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U Keilholz

Publications and source records attributed to U Keilholz.

At least 37 records · Page 2Linked to original sources
PubMed2001

Application of the IFN-gamma ELISPOT assay to quantify T cell responses against proteins.

The ELISPOT assay has been established for the direct ex vivo quantification of peptide-reactive T lymphocytes from peripheral blood mononuclear cells (PBMC). In this report we studied the ELISPOT assay conditions for the detection of T cell responses against protein antigens including Tetanus toxoid (TT), purified-protein-derivative (PPD) and a synthetic 30 mer peptide derived from influenza matrix protein (IMP) containing the HLA-A*0201-restricted 9 mer peptide epitope GILGFVFTL as a model for a viral protein. We found several aspects to be crucial for a sensitive detection of T cell responses against proteins including a pellet preincubation step and the monocyte concentration. Using optimized assay conditions specific CD4+ T cell responses against TT and PPD as well as CD8+ T cell responses against IMP can be quantified directly ex vivo from peripheral blood mononuclear cells.

Amino Acid Sequence↗
PubMed2001

Minimal residual disease in melanoma.

A number of specific genes encoding for melanosomal proteins are selectively expressed in melanocytes and melanomas. For detection of circulating melanoma cells, the expression of the tyrosinase gene is most widely used. Several cohorts of melanoma patients from single institutions have been analyzed by various research groups for the presence of circulating melanoma cells in all stages of disease. The percentage of patients with evidence of occult tumor dissemination has been correlated with stage of disease in several, but not all, reports. Two prospective analyses suggest that the PCR result is of prognostic value in melanoma. Several laboratories have found PCR evidence for circulating melanoma cells in the vast majority of untreated patients with stage IV disease, although other groups have reported much lower frequencies. Taken together, there is a wide range of results. Methodological differences likely account for this discrepancy. With the availability of true quantitative RT-PCR systems, such as the Light Cycler system, accurate quantification of tyrosinase transcripts over a range of 1-10,000 tumor cells per ml of blood is possible. Quantitative RT-PCR systems also dramatically improve quality control, since exact quantitation of housekeeping gene mRNA facilitates determination of sample quality.

Humans↗
PubMed2001

Nested quantitative real time PCR for detection of occult tumor cells.

Quantification of tumor mRNA markers expressed by occult circulating tumor cells may be of prognostic value in a variety of neoplasms and disease stages. We therefore developed a novel real time nested polymerase chain reaction (PCR) assay to quantify rare transcripts using the light cycler system. Tyrosinase mRNA expressed by melanocytes and melanoma cells was used as a model. Ten-milliliter samples of ethylenediaminetetra-acetate (EDTA) blood from healthy volunteers were spiked with 10-10(4) RVH melanoma cells. Following RNA extraction and cDNA synthesis, a nested PCR with specific primers was performed. Resonance energy transfer between fluorescently labeled hybridization probes specific for the amplified portion of the tyrosinase transcript allowed continuous monitoring of the second round of PCR. The number of preamplification cycles in the first round was varied between 20 and 35. Our results show that nested PCR revealed quantitative data, regardless of the number of cycles used in the first round. The range of real time data can span four logs of target mRNA concentrations (1-10(3) tumor cells/ml of blood) and the x-intercepts of the log phases were always consistent with the amount of transcripts in the sample. These experiments indicate that nested PCR, which is much more sensitive than single-round PCR, is a useful tool for the quantification of rare transcripts--a result with important implications for the study of minimal residual disease. The assay is currently being adapted for other tumor types including leukemias as well as other solid tumors.

DNA Primers↗
PubMed2001

Pilot study of hepatic intraarterial fotemustine chemotherapy for liver metastases from uveal melanoma: a single-center experience with seven patients.

BACKGROUND: Uveal melanoma is characterized by a high frequency of hepatic metastases. For patients with liver metastases, who have a median survival of 5 to 7 months, surgery and systemic conventional chemotherapy have little to offer. METHODS: Between February 1995 and July 1999, seven patients with isolated hepatic metastases from uveal melanoma were enrolled into a pilot trial of intraarterial fotemustine therapy. An implantable Port-A-Cath catheter was inserted into the hepatic artery for regional chemotherapy via the gastroduodenal artery. Fotemustine 100 mg/m2 was administered intraarterially over a 4-h period. The induction phase consisted of one administration per week for 4 weeks, followed by a 5-week rest period. Maintenance therapy with administration of fotemustine every 3 weeks continued until progression or toxicity. RESULTS: Ten patients were evaluated for the trial. One patient was not eligible because of impaired liver function, and in two patients implantation of the port system was not possible for anatomic reasons. Seven patients received a median of 16 treatment cycles (range, 4-28) and all were evaluable for response. Two patients achieved a partial response (PR), three had stable disease (SD), and tumor progressed in two patients (PD). The median survival time from diagnosis of liver metastasis was 24 months (range, 4 to 50+ months). Two patients survived for more than 2 years and two patients are still alive. The toxicity was low and the treatment could be administered on an outpatient basis. CONCLUSION: Intraarterial fotemustine treatment of uveal melanoma metastatic to the liver is well tolerated, and in some patients is associated with prolonged survival.

Adult↗
PubMed2001

Cyclophosphamide, adriamycin and dexamethasone (CAD) is a highly effective therapy for patients with advanced multiple myeloma.

BACKGROUND: Patients with advanced multiple myeloma (stage III or progressive myeloma) received the CAD protocol every three weeks: cyclophosphamide 200 mg/m2 i.v./orally days 1-4, adriamycin 30 mg/m2 i.v. on day 1 and dexamethasone 40 mg p.o. days 1-4. PATIENTS AND METHODS: Forty-six patients with a median age of sixty years (range 34-84 years) were enrolled. According to Durie-Salmon 44 patients were in stage III, 2 in stage II; 6 patients had renal insufficiency (stage B). Twenty-three patients were pre-treated at least with melphalane/prednisone. RESULTS: Remission rates were as follows: complete remission 4%, partial remission 70%, minimal change 11%, no change 11%, progressive disease 4%. After an observation time of 14 months the median progression free interval for 33 patients not treated with subsequent high-dose chemotherapy with stem-cell support was more than 14 months. Overall, treatment was well tolerated. After 209 cycles given febrile neutropenia occurred in 11% of cycles including one fatal outcome. Neutropenia or thrombocytopenia grade 3-4 WHO was recorded in 18% and 6% of the cycles, respectively. CONCLUSIONS: This study shows that CAD is an effective regimen with an overall remission rate of 74%. The CAD protocol should be further evaluated in prospective trials.

Administration, Oral↗
PubMed2001

Reliability of PCR-based detection of occult tumour cells: lessons from real-time RT-PCR.

For the molecular detection of rare tumour cells in clinical samples, real-time reverse transcription-polymerase chain reaction (RT-PCR) offers two important advantages over conventional RT-PCR assays: the results are quantitative and, perhaps more importantly, it facilitates exact sensitivity controls on a per sample basis as well as exact comparison of different assay protocols. We report here on quantitative results obtained with different protocols for RNA isolation and cDNA synthesis for amplification of beta2-microglobulin transcripts using the light cycler system. Furthermore, housekeeping gene-specific PCRs were compared with PCRs specific for an artificial transcript (internal standard) detected simultaneously at a level comparable to the wild-type sequence. Artificial tyrosinase transcripts derived from a vector construct stably transfected into a human lymphoma cell line were used as a model to test the usefulness of artificial internal standards as an alternative to housekeeping genes. The highest RNA yields were obtained using a combination of phenol-chloroform extraction and the High Pure RNA Isolation Kit. Analysing beta2-microglobulin transcript-specific RT-PCRs, the highest sensitivity was obtained for cDNAs generated with Omniscript reverse transcriptase and oligo-p(dT)15 primer. Regarding patient blood samples, RT-PCRs specific for beta2-microglobulin, porphobilinogen deaminase and artificial tyrosinase transcripts provided quantitative data for all, for 18 out of 21, and for 10 out of 21 samples, respectively. Quantification of beta2-microglobulin transcripts by the light cycler system defined the protocol revealing the highest cDNA quality. Comparisons of quantitative data from RT-PCRs specific for beta2-microglobulin, porphobilinogen deaminase and artificial tyrosinase transcripts enabled us to determine a close range for crossing points within which sufficient cDNA quality can be guaranteed, even for the detection of rare transcripts. PCRs specific for the artificial internal standard are ideally suited for cDNA quality assessment on a per sample basis.

DNA, Complementary↗
PubMed2001

The EORTC melanoma group translational research program on prognostic factors and ultrastaging in association with the adjuvant therapy trials in stage II and stage III melanoma. European Organization for Research and Treatment of Cancer.

Last year the Melanoma Group of the European Organization for Research and Treatment of Cancer (EORTC-MG) completed accrual (1418 patients) for trial EORTC 18952, a three-arm phase III trial evaluating adjuvant therapy with two different intermediate doses of interferon (IFN) alfa-2b versus observation for stage IIB-III melanoma. About 25% of the patients entered the trial with tumor-positive sentinel nodes (SNs). Prognosis was significantly better in SN-positive patients than in patients with palpable regional node involvement (P < .00001). Subsequently the EORTC-MG embarked on two large phase III trials of adjuvant therapy based on the tumor status of the SN. In trial EORTC 18961 for stage II melanoma, GM2-KLH/QS-21 vaccination is compared with observation (1300 patients); in trial EORTC 18991 for stage III melanoma, 5-year treatment with pegylated interferon alfa-2b (PEG-Intron) is compared with observation (900 patients). Translational research projects will compare SN assessment by hematoxylin and eosin (H&E) staining, immunohistochemistry (IHC), and reverse transcriptase-polymerase chain reaction (RT-PCR) to determine the relative accuracy of each method and its correlation to relapse and survival of patients with stage II melanoma. In stage III patients, a similar workup of the most proximal nonsentinel node in the full lymph-node dissection specimen will indicate the accuracy of each methodology to detect nodal metastasis beyond the SN and the prognostic significance thereof. These findings will be correlated to the results of sequential blood testing by RT-PCR and by tumor marker assays for S100, TA90, and angiostatin. In addition, tumor-positive and tumor-negative SNs will be assessed for activated cytotoxic T lymphocytes and downregulation of dendritic cell functions.

Antineoplastic Agents↗
PubMed2000

Quantitation of antigen-reactive T cells in peripheral blood by IFNgamma-ELISPOT assay and chromium-release assay: a four-centre comparative trial.

The ELISPOT assay is increasingly being used for the monitoring of the induction of antigen-reactive T cells in cancer vaccination trials. In order to evaluate the reliability of T cell frequency analysis with the ELISPOT assay, a comparative study was performed in four European laboratories. Six samples from healthy subjects were analyzed for the frequency of influenza-reactive CD8+ T cells in peripheral blood mononuclear cells (PBMC) by IFNgamma-ELISPOT assay. In addition, one laboratory determined cytotoxic T cell precursor (CTL) frequencies in these samples by limiting dilution chromium-release assay (LDA), and three laboratories performed a variant of the LDA, the multiple microculture assay (MMA). Consistent frequencies of influenza peptide-reactive T cells were obtained with the ELISPOT assay in all four laboratories. The numbers detected by ELISPOT assay correlated closely with those determined by LDA. In contrast, the frequencies obtained with the MMA differed considerably and showed little correlation with the other two assays. This study shows that it is possible to use the ELISPOT assay to determine with reliability antigen-reactive T cells in a multicenter setting. We suggest that this assay may be suitable for monitoring cancer vaccine trials.

Antigens, Viral↗
PubMed2000

High frequencies of circulating melanoma-reactive CD8+ T cells in patients with advanced melanoma.

To determine whether circulating tumor-reactive T cells are present in melanoma patients, unstimulated T cells from peripheral blood were tested for recognition of HLA-A2- or HLA-A1-matched melanoma cell lines using the ELISPOT assay. Eleven out of 19 patients with metastatic melanoma had a T-cell response with up to 0.81%, 0.78%, 0. 53%, 0.12%, 0.10%, 0.09%, 0.07%, 0.06%, 0.06%, 0.04%, and 0.04% of peripheral blood mononuclear cells (PBMC) secreting IFNgamma upon exposure to various HLA-A2- or HLA-A1-matched melanoma cell lines. These T-cell responses were mediated by CD8+ T cells and could specifically be blocked by an anti-HLA-A2 antibody in HLA-A2-positive patients. Separation experiments performed in one melanoma patient showed tumor-reactive T cells in both the CD8+ effector T cell (CD45RA+/IFNgamma+) as well as the CD8+ memory T-cell compartment (CD45RO+/IFNgamma+). In 3 out of 5 patients, in whom autologous cell lines were available, similar frequencies of T cells in response to HLA-A1- or HLA-A2-matched allogeneic and autologous tumor cells were observed, while 2 patients had a T-cell response restricted to either the autologous or the allogeneic cell lines. These results give evidence for the presence of tumor-reactive CD8+ T cells in more than half of melanoma patients tested. Although some of these patients have clinical evidence for an immunological-mediated tumor control, several patients have growing tumors suggesting presence of escape mechanisms.

Antigens, Neoplasm↗
PubMed2000

Natural T-cell response against MHC class I epitopes of epithelial cell adhesion molecule, her-2/neu, and carcinoembryonic antigen in patients with colorectal cancer.

The antigens epithelial cell adhesion molecule (Ep-CAM), her-2/neu, and carcinoembryonic antigen (CEA) are potential T-cell targets in antigen-specific vaccination-based cancer therapy. We performed this study to evaluate whether a natural specific T-cell response against these tumor-associated antigens (TAAs) already exists in patients with colorectal carcinoma (CRC). We used the IFN-gamma ELISPOT assay to detect circulating TAA-reactive T cells directly ex vivo in unstimulated peripheral blood mononuclear cells. We analyzed the T-cell response in peripheral blood mononuclear cells of 22 HLA-A2-positive patients with CRC and 8 HLA-A2-positive healthy subjects against 3 HLA A2-restricted peptide epitopes of the TAAs Ep-CAM (GLKAGVIAV), her-2/neu (IISAVVGIL), and CEA (YLSGANLNL). Seven of 22 patients but none of the 8 healthy subjects had T cells specifically secreting IFN-gamma in response to one to three of these antigens (n = 4, Ep-CAM; n = 5, her-2/neu; n = 6, CEA). In three of the seven responding patients, TAA-reactive T cells were further characterized by flow cytometry. In all three patients, the majority of these T cells have a CD3+CD8+IFN-gamma+CD69+CD45RA+ phenotype, resembling activated effector-type T cells. T-cell responses occurred only in patients with metastatic disease (Dukes' stages C and D). The results of this study indicate that natural T-cell responses against TAAs occur in approximately one-half of CRC patients with involvement of lymph nodes or distant metastases, but not in CRC patients with disease confined to the intestinum.

Antibodies, Monoclonal↗
PubMed2000

Correlation of positive RT-PCR for tyrosinase in peripheral blood of malignant melanoma patients with clinical stage, survival and other risk factors.

The clinical value of the reverse transcription polymerase chain reaction (RT-PCR) assay for tyrosinase in peripheral blood of melanoma patients is still under debate. A total of 212 blood samples from 212 melanoma patients in all clinical stages (AJCC) were examined. Erythrocytes were lysed prior to RNA extraction by phenol precipitation from 2.7 ml of blood. cDNA for tyrosinase PCR was synthesized using random hexamers. Positive tyrosinase RT-PCR results were obtained in 11% of 106 stage I patients, 18% of 56 stage II patients, 31% of 26 stage III patients and 67% of 24 stage IV patients. After a median follow-up of 36 months (range 26-41), stage III patients with positive RT-PCR for tyrosinase had a shortened disease-free interval as compared to negative patients (P < 0.01). In stage IV patients, median overall survival was 8 months in case of a positive RT-PCR in contrast to 12 months in case of a negative test. While univariate analysis showed sex and primary tumour location associated with positive RT-PCR, multiple regression analysis revealed clinical stage and detection of tyrosinase transcripts in peripheral blood as best prognostic factors. Hazard ratios for disease-free survival were 19.7 (confidence interval (CI) 8.53-45.5, P = 0.0001) for metastatic vs primary disease and 2.96 (CI 1.49-5.89, P = 0.002) for positive vs negative tyrosinase RT-PCR. The corresponding hazard ratios for overall survival were 97.0 (CI 12.7-741, P = 0.0001) and 4.33 (CI 1.69-11.1, P= 0.002). Our results emphasize the importance of tyrosinase RT-PCR testing in peripheral blood.

Adult↗
PubMed2000

The emerging role of cytokines in the treatment of advanced melanoma. For the EORTC Melanoma Cooperative Group.

Cytokine-based treatment regimens have been evaluated for advanced melanoma in a number of phase I and phase II trials within the last decade. Treatment with interleukin-2 (IL-2) as a single agent has resulted in response rates of approximately 15%, if a high dose of IL-2 is administered. Combination of interferon-alpha (IFNalpha) and high dose IL-2 yields response rates ranging from 10 to 41%. Response rates exceeding 50% have been reported with chemoimmunotherapy, if the treatment regimens included at least the three agents IL-2, IFNalpha and cisplatin. Recent randomized trials have evaluated the impact of these drugs on the survival of patients with advanced melanoma. The current 'state of the art' is discussed in this review.

Antineoplastic Agents↗
PubMed2000

The role of interleukin-2 in the management of stage IV melanoma: the EORTC melanoma cooperative group program.

PURPOSE: To review the current information available from the European Organization for Research and Treatment of Cancer (EORTC) programs on the use of interleukin (IL)-2 in stage IV melanoma patients. PATIENTS AND METHODS: A database from 631 patients treated within 27 trials with high-dose IL-2-based regimens was compiled to develop hypotheses and valid stratification factors for randomized trials. Subsequently, 126 patients were enrolled in a trial evaluating interferon alfa (IFN-alpha) and IL-2 with or without cisplatin, and 325 patients were enrolled in an ongoing EORTC trial (18951) to evaluate dacarbazine, cisplatin, and IFN-alpha, with or without IL-2. RESULTS: The database suggests long-term survival rates of 23% and a 5-year survival rate of 13% for patients receiving a combination of IFN-alpha and IL-2 with or without chemotherapy. The addition of chemotherapy improved response rate but not survival. The first randomized trial testing the role of cisplatin in a chemoimmunotherapy regimen for advanced melanoma revealed a palliative effect for cisplatin but no survival benefit. The current trial (EORTC 18951), which is testing the impact of IL-2 on survival, is still immature. In the translational research program, we have evidence that patients in continuous complete remission after IL-2-based treatment have evidence of residual disease by polymerase chain reaction assay and, at the same time, melanoma-reactive T cells are present in the peripheral blood. CONCLUSION: Mature results defining the role and, to some extent, the mechanism of IL-2 in advanced melanoma are emerging.

Cisplatin↗
PubMed1999

Induction of tyrosinase-reactive T cells by treatment with dacarbazine, cisplatin, interferon-alpha +/- interleukin-2 in patients with metastatic melanoma.

We have shown the presence of tyrosinase-reactive T cells in the peripheral blood of melanoma patients, who had been in remission after treatment with IL-2-containing regimens. In this consecutive study, we analyzed the T-cell response to various peptides derived from tyrosinase in serial blood samples obtained from 7 stage-IV melanoma patients before, during and following treatment. All patients were treated within a randomized trial (EORTC 18951) with cisplatin (CDDP), dacarbazine (DTIC), interferon-alpha (IFN-alpha) +/- interleukin-2 (IL-2). Using an ELISPOT assay detecting peptide-specific IFN-gamma release, we measured the T-cell response to 4 different HLA class I-binding peptide epitopes derived from tyrosinase containing an HLA-A2.1-, HLA-A24- or HLA-B44-binding motif in peripheral-blood mononuclear cells (PBMC). In one patient, tyrosinase-reactive T cells were detected before therapy. In 4 out of 7 patients, tyrosinase-reactive T cells against both HLA-A2.1-binding peptides and the B44-binding peptide became detectable at frequencies of up to 30 in 5 x 10(5) lymphocytes following treatment. These patients received CDDP, DTIC and IFN-alpha, 2 of them without IL-2 and 2 with IL-2, resulting in one complete remission and 3 partial remissions. Two patients relapsed 8 and 9 months after treatment. At the time of relapse, no T cells reactive with tyrosinase were detectable. Our results show that high frequencies of tyrosinase-reactive T cells in the peripheral blood of melanoma patients can be induced by chemotherapy in combination with IFN-alpha, regardless of concomitant IL-2 administration.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1998

Reliability of reverse transcription-polymerase chain reaction (RT-PCR)-based assays for the detection of circulating tumour cells: a quality-assurance initiative of the EORTC Melanoma Cooperative Group.

Reverse transcription-polymerase chain reaction (RT-PCR)-based assays detecting occult neoplastic cells are increasingly being used for the study of tumour dissemination and minimal residual disease. However, different methods are employed by various research groups and the results are heterogenous. We prospectively assessed the results from nine laboratories performing tyrosinase RT-PCR assays for the detection of melanoma cells on a series of blind samples. After complete analysis, the results were compared for sensitivity and specificity. All laboratories reported correct results for cDNA standards. Five laboratories attained acceptable specificity and a sensitivity detecting 10 cells in 10 ml of whole blood. Four laboratories had unacceptable specificity and/or sensitivity. This blind study highlights the difficulty of RT-PCR data interpretation and the need for quality assurance between laboratories. Measures to increase the reliability of RT-PCR assays are proposed, which have to be prospectively evaluated in future studies.

DNA, Neoplasm↗
PubMed1998

New prognostic factors in melanoma: mRNA tumour markers.

Circulating tumour cells in the peripheral blood may be important for haematogenous spread of malignant disease. Monitoring these cells may therefore be of prognostic value. Reverse transcriptase-polymerase chain reaction (RT-PCR)-based assays to detect occult neoplastic cells offer the highest sensitivity for the study of tumour dissemination and minimal residual disease. This review summarises technical considerations and clinical investigations in melanoma patients of various disease stages. The clinical data are promising, but to clearly define the clinical usefulness of messenger RNA (mRNA) tumour markers, methodological issues must be resolved and the clinical value must be assessed prospectively in sufficiently large patient cohorts.

Biomarkers, Tumor↗