does not correlate with disease progression and does not carry long-term prognostic implications.     

PCR-heteroduplex analysis of T-cell receptor gamma gene rearrangement in paraffin-embedded skin biopsies

We developed a rapid, simple, and sensitive method for the detection of T-cell receptor-gamma (TCRgamma) gene rearrangements in paraffin-embedded skin biopsies. Available techniques often require either fresh tissue, several primer pairs, nested amplifications, or specialized electrophoresis steps such as denaturing gradient gel electrophoresis. Our method is based on heteroduplex analysis of polymerase chain reaction (PCR) products of the TCRgamma in a nondenaturing modified polyacrylamide gel using a single pair of primers and is adapted for paraffin-embedded tissue. When tested against Southern blot analysis, the PCR results correlated in 8 of 9 cases. Six mature cutaneous B-cell lymphomas and 29 inflammatory skin disorders all resulted in a polyclonal amplification pattern. When analyzing 3-mm or 4-mm punch biopsies of 51 cases of cutaneous T-cell lymphoma, 37 (72.5%) showed a clonal rearrangement with this technique. For 7 cases of patch stage mycosis fungoides, frozen tissue and formalin-fixed and paraffin-embedded tissue was available, and in 5 of 7 cases (71%), the results in frozen and paraffin-embedded tissue were concordant. One case showed a clonal pattern in frozen tissue but not in paraffin-embedded tissue, and one case was polyclonal in frozen tissue but monoclonal in paraffin-embedded tissue. Using serial dilutions of DNA from a T-cell ALL in a polyclonal background (tonsil), we established a sensitivity of 0.5%. Heteroduplex PCR of the TCRgamma is a rapid, sensitive, and inexpensive screening procedure as well as a useful adjunct to histologic analysis and immunophenotyping of cutaneous T-cell proliferations.     

皮肤T细胞淋巴瘤的T细胞受体基因克隆性重排

目的 研究皮肤T细胞淋巴瘤（CTCL）早期诊断。方法 利用PCR方法，设计PCRVγ1-8,Vγ9,Jγ1/γ2特异引物，分析24例各类型CTCL，2例可疑CTCL及4例非特异性红皮病患者的33份示TCRVγ1-8,37份示Vγ9呈MCGR，BCGR或OCGR，尤其是13例早期蕈样肉芽肿，2例可疑CTCL和4例非特异性红皮病均呈TCRγ-GR克隆性扩增带，6例炎性病变标本示TCRγ-GR克隆性，提示他们为“克隆性皮炎”，需长期随访。结论 用PCR发现早期CTCL的PCRγ-GR克隆性，为诊断提供依据。     

T-cell receptor gene analysis in cutaneous T-cell lymphomas

An essential property of the immune system is its ability to generate diverse antibody and T-cell mediated responses to virtually any potential foreign particle, The basic molecular mechanisms responsible for producing this extensive diversity have now been elucidated. Each T cell expresses a unique membrane hound T-cell antigen receptor (TCR) which combines with specific antigenic peptides and major histocompatibility complex molecules. The characterization of TCR usage now represents a focal point for many studies of inflammatory and neoplastic disorders. Such studies are helping to clarify the pathogenesis of T-cell mediated diseases and provide the basis for the development of specific therapies. This paper will review several techniques used to identify neoplastic T-cell clones in cutaneous T-cell lymphoma. Similar methods may be used to analyse TCR gene usage in cutaneous inflammatory dermatoses.     

T-cell receptor-gamma gene analysis in evolving to advancing cutaneous T-cell lymphoma

The diagnosis of cutaneous T-cell lymphoma is often a challenge for the dermatopathologist. Early stages can mimic inflammatory dermatoses. Our aim was to explore the applicability of a standard T-cell receptor-gamma polymerase chain reaction in various subtypes of cutaneous T-cell lymphomas. Ninety-six biopsy specimens from 38 patients were selected. These included 72 specimens of mycosis fungoides, 12 specimens of non-mycosis fungoides T-cell lymphomas, and 12 specimens in which histology was non-specific or equivocal in patients who were later diagnosed to have lymphoma. T-cell clones were detected in 53 of 72 specimens of mycosis fungoides and eight of 12 specimens of non-mycosis fungoides lymphomas. Of the 72 specimens of mycosis fungoides, T-cell clones were detected in eight of 10 specimens of mycosis fungoides-associated follicular mucinosis and pigmented purpura-like mycosis fungoides. Four specimens from the 12 prediagnostic for cutaneous T-cell lymphomas showed presence of T-cell clones, identical to subsequent clones detected when lymphoma was fully established. In specimens where histology is not diagnostic and T-cell receptor-gamma gene analysis is positive, patients should be followed up closely. T-cell receptor-gamma gene analysis is a useful adjunct to histological diagnosis of early stage and variant types of mycosis fungoides.     

Molecular analysis of T-cell receptor beta genes in cutaneous T-cell lymphoma reveals Jbeta1 bias

Molecular characterization of T-cell receptor junctional region sequences in cutaneous T-cell lymphoma had not been previously reported. We have examined in detail the features of the T-cell receptor beta (TCRB) gene rearrangements in 20 individuals with well-defined stages of cutaneous T-cell lymphoma (CTCL) comprising 10 cases with early-stage mycosis fungoides (MF) and 10 cases with late-stage MF or Sezary syndrome. Using BIOMED-2 PCR primers, we detected a high frequency of clonally rearranged TCR gamma and TCRB genes (17/20 and 15/20 cases, respectively). We carried out sequencing analysis of each complete clonal variable (V)beta-diversity (D)beta-joining(J)beta fingerprint generated by PCR amplification, and determined the primary structure of the Vbeta-Dbeta-Jbeta junctional regions. We observed considerable diversity in the T-cell receptor Vbeta gene usage and complementarity-determining region 3 loops. Although we found that TCRB gene usage in CTCL and normal individuals share common features, our analysis also revealed preferential usage of Jbeta1 genes in all cases with advanced stages of disease.     

Nasal-type T/natural killer cell angiocentric lymphoma, Epstein-Barr virus-associated, and showing clonal T-cell receptor gamma gene rearrangement

Nasal-type T/natural killer (NK) cell lymphoma, which often shows an angiocentric growth pattern, is a distinct clinicopathological entity highly associated with the Epstein-Barr virus (EBV). This tumour has a characteristic immunophenotype, whereas the cytological spectrum is broad. It is known that a clonal T-cell receptor (TCR) gene rearrangement is not found in this tumour. However, it is still unresolved as to whether the finding of a clonal TCR gene rearrangement excludes the diagnosis of nasal-type T/NK cell lymphoma. We describe a case of nasal-type T/NK cell angiocentric lymphoma, EBV-associated, and showing clonal TCR gamma gene rearrangement. The patient died of sepsis 5 months after diagnosis in spite of aggressive chemotherapy.     

New techniques in the evaluation of cutaneous T-cell lymphoma

The prognosis of mycosis fungoides and Sézary syndrome can be improved when antitumor therapies such as total skin electron beam irradiation, topical nitrogen mustard, or systemic cytostatic agents are given as soon as the diagnosis is established. However, differentiation of early lesions of mycosis fungoides and Sézary syndrome from chronic benign dermatoses remains very difficult. To aid in the differential diagnosis, more objective techniques have been developed, including DNA cytophotometry, chromosome analysis, quantitative electron microscopy, and monoclonal antibody staining. Using these methods, skin infiltrates and lymph nodes can be classified more precisely as malignant or benign at an earlier stage of the disease. It must be stressed that these methods can be used only in conjunction with other clinical and histomorphologic criteria. They should never be used alone, however, because reactive processes may sometimes cause problems in interpreting the results.     

T-cell receptor gene rearrangement in regressing atypical histiocytosis

A case of regressing atypical histiocytosis having characteristic clinical and light microscopic findings was studied immunologically for immunoglobulin and T-cell receptor gene rearrangement and for DNA ploidy analysis. Immunologic phenotyping and rearrangement of T-cell receptor Beta- and gamma-chain genes indicated that this primary cutaneous neoplasm, previously considered "histiocytic" in origin, is most probably of T-cell lineage.     

TCRgamma-chain gene rearrangement by PCR-based GeneScan: diagnostic accuracy improvement and clonal heterogeneity analysis in multiple cutaneous T-cell lymphoma samples

Cutaneous T-cell lymphomas are a heterogeneous group of lymphomas where the tumor population emerges within a multiple subclone pattern ("clonal heterogeneity"). PCR analysis has been shown to be useful in the diagnosis of mycosis fungoides (MF) and Sézary Syndrome (SS). Focusing the attention on clonal heterogeneity, the efficacy of the multiplex/heteroduplex (HD) PCR and the GeneScan (GS) capillary electrophoresis analysis was compared in the early diagnosis of MF/SS, using a multiple sample approach. Indeed, GS demonstrated TCRgamma gene rearrangement (GR) in all the 57 SS (100%) and in 123/146 (84%) of the MF samples, whereas the multiplex/HD PCR was less sensitive. An increase in clonality was observed in connection with both a worsening of the cutaneous disease (79% T1/T2; 100% T3/T4) and an increase in the histopathological score (HS < 5, 76%; HS > or = 5, 94%). Clonal heterogeneity with adjunctive reproducible skin TCRgamma-GRs was also observed. "Clonal instability," with different GRs, was present in a small percentage of patients. Therefore, it can be concluded that GS analysis in TCRgamma-GR is able to improve diagnosis in MF/SS patients and the multiple sample approach is helpful for a correct interpretation of clonal patterns in skin lesions, especially in early-stage MF and in SS skin/blood samples.     

原发性皮肤T细胞淋巴瘤T细胞受体基因重排检测

Southern印迹分析（SBA）和取和合酶链反应（PCR）法检测早期（IA）和组织学上未确诊的蕈样肉芽肿（MF）皮肤损害的T细胞受体基因重排（TCRGR）阳性率分别为50％以上和19％,有助于诊断。IIA期MF患者特别是伴浅表淋巴结肿大（组织学上大都是反应性增生）的外周血中TCRGR阳性率较高（65％－80％）,支持MF早期即为一系统性疾病的理论,但外周血中克隆T细胞和预后相关性则难于确定。Sezary综合征（SS）的TCRGR的发生率在皮肤、淋巴结和外周血中分别为70％、100％和86％。非MF／SS皮肤T细胞淋巴瘤的皮肤 损害和外周血中TCRGR亦较常见。淋巴瘤样丘疹病的皮肤损害和外周血中也可见TCRGR。     

Lymphomatoid papulosis. Development into cutaneous T-cell lymphoma

Lymphomatoid papulosis is usually considered to have a benign course, but many reports of subsequent evolution into systemic lymphoma have been reported. By measuring single-cell DNA content by flow cytometry, it may be possible to predict those cases that have the potential for the development of a malignant neoplasm. Two cases that differ from classic benign lymphomatoid papulosis had a more "malignant" clinical picture, with nodules and tumors, and the finding of aneuploidy (abnormal DNA content) from several skin lesion specimens and also from a lymph node specimen in one of the cases. Clinically evident malignant neoplasms have not yet developed in the two patients, but we suggest that the finding of aneuploidy predicts those cases that later could become malignant.     

Analysis of T-cell receptor gene rearrangement for predicting clinical outcome in patients with cutaneous T-cell lymphoma: a comparison of Southern blot and polymerase chain reaction methods

OBJECTIVE: To extend previous observations regarding the prognostic value of analyzing lymph node DNA from patients with cutaneous T-cell lymphoma for the presence of a monoclonal T-cell population by Southern blot vs polymerase chain reaction (PCR) methods. DESIGN: Inception cohort study from 1982 to 1998. Recruitment of new patients ended in 1994. SETTING: A tertiary care referral center in Seattle, Wash.Patients Fifty-five uniformly staged patients with the diagnosis of cutaneous T-cell lymphoma who underwent a lymph node biopsy, 21 with clinically abnormal nodes and 34 with normal nodes.Interventions Lymph nodes were evaluated for T-cell receptor (TCR) gamma-chain gene rearrangement by 2 PCR methods: capillary electrophoresis and denaturing gradient gel electrophoresis. The same lymph nodes were evaluated by Southern blot analysis for TCR beta-chain gene rearrangement and examined histopathologically on the basis of the National Cancer Institute lymph node classification system. Patients were observed clinically for a mean of 9.5 years. MAIN OUTCOME MEASURES: Skin stage, clinical lymph node examination, lymph node histologic examination, Southern blot analysis, and PCR analyses were evaluated as potential prognostic predictors by univariate and multivariate analyses. The statistical association of TCR analysis and clinical outcome was determined among all patients. Hazard ratios (HRs) by Cox proportional hazards regression analysis were used to estimate the risk of a poor clinical outcome. Cumulative survival rates were analyzed by the Kaplan-Meier method. RESULTS: A skin stage of T3 (tumors) or T4 (erythroderma) was the most powerful predictor of a poor clinical outcome (HR, 31.3 vs T1; P<.001). Patients with detectable TCR gamma-chain gene rearrangement in lymph node DNA by PCR also were more likely to have a poor outcome (HR, 5.1; P<.001), but it was a less powerful predictor than skin stage. Even when the skin stage, presence or absence of lymphadenopathy, and histologic lymph node score were known for the patient, Southern blot analysis still added to prediction of a poor outcome (HR, 9.3; P = .007), whereas PCR provided no statistically significant additional information on outcome. CONCLUSIONS: Detection of a monoclonal T-cell population by PCR in lymph nodes of patients with cutaneous T-cell lymphoma does not enhance prediction of clinical outcome and probability of survival beyond what can be determined from clinical examination and histologic lymph node scores. Skin stage and the presence or absence of lymphadenopathy remain the most important determinants of clinical outcome.     

T-cell receptor variable region gene expression in cutaneous T-cell lymphomas

The cutaneus T-cell lymphomas (CTCL) arc a group of diseases characterized by malignant proliferations of CD4 positive T-cells having monoclonally rearranged T-cell receptor (TCR) genes. A recent study using monoclonal antibodies to two TCR β-chain variable (V) region gene products showed preferential expression of the Vβ8 gene product in these tumors. The finding of predominant usage of a single Vβ gene would imply that selection by antigen is important in the etiology of these tumors. We have studied eight cases of cutaneous T-cell lymphoma and one cell line derived from a patient with mycosis fungoides/Sezary syndrome, using an extended panel of antibodies to V region gene products. Contrary to the previous report, in our study expression of the Vβ8 gene product by tumor cells was not observed in any of the cases of CTCL or in the tumor cell line studied; preferential use of any of the variable region genes recognized by the antibodies in the panel was not observed.     

A rare case of the cutaneous form of adult T-cell leukaemia/lymphoma: assessment of remission by PCR for clonal T-cell receptor gamma gene rearrangements in an electron beam-irradiated cutaneous lesion

Adult T-cell leukaemia/lymphoma is a lymphoproliferative disorder aetiologically associated with human T-cell lymphotropic virus type I infection. A cutaneous lesion often develops in the disease, and in rare cases, is even the only manifestation. Here we report a rare case of 'cutaneous' adult T-cell leukaemia/lymphoma with neither atypical cells in the peripheral blood nor lymph node involvement. All nodular lesions were completely eliminated after local electron beam irradiation (20 Gy/nodule in total). To evaluate whether or not there were residual lymphoma cells in the skin, we performed PCR to detect clonal T cell receptor gamma gene rearrangements. The sample from the nodule before irradiation showed evidence of a rearranged band, which was not detected at the same site after treatment nor in any peripheral blood. The findings suggest that this procedure is useful for the evaluation of therapeutic effects and the early detection of lymphoma recurrence.     

皮肤T细胞淋巴瘤表观遗传学研究进展

表观遗传学主要是研究在不改变DNA序列的情况下,发生的基因表达水平可遗传的变化,主要包括DNA甲基化、组蛋白修饰和非编码RNA调控.皮肤T细胞淋巴瘤是一组原发于皮肤的T淋巴细胞恶性增殖性疾病,蕈样肉芽肿和Sezary综合征是最常见的皮肤T细胞淋巴瘤.近年来研究表明,表观遗传学不仅在皮肤T细胞淋巴瘤的发生发展中起重要作用,而且可以通过改变表观遗传达到治疗皮肤T细胞淋巴瘤目的.     

Aneuploidy in cutaneous T-cell lymphoma

A new method is described which makes it possible using skin specimens to perform flow cytometric analysis of DNA content. DNA content analysis was performed on 28 skin specimens and 9 blood samples from 18 patients with mycosis fungoides and Sézary syndrome. The reproducibility was fair, with almost identical results in 6 cases (mycosis fungoides and Sézary syndrome) where two samples (skin specimens or blood samples) were taken 6 hours to 10 days apart. Hyperdiploidy was found in 7 of 11 skin specimens from patients with mycosis fungoides stage I with negative histology. In 13 skin specimens and 3 blood samples from patients with mycosis fungoides stages II and IV, abnormalities including hyperdiploidy, near-tetraploidy and near-hexaploidy were found in 8 of 13 skin specimens and in 2 of 3 blood samples. Four patients with Sézary syndrome were studied: 2 patients in remission showed normal DNA histograms (2 skin specimens, 3 blood samples) and 2 patients with active disease showed aneuploidy in the 2 skin specimens examined and in 1 of 3 blood samples. These studies demonstrate: 1) the importance of flow cytometry as a diagnostic tool for use on skin specimens in the early stages of mycosis fungoides where routine histology is non-diagnostic; 2) the diagnostic and prognostic aid of flow cytometry during the course of mycosis fungoides and Sézary syndrome in addition to the probability of measuring the effect of treatment.     

Mapping toll-like receptor activity in different stages of cutaneous T-cell lymphoma

ABSTRACT:: It is known that human keratinocytes (KCs) express Toll-like receptors (TLRs). However, published reports conflict regarding TLR expression in cutaneous T-cell lymphoma patient's KCs. To define the pattern of expression and detect any differences of TLRs 1-9 and p65 expression in epidermal KCs, tumor infiltrate, and endothelial cell types using immunohistochemical stains on fixed and paraffin-embedded sections of mycosis fungoides (MF) in patch, plaque, and nodular stages. MF cases showed no change in pattern of TLRs expressed through different stages but increased epidermal staining of TLRs 2, 3, 4, 5, 6, and 8 with higher scores associated with more aggressive stages. Endothelial cell staining was increased for TLR 4 and 6. Tumor infiltrate staining was strongest with TLRs 5 and 7. Individual cases with disease progression showed increased intensity of TLRs 4, 5, and 6 staining in the epidermis, tumor infiltrate, and endothelial cell. p65 verified nuclear factor kappa B activation of the TLR pathway with trace staining of the epidermis and 1-2+ staining of tumor infiltrate. MF cases showed increased epidermal expression of TLRs and increased endothelial cell staining compared with controls. TLR expression may be driven by antigenic stimulation and may play a role in the activation of neoplastic T cells in the skin. Further definition of TLR patterns may refine the use of TLR modifiers for treatment.