Long-term outcome of intravenous therapy with rituximab in patients with primary cutaneous B-cell lymphomas.

BACKGROUND: The monoclonal antibody rituximab directed against the B-cell antigen CD20 was approved for the treatment of B-cell lymphomas and maintenance therapy in follicular lymphomas more than a decade ago. However, median follow-up in case series of intravenous rituximab therapy in primary cutaneous B-cell lymphomas (CBCL) lasts only up to 3 years. We retrospectively analysed a cohort of CBCL patients treated with rituximab to gain more long term information. PATIENTS AND METHODS: Eighteen patients, treated intravenously with rituximab for a primary cutaneous B-cell lymphoma [follicle centre lymphoma (PCFCL), n = 11; diffuse large B-cell lymphoma, leg type (PCLBCL, leg type), n = 5; marginal zone B-cell lymphoma (PCMZL), n = 2] were included. The response rate (RR), time to relapse (TTR), and course of the disease after treatment were analysed. RESULTS: The overall RR was 89% (16 of 18 patients). Within the median follow-up time of 52 months, 81% (13 of 16) of patients experienced a relapse; the median TTR was 25 months. The duration of remission was significantly shorter in patients presenting with generalized skin lesions at start of therapy. Both nonresponding patients suffered from PCLBCL, leg type, with extracutaneous manifestations. In responders severe adverse events, the occurrence of extracutaneous dissemination or nodal lymphomas were not observed during follow-up. CONCLUSIONS: Therapy with rituximab is effective and safe for the treatment of PCFCL, but relapses, in particular in patients with generalized skin involvement, are commonly observed. However, all patients with relapses responded well to treatment and therefore maintenance therapy does not seem to be indicated. Patients with PCLBCL, leg type, should receive chemotherapy in addition to rituximab.

Efficacy and safety of bexarotene combined with psoralen-ultraviolet A (PUVA) compared with PUVA treatment alone in stage IB-IIA mycosis fungoides: final results from the EORTC Cutaneous Lymphoma Task Force phase III randomized clinical trial (NCT00056056).

BACKGROUND: Psoralen plus ultraviolet A (PUVA) is the standard treatment for early stages of mycosis fungoides. There have been no adequate randomized controlled trials with sufficient power comparing this modality with other therapies. OBJECTIVE: To assess disease response and to compare the response rates of patients treated with PUVA alone or PUVA and bexarotene. METHODS: EORTC 21011 (NCT 00056056) was a randomized phase III study comparing combined bexarotene (Targretin(®) ) and PUVA vs. PUVA alone in patients with stage IB and IIA mycosis fungoides (MF). The primary endpoint was the overall response rate [complete clinical response (CCR) plus partial response (PR)]. RESULTS: The study was prematurely closed due to low accrual after 93 of 145 required patients (65%) were randomized. Of the 93 randomized patients, 87 started treatment, 41 received PUVA and 46 received PUVA + bexarotene. Total UVA doses received were 107 J cm(-2) (range 1·4-489·9) in the PUVA arm vs. 101·7 J cm(-2) (0·2-529·9) in the combination arm. The safety profile was acceptable with few grade 3-4 toxicities observed in either arm. More drop-outs due to toxicity were observed in the combination arm compared with the PUVA-alone arm. The best overall response (CCR + PR) rate was 71% for PUVA alone and 77% for the combination arm (P = 0·57). The median duration of response was 9·7 months for PUVA vs. 5·8 months for the combination arm (P = 0·33). CCR was seen in 25 patients of whom 10 received PUVA alone (CCR 22%) and 15 received combination therapy (CCR 31%) (P = 0·45). CCR was sustained in 25% of patients regardless of therapy. There was a trend towards fewer PUVA sessions needed to achieve CCR in the combination arm (median 22) compared with the PUVA arm (median 27·5) (P = 0·11). Similarly, a trend towards lower UVA dose required to achieve CCR in the combination arm (median 55·8 J cm(-2) ) compared with the PUVA arm alone (median 117·5 J cm(-2) ) (P = 0·5) was observed. CONCLUSIONS: No significant difference in response rate or response duration was observed in this study. However, there was a trend towards fewer PUVA sessions and lower UVA dose required to achieve CCR in the combination arm (PUVA + bexarotene) but this did not achieve statistical significance due to insufficient power.

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.

Polyclonal expansion of T cells with the TCR V beta type of the tumour cell in lesions of cutaneous T-cell lymphoma: evidence for possible superantigen involvement.

The involvement of superantigens in the pathology of cutaneous T-cell lymphomas (CTCL) has been suggested before, but without unequivocal evidence for superantigen activity in the patients. Seeking evidence for superantigen activity we analysed clones and microdissected single cells isolated from the epidermis of early-stage lesions of a CTCL patient for their T-cell receptor (TCR) V beta expression and TCR V gamma gene rearrangements. The vast majority of these T cells expressed the TCR V beta family type of the tumour. From their TCR gamma gene rearrangements, however, these cells were polyclonal. The tumour cell clone accounted for about 60% of these cells, about 40% were of heterogeneous origin. This dominance of a single V beta family in the polyclonally expanded dermal T-cell populations implies superantigen activity in the CTCL lesions.

Bexarotene--an alternative therapy for progressive cutaneous T-cell lymphoma? First experiences.

BACKGROUND: A standard therapy for advanced cutaneous T-cell lymphomas has not yet been defined. Bexarotene is a new retinoid x receptor-specific retinoid that has been approved for systemic second-line therapy for cutaneous T-cell lymphomas in the USA and Europe. In order to evaluate the efficacy of bexarotene in cutaneous T-cell lymphomas, a pilot trial was initiated. PATIENTS AND METHODS: In a pilot project 10 patients with advanced cutaneous T-cell lymphomas, who had received a variety of previous treatments, were treated with bexarotene at the departments of dermatology in Münster, Minden and Charité Berlin, Germany. The patients received bexarotene at a dose of 300 mg/m2 body surface daily. According to the percentage of tumour reduction and affected body surface, the response rates were divided in complete and partial remission, stable disease and progressive disease. Laboratory parameters i.e. cholesterol, triglycerides transaminases, T3, T4, and TSH were screened regularly. RESULTS: In 2 patients a short partial remission was achieved; however, after a few weeks progression followed. In 4 patients a lasting stabilisation was obtained. The other 4 patients showed a progressive disease during therapy. 6 patients developed hypertriglyceridemia with levels up to 2000 mg/dl; therapy had to be suspended in 3 patients because of these adverse drug events. CONCLUSION: Weighing benefits and risks, bexarotene can at present not be recommended as standard therapy in the treatment of patients with progressive cutaneous lymphomas.

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.

Mimotopes for tumor-specific T lymphocytes in human cancer determined with combinatorial peptide libraries.

Mimotopes of a tumor-associated T cell epitope were determined using randomized and combinatorial peptide libraries and a CD8(+) T cell clone specific for the cutaneous T cell lymphoma cell line MyLa. Antigen recognition by this clone was found to be HLA-B8 restricted. More than 80 % of HLA-matched patients with cutaneous T cell lymphoma had mimotope-specific CD8(+) T cells in their peripheral blood. Mimotope-specific T cells isolated and expanded from a patient lysed MyLa cells in in vitro assays thus demonstrating their cytolytic capacity and tumor specificity. Mimotope vaccination of a patient without detectable mimotope-specific T cells induced frequencies of these cells of up to 1.82 % of the peripheral blood CD8(+) T cells.

Primary cutaneous follicle center cell lymphomas and large B cell lymphomas of the leg descend from germinal center cells. A single cell polymerase chain reaction analysis.

Primary cutaneous B cell lymphomas are defined as non-Hodgkin lymphomas that occur in the skin without extracutaneous involvement for 6 mo after diagnosis. They are characterized by a less aggressive course and better prognosis than their nodal counterparts. According to the European Organization for Research and Treatment of Cancer classification, the major subentities of primary cutaneous B cell lymphoma are follicle center cell lymphomas, immunocytomas, and large B cell lymphomas of the leg, which differ considerably regarding their clinical behavior, the former two being indolent, the latter being of intermediate malignancy. In this study, we applied a single cell polymerase chain reaction approach to analyze immunoglobulin V(H)/V(L) genes in 532 individual B lymphocytes from histologic sections of four follicle center cell lymphomas localized on the head and trunk, and four large B cell lymphomas on the leg. We found: (i) in six of eight patients a clonal heavy chain, and in seven of eight patients a clonal light chain rearrangement, all being potentially productive; (ii) no bias in VH gene usage, in four of seven light chain rearrangements the V kappa germline gene IGVK3-20*1 was used; (iii) no biallelic rearrangements; (iv) all V(H)/V(L) genes are extensively mutated (mutation rate 5.4-16.3%); (v) intraclonal diversity in six of eight cases (three of each group); and (vi) low replacement vs silent mutation ratios in framework regions indicating preservation of antigen-receptor structure, as in normal B cells selected for antibody expression. Our data indicate a germinal center cell origin of primary cutaneous follicle center cell lymphomas and large B cell lymphomas independent of those belonging to one of these subentities.

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.

IL-15 and IL-16 overexpression in cutaneous T-cell lymphomas: stage-dependent increase in mycosis fungoides progression.

Cytokines are of major importance for the pathogenesis of cutaneous T-cell lymphomas (CTCL). Recent data suggested that IL-15 and IL-16 are survival/growth factors for the malignant T cells in these entities. To investigate the expression of IL-15 and IL-16 in mycosis fungoides (MF) and CD30+ pleomorphic T-cell lymphoma in vivo, we established a competitive RT-PCR technique. Analyzing skin biopsies from CTCL patients at different stages in comparison to psoriatic and healthy skin, we found IL-15 and IL-16 mRNA overexpression in both CTCL entities. Remarkably, there was some evidence for a stage-dependent increase during MF progression. We found only slight overexpression in early stage MF, when only few tumor cells are detectable within the infiltrates, whereas marked overexpression was found in more advanced lesions, which are characterized by a higher density of malignant cells. These results suggested that CTCL cells themselves might produce the cytokines. To further elucidate this hypothesis, two CTCL cell lines were analyzed but gave conflicting results. Therefore, the cellular origin of the IL-15 and IL-16 overexpression in CTCL remains unclear. Considering the significant overexpression of IL-15 and IL-16 and their biological capacities it is likely that these cytokines contribute to the tumor development. So, they might be involved in growth and skin homing of CTCL cells.

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.

Clonal evolution in a primary cutaneous follicle center B cell lymphoma revealed by single cell analysis in sequential biopsies.

B cell neoplasias descending from germinal center cells harbor the hallmark of intraclonal diversity resulting from ongoing mutation in the variable parts of their immunoglobulin-encoding genes. To characterize a primary cutaneous follicle center B cell lymphoma in more detail, we analyzed the respective VH and VL genes in single cells mobilized from four sequential biopsies, three taken from the skin and one obtained after internal dissemination from a retrobulbar infiltrate. The lymphoma cells were found to contain V5-51/D6-12/JH5b (heavy chain) and A27/Jkappa2 (light chain) gene rearrangements detected on both the genomic and the transcriptional level. To provide an accurate mutation analysis, the specific VH gene counterpart (V5-51UK) was cloned from the patient's germline. Analyzing 226 single cells, we found: (i) complete nucleotide identity when VH and VL genes of lymphoma cells from one particular biopsy were compared among each other; (ii) intraclonal diversity due to ongoing mutation comparing the sequences obtained from sequential biopsies; (iii) both VH and VL genes to be highly mutated. Deducing from the sequence data, we propose a scenario of the clonal evolution of the B cell tumor in this patient. From the molecular-biological point of view, this primary cutaneous follicle center B cell lymphoma shows the features of a germinal center cell lymphoma. To draw this conclusion from single cell PCR data, however, a sample of sequential biopsies had to be analyzed.

A T-cell epitope determined with random peptide libraries and combinatorial peptide chemistry stimulates T cells specific for cutaneous T-cell lymphoma.

BACKGROUND: Mycosis fungoides is the most frequent T-cell lymphoma of the skin. Despite numerous attempts, no tumour antigens have yet been identified. Only in one case has an idiotype-derived peptide been found to trigger CTL of the respective patient. The identification of natural antigens requires the cultivation of large amounts of tumour cells in vitro, which has been possible in two exceptional cases. The identification of synthetic epitopes for tumour-specific CTL with random peptide libraries can overcome this limitation and is a powerful tool for application in the development of immune therapies for a wide range of patients. MATERIALS AND METHODS: The critical amino acids for the construction of epitopes for the CTCL-specific CTL clone My-La CTL were determined with synthetic peptide libraries in positional scanning OX8 format in a standard 61chromium release assay. Sixteen different peptides could be synthesized from the combinatoric of these amino acids with the canonical anchor amino acids for MHC binding. These peptides were tested for their capacity to stimulate My-La CTL and PBMC of an HLA-matched CTCL patient. RESULTS: A synthetic epitope could be identified for My-La CTL, which was recognized in a HLA-restricted manner. The response towards this epitope was comparable to the response towards their natural target My-La. Using these synthetic epitopes, T cells of a HLA-matched patient could be induced in vitro and led to the establishment of different cell lines and clones. Some of these lines recognized the peptides as well as the allogenic but HLA-matched tumour cell line My-La, indicating that they are specific for a naturally expressed tumour antigen. CONCLUSIONS: The identification of synthetic epitopes for tumour-specific CTL clones can be used for the development of vaccines for immune therapies of cancer; such peptides can be applied inter-individually. Synthetic epitopes must not correspond to the natural ones, but they can be even more potent as stimulation of specific T cells and can be fine-tuned to increase the success of the therapy.

Cytokine expression in primary cutaneous germinal center cell lymphomas.

Physiologically, B-lymphocytes are not present in the skin. Even in pathological situations they rarely occur. In contrast, primary cutaneous B-cell lymphomas (CBCL) are characterized by proliferation of B lymphocytes within the skin. This suggests the existence of a certain microenvironment supporting homing and expansion of clonal B cells. Cytokines were demonstrated to be involved in the pathogenesis of cutaneous lymphomas of T-cell origin. Cytokine expression in cutaneous B-cell lymphoma lesions, however, has not been investigated so far. Therefore, the mRNA level of several cytokines was analyzed in biopsies from 7 patients with CBCL and compared to pleomorphic T-cell lymphoma (n = 6), psoriasis (n = 9), and healthy skin (n = 7), using a competitive RT-PCR approach. An overexpression of TNF-alpha, IL-10, and IL-6 was found. Enhanced IL-8 mRNA expression was detected in 2/7 cases. The overexpression of IL-6 and IL-10 in CBCL might be of particular importance, since these cytokines are considered to support B-cell growth. Additionally, the overexpression of IL-10 may contribute to tumor progression since this immunosuppressive cytokine might be involved in downregulation of immunological tumor surveillance, in part by inhibiting type 1 cytokine formation. In fact, we did not detect IFN-gamma and IL-2 expression. Taken together, we found a cytokine pattern in CBCL lesions which might contribute to tumor B-cell growth.

Mainly unmutated V(H) genes rearranged in B cells forming germinal centers in a cutaneous pleomorphic T-cell lymphoma.

B cells in skin lesions of a pleomorphic cutaneous T-cell lymphoma with reactive germinal center hyperplasia were analyzed for their immunoglobulin V(H)DJ(H) gene rearrangements by micromanipulation and single cell polymerase chain reaction (PCR) analysis. In B lymphocytes located in germinal center-like structures, we found in 11/16 different V(H)DJ(H) rearrangements completely unmutated VH genes, suggesting that those cells did not undergo antigen-driven selection. Two V(H) genes showed more than 98% germ-line identity. In only three cells V(H) segments were somatically mutated to a higher extent, but two of these rearrangements were non-productive. These results differ markedly from what we have previously detected in B cells present in mycosis fungoides, another entity of cutaneous T-cell lymphomas where the Ig gene repertoire resembles the situation in peripheral blood with a significantly higher proportion of mutated V(H) genes. When investigating the large atypical B cells strongly expressing CD30 which were detected within the T-cell zone outside the germinal centers, we found again, in most cases, that the rearranged VH genes were completely unmutated. The B cells were of polyclonal origin. Due to this comparable Ig gene repertoire and mutational pattern, we suggest that these cells descend from the germinal center centroblasts which migrated into the T-cell zone and obviously became stimulated to express the CD30 marker. The micromanipulation technique and molecular analysis on the single cell level may provide an important input into our understanding of the mechanisms of immune regulation in cutaneous lymphomas.