T-cell receptor gene rearrangement analysis of sequential biopsies in cutaneous T-cell lymphomas with the Biomed-2 PCR reveals transient T-cell clones in addition to the tumor clone.

Detection of a dominant T-cell clone by T-cell receptor (TCR) gene rearrangement analysis is often essential for the diagnosis of cutaneous T-cell lymphomas (CTCL). The occurrence of T-cell clones in addition to the diagnostic T-cell clone during the course of CTCL has been reported, but the data of these studies have been contradictory. We retrospectively evaluated the data of 114 lesional skin biopsies from 26 patients with Mycosis fungoides and two patients with primary cutaneous anaplastic large cell lymphoma, which were analysed with the standardized Biomed-2 PCR for the TCRγ and TCRß locus. A dominant T-cell clone was repetitively detected in 93% (26/28) of patients. Additional T-cell clones appeared temporarily in 39% (11/28) of patients. Correlation with the clinical data did not show an association of the presence of additional T-cell clones with age, number of treatments, progression of disease or survival. Our findings demonstrate that a persistent T-cell clone, most likely the disease causing tumor clone, is detectable in almost all CTCL patients. In addition, transiently appearing T-cell clones frequently occur during the course of disease. The biological relevance of these additional clones has still to be determined. However, it is important to take the possibility of additional T-cell clones into account for diagnostic analyses.

Deficiency of IL-22 contributes to a chronic inflammatory disease: pathogenetic mechanisms in acne inversa.

Overexpression of the T cell cytokine IL-22 is linked to the development of some chronic diseases, but little is known about IL-22 deficiency in humans. As demonstrated in this study, acne inversa (AI; also designated as Hidradenitis suppurativa) lesions show a relative deficiency of IL-22 and IL-20, but not of IL-17A, IL-26, IFN-γ, IL-24, or IL-1ß. Moreover, AI lesions had reduced expression of membranous IL-22 and IL-20 receptors and increased expression of the natural IL-22 inhibitor, IL-22 binding protein. AI is a chronic inflammatory skin disease with prevalence up to 4% of the population and in which cutaneous bacterial persistence represents an important pathogenetic factor. Accordingly, we also found a relative deficiency of antimicrobial proteins (AMPs) in AI lesions and a positive correlation between lesional IL-22 and IL-20 versus AMP levels. IL-22, like its tissue cell downstream mediator IL-20, upregulated AMPs in reconstituted human epidermis and was critical for increased AMP levels under inflammatory conditions. The relative IL-22 deficiency in AI was not linked to lesional T cell numbers or Th22/Th1/Th17 subset markers and -inducing cytokines. However, IL-10 was highly expressed in AI lesions and correlated negatively with IL-22 expression. Moreover, IL-10 inhibited IL-22 but not IL-17 production in vitro. The IL-10 overexpression, in turn, was not associated with an elevated presence of regulatory T cells but with the enhanced presence of an IL-10-inducing cytokine. We conclude that IL-22 deficiency may contribute to the pathogenesis of certain chronic disorders as postulated in this paper for AI.

Prevalence of the MspI and Ile462Val SNPs of cytochrome P-450 1A1 in hidradenitis suppurativa.

Hidradenitis Suppurativa (HS) is a chronic inflammatory skin disease that affects the hair follicles in the axillary, perianal and inguinal area. Its cause and pathogenesis are unknown, but cigarette smoking increases the risk of developing HS conceivably by accumulating toxic metabolites in sweat. The xenobiotic compounds from tobacco are metabolized by the cytochromes P-450. The cytochrome P-450 1A1 (CYP1A1), one of the most active isoenzymes, harbours several polymorphisms. Two of them, MspI and Ile(462)Val single nucleotide polymorphism (SNP), are associated with enhanced activity and inducibility. Performing direct DNA sequencing, we investigated the frequencies of these SNP in 51 patients with HS, 45 of these were smokers. We found similar overall SNP rates in our patients in comparison with previous data for Caucasian or German controls. Obviously, there is no relation between the occurrence of these SNPs and the risk of developing HS.

Evaluation of T-cell clonality in archival skin biopsy samples of cutaneous T-cell lymphomas using the biomed-2 PCR protocol.

Recently, several European centers of lymphoma diagnosis and research developed various polymerase chain reaction (PCR) methods for clonality analysis in suspect T-cell and B-cell proliferations (Biomed-2 Concerted Action). They have mainly been applied to frozen material of systemic B-cell and T-cell malignancies. Thus far, only limited data exist with regard to cutaneous T-cell lymphoma (CTCL) and paraffin-embedded material. Thus, we applied the Biomed-2 T-cell receptor (TCR) gamma and TCRbeta PCR as well as an in-house TCRgamma PCR to a collection of 107 archival skin samples (84 CTCL, 3 systemic TCL and 20 controls). As a result, the Biomed-2 TCRgamma PCR revealed 81% clonality, the in-house TCRgamma method revealed 86% clonality, and the Biomed-2 TCRbeta revealed 78% clonality in CTCL samples generating at least the 300 bp fragment in the Biomed-2 control PCR. We found clonal TCRbeta rearrangements in 5 of 17 CTCL samples that were polyclonal in the Biomed-2 TCRgamma PCR. By combining all Biomed-2 assays, one or more clonal rearrangements were detected in 87% of CTCL and in all 3 systemic TCLs. By combining all TCR PCR assays applied here, clonality was shown in 90% of the CTCL cases. In conclusion, we showed that the Biomed-2 TCR PCR worked well with DNA from paraffin-embedded tissue, revealing a high-clonality detection rate in CTCL, and thus should be highly recommended for routine molecular analysis. In addition, the performance of our in-house TCRgamma assay verifies our previously published findings on clonally expanded T-cells in CTCL.

Prevalence of genetically defined tumor cells in CD7 as well as CD26 positive and negative circulating T-cell subsets in Sézary syndrome.

For diagnosis and monitoring of Sézary syndrome flow cytometric quantification of CD7- and CD26- T-cells is widely used. Because antigen loss is a characteristic but not disease-specific finding we investigated the significance of this approach. Therefore we analyzed the prevalence of tumor cells in FACS-sorted CD7+/- as well as CD26+/- circulating T-cells applying a clone-specific qualitative and quantitative T-cell receptor PCR. Tumor cells varied considerably in the CD7+ and CD7- cell subset but were largely confined to the CD26- population. We conclude that quantification of CD26- T-cells reflects the tumor cell amount more accurate and should be preferred in the clinical setting.

Dominance of nonmalignant T-cell clones and distortion of the TCR repertoire in the peripheral blood of patients with cutaneous CD30+ lymphoproliferative disorders.

The CD30-positive cutaneous lymphoproliferative disorders (CLPD) include lymphomatoid papulosis (LyP) and primary cutaneous anaplastic large T-cell lymphoma (cALCL). Despite the malign-appearing histology, an excellent prognosis and spontaneous regression of single lesions characterize LyP. Even after years of clinical remission newly erupting lesions often harbor a T-cell clone identical to the initial one. This fact raises the question whether the clonal T-cell population persists in the peripheral blood. Therefore we investigated genomic DNA of 126 samples of lesional skin and peripheral blood from 31 patients with CLPD, obtained during both active disease and clinical remission. We performed molecular genetic analysis by combining T-cell receptor (TCR)-gamma PCR with the GeneScan technique and assessed the TCR repertoire in selected blood samples by beta-variable complementarity-determining region 3 (CDR3) spectratyping qualitatively and quantitatively. We were able to detect a clonal T-cell population in 36/43 (84%) skin samples and in 35/83 (42%) blood samples. Comparison of the compartments in each patient demonstrated different T-cell clones in skin and blood, suggesting a reactive nature of the clonal T cells in the blood. Moreover, CDR3 spectratyping revealed a restricted T-cell repertoire in the blood, suggesting T-cell stimulation by an unknown antigen.

Impact of molecular staging methods in primary melanoma: reverse-transcriptase polymerase chain reaction (RT-PCR) of ultrasound-guided aspirate of the sentinel node does not improve diagnostic accuracy, but RT-PCR of peripheral blood does predict survival.

PURPOSE: This study analyzes (1) the value of tyrosinase reverse-transcriptase polymerase chain reaction (RT-PCR) of aspirates obtained by ultrasound-guided fine-needle aspiration cytology (US-FNAC) of sentinel nodes (SNs) in patients with melanoma before sentinel lymph node biopsy (SLNB) and (2) the value of RT-PCR of blood samples of all SLNB patients. PATIENTS AND METHODS: Between 2001 and 2003, 127 patients with melanoma (median Breslow depth, 2.1 mm) underwent SLNB. FNAC was performed in all SNs of all patients pre- and post-SLNB. The aspirates were partly shock-frozen for RT-PCR and were partly used for standard cytology. Peripheral blood was collected at the time of SLNB and at every outpatient visit thereafter. RESULTS: Thirty-four (23%) of 120 SNs were positive for melanoma. SN involvement was predicted by US-FNAC with a sensitivity of 82% and a specificity of 72%. Additional tyrosinase RT-PCR revealed the same sensitivity of 82% and a specificity of 72%. At a median follow-up time of 40 months from first blood sample, peripheral-blood RT-PCR was a significant independent predictor of disease-free survival (DFS) and overall survival (OS; P < .001). CONCLUSION: US-FNAC is highly accurate and eliminates the need for SLNB in 16% of all SLNB patients. RT-PCR of the aspirate or excised SN does not improve sensitivity or specificity. RT-PCR of blood samples predicts DFS and OS.

Complete clinical remission of tumor-stage mycosis fungoides after acute extensive skin necroses, granulomatous reaction, and fever under treatment with bexarotene, vorinostat, and high-dose fenofibrate.

Mycosis fungoides and its variants are a distinct entity with a variable, but well-characterized clinical course. We report on a 51-year-old patient with tumor-stage mycosis fungoides who developed several unusual features such as extensive necrosis of lymphoma lesions, granulomatous reaction, and venular thromboses while under treatment with bexarotene, vorinostat, and high-dose fenofibrate. After surgical removal of skin necroses, the patient recovered and was in complete clinical remission. Possible causal factors such as blastic transformation; hematophagic syndrome; or bacterial, fungal, or viral infection could be excluded. We hypothesize that combination of the high-dose fenofibrate (400 mg) with the retinoid X receptor ligand bexarotene and vorinostat might have induced an increased rate of apoptosis in lymphoma cells in our patient resulting in an extensive release of lymphoma antigens. Augmented antigen release along with changes in local cytokine milieu might have induced macrophage activation and granuloma formation.

Differential expression of TRAF1 aids in the distinction of cutaneous CD30-positive lymphoproliferations.

Lymphomatoid papulosis (LyP), primary cutaneous anaplastic large T-cell lymphoma (cALCL), and cutaneous infiltrates of systemic anaplastic large cell lymphoma (sALCL) are CD30-positive lymphoproliferative disorders of the skin that overlap clinically, histopathologically, immunophenotypically, and genetically but differ considerably in their prognosis. In particular, lesions of LyP regress spontaneously, whereas those of cALCL and sALCL persist and may progress and spread to extracutaneous sites. In contrast to patients with cALCL, LyP patients do not benefit from an aggressive radio- and/or chemotherapeutic approach. We generated a novel tumor necrosis factor receptor (TNFR)-associated factor 1 (TRAF1) antibody that recognizes a formalin-resistant epitope (Ber-TRAF1A) and investigated the expression of TRAF1, an intracellular component of TNFR signaling, in LyP and ALCL. We could show a strong TRAF1 expression in the tumor cells of most LyP cases (42/49, 84%). In contrast, tumor cells of primary and secondary cALCL revealed TRAF1 expression in only a few cases (3/41, 7%) as shown for sALCL without skin manifestation. The data indicate that TRAF1 expression reliably distinguishes LyP from primary or secondary cALCL. This might be of crucial diagnostic importance and has a strong impact on the treatment decision for patients with cALCL and LyP.

Absence of microsatellite instability and lack of evidence for subclone diversification in the pathogenesis and progression of mycosis fungoides.

Mutator phenotypes with microsatellite instability (MSI) correlated with defects in the mismatch repair system are characteristic for a subset of solid neoplasms, but are rare in non-Hodgkin lymphomas. In mismatch repair-deficient mice, however, mutator-type non-Hodgkin lymphomas are the most frequent tumors. To determine the role of MSI in mycosis fungoides, we compared the states of the eight dinucleotide microsatellite loci DXS418, DXS453, DXS556, DXS1060, D1S201, D6S260, D9S162, and D10S215 in tumor cells of 12 well-characterized patients at early- and advanced-stage diseases to matched healthy tissue. We did not find any MSI, although all but one patient had progressed to advanced-stage disease within the timeframe of the study. Concordantly, the expression of mismatch repair genes was normal. These results suggest that progressive accumulation of mutations as detected by MS analysis does not play a major role in the pathogenesis or in the progression of mycosis fungoides.

Ultrasound-guided fine needle aspiration cytology prior to sentinel lymph node biopsy in melanoma patients.

BACKGROUND: Sentinel lymph node biopsy (SLNB) allows early detection of metastases, thereby enabling early treatment in melanoma patients likely to benefit from adjuvant therapies. This prospective study analyzes the possible benefits of additional ultrasound (US) and fine needle aspiration cytology (FNAC) of sentinel nodes (SN) prior to SLNB. METHOD: Over a 2-year period 127 melanoma patients with 151 SN were scheduled for SLNB. All SN were initially identified with lymphoscintigraphy, then identified and evaluated by US and the cells aspirated for cytology (FNAC). US findings and FNAC results were compared to surgical findings. RESULTS: Of 127 patients, 114 had one SN each, 12 had two, and one had three. In vivo US achieved a sensitivity of 79% (95% CI: 62-91%) and a specificity of 72% (95% CI: 62-81%). FNAC showed a sensitivity of 59% (95% CI: 41-76%) and a specificity of 100% (95% CI: 95-100%). The combination of these two in vivo methods achieved an overall sensitivity of 82% (95% CI: 65-93%) and an overall specificity of 72% [95% CI: 62-81%]. CONCLUSION: Combined US and FNAC provides important information prior to SLNB in that both procedures identify metastases in the lymph nodes (sensitivity > 80%). Patients with positive FNAC may proceed directly to complete lymph node dissection (cLND) instead of having initial SLNB. Thus, combined US and FNAC may prevent unnecessary anesthesia and surgical management as well reduce costs. In our study 16% (19/121) fewer SLNB procedures were carried out, subsequently replaced by cLND. For patients with a negative combination of in vivo US and FNAC, SLNB remains the best diagnostic option.

Evaluation of B-cell clonality in archival skin biopsy samples of cutaneous B-cell lymphoma by immunoglobulin heavy chain gene polymerase chain reaction.

Polymerase chain reaction (PCR)-based detection of clonal T- and B-cells is widely used in the diagnosis of various lymphomas, including those of the skin. A large number of corresponding methods have been published. Recently, for the first time, standardized PCR protocols were developed in common by 14 European centers of lymphoma diagnosis and research (Biomed-2 protocols). Here, we have applied Biomed-2 immunoglobulin heavy chain (IgH)-PCR for clonality detection in primary cutaneous B-cell lymphoma (CBCL) and compared it with previously established methods. The DNA of 43 paraffin-embedded lesional skin biopsies of confirmed CBCL cases [27 follicle center cell lymphoma (FCCL), 11 marginal zone B-cell lymphoma/immunocytoma (MZL/IC) and five large CBCL of the lower leg (CBCL-LL)] were amplified by the Biomed-2 IgH-PCR protocols as well as using four other assays, priming also the three IgH framework regions (FR) 1-3. All PCR products were analysed by fluorescence fragment analysis. Twenty-nine of 43 (67%) CBCL samples (5/5, 100% of CBCL-LL; six of 11, 54.5% of IC/MZL; 18 of 27, 66.7% of FCCL) showed monoclonal B-cell presence complementary in all of the IgH-PCR. The three Biomed-2 PCR indicated together clonality in 24 of 43 samples (56%). Considering each method separately, the Biomed-2 FR3-PCR showed the highest rate of clonality detection (20 of 43, 47%). In conclusion, the Biomed-2 FR3-PCR is recommended for detecting B-cell clonality in archival skin samples of CBCL but should be completed by FR1- and/or FR2-PCR.

Monoclonal T-cell dyscrasia of undetermined significance associated with recalcitrant erythroderma.

BACKGROUND: Erythroderma is a diffuse, inflammatory skin reaction that, in rare instances, is associated with hematologic malignancies such as cutaneous T-cell lymphoma (erythrodermic mycosis fungoides) or T-cell leukemia (Sezary syndrome or adult T-cell leukemia/lymphoma). OBSERVATIONS: We screened 30 patients with erythroderma (20 patients with erythroderma of known etiology and 10 patients with idiopathic erythroderma) for the presence of circulating monoclonal T-lymphocyte populations using T-cell receptor (TCR)-gamma gene-specific polymerase chain reaction and automated capillary DNA electrophoresis. Moreover, the phenotypic analysis of peripheral blood CD4+ lymphocytes was performed using the following surface markers: CD3, CD7, CD8, CD25, CD26, CD27, CD28, CD29, CD30, CD45RO, CD45RA, CD56, CD134, HLA-DR, TCRalphabeta, TCRgammadelta, and cutaneous lymphocyte antigen (CLA). In 5 patients with idiopathic erythroderma we detected T-cell clones in peripheral blood (in 1 case, associated with the presence of the same clone in the skin) and a 2-fold increase in the proportion of CD3+ CD4+ CD7- CD26- cells. Cell depletion studies indicated that the monoclonal T cells were present within the CD4+ CD7- cell population. Clinically, all patients had chronic, recalcitrant erythroderma but none developed any hematological malignancy during their lifetimes or fulfilled the criteria for cutaneous lymphoma or Sezary syndrome. CONCLUSIONS: A proportion of patients with chronic erythroderma present with the monoclonal expansion of CD4+ CD7- CD26- lymphocytes in their blood. This condition represents a probably benign T-cell dyscrasia, or one of very low malignancy. Alongside monoclonal gammapathy of undetermined significance (MGUS) and monoclonal (B-cell) lymphocytosis of undetermined significance (MLUS), we propose using monoclonal T-cell dyscrasia of undetermined significance (MTUS) to underline a conceptual similarity between this disorder and the more common types of lymphocytic dyscrasia.

Cellular coincidence of clonal T cell receptor rearrangements and complex clonal chromosomal aberrations-a hallmark of malignancy in cutaneous T cell lymphoma.

Detection of a clonal T cell receptor (TCR) gene rearrangement is used in the diagnosis of primary cutaneous T cell lymphomas (CTCL) whereas chromosomal aberrations serve as a diagnostic tool for leukaemias and nodal lymphomas. To what extent both approaches specify the same cell population remains unknown. We investigated the coincidence of TCR clonality with complex clonal chromosomal aberrations, indicating qualitative alteration of the affected cells, in 17 CTCL patients. Out of 41 skin, blood, and lymph node samples studied, 34 gave results in chromosome and TCR analyses. With 88%, most specimens revealed corresponding results by both techniques (27 of 34 clonal, three of 34 non-clonal). In two patients, analysis of micro-dissected cells demonstrated that neoplastic T cells bear both a dominant TCR rearrangement and a complex chromosomal aberration. The cutaneous clone was found in blood samples of 11 of 12 patients (including early stages), and investigation of follow-up skin and blood samples indicated persistence of the T cell clone in 11 of 14 cases. In conclusion, we show that dominant TCR clones and chromosomal clones converge in all stages of CTCL. These clones disseminate into blood and skin at early disease stages and persist despite therapy. The coexistence of a dominant TCR clone and a clonal chromosomal aberration can thus be used as a hallmark of malignancy.

Anti-tumor immune responses and tumor regression induced with mimotopes of a tumor-associated T cell epitope.

Mimotopes provide an alternative to natural T cell epitopes for cancer immune therapy, as they can recruit and stimulate T cell repertoires that deviate from the repertoires engaged with the tumor and exposed to disease-related immune suppression. Here, mimotopes of a shared tumor-associated T cell epitope in cutaneous lymphoma were tested for their capacities to induce clinical and immunological responses in cancer patients. The mimotope sequences had been determined by a combinatorial peptide library approach without knowledge of the corresponding natural tumor-associated antigen. Vaccination with these mimotopes together with helper T cell-inducing antigens led to complete tumor remission in the two patients tested. After each booster vaccination, enhanced frequencies of mimotope-specific CD8+ T cells were detected in the peripheral blood of the patients, and the CTL proved to be cytotoxic and tumoricidal when tested in vitro. These data provide a first indication of clinical efficacy of mimotopes in cancer patients.

Peripheral blood T-cell clonality in mycosis fungoides and nonlymphoma controls.

In mycosis fungoides (MF), T-cell clonality is reported in about 90% of skin and 40% of blood samples. However, identity of blood and cutaneous T-cell clone and prognostic relevance of blood T-cell clonality remain controversial. By PCR/fluorescence fragment analysis with estimation of clonal fragment lengths and relative peak heights, we objectively identified T-cell clonality unrelated to malignant lymphoproliferation in healthy donors (5/38), autoimmune dermatoses (3/8), and nonlymphoma skin cancer (9/39). This T-cell expansion of undetermined significance (TEXUS) was also found in 8/64 MF patients. Dissemination of neoplastic cells into blood, as identified by identical clonal fragment lengths in blood and skin, was detected in 23/64 MF patients. When monitoring for progression at TNM stage for a mean of 45.7 months, univariate analysis identified age of >60 years and detection of a related blood T-cell clone to be of prognostic relevance, whereas detection of TEXUS, sex, TNM stage at initial diagnosis, and detection of a cutaneous T-cell clone were irrelevant. Although multivariate analysis was not possible, further stratification clearly indicated an age of >60 years to be the predominating prognostic factor. In conclusion, investigation of T-cell clonality in skin and blood samples at the initial diagnosis cannot predict the clinical course of MF and the occurrence of TEXUS should be considered when assessing blood T-cell clonality.