Allogeneic gene-modified tumor cells (RCC-26/IL-7/CD80) as a vaccine in patients with metastatic renal cell cancer: a clinical phase-I study.

Despite novel targeted agents, prognosis of metastatic renal cell cancer (RCC) remains poor, and experimental therapeutic strategies are warranted. Transfection of tumor cells with co-stimulatory molecules and/or cytokines is able to increase immunogenicity. Therefore, in our clinical study, 10 human leukocyte antigen (HLA)-A(*)0201(+) patients with histologically-confirmed progressive metastatic clear cell RCC were immunized repetitively over 22 weeks with 2.5-40 × 10(6) interleukin (IL)-7/CD80 cotransfected allogeneic HLA-A(*)0201(+) tumor cells (RCC26/IL-7/CD80). Endpoints of the study were feasibility, safety, immunological and clinical responses. Vaccination was feasible and safe. In all, 50% of the patients showed stable disease throughout the study; the median time to progression was 18 weeks. However, vaccination with allogeneic RCC26/IL-7/CD80 tumor cells was not able to induce TH1-polarized immune responses. A TH2 cytokine profile with increasing amounts of antigen-specific IL-10 secretion was observed in most of the responding patients. Interferon-γ secretion by patient lymphocytes upon antigen-specific and non-specific stimulation was substantially impaired, both before and during vaccination, as compared with healthy controls. This is possibly due to profound tumor-induced immunosuppression, which may prevent induction of antitumor immune responses by the gene-modified vaccine. Vaccination in minimal residual disease with concurrent depletion of regulatory cells might be one strategy to overcome this limitation.

Diagnostic tools in mycosis fungoides.

Cutaneous T-cell lymphomas (CTCL) represent clonal proliferations of neoplastic skin homing T-cells. Within the group of primary CTCL, mycosis fungoides (MF) is the most common entity, affecting the skin as a primary site. MF initially presents in the skin with a slow indolent course of a characteristic stepwise progression from patches to plaques and tumors accompanied by distinctive histological changes. Routine diagnosis is based on these clinical and histological features. However, due to similarities with benign lymphoproliferative or reactive skin diseases, especially at the initial presentation of the disease, diagnosis can be difficult. Although the etiology of mycosis fungoides is still unknown, important insights have been gained in the immunological and genetic perturbations, which are associated with the disease. In the last years the emergence of molecular genetic techniques allowing to analyze the clonality status in lymphocytic infiltrates, has provided new tools with the potential to increase the accuracy of diagnosis, staging and therefore stage-adapted treatment. Nevertheless, it is important to notice that some limitations restrict the predictive value of the results obtained by these analyses. Diagnostic tool of MF, including clinical, histo- and immunohistological findings as well as molecular genetic analysis will be covered in this review.

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.

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.

Detection of melanoma micrometastases in the sentinel lymph node and in nonsentinel nodes by tyrosinase polymerase chain reaction.

The aim of our study was to investigate the metastatic pathways of melanoma cells in sentinel and other regional lymph nodes. The term "sentinel lymph node" means that the first lymph node of the draining site of a primary tumor is never bypassed in malignant melanoma. In this case lymph node dissection would be necessary only when melanoma cells are detected in the sentinel node. Tyrosinase reverse transcriptase-polymerase chain reaction was applied to search for metastatic melanoma in the sentinel lymph node and in further lymph nodes of a complete lymph node basin in patients who underwent lymph node dissection. In 24 patients with malignant melanoma the draining site of the tumor was marked by lymphoscintigraphy and by intraoperative injection of patent blue V in the area around the primary tumor. The lymph nodes of the affected basin were excised and prepared for histopathologic, immunohistochemical, and molecular biologic examinations. Regarding the sentinel lymph node, 10 of 24 patients showed morphologic evidence for metastases, three additional patients showed only tyrosinase transcripts. In 11 of these 13 cases we found one or more nonsentinel lymph nodes with morphologically detectable melanoma cells and/or tyrosinase mRNA. Interestingly, in seven of 24 patients a positive tyrosinase reverse transcriptase-polymerase chain reaction was received in nonsentinel lymph nodes, whereas the sentinel lymph node was negative, not only for all histologic examinations but also by tyrosinase reverse transcriptase-polymerase chain reaction. In five of seven patients of the latter group, gp100 reverse transcriptase-polymerase chain reaction was carried out, showing also gp100 mRNA in nonsentinel lymph nodes only. Our data indicate that the concept of the sentinel lymph node may miss micrometastases. Whether such micrometastases cause a recurrence or a metastasis of malignant melanoma, or can be destroyed by the immune system, remains to be clarified.

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 monoclonal T-cells with TCR gamma PCR in mycosis fungoides]. / Nachweis von monoklonalen T-Zellen mittels TCR gamma-PCR bei Mycosis Fungoides (MF).

The monoclonal dominant malignant T cells in mycosis fungoides (MF) carry identical TCR gamma rearrangements. Their detection is a useful diagnostic tool. Thus, in routine diagnosis we investigated the occurrence of monoclonal T cells in skin biopsy samples of MF-patients by TCR gamma-PCR, followed by temperature gradient gel electrophoresis (TGGE). In 188 out of 208 MF patients, at least one skin sample with sufficient DNA quality for PCR analysis was obtained. Applying a consensus PCR for the TCR gamma genes V gamma I and J gamma 1/2, we detected monoclonal T cells in 122 cases (65%). In the remaining 66 cases, we performed two multiplex-PCRs, covering rearrangements of the other TCR gamma genes. Here we found in 11 cases (6%) predominant clonal rearrangements of V gamma II-IV and J gamma 1/2 and in 2 (1%) those of V gamma I-IV and J gamma P 1/2. In patients with MF, detecting rearrangements of only V gamma I and J gamma 1/2 is sufficient for PCR screening analysis. In 53 of the patients (28%) the applied methods revealed no monoclonal T cells. This may be due to a low cell number, oligoclonal nature, chromosomal abberations or remaining of TCR in germline configuration.

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.

Demonstration of frequent occurrence of clonal T cells in the peripheral blood of patients with primary cutaneous T-cell lymphoma.

Clonal T cells have been demonstrated in skin lesions of all stages of cutaneous T-cell lymphomas (CTCLs). However, there are conflicting data regarding the CTCL stage at which dissemination of clonal cells into peripheral blood occurs. Although the multifocal occurrence of cutaneous CTCL lesions and T-cell recirculation suggest an early appearance of neoplastic cells in the blood, circulating clonal T cells have only been detected in advanced stages. We investigated their occurrence by a highly sensitive polymerase chain reaction (PCR) assay amplifying T-cell receptor gamma rearrangements and subsequent heteroduplex temperature gradient gel electrophoresis (HD-TGGE) of the amplification products. Circulating clonal T cells were found in 26 of 45 patients with mycosis fungoides (MF), six of seven with Sezary's syndrome (SS), 10 of 13 pleomorphic CTCLs, and three of four unclassified CTCLs. Corresponding skin specimens carried clonal T cells in 29 of 40 MF, three of four SS, 12 of 12 pleomorphic, and two of two unclassified CTCL patients. Except for the blood specimen of a psoriatic patient, all samples of 60 controls (psoriasis vulgaris, atopic dermatitis, and healthy volunteers) revealed polyclonal amplification products. In 30 of 32 CTCL patients carrying a clonal rearrangement in blood and skin, identity of both clones was indicated by HD-TGGE and confirmed by sequencing six of these cases. We found an unexpected high frequency of identical clonal T cells in peripheral blood and skin of CTCL patients, including early stages of MF. This supports the concept of an early systemic disease in CTCL and raises new questions concerning the pathogenesis.

Search for Kaposi's sarcoma-associated virus DNA in hemangioproliferative disorders and cutaneous malignant lymphoma.

Recently, a Kaposi's sarcoma-associated herpesvirus (KSHV) was discovered. We evaluated by PCR 14 paraffin-embedded specimens with the histological diagnosis of endemic, classic and HIV-associated Kaposi's sarcoma (KS) for the presence of the KSHV DNA sequence. In addition, biopsies of adjacent, histologically unaffected skin, peripheral-blood mononuclear cells (PBMCs) of HIV-infected KS patients, PBMCs of one classic KS patient, and specimens of patients with hemangioproliferative disorders other than KS as well as samples of cutaneous T- and B-cell lymphoma were analyzed for KSHV. In all cases of KS, independent of the KS subtype, KSHV was detected in lesional skin. No KSHV was found in biopsies of the adjacent unaffected skin or PBMCs of HIV-infected KS patients. We found KSHV in the PBMCs of a patient with classical KS. All specimens of cutaneous T- and B-cell lymphomas or lymphomatoid papulosis were negative for KSHV. In addition, the samples with hemangioproliferative disorders other than KS were negative for KSHV. There was one borderline case of KS or acroangiodermatitis that was positive for KSHV. Additional histological sections and clinical evaluation confirmed the diagnosis of classic KS. In summary, the data indicate that PCR for KSHV should be a useful diagnostic tool in cases of hemangioproliferative disorders.

[The diagnostic value of antinuclear antibodies]. / Zur diagnostischen Bedeutung von antinukleären Antikörpern.

In this review we focus on the diagnostic importance of antinuclear antibodies (ANA), the biological function of the relevant autoantigens and on some methodological questions regarding the detection of ANA. The qualitative and quantitative evaluation of ANA has improved significantly in recent years with the introduction of several new test kits. A precondition for the rational use of those assays is knowledge of the diagnostic validity of the detected ANA with regard to the method used. ANA are autoantibodies that react with nucleic acids, protein-nucleic acid complexes and proteins of nuclei. The reasons for their in vivo production are unknown. ANA characterize several of the so-called connective tissue autoimmune diseases and their subtypes, either alone or in typical combinations. They differ significantly with regard to their prevalence and thus in their diagnostic validity. Specificity and prevalence do not correlate. ANA are not markers of disease activity except for antibodies against dsDNA. ANA levels can be interpreted correctly only in connection with clinical symptoms and other laboratory findings.

Serologic characteristics in primary biliary cirrhosis associated with sicca syndrome.

BACKGROUND: Patients with primary biliary cirrhosis (PBC) may have coincidental sicca syndrome (SS). The frequencies of SS and the occurrence of the Sjögren's-associated anti-Ro and -La antibodies in PBC patients have been reported at widely varying prevalences. This study investigated whether distinctive serologic characteristics are associated with SS in PBC. METHODS: Forty PBC patients and thirty patients with other types of liver cirrhosis were tested for SS and associated autoantibodies (ANA, AMA 2, anti-Ro, anti-La, anti-U1RNP-A, -C, -68 kD, and rheumatoid factors). RESULTS: Fourteen PBC patients (35%) complained of sicca symptoms, of whom 10 (25%) had a positive Schirmer-I test, and 7 (17.5%) had serologic characteristics similar to those of Sjögren's syndrome. Anti-52-kD Ro antibodies were positive in seven PBC/SS cases (p < 0.025). There was no anti-Ro positive PBC patient without SS. Three patients with PBC/SS with anti-52-kD Ro and anti-smooth-muscle antibodies developed lung fibrosis. No patient in the other cirrhosis group had SS or its characteristic autoantibody findings. CONCLUSIONS: It was suggested that PBC and SS are frequently associated. Anti-52-kD Ro antibodies seem to be a characteristic serologic finding for SS in PBC, suggesting their pathogenic role in autoimmune sialadenitis.

Precursor frequencies for DNA-specific B lymphocytes in patients with systemic lupus erythematosus (SLE).

Precursor frequencies for anti-DNA-secreting B cells were estimated in six healthy donors and 18 SLE patients with active and inactive disease. Precursors for IgG anti-dsDNA-secreting B cells were exclusively detected in SLE patients (73% of active patients and one inactive patient, 0.01-0.99% of IgG-producing B cells). These frequencies were in the same order of magnitude as frequencies of precursors for IgG anti-tetanus toxoid, which were detectable in three healthy volunteers after booster vaccination (0.07-0.8% of IgG-producing B cells), but not before (< 0.01%). Precursors for IgG anti-ss-DNA secreting B cells were observed in 33% of healthy donors and in 78% of SLE patients (0.01-0.32% of IgG-producing B cells). Only patient-derived IgG anti-DNA clones cross-reacted with (33%) or were monoreactive to dsDNA (12%). Precursors for IgM anti-DNA-secreting B cells were observed in healthy donors and SLE patients in comparable frequencies and with similar reactivities with ssDNA and dsDNA. Segregation analyses and sorting experiments showed that > 94% of clones secreting IgG anti-DNA were derived from in vivo sIgG+ B cells. sIgM+ B cells were induced to switch in vitro; however, only twice were cultures containing IgM and IgG anti-DNA antibodies observed under clonal conditions. In conclusion, our results indicate that precursor B cells for IgG anti-dsDNA in SLE patients are similarly selected and expanded as are precursor B cells specific for foreign antigens such as tetanus toxoid.