Journal of Translational Medicine - Latest articles

Clinical and Immunological Evaluation of Anti-apoptosis Protein, Survivin-derived Peptide Vaccine in Phase I Clinical Study for Patients with Advanced or Recurrent Breast Cancer

2008-05-10

Background: We previously reported that survivin-2B, a splicing variant of survivin, was expressed in various types of tumors and that survivin-2B peptide might serve as a potent immunogenic cancer vaccine. The objective of this study was to examine the toxicity of and to clinically and immunologically evaluate survivin-2B peptide in a phase I clinical study for patients with advanced or recurrent breast cancer. Methods: We set up two protocols. In the first protocol, 10 patients were vaccinated with escalating doses (0.1-1.0 mg) of survivin-2B peptide alone 4 times every 2 weeks. In the second protocol, 4 patients were vaccinated with the peptide at a dose of 1.0 mg mixed with IFA 4 times every 2 weeks. Results: In the first protocol, no adverse events were observed during or after vaccination. In the second protocol, two patients had induration at the injection site. One patient had general malaise (grade 1), and another had general malaise (grade 1) and fever (grade 1). Peptide vaccination was well tolerated in all patients. In the first protocol, tumor marker levels increased in 8 patients, slightly decreased in 1 patient and were within the normal range during this clinical trial in 1 patient. With regard to tumor size, two patients were considered to have stable disease (SD). Immunologically, in 3 of the 10 patients (30%), an increase of the peptide-specific CTL frequency was detected. In the second protocol, an increase of the peptide-specific CTL frequency was detected in all 4 patients (100%), although there were no significant beneficial clinical responses. ELISPOT assay showed peptide-specific IFN-g responses in 2 patients in whom the peptide-specific CTL frequency in tetramer staining also was increased in both protocols. Conclusions: This phase I clinical study revealed that survivin-2B peptide vaccination was well tolerated. The vaccination with survivin-2B peptide mixed with IFA increased the frequency of peptide-specific CTL more effectively than vaccination with the peptide alone, although neither vaccination could induce efficient clinical responses. Considering the above, the addition of another effectual adjuvant such as a cytokine, heat shock protein, etc. to the vaccination with survivin-2B peptide mixed with IFA might induce improved immunological and clinical responses.

Identification of heterogeneity among soft tissue sarcomas by gene expression profiles from different tumors

Keith M. Skubitz, Stefan Pambuccian, J. Carlos Manivel and Amy P. N. Skubitz — 2008-05-06

The heterogeneity that soft tissue sarcomas (STS) exhibit in their clinical behavior, even within histological subtypes, complicates patient care. Histological appearance is determined by gene expression. However, metastatic propensity, tumor growth, and response to chemotherapy may be determined by gene expression patterns that do not correlate well with morphology. One approach to identify heterogeneity is to search for markers that correlate with differences in tumor behavior. Alternatively, subsets may be identified based on gene expression patterns alone, independent of knowledge of clinical outcome. We have reported gene expression patterns that distinguish two subgroups of clear cell renal carcinoma (ccRCC), and other gene expression patterns that distinguish heterogeneity of serous ovarian carcinoma (OVCA) and aggressive fibromatosis (AF). In this study, gene expression in 53 samples of STS and AF [including 16 malignant fibrous histiocytoma (MFH), 9 leiomyosarcoma, 12 liposarcoma, 4 synovial sarcoma, and 12 samples of AF] was determined at Gene Logic Inc. (Gaithersburg, MD) using Affymetrix GeneChip(R) U_133 arrays containing approximately 40,000 genes/ESTs. Gene expression analysis was performed with the Gene Logic Genesis Enterprise System(R) Software and Expressionist software. Hierarchical clustering of the STS using our three previously reported gene sets, each generated subgroups within the STS that for some subtypes correlated with histology, and also suggested the existence of subsets of MFH. All three gene sets also recognized the same two subsets of the fibromatosis samples that we had found in our earlier study of AF. These results suggest that these subgroups may have biological significance, and that these gene sets may be useful for sub-classification of STS. In addition, several genes that are targets of some anti-tumor drugs were found to be differentially expressed in particular subsets of STS.

Detailed analysis of immunologic effects of the cytotoxic T lymphocyte-associated antigen 4-blocking monoclonal antibody tremelimumab in peripheral blood of patients with melanoma

2008-05-01

Background: CTLA4-blocking antibodies induce tumor regression in a subset of patients with melanoma. Analysis of immune parameters in peripheral blood may help define how responses are mediated. Methods: Peripheral blood from HLA-A*0201-positive patients with advanced melanoma receiving tremelimumab (formerly CP-675,206) at 10 mg/kg monthly was repeatedly sampled during the first 4 cycles. Samples were analyzed by 1) tetramer and ELISPOT assays for reactivity to CMV, EBV, MART1, gp100, and tyrosinase; 2) activation HLA-DR and memory CD45RO markers on CD4+/CD8+ cells; and 3) real-time quantitative PCR of mRNA for FoxP3 transcription factor, preferentially expressed by T regulatory cells. The primary endpoint was difference in MART1-specific T cells by tetramer assay. Immunological data were explored for significant trends using clustering analysis. Results: Three of 12 patients eligible for immune monitoring had tumor regression lasting > 2 years without relapse. There was no significant change in percent of MART1-specific T cells by tetramer assay. Additionally, there was no generalized trend toward postdosing changes in other antigen-specific CD8+ cell populations, FoxP3 transcripts, or overall changes in surface expression of T-cell activation or memory markers. Unsupervised hierarchical clustering based on immune monitoring data segregated patients randomly. However, clustering according to T-cell activation or memory markers separated patients with clinical response and most patients with inflammatory toxicity into a common subgroup. Conclusions: Administration of CTLA4-blocking antibody tremelimumab to patients with advanced melanoma results in a subset of patients with long-lived tumor responses. T-cell activation and memory markers served as the only readout of the pharmacodynamic effects of this antibody in peripheral blood. Clinical trial registration number: NCT00086489

Whole tumor antigen vaccination using dendritic cells: Comparison of RNA electroporation and pulsing with UV-irradiated tumor cells

Fabian Benencia, Maria C Courreges and George Coukos — 2008-04-29

Because of the lack of full characterization of tumor associated antigens for solid tumors, whole antigen use is a convenient approach to tumor vaccination. Tumor RNA and apoptotic tumor cells have been used as a source of whole tumor antigen to prepare dendritic cell (DC) based tumor vaccines, but their efficacy has not been directly compared. Here we compare directly RNA electroporation and pulsing of DCs with whole tumor cells killed by ultraviolet (UV) B radiation using a convenient tumor model expressing human papilloma virus (HPV) E6 and E7 oncogenes. Although both approaches led to DCs presenting tumor antigen, electroporation with tumor cell total RNA induced a significantly higher frequency of tumor-reactive IFN-gamma secreting T cells, and E7-specific CD8+ lymphocytes compared to pulsing with UV-irradiated tumor cells. DCs electroporated with tumor cell RNA induced a larger tumor infiltration by T cells and produced a significantly stronger delay in tumor growth compared to DCs pulsed with UV-irradiated tumor cells. We conclude that electroporation with whole tumor cell RNA and pulsing with UV-irradiated tumor cells are both effective in eliciting antitumor immune response, but RNA electroporation results in more potent tumor vaccination under the examined experimental conditions.

Preserving a legacy for our patients: The bedside-to-bench award in translational research

Francesco M Marincola — 2008-04-22

The Journal of Translational Medicine is pleased to announce a prize to recognize outstanding contributions in the field of translational medicine. This year, the Bedside-to-Bench Award was provided by an anonymous donor and supported by the Journal of Translational Medicine Editorial Board. Applications should be submitted directly to the Journal of Translational Medicine 1.

Foxp3 expression in human cancer cells

2008-04-22

ObjectiveTranscription factor forkhead box protein 3 (Foxp3) specifically characterizes the thymically derived naturally occurring regulatory T cells (Tregs). Limited evidence indicates that it is also expressed, albeit to a lesser extent, in tissues other than thymus and spleen, while, very recently, it was shown that Foxp3 is expressed by pancreatic carcinoma. This study was scheduled to investigate whether expression of Foxp3 transcripts and mature protein occurs constitutively in various tumor types. Material and methodsTwenty five tumor cell lines of different tissue origins (lung cancer, colon cancer, breast cancer, melanoma, erythroid leukemia, acute T-cell leukemia) were studied. Detection of Foxp3 mRNA was performed using both conventional RT-PCR and quantitative real-time PCR while protein expression was assessed by immunocytochemistry and flow cytometry, using different antibody clones. Results: Foxp3 mRNA as well as Foxp3 protein was detected in all tumor cell lines, albeit in variable levels, not related to the tissue of origin. This expression correlated with the expression levels of IL-10 and TGFb1. Conclusion: We offer evidence that Foxp3 expression, characterizes tumor cells of various tissue origins. The biological significance of these findings warrants further investigation in the context of tumor immune escape, and especially under the light of current anti-cancer efforts interfering with Foxp3 expression.

Practical aspects in the management of hypokalemic periodic paralysis

Jacob O Levitt — 2008-04-21

Management considerations in hypokalemic periodic paralysis include accurate diagnosis, potassium dosage for acute attacks, choice of diuretic for prophylaxis, identification of triggers, creating a safe physical environment, peri-operative measures, and issues in pregnancy. A positive genetic test in the context of symptoms is the gold standard for diagnosis. Potassium chloride is the favored potassium salt given at 0.5–1.0 mEq/kg for acute attacks. The oral route is favored, but if necessary, a mannitol solvent can be used for intravenous administration. Avoidance of or potassium prophylaxis for common triggers, such as rest after exercise, high carbohydrate meals, and sodium, can prevent attacks. Chronically, acetazolamide, dichlorphenamide, or potassium-sparing diuretics decrease attack frequency and severity but are of little value acutely. Potassium, water, and a telephone should always be at a patient's bedside, regardless of the presence of weakness. Perioperatively, the patient's clinical status should be checked frequently. Firm data on the management of periodic paralysis during pregnancy is lacking. Patient support can be found at http://www.periodicparalysis.org.

GM-CSF/IL-3/IL-5 receptor common β chain (CD131) expression as a biomarker of antigen-stimulated CD8+ T cells

2008-04-15

Background: Upon Ag-activation cytotoxic T cells (CTLs) produce IFN-γ GM-CSF and TNF-α, which deliver simultaneously pro-apoptotic and pro-inflammatory signals to the surrounding microenvironment. Whether this secretion affects in an autocrine loop the CTLs themselves is unknown. Methods: Here, we compared the transcriptional profile of Ag-activated, Flu-specific CTL stimulated with the FLU M1:58-66 peptide to that of convivial CTLs expanded in vitro in the same culture. PBMCs from 6 HLA-A*0201 expressing donors were expanded for 7 days in culture following Flu M1:58-66 stimulation in the presence of 300 IU/ml of interleukin-2 and than sorted by high speed sorting to high purity CD8+ expressing T cells gated according to FluM1:58-66 tetrameric human leukocyte antigen complexes expression. Results: Ag-activated CTLs displayed higher levels of IFN-γ, GM-CSF (CSF2) and GM-CSF/IL-3/IL-5 receptor common β- chain (CD131) but lacked completely expression of IFN-γ receptor-II and IFN-stimulated genes (ISGs). This observation suggested that Ag-activated CTLs in preparation for the release of IFN-γ and GM-CSF shield themselves from the potentially apoptotic effects of the former entrusting their survival to GM-SCF. In vitro phenotyping confirmed the selective surface expression of CD131 by Ag-activated CTLs and their increased proliferation upon exogenous administration of GM-CSF. Conclusion: The selective responsiveness of Ag-activated CTLs to GM-CSF may provide an alternative explanation to the usefulness of this chemokine as an adjuvant for T cell aimed vaccines. Moreover, the selective expression of CD131 by Ag-activated CTLs proposes CD131 as a novel biomarker of Ag-dependent CTL activation.

Comparison between conventional and "clinical" assessment of experimental lung fibrosis

2008-04-10

Background: Idiopathic pulmonary fibrosis (IPF) is a treatment resistant disease with poor prognosis. Numerous compounds have been demonstrated to efficiently prevent pulmonary fibrosis (PF) in animal models but only a few were successful when given to animals with established fibrosis. Major concerns of current PF models are spontaneous resolution and high variability of fibrosis, and the lack of assessment methods that can allow to monitor the effect of drugs in individual animals over time. We used a model of experimental PF in rats and compare parameters obtained in living animals with conventional assessment tools that require removal of the lungs. Methods: PF was induced in rats by adenoviral gene transfer of transforming growth factor-beta. Morphological and functional changes were assessed for up to 56 days by micro-CT, lung compliance (measured via a mechanical ventilator) and VO2max and compared to histomorphometry and hydroxyproline content. Results: Standard histological and collagen assessment confirmed the persistent fibrotic phenotype as described before. The histomorphological scores correlated both to radiological (r2 = 0.29, p < 0.01) and functional changes (r2 = 0.51, p < 0.0001). VO2max did not correlate with fibrosis. Conclusion: The progression of pulmonary fibrosis can be reliably assessed and followed in living animals over time using invasive, non-terminal compliance measurements and micro-CT. This approach directly translates to the management of patients with IPF and allows to monitor therapeutic effects in drug intervention studies.

The pituitary tumor transforming gene 1 (PTTG-1): An immunological target for multiple myeloma

2008-04-02

Background: Multiple Myeloma is a cancer of B plasma cells, which produce non-specific antibodies and proliferate uncontrolled. Due to the potential relapse and non-specificity of current treatments, immunotherapy promises to be more specific and may induce long-term immunity in patients. The pituitary tumor transforming gene 1 (PTTG-1) has been shown to be a novel oncogene, expressed in the testis, thymus, colon, lung and placenta (undetectable in most other tissues). Furthermore, it is over expressed in many tumors such as the pituitary adenoma, breast, gastrointestinal cancers, leukemia, lymphoma, and lung cancer and it seems to be associated with tumorigenesis, angiogenesis and cancer progression. The purpose was to investigate the presence/rate of expression of PTTG-1 in multiple myeloma patients. Methods: We analyzed the PTTG-1 expression at the transcriptional and the protein level, by PCR, immunocytochemical methods, Dot-blot and ELISA performed on patient's sera in 19 multiple myeloma patients, 6 different multiple myeloma cell lines and in normal human tissue. Results: We did not find PTTG-1 presence in the normal human tissue panel, but PTTG-1 mRNA was detectable in 12 of the 19 patients, giving evidence of a 63% rate of expression (data confirmed by ELISA). Four of the 6 investigated cell lines (66.6%) were positive for PTTG-1. Investigations of protein expression gave evidence of 26.3% cytoplasmic expression and 16% surface expression in the plasma cells of multiple myeloma patients. Protein presence was also confirmed by Dot-blot in both cell lines and patients. Conclusion: We established PTTG-1's presence at both the transcriptional and protein levels. These data suggest that PTTG-1 is aberrantly expressed in multiple myeloma plasma cells, is highly immunogenic and is a suitable target for immunotherapy of multiple myeloma.