[Complete treatment of basal cell carcinoma; cost effectiveness of Mohs micrographic surgery versus conventional excision]. / Radicale behandeling van basaalcelcarcinoom.

OBJECTIVE: To investigate whether Mohs micrographic surgery (MMS) in accordance with the indications in the revised guideline on basal cell carcinoma (BCC) more often leads to complete treatment than conventional excision (CE) and whether the costs are comparable, and to analyse whether this also applies to all primary BCC of the face. DESIGN: Retrospective analysis. METHOD: We gathered data on 3374 MMS procedures and calculated per localisation, subtype and size: the percentage of CE that would have been incomplete after applying the recommended surgical margin; the surgical margin necessary to achieve complete excision in > 90% of cases; the final defect after CE and after MMS; the cost of MMS and the cost of CE with postponed reconstruction or, in the case of incomplete CE, subsequent MMS. RESULTS: For the new MMS indications we can show that using MMS seems to prevent incomplete CE in 12-45% of cases. CE will also lead to incomplete excision in ≥ 10% of cases in most subgroups of primary facial BCC, with the exception of some small superficial or nodular BCCs in the H-zone. The final defect was always smaller after MMS than after CE. MMS can be used with comparable costs for primary BCCs > 5 mm in the H-zone and > 20 mm elsewhere in the face. CONCLUSION: The surgical margins recommended in the guidelines are inadequate to achieve complete excision rates in > 90% of facial CEs. MMS is an efficient alternative for CE for primary BCCs > 5 mm in the H-zone and > 20 mm elsewhere in the face.

Systemic eight-cycle anti-CD20 monoclonal antibody (rituximab) therapy in primary cutaneous B-cell lymphomas--an applicational observation.

BACKGROUND: Primary cutaneous B-cell lymphomas (PCBCLs) are characterized by restriction to the skin and a variable but mostly favourable prognosis. Since 1997 the recombinant, chimeric anti-CD20 antibody rituximab has been used in patients suffering from non-Hodgkin's B-cell lymphomas. Different studies have shown that the effectiveness and safety in the treatment of patients with low-grade follicular lymphoma is comparable to or even higher than the standard CHOP chemotherapy. So far it has been unclear whether an extended duration of therapy leads to a benefit for the patients with PCBCL. OBJECTIVES: To evaluate the objective response rate, time to progression, remission quality and histological changes and to compare our data with the literature. PATIENTS/METHODS: Ten patients with PCBCL [eight with follicle centre cell lymphoma (FCCL), one with marginal zone lymphoma (MZL) and one with diffuse large B-cell lymphoma of the leg (DLBCL)] were treated by intravenous application of a chimeric antibody against the CD20 transmembrane antigen (rituximab) with a dosage of eight cycles, 375 mg m(-2) body surface, weekly. RESULTS: The treatment regimen resulted in clinical overall response in 9 of 10 patients, in particular there were seven complete responses (70%) plus two partial responses (20%). The median duration of remission (durable remission, DR) is 23 months (4-30 months) to date. Histological assessment of responses in four patients showed no tumour-specific infiltration. In two patients histology revealed a residual infiltration and in one patient an increasing infiltration. In two patients no histology was taken after treatment; one patient developed a new lesion. No severe side-effects occurred. Observed side-effects were two bacterial infections, two patients with shivering during infusion, one patient with sweating for months and one patient with persisting itching. As expected the B-cell count in peripheral blood was depressed in all patients after infusion. CONCLUSIONS: Intravenous therapy with eight cycles of the anti-CD20 antibody rituximab is a non-toxic and effective treatment for a subset of patients with PCBCL (relapsed, aggressive entity, old patients, multiple lesions) with a long DR.

Palpable arciform migratory erythema in an HIV patient, a CD8+ pseudolymphoma.

BACKGROUND: Palpable arciform migratory erythema (PAME) is characterized by large, elevated, reddish annular lesions localized on the upper trunk. As its infiltrate consists predominantly of dense infiltrates of CD4+ lymphocytes with polyclonal T-cell receptor (TCR) gene rearrangement, it has been grouped as a rare member of the T-cell pseudolymphomas. METHODS: We performed histology, immunophenotyping, and TCR-gamma gene rearrangement studies in an human immunodeficiency virus (HIV)-positive patient, CDC stage IIIB, who showed a clinically typical PAME. RESULTS: While TCR-gamma gene rearrangement studies showed a polyclonal infiltrate confirming a pseudolymphoma, 85% of skin-infiltrating lymphocytes were CD8+ T cells. CONCLUSION: PAME may also occur in HIV-positive patients with CD4+ deficiency. Our case demonstrates that regular CD4 counts and immunocompetence are not necessary for its pathogenesis.

Vaccination therapy for cutaneous T-cell lymphoma.

Primary cutaneous T-cell lymphomas (CTCL) are defined as clonal proliferation of skin-infiltrating T lymphocytes. Despite their heterogeneity, CTCL are generally incurable, which has led to the development of various treatment strategies including vaccination. Here, the attempts to vaccinate against lymphoma will be reviewed with special emphasis on CTCL. Because an universal tumour antigen is not available so far, different targets - including whole tumour cells, idiotypes, cancer/testis antigens, proteins derived from tumour-associated mutations, and mimotopes - have been investigated for their applicability in CTCL vaccination. The antigenic information can be delivered in different ways. So far, tumour cells fused to dendritic cells, idiotypic proteins/peptides and DNA/RNA preparations have been applied in lymphoma. As most targets are weak immunogens, adjuvants and other helpers - including dendritic cells, immunogenic peptides and oligonucleotides, cytokines, and viral vectors - are required to enable proper presentation of the antigens and sufficient activation of the immune system. Although first data from CTCL patients prove the suitability of vaccination in CTCL therapy, the number of available antigens, carriers, adjuvants and application schemes creates a multitude of vaccine formulations; identification of the best-suited approach remains nearly impossible. Furthermore, the relationship between lymphoma and the host immune system is complex and incompletely understood. As a result, CTCL vaccination still requires a lot of research.

CD4+CD7- leukemic T cells from patients with Sézary syndrome are protected from galectin-1-triggered T cell death.

In early stages of cutaneous T cell lymphoma (Sézary syndrome) both CD4+CD7- and CD4+CD7+ T cells clonally expand whereas in late stages of the disease CD7- cells are predominant in number, giving rise to the question whether CD7- T cells have a survival advantage in the skin. Galectin-1, a cell-bound lectin, was recently reported to trigger apoptosis in activated CD7+ T cells. Here, we demonstrate that in contrast to activated CD7(+) T cells, quiescent and activated CD69+ CD7- T cells from healthy donors and from Sézary patients are resistant to galectin-1-mediated cell death. CD7- T cells are apoptosis-resistant even during coculture with IFN-gamma-stimulated endothelial cells that constitutively express galectin-1 in high amounts. These data imply that resistance of CD7- T cells to galectin-1-induced apoptosis may contribute to the accumulation of CD7- Sézary T cells during progression of the disease.

T cells engrafted with a recombinant anti-CD30 receptor target autologous CD30(+) cutaneous lymphoma cells.

T cells can be directed to antigen-specific, MHC-independent target cell lysis by grafting with a recombinant receptor with antibody-like specificity. Here, we asked whether T cells from the peripheral blood of a patient with cutaneous T cell lymphoma can be recruited for an immune response against autologous tumor cells. Lymphoma cells with a CD3(-) CD4(+) CD30(+) phenotype and clonal TCR-Vbeta7 rearrangement were isolated from a cutaneous lesion. The lymphoma lesion additionally harbored CD3(+) CD25(+) activated normal T cells despite ongoing tumor progression. Peripheral blood-derived T cells from the lymphoma patient were retrovirally engrafted with a recombinant anti-CD30-scFv-gamma receptor. Upon cocultivation with autologous CD30(+)lymphoma cells, grafted T cells increase IFN-gamma secretion and lyse specifically lymphoma cells with high efficiency, even at an effector to target cell ratio of as low as 1:20. Our data demonstrate that the recombinant anti-CD30-gamma receptor overcomes T cell tolerance for tumor cells and directs T cells specifically against autologous lymphoma cells.

[Anti-CD20 antibodies in primary cutaneous B-cell lymphoma. Initial results in dermatologic patients]. / Der Anti-CD20-Antikörper bei primär kutanen B-Zell-Lymphomen. Erste Erfahrungen in der dermatologischen Anwendung.

BACKGROUND AND OBJECTIVE: Primary cutaneous B cell lymphomas (pCBCL) are rare extra-cutaneous non-Hodgkin lymphomas which occur on the trunk as follicle center cell lymphoma or on the leg as large B cell lymphoma. The currently accepted therapy of pCBCL (excision and/or radiotherapy, systemic interleukin 2 and interferon alpha 2a, local injection of cisplatin or multiagent chemotherapy, i.e. CHOP) is insufficient for treatment of multifocal pCBCL and secondary extracutaneous involvement. For this reason, the new synthetic chimeric, monoclonal anti-CD20 antibody Rituximab is an alternative treatment for patients with pCBCL. PATIENTS/METHODS: Four patients with pCBCL localized to the trunk or head were treated with Rituximab (375 mg/kg weekly for 4-8 weeks, then maintenance therapy every 4 weeks for 6 months). RESULTS: All 4 patients showed a response (2/4 partial; 2/4 complete). Side effects were minimal. CONCLUSIONS: Rituximab is an alternative immunotherapeutic drug for the treatment of pCBCL. Our initial experience with this new modality are presented and discussed.

CD4(+)CD7(-) T cells compose the dominant T-cell clone in the peripheral blood of patients with Sézary syndrome.

BACKGROUND: Absence of CD7 antigen expression in T cells defines a subset of normal CD4(+) CD45RO(+) CD45RA(-) memory cells and is furthermore observed in Sézary syndrome (SS). OBJECTIVE: Our purpose was to identify circulating T-cell clones in patients with SS and to elucidate whether the dominant T-cell clones express the CD7 antigen. METHODS: Peripheral blood lymphocytes of patients with SS were analyzed by two-color flow cytometry using antibodies to the V beta region of the T cell receptor (TCR) in combination with an antibody to CD7. In addition, T cells were analyzed for TCR-gamma gene rearrangement by polymerase chain reaction (PCR) techniques. RESULTS: Clonal T-cell expansion was detected in 7 patients with SS by immunostaining of the TCR V beta regions. PCR analysis confirmed the presence of dominant T cell clones. Double-immunostaining revealed that in each case cells of the clonal V beta TCR rearrangement homogeneously express the CD4(+)CD7(-) phenotype. Furthermore, CD4(+)CD7(-) cells express the CD15s antigen but lack expression of CD26 and CD49d. CONCLUSION: Expansion of clonal T cells strongly correlates with the expansion of CD4(+)CD7(-) T cells in 7 tested patients with SS. This supports our model that a subset of late differentiated, normal CD4(+)CD7(-) memory T cells may represent the physiologic counterpart of Sézary cells. Monitoring of circulating T cells with the CD4(+)CD7(-)CD15s(+)CD26(-)CD49d(-) phenotype proved to be useful for the identification of clonal T cells in patients with SS.

An approach to the sensitivity of temperature-gradient gel electrophoresis in the detection of clonally expanded T-cells in cutaneous T-cell lymphoma.

Detection of T-cell clonality by polymerase chain reaction (PCR) and high-resolution electrophoresis facilitates differentiation of early stages of cutaneous T-cell lymphoma (CTCL) from benign T-cell-rich dermatoses. However, data regarding the sensitivity of the various electrophoresis techniques differ remarkably. In the present study, the capacity of heteroduplex (HD)-loaded temperature-gradient gel electrophoresis (TGGE) to detect clonally expanded T-cells was assessed systematically and modifications to the procedure were defined. Using our standard protocol, HD-TGGE detected clonal T-cell receptor (TCR)-gamma PCR products, generated from the Jurkat cell line, down to a total of 2 ng/microL (14 ng) DNA. However, slowly migrating single strands of the clonal PCR product reduced the amount of the clonality indicating homoduplices. To overcome this single-strand formation, thus decreasing the detection limit, the urea concentration in the gel and the temperature ramp for the HD-formation were altered, as well as the temperature gradient in the gel. Application of the modified protocol resulted in a tenfold lower detection limit of 0.15 ng/microL (1.05 ng) DNA in the clonal band. The sensitivity of the adapted HD-TGGE was investigated by dilution experiments using the well established T-cell lines Jurkat, Molt-4, MyLa and SeAx. By these approaches clonal PCR products diluted in nonclonal PCR products were detectable down to concentrations of 5-10%. Comparably, in the case of mixtures of clonal in nonclonal DNA the detection limit reached 5-10% clonal DNA. However, by dilution of clonal cells in nonclonal peripheral blood mononuclear cells, which corresponds to in vivo conditions, a lower detection limit of approximately 1-5% was observed.

Microanatomical compartments of clonal and reactive T cells in mycosis fungoides: molecular demonstration by single cell polymerase chain reaction of T cell receptor gene rearrangements.

Mycosis fungoides (MF) is a cutaneous T cell lymphoma, clinically characterized by patches, plaques and tumors occurring in successive stages of the disease. In early MF, an infiltrate consisting of mainly reactive T cells is seen in the papillary dermis while tumor cells are mostly confined to the epidermis. By contrast, later stages show nodular infiltrates formed mostly of tumor cells in the dermis while the epidermis is relatively devoid of tumor cells; however, knowledge of the localization of clonal T cells has been based on histomorphologic features and immunohistochemical stainings visualizing certain V-beta subfamilies of the T cell receptor (TCR). As these techniques do not allow for an unequivocal identification of clonal tumor cells, we used micromanipulation and single cell PCR amplifying the TCR chain gene rearrangement. A total number of 387 single T cells was isolated from six skin biopsies in five patients in patch, plaque, and tumor stages. Of these, 180 T cells were picked from the epidermis and 207 from the dermal infiltrate. The rearranged TCR-gamma DNA could be sequenced from 181 of 387 T cells. In three of six patients representing all three stages, epidermal T cells with a clonal rearrangement could be amplified. In early plaque stage a higher degree of epidermal T lymphocytes was found than in initial patch, later plaque, and tumor stages with an inverse distribution found for reactive T lymphocytes. In two patients a biallelic rearrangement was demonstrated that had not been detected in prior PCR analysis from blood and skin samples. These data show that clonal (neoplastic) and non-clonal (reactive) T lymphocytes in MF preferentially infiltrate different microanatomical compartments of the skin, depending on the stage of disease. The microanatomically distinct localization of reactive and clonal T cells suggests that the absence of direct contact between tumor and host-defense lymphocytes may contribute to tumor persistence and progression in epidermis, peripheral blood, and deep dermal tumor cell nests, respectively.

Detection of expanded T cell clones in skin biopsy samples of patients with lichen sclerosus et atrophicus by T cell receptor-gamma polymerase chain reaction assays.

Lichen sclerosus et atrophicus is a chronic dermatosis of unknown etiology and pathogenesis. Lichen sclerosus et atrophicus associated skin lesions show T cell enriched infiltrates, sometimes resembling the histologic picture of early mycosis fungoides. It is supposed that the infiltrating T cells participate in the pathogenesis of atrophy and sclerosis. We investigated skin biopsies from 39 lichen sclerosus et atrophicus patients by histology, immunohistochemistry and, in order to establish the status of T cell clonality, by polymerase chain reaction amplifying the T cell receptor-gamma rearrangements. A stage-dependent shift of the CD3-positive T cells was observed from a predominantly CD4-positive to a predominantly CD8-positive phenotype. The increase of CD8-positive cells was associated with more pronounced epidermotropism and basal degeneration. Nearly all CD8-positive cells expressed cytotoxic granules (TIA1), possibly causing the basal destruction. In the late fibrotic stage of the disease, only a weak or no infiltrate was found. Regarding the T cell receptor-gamma polymerase chain reaction, the presence of clonally expanded T cells was demonstrated in 19 of 39 patients (49%) by at least one of two different high resolution electrophoresis techniques applied to separate the amplification products. Thus, for the first time clonally expanded infiltrating T cells were detected in lichen sclerosus et atrophicus. Furthermore, this is one of the first reports on the detection of clonally expanded infiltrating T cells in an inflammatory skin disease. The clonal T cells could not be assigned to the CD4 or CD8 subtype. Most likely, their presence is not the result of a malignant transformation but a response to an as yet unknown lichen sclerosus et atrophicus associated antigen.

Treatment of cutaneous T-cell lymphomas.

Primary cutaneous T-cell lymphomas (CTCL), representing a heterogeneous group of non-Hodgkin's lymphomas (NHL), can be defined as clonal proliferation of skin-infiltrating T lymphocytes primarily presenting in the cutaneous compartment. They show a considerable variation in clinical presentation, histology, immunophenotype, and prognosis, which is best reflected by the proposal of the Cutaneous Lymphoma Study Group of the European Organization for Research and Treatment of Cancer (EORTC). Due to the heterogeneity of CTCL and the lack of curative therapy regimens, multiple strategies have been proposed for the management of the different CTCL entities. This includes topical application of corticosteroids, nitrogen mustard or carmustine (BCNU), radiotherapy, including total skin electron beam irradiation, photo(chemo)therapy, biological response modifiers, cytostatic chemotherapy, and combined regimens. More recently, fusion proteins and peptide vaccines have been introduced in the management of CTCL. Classification, staging, and treatment modalities are discussed in detail and summarized in a stage-adapted therapy regimen for CTCL.

Demonstration of frequent occurrence of clonal T cells in the peripheral blood but not in the skin of patients with small plaque parapsoriasis.

Clinical, immunohistological, and molecular biological data suggest the chronic dermatosis small plaque parapsoriasis (SPP) to be a precursor of mycosis fungoides (MF). However, most data are contradictory and confusing due to inexact definition of SPP. Recently, clonal T cells were detected in skin and blood samples of early MF. Because demonstration of identical T-cell clones in skin and blood of SPP patients would indicate a close relationship of SPP to MF, we investigated the clonality of skin and blood specimens from 14 well-defined SPP patients. By a polymerase chain reaction (PCR) amplifying T-cell receptor gamma rearrangements and subsequent high-resolution electrophoresis, clonal T cells were detected in 9 of 14 initial and 32 of 49 follow-up blood samples, but in 0 of 14 initial skin specimens. Even a clone-specific PCR showing the persistence of the initial blood T-cell clone in 20 of 20 follow-up samples, failed to detect the T-cell clone in the skin. In 2 patients, the clonal T cells were shown to be CD4(+). For the first time, the majority of SPP patients was shown to carry a T-cell clone in the peripheral blood. Although a relation between circulating clonal T cells and SPP cannot directly be proven by the applied techniques, our results indicate blood T-cell clonality to be a characteristic feature of SPP and CTCL because analysis of multiple controls and clinical workup of our SPP patients excluded other factors simulating or causing a clonal T-cell proliferation. A sufficient cutaneous antitumor response but also an extracutaneous origin of the T-cell clones might explain the failure to detect skin infiltrating clonal T cells.

Detection of clonal T cells in cutaneous T cell lymphoma by polymerase chain reaction: comparison of mutation detection enhancement-polyacrylamide gel electrophoresis, temperature gradient gel electrophoresis and fragment analysis of sequencing gels.

Cutaneous T cell lymphomas (CTCL) can be differentiated from benign inflammatory dermatoses by the demonstration of clonal T cells in skin biopsy. As a marker of the T cell clonality, the rearrangement of the T cell receptor (TCR) genes is amplified by polymerase chain reaction (PCR) and subsequently analyzed by several electrophoresis techniques. Since the validity of this approach depends substantially on the separating capacity of the applied electrophoresis technique, we investigated in the present study the lower detection limit and the sensitivity of heteroduplex-loaded polyacrylamide gel electrophoresis on MDE (mutation detection enhancement) gels (HD-MDE PAGE), of heteroduplex-loaded temperature gradient gel electrophoresis (HD-TGGE) and fragment analysis (FA) on sequencing gels. Genomic DNA from formalin-fixed, paraffin-embedded skin biopsies of 53 CTCL specimens and 27 samples of benign dermatoses was analyzed by TCRy PCR followed by electrophoretic separation. Clonality was detected by HD-MDE PAGE in 22, by HD-TGGE in 34, and by FA in 33 of the 53 CTCL cases. Additionally, FA revealed an oligoclonal fragment profile in seven CTCL specimens. In the 27 samples from benign dermatoses, HD-MDE PAGE and HD-TGGE showed the expected polyclonal pattern in 26, and FA in 25 specimens. HD-TGGE and FA detected a clonal pattern down to a dilution of 10(3) monoclonal cells in 10(6) peripheral blood mononuclear cells (PBMC), while HD-MDE PAGE revealed a detection limit of 10(4) monoclonal cells in 10(6) PBMC. In conclusion, HD-TGGE and FA possess a higher sensitivity and lower detection limit than HD-MDE PAGE. Therefore, both former techniques are useful tools for the routine diagnostic procedure. With regard to time and cost, we recommend HD-TGGE.