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ABSTRACT: Cellular inflammation of the nasal mucosa demonstrates a local immune response which plays an important role in allergic rhinitis. The aim of the present study was to characterize nasal mucosal lymphocytes regarding their activation and differentiation state by direct ex vivo flowcytometric analysis. Lymphocytes from the inferior turbinates were isolated by a mechanical method of preparation and, for comparison, from peripheral blood by Ficoll gradient centrifugation. Patients suffering from rhinitis or difficulty in nasal breathing were divided into an allergic (pollen-allergy, n = 13) and non-allergic group (n = 24). Expression of different T- and B-cell markers was determined by flowcytometric analysis. CD4+ T-cells from the nasal mucosa exhibited a memory phenotype (CD45RO+, 97%), were highly activated (CD69+, 43-73%), and showed low expression of the cutaneous lymphocyte antigen (CLA+, 5%). Nasal CD20+ B-lymphocytes expressed significantly higher levels of mIgE and lower levels of CD23 and CD80 than peripheral B-cells. Subsets of CD80+ (4%) and CD86+ (6%) CD20+ B-lymphocytes were identified in the nasal mucosa. No significant differences between allergic and non-allergic individuals were determined. As expected, the data show profound phenotypical differences between circulating peripheral blood and nasal mucosal lymphocytes. Activated memory lymphocytes are present in the nasal mucosa from allergic, but also non-allergic patients and may indicate to a significant role of a local inflammatory state without systemic criteria for allergy. In our study, we show that direct ex vivo isolation of lymphocytes is practicable method and offers a new technique to examine the local nasal allergic immune response using a multiparametric phenotypical analysis.
Archives of Oto-Rhino-Laryngology 10/2008; 266(5):677-83. · 1.29 Impact Factor
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ABSTRACT: Synthetic oligodeoxynucleotides (ODNs), such as PF-3512676, that contain unmethylated cytosine-guanine motifs (CpG ODN) have been identified as highly potent immune activators by in vitro examinations and in murine models. CpG ODNs induce innate and adaptive immune responses by triggering Toll-like receptor 9 expressed by human B cells and plasmacytoid dendritic cells. A phase 1 study was initiated to investigate safety, tolerability, serum cytokine levels, cellular immune responses, and clinical activity of intralesional treatment with PF-3512676 in patients with basal cell carcinoma (BCC) or cutaneous or subcutaneous melanoma metastases. Intrapatient escalating doses of PF-3512676 (up to 10 mg) were injected intralesionally every 14 days in 5 patients with BCC and in cutaneous or subcutaneous metastases of 5 patients with melanoma. PF-3512676 was well tolerated. Local swelling and erythema occurred at the injection site in 9/10 patients. There was only 1 incidence of a grade III hematologic adverse event (lymphocytopenia). Local tumor regressions were observed in patients with BCC (1 complete regression, 4 partial regressions) and metastatic melanoma (1 complete regression). After treatment with PF-3512676, interleukin-6 was increased in all patients, interferon-gamma induced protein-10 in 8/10 patients, interleukin-12p40 in 7/10 patients, and tumor necrosis factor-alpha levels in 6/10 patients. All patients had biopsies; moderate to abundant cellular infiltrates of lymphocytes were found posttreatment in most lesions of both histologic types. Intralesional treatment of skin tumors with PF-3512676 was safe and well tolerated. Despite the relatively low dosage, clinical activity was demonstrated both in patients with BCC and with cutaneous or subcutaneous metastatic melanoma lesions.
Journla of Immunotherapy 07/2008; 31(5):520-7. · 3.27 Impact Factor
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ABSTRACT: Previous studies suggest that drug delivery systems based on particles can be used to deposit active compounds in hair follicles and to target hair follicle-associated cell populations. The development of application protocols is complicated by the fact that there is no information available on the size and the position of key target structures in the different hair follicle types and their intra- and interindividual variation. Therefore, we performed morphometric measurements on histological sections of human terminal (THF) and vellus hair follicles (VHF) from the scalp and the retroauricular region. With 3864 +/- 605 microm and 580 +/- 84 microm in THF compared to 646 +/- 140 microm and 225 +/- 34 microm in VHF, the total length and the length of the infundibulum differed significantly as determined by paired t-test (P < 0.0001). The same level of significance was observed for the position and the length of the bulge region. The thickness of the epithelial lining was lowest in VHF (45 +/- 14 microm at 100 microm from skin surface) compared to 65 +/- 20 microm at 150 microm in THF, while the thickness of the interfollicular epidermis ranged between 64 +/- 12 microm and 99 +/- 18 microm in VHF-bearing skin and 72 +/- 16 microm and 136 +/- 37 microm in THF-bearing skin. In addition, the diameter of the hair follicle opening was determined at 50 microm intervals from the skin surface. Our data suggest that hair follicle types in defined body regions represent rather homogenous groups and that particle-based drug delivery may be a feasible approach, also in larger numbers of individuals. We provide precise information on the size and the position of key target structures in VHF and THF.
Experimental Dermatology 11/2007; 16(11):946-50. · 3.54 Impact Factor
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ABSTRACT: Neurocutaneous melanosis is often associated with melanoma of the central nervous system. Due to the inconspicuously looking cutaneous melanosis additional examinations are often not performed. We describe a patient with cutaneous melanosis who presented with neurological symptoms due to a large primary meningeal melanoma. The diagnosis of neurocutaneous melanosis was made. This case is an illustration of melanoma development in the central nervous system in a patient with cutaneous melanosis. This phenomenon should be kept in mind when observing patients with such skin lesions, even if they are of minor extent.
Clinical Neurology and Neurosurgery 07/2007; 109(5):448-51. · 1.58 Impact Factor
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ABSTRACT: Optical, non-invasive methods, such as fluorescence laser scanning microscopy (LSM) and optical coherent tomography (OCT), have become efficient tools for the characterization of the skin structure in vivo, as well as real-time investigation of distribution and penetration of topically applied substances.
In the present paper, the results obtained with both non-invasive methods - OCT and LSM - were compared to conventional light microscopy of histological sections. Skin structure and the distribution of topically applied particulate and non-particulate substances on the skin surface and in the epidermis were analyzed.
None of the methods used are suitable for the realization of all diagnostic tasks, however, each method has advantages for particular applications. Fluorescence LSM is well suited for the investigation of the upper 150 microm of the skin as well as for the investigation of the kinetics of substances applied onto or into the epidermis. OCT can be applied for the investigation of vertical cross-sections of the skin up to a depth of 2 mm, albeit at lower resolution than achieved by LSM or conventional light microscopy. Conventional light microscopy of histological sections of biopsy specimens produces familiar high-resolution images of deeper tissue layers. However, the analysis of the kinetic processes is limited in this case.
LSM- and OCT-measurements are efficient non-invasive tools for the characterization of morphological structures of the skin. On the one hand, the optical methods have a clear advantage in the case of kinetic measurements. On the other hand, histological investigations are characterized by a high information density and a high resolution, also in deep tissue layers. The selection of the best method for the analysis of the skin morphology depends on the target and the task of the investigation.
Skin Research and Technology 06/2007; 13(2):119-32. · 1.71 Impact Factor
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ABSTRACT: Porcine ear skin is used in studies of percutaneous penetration as a substitute for human skin. The structure of this tissue, including hair follicles, was studied qualitatively and quantitatively in comparison with human skin.
Sections of shock-frozen biopsies, biopsies embedded in paraffin and cyanoacrylate skin surface biopsies were investigated using microscopy. The thickness of the different skin layers and the follicular characteristics were determined.
The thickness of the stratum corneum was 17-28 microm, whereas the viable epidermis was 60-85 microm thick. On 1 cm(2), 11-25 hairs were detected, showing a diameter of 58-97 microm and a maximal extension depth of 0.96-1.38 mm into the skin. The orifices of the porcine infundibula showed a diameter of approximately 200 microm.
The results obtained are similar to those of human skin, indicating the suitability of this porcine tissue as a model for human skin.
Skin Research and Technology 03/2007; 13(1):19-24. · 1.71 Impact Factor
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ABSTRACT: 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.
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.
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%].
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.
Annals of Surgical Oncology 12/2006; 13(12):1682-9. · 4.17 Impact Factor
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ABSTRACT: Merkel cell carcinoma (MCC) is the most aggressive of the cutaneous malignancies, showing a propensity to spread to regional lymph nodes (LNs). The aim of this prospective study was to examine the feasibility and clinical impact of sentinel lymph node biopsy (SLNB) in this cutaneous malignancy.
The study population comprised 23 patients with stage I MCC (median age 70 years, range 50-85 years). Lymphoscintigraphic mapping with( 99 m)Tc-nanocolloid was performed in all patients. Sentinel lymph nodes (SLNs) were identified, excised and analysed in serial sections by conventional histopathology and cytokeratin-20 immunohistochemistry.
Metastatic disease was determined in the SLNs of 11 patients (47.8%). Elective lymph node dissection (ELND) was performed in eight of these 11 patients, four of whom had additional positive LNs. During follow-up (median 36.1 months, range 3-79 months), seven of the 23 patients (30%) relapsed: four had a local recurrence and three, in-transit metastases. Recurrence developed in two SLN-negative patients with local LN metastases and in one SLN-positive patient with distant metastases. This patient died, representing the only tumour-related death in our sample. Median survival was 49.1 and 35.5 months for SLN-negative and SLN-positive patients, respectively. This difference was not statistically significant (p=0.3452).
SLNB allows for exact nodal staging in patients with MCC. Whether additional ELND is of further benefit remains unclear.
European journal of nuclear medicine and molecular imaging 05/2006; 33(4):433-40. · 4.99 Impact Factor
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ABSTRACT: Because of its known heterogeneity, the analysis of antigen expression is crucial prior to the initiation of antigen-specific immunotherapy for melanoma. The melanoma differentiation antigens gp100, MART-1 and tyrosinase are involved in a common pathway of melanin synthesis. Peptides derived from these melanoma differentiation antigens are used in the immunotherapy of melanoma and antibodies recognizing these antigens are commonly applied to detect melanocytic lesions. One hundred and ninety-one paraffin-embedded melanoma metastases from 28 patients with 2-19 lesions (mean, 6.8) developing synchronously (n = 67) or asynchronously (n = 124) were analysed by immunohistochemistry for the expression of the melanoma differentiation antigens, as well as cancer/testis antigens of the melanoma antigen-A (MAGE-A) family (monoclonal antibodies 77B and 57B), anti-S100 and SM5-1. The overall reactivities were 81.6% (gp100), 79.5% (MART-1), 59.6% (tyrosinase), 59.1% (77B), 60.7% (57B), 93.2% (S100) and 91.6% (SM5-1). Twenty-seven lesions (14.1%) were positive for all tumour-associated antigens, 75 lesions (39.2%) were negative for one antigen and 87 lesions (45.5%) were negative for several tumour-associated antigens. Co-ordinated loss was found for lesions negative for gp100 and MART-1 (9.4%, P < 0.0005), gp100 and tyrosinase (11.0%, P = 0.009), MART-1 and tyrosinase (15.2%, P < 0.0005) and gp100, MART-1 and tyrosinase (8.9%, P < 0.0005), which is up to six times higher than the expected calculated loss. This co-ordinated loss of melanoma differentiation antigens in melanoma did not include cancer testis antigens and S100 or SM5-1. On average, the melanoma differentiation antigens stained 50-65% of cells within a lesion, and 10-39% of synchronous clusters were heterogeneous for melanoma differentiation antigen expression. In conclusion, broader polypeptide vaccines should be used for melanoma immunotherapy.
Melanoma Research 04/2006; 16(2):137-45. · 2.19 Impact Factor
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ABSTRACT: The new monoclonal antibody SM5-1 has been shown to have significant advantages in immunohistochemistry of melanoma over currently used antibodies such as HMB-45 or anti-S100. In this study we compared the immunohistological staining pattern of SM5-1 with that of the more recently described antibodies A103 (anti-MART-1) and T311 (anti-Tyrosinase) in 344 paraffin-embedded melanoma specimens, consisting of 101 primary melanomas (77 SSM, 16 NM, 6 ALM, 2 LMM) and 243 melanoma metastases. The overall reactivity of SM5-1 for all the specimens was 92% (318/344) compared with 83% (285/344) for MART-1 and 71% (245/344) for Tyrosinase. Staining of melanoma metastases with SM5-1 was found in 91% (222/243), but only in 77% (187/243) with A103 and 63% (154/243) with T311, respectively. Staining with SM5-1 was more homogenous with 196 of 243 (80%) of metastatic lesions showing 50% or more positively stained cells within the lesions, whereas A103 and T311 did so in 141 of 243 (58%) or 117 of 243 (48%) of the lesions. With regard to staining intensity of SM5-1, 157 of 243 (64%) showed a strong or very strong staining intensity, whereas A103 and T311 did so in 85 of 243 (35%) or 70 of 243 (29%) of the lesions. Staining intensity and percentage positivity correlated well for SM5-1, because from the 58 very strong positive metastases 55 showed staining in more than 75% of the cells within a lesion. Importantly, 52 of 56 MART-1-negative metastases and 81 of 89 Tyrosinase-negative metastases were positive for SM5-1. Thirty-eight metastases (15.6%) were negative for both A103 and T311. Of those, 35 (92.1%) were positive for SM5-1, demonstrating the value of SM5-1 in identifying melanoma-associated antigen-negative lesions. We conclude that SM5-1 could be of value in immunohistochemistry of melanoma.
American Journal of Dermatopathology 11/2005; 27(5):401-6. · 1.20 Impact Factor
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British Journal of Dermatology 01/2005; 152:827. · 3.67 Impact Factor
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Journal of Investigative Dermatology 04/2004; 122(3):859-61. · 6.31 Impact Factor
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ABSTRACT: P16INK4a overexpression has been identified as a specific biomarker in high-risk human papillomavirus (HPV)-infected cervical (pre)cancer lesions.
To evaluate the overexpression of this cyclin-dependent kinase inhibitor in skin tumors depending on HPV infections, we analyzed normal skin, benign skin disease, and skin cancer specimens.
Biopsies of 23 patients with normal histology (3), psoriasis (2), verrucae vulgaris (2), actinic keratoses (5), squamous cell carcinoma (SCC) in situ (3), Bowen's carcinoma (1), and SCC (7) were analyzed. Specimens of 23 patients were immunostained using the monoclonal antibody E6H4 specific for p16INK4a. HPV status was assessed by a polymerase chain reaction (PCR) system to detect all currently known HPV types. MY (MY09/MY11 and MYN9/MYN10)-, CP (CP65/CP70 and CP66/CP69)-nested PCR, and three single PCR methods CN1, CN3, and CN4 were used in a first step, and HPV typing was performed by restriction fragment length polymorphism analysis. Only beta-globin-positive patients were included in this study.
HPV DNA was detected in all actinic keratoses, SCC in situ, Bowen's carcinoma, and SCC, in 50% (one of two) of verrucae vulgaris, in 66% (two of three) of normal skin, and in none of two psoriasis. P16INK4a expression was not detected in normal skin, psoriasis, and verrucae vulgares. Overexpression of p16INK4a was detected in a subset of dysplastic cells (10% to 80%) of all skin (pre)cancer lesions such as actinic keratoses, SCC in situ, Bowen's carcinoma, and SCC infected with HPV independent of sun exposure.
P16INK4a appears to be overexpressed in a portion of dysplastic cells from actinic keratoses and SCC. Further studies to examine the association of HPV infection and the overexpression of p16INK4a are warranted.
Dermatologic Surgery 04/2004; 30(3):409-14. · 1.80 Impact Factor
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ABSTRACT: Information on chromosomal aberrations in cutaneous T cell lymphomas (CTCL), is scarce. In this study, comparative genomic hybridization (CGH) was used to analyze chromosomal imbalances (CI) in 32 patients with CTCL. CI were detected in 21 patients (66%). Euchromatic loss (dim) was localized most frequently (>16%) at the chromosomal regions 17p (28%), 13q (25%), 10q (16%), and 6q (19%), and gain of chromatin (enh) at 7 (25%), 8q (25%), and 17q (16%). The pattern dim6q-enh7-enh8-dim13 was the most frequent combination of CI. The number of aberrations per tumor sample varied between 0 and 19 and correlated with clinical tumor stages: from none in stage Ia to 8.75+/-1.8 (mean+/-SEM) in stage IVa. CI occurred more frequently in aggressive subtypes (9.33+/-2.16) than in indolent (2.88+/-0.8) subtypes. A high number of CI (>/=5) was associated with shorter survival. Gain of chromatin in 8q and loss of 6q and 13q correlated with a significantly shorter survival, whereas the most frequently observed aberrations (loss in 17p and gain in 7) did not influence the prognosis. In summary, CGH analysis revealed a characteristic pattern of recurring chromosomal gains and losses in CTCL. The association of the imbalances with the clinical course of the disease suggests that genes encoded at these loci may influence tumor development and progression.
Journal of Investigative Dermatology 04/2004; 122(3):579-86. · 6.31 Impact Factor
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ABSTRACT: The Stewart-Treves syndrome (STS) is a lymphedema-associated angiosarcoma which usually develops in female patients after mastectomy and axillary lymph node dissection. A 55-year old woman developed STS in her lymphedematous left arm seven years after breast-preserving surgery with axillary lymph node dissection and radiotherapy. Various therapies which have been employed are radiotherapy and isolated limb perfusion with cytostatic agents. Since our patient had a huge lesion with additional chest wall involvement, neither approach represented a good option. Radiological staging showed no evidence of further lesions or metastases. We started infusion therapy with liposomal doxorubicin (20 mg/m2 body surface) fort six cycles at regular intervals of 14 days. The patient tolerated the therapy well. Palmar-plantar erythrodysesthesia, a well-known side effect of doxorubicin, did develop. Because the disease was stable, the therapy interval was increased to six weeks after the 6th cycle. The patient has shown no recurrence for eight months. STS is a very rare variant of an angiosarcoma with poor prognosis. The case report shows that liposomal doxorubicin provides an effective therapeutic option.
Journal der Deutschen Dermatologischen Gesellschaft 02/2004; 2(1):49-52. · 1.47 Impact Factor
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Dermatology 01/2004; 209(4):350-2. · 2.05 Impact Factor
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ABSTRACT: We analyzed the expression of 13 chemokine receptors in mycosis fungoides, in order to assess the contribution of chemotaxis to the pathogenesis of the disease. Material from skin biopsies of six patients with early disease and six patients at the tumor stage of mycosis fungoides was analyzed by immunohistochemistry and partly also by flow cytometry. The receptors CCR1, CCR2, CCR3, CCR5, CCR6, CXCR1, CXCR2, CXCR5, and CX3CR1 were rarely and inconsistently detected in lesional skin and thus their participation in mycosis fungoides could largely be ruled out. In contrast, CCR4, CXCR3, and CXCR4 were substantially expressed on both mycosis fungoides cells and the surrounding reactive T cells in the early patch and plaque stages of the disease, indicating an involvement of these chemokine receptors in the disease process. In the tumor stage of mycosis fungoides, we interestingly observed a loss of a relevant chemokine receptor in four out of six patients. In three patients CXCR3 and in one patient CCR4 was absent on tumor mycosis fungoides cells, whereas the reactive T cells showed normal levels of expression. Within these samples, tumor mycosis fungoides cells exhibited high levels of CCR7, a chemokine receptor central for the entry of T cells to lymphatic tissue. Taken together, our data suggest that the loss of one or more of the chemokine receptors involved in the homing of the mycosis fungoides cells to the skin may trigger the latent potential of these cells to metastasize into regional lymphatic tissue.
Journal of Investigative Dermatology 12/2003; 121(5):1045-52. · 6.31 Impact Factor
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ABSTRACT: 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.
European Journal of Immunology 12/2003; 33(11):3175-85. · 5.10 Impact Factor
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ABSTRACT: 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.
Diagnostic Molecular Pathology 10/2003; 12(3):142-50. · 2.26 Impact Factor
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ABSTRACT: 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.
Mycosis fungoides (MF) represents the prototype of cutaneous T-cell lymphomas (CTCL), which are defined as a neoplastic proliferation of skin-homing T lymphocytes. 1 By PCR amplification of T-cell receptor (TCR) rearrangements and subsequent high-resolution electrophoresis, a single expanded (predominant) T-cell clone can be demonstrated in skin biopsy samples of up to 90% of the MF cases, 2–4 and detection of a predominant T-cell clone at the initial diagnosis has been found to be an independent negative predictive marker of treatment response in MF. 5 Since, with respect to the current pathogenetic model of lymphoproliferative diseases, the T-cell clone detected by PCR is generally assumed to represent the malignant T cells, PCR methods have been applied for the detection of clonal, ie, neoplastic T cells in extracutaneous sites of MF patients.
Regarding the peripheral blood, a predominant T-cell expansion has been found in 46% to 57% of the investigated MF cases. 6–8 However, the identity of the circulating T-cell clone to the cutaneous tumor and the relevance of its detection to the prognosis of MF patients remain controversial.
Clonal T-cell expansions have also been found within subsets of peripheral blood T lymphocytes in nonmalignant conditions, eg, in Vβ subsets derived from peripheral T cells of healthy individuals 9–14 and patients suffering from autoimmune diseases. 15–17 Here, the accumulation of expanded T-cell clones was found to be age dependent and started earlier, as well as being more pronounced in the CD8-positive fraction than in CD4-positive cells. 14 With regard to MF, Delfau-Larue and colleagues, 18 after detecting circulating clonal T cells in 40/88 patients by TCRγ-PCR and denaturing gradient gel electrophoresis, reported 29/40 peripheral blood T-cell clones to differ from the cutaneous tumor. In another study, which detected clonal T cells in the peripheral blood of 33/67 MF patients by means of the same approach, T-cell clones were distinct from the skin tumor in 16/33 cases. 19 In contrast, and with the use of TCRγ-PCR with temperature gradient gel electrophoresis, we detected circulating T-cell clones in 26/45 MF patients and skin and blood clones differed in only 1 case. Out of 5 cases, in which the clonal TCRγ junctional region had been sequenced, 4 revealed identical sequences in both compartments. 7 Dippel et al, 20 by applying TCRγ-PCR in combination with fluorescence fragment analysis (FFA), were even not able to detect nonidentical peripheral blood T-cell clones in their cohort of 19 CTCL patients.
Already in 1992, the presence of a peripheral blood T-cell clone as detected by Southern blot analysis has been associated with a rapidly fatal disease in MF patients with lymph node involvement. 1 More recently, the detection of circulating clonal T cells has been associated with a worse prognosis in MF by Fraser-Andrews et al, 6 using Southern blot and TCRγ-PCR with subsequent single-strand conformational polymorphism analysis in 66 MF patients, and by Beylot-Barry et al, 19 applying TCRγ-PCR and denaturing gradient gel electrophoresis in 67 cases. However, our group 22 could not confirm such correlation when investigating samples of 67 MF patients with TCRγ-PCR and temperature gradient gel electrophoresis. Accordingly, Laetsch et al, 23 after analyzing 51 CTCL patients with TCRγ-PCR and denaturing gradient gel electrophoresis, reported that the clinical course of patients with demonstrable blood involvement did not differ from PCR-negative cases.
Controversies in the above-cited studies are caused by the following facts: Determination of T-cell clonality by assessing the migration pattern in gradient gels is very subjective. Estimation of the identity of T-cell clones found in different samples by comparing the position of clonal band(s) in such gels is even more prone to errors. Different outcome criteria for the investigation of the prognostic relevance of peripheral blood T-cell clonality were chosen, ie, survival, 6 progression at TNM stage, 22 correlation to TNM stage, 23 and response to treatment. 19 Cohorts with differently distributed MF stages were analyzed: Unlike stage T1, which is unlikely to progress, 24 and stage 3/4, which is likely to progress rapidly, stage T2 MF has the most variable prognosis and, therefore, would benefit most of all from additional prognostic markers.
To solve the controversies, a technique is required that allows for an objective determination of T-cell clonality, as well as for a simple and objective comparison of T-cell clones detected in different samples. We are reporting here on the first results of an extended study that has utilized FFA to objectively compare composition and length of the clonal TCRγ junctional region in blood and skin samples taken from 72 MF patients at the time of initial diagnosis, and from 85 controls derived from healthy volunteers and patients suffering from autoimmune dermatoses (AID) or nonlymphoma skin cancer (NLSC). Progression at TNM stage was chosen to address the prognostic relevance of peripheral blood T-cell clonality in our T2 predominated MF cohort.
Diagnostic Molecular Pathology 08/2003; 12(3):142-150. · 2.26 Impact Factor
