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Vaccine Feb 2010It is commonly believed that delivery of antigen into the class I antigen presentation pathway is a limiting factor in the clinical translation of DNA vaccines. This is...
It is commonly believed that delivery of antigen into the class I antigen presentation pathway is a limiting factor in the clinical translation of DNA vaccines. This is of particular concern in the context of cancer vaccine development as many immunodominant peptides derived from self tumor antigens are not processed and presented efficiently. To address this limitation, we have engineered completely assembled peptide/MHC class I complexes whereby all three components (class I heavy chain, beta(2)m, and peptide) are attached by flexible linkers and expressed as a single polypeptide (single chain trimers or SCT). In this study, we tested the efficacy of progressive generations of SCT DNA vaccines engineered to (1) enhance peptide binding, (2) enhance interaction with the CD8 coreceptor, and/or (3) activate CD4(+) helper T cells. Disulfide trap SCT (dtSCT) have been engineered to improve peptide binding, with mutations designed to create a disulfide bond between the class I heavy chain and the peptide linker. dtSCT DNA vaccines dramatically enhance the immune response to model low affinity antigens as measured by ELISPOT analysis and tumor challenge. SCT engineered to enhance interaction with the CD8 coreceptor have a higher affinity for the TCR/CD8 complex, and are associated with more robust CD8(+) T cell responses following vaccination. Finally, SCT constructs that coexpress a universal helper epitope PADRE, dramatically enhance CD8(+) T cell responses. Taken together, our data demonstrate that dtSCT DNA vaccines coexpressing a universal CD4 epitope are highly effective in generating immune responses to poorly processed and presented cancer antigens.
Topics: Animals; Antigen Presentation; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; CHO Cells; Cell Line, Tumor; Cricetinae; Cricetulus; Epitopes, T-Lymphocyte; Genes, MHC Class I; HLA-A2 Antigen; Humans; Lymphocyte Activation; Mammaglobin A; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mutagenesis, Site-Directed; Neoplasm Proteins; Ovalbumin; Protein Binding; Protein Engineering; Uteroglobin; Vaccines, DNA; beta 2-Microglobulin
PubMed: 20188246
DOI: 10.1016/j.vaccine.2009.10.096 -
International Journal of Molecular... Jan 2013It is widely known that cells from epithelial tumors, e.g., breast cancer, detach from their primary tissue and enter blood circulation. We show that the presence of...
It is widely known that cells from epithelial tumors, e.g., breast cancer, detach from their primary tissue and enter blood circulation. We show that the presence of circulating tumor cells (CTCs) in samples of patients with primary and metastatic breast cancer can be detected with an array of selected tumor-marker-genes by reverse transcription real-time PCR. The focus of the presented work is on detecting differences in gene expression between healthy individuals and adjuvant and metastatic breast cancer patients, not an accurate quantification of these differences. Therefore, total RNA was isolated from blood samples of healthy donors and patients with primary or metastatic breast cancer after enrichment of mononuclear cells by density gradient centrifugation. After reverse transcription real-time PCR was carried out with a set of marker genes (BCSP, CK8, Her2, MGL, CK18, CK19). B2M and GAPDH were used as reference genes. Blood samples from patients with metastatic disease revealed increased cytokine gene levels in comparison to normal blood samples. Detection of a single gene was not sufficient to detect CTCs by reverse transcription real-time PCR. Markers used here were selected based on a recent study detecting cancer cells on different protein levels. The combination of such a marker array leads to higher and more specific discovery rates, predominantly in metastatic patients. Identification of CTCs by PCR methods may lead to better diagnosis and prognosis and could help to choose an adequate therapy.
Topics: Biomarkers, Tumor; Breast Neoplasms; Female; Gene Expression Regulation, Neoplastic; Humans; Keratin-18; Keratin-19; Keratin-8; Keratins; Mammaglobin A; Neoplasm Metastasis; Neoplasm Proteins; Neoplastic Cells, Circulating; Prognosis; RNA, Messenger; Receptor, ErbB-2; Reproducibility of Results; Reverse Transcriptase Polymerase Chain Reaction; Sensitivity and Specificity; gamma-Synuclein
PubMed: 23299436
DOI: 10.3390/ijms14011093 -
Cancer Science Jan 2003Human mammaglobin (hMAM) mRNA is considered to be a promising candidate for a sensitive molecular marker for breast cancer. In this study, we attempted to relate the... (Comparative Study)
Comparative Study
Human mammaglobin (hMAM) mRNA is considered to be a promising candidate for a sensitive molecular marker for breast cancer. In this study, we attempted to relate the presence of hMAM mRNA in the peripheral blood with certain established clinicopathological features of breast cancer in order to validate its clinical utility. A total of 139 subjects including 79 with localized cancer, 33 with metastatic disease, and a control group of 27 individuals were studied. hMAM mRNA expression was assessed by reverse transcriptase polymerase chain reaction on cells from peripheral blood. The expression of hMAM mRNA was found in 0 of the 27 control subjects, 1 of the 8 stage 0 (12.5%) patients, 4 of the 16 stage I (25%) patients, 13 of the 40 stage II (32.5%) patients, 5 of the 15 stage III (33.3%) patients, and 18 of the 33 (54%) cases of metastatic disease. There was a statistically significant (P=0.045) difference in frequency between patients with localized disease (29%) and those with metastatic disease. Although trends of increasing frequency of hMAM mRNA expression existed in patients with unfavorable prognostic factors, including primary tumor size, T stage, N stage, overall stage, presence of angiolymphatic permeation, negative estrogen receptor, high 5-phase fraction (>7%), and aneuploid DNA index, none of the differences was significant. In conclusion, the clinical utility of hMAM mRNA may not be in screening or staging of breast cancer, but in following patients after surgery to detect recurrence. Further evaluation of hMAM mRNA in combination with other molecular markers to follow post-operative breast cancer patient is warranted.
Topics: Adult; Aged; Aged, 80 and over; Aneuploidy; Biomarkers, Tumor; Breast Neoplasms; Female; Gene Expression Regulation, Neoplastic; Humans; Mammaglobin A; Middle Aged; Neoplasm Metastasis; Neoplasm Proteins; Neoplasm Recurrence, Local; Neoplasm Staging; Prognosis; RNA, Messenger; RNA, Neoplasm; Receptor, ErbB-2; Receptors, Estrogen; Receptors, Progesterone; Risk Factors; S Phase; Tumor Cells, Cultured; Uteroglobin
PubMed: 12708482
DOI: 10.1111/j.1349-7006.2003.tb01359.x -
The International Journal of Biological... Sep 2014The aim of this study was to evaluate tumor markers of molecular abnormalities that display tissue specificity, as to detect circulating tumor cells (CTCs) in breast...
The aim of this study was to evaluate tumor markers of molecular abnormalities that display tissue specificity, as to detect circulating tumor cells (CTCs) in breast cancer patients. Quantitative real-time RT-PCR was used to determine h-MAM, BCSG1, CK19, and c-erbB2 mRNA levels in peripheral blood (PB) of breast cancer patients. Results were compared with other epithelial cancers (lung or esophagus cancer), benign breast disease, and healthy individuals. We found that h-MAM mRNA was only detectable in the PB of patients with breast cancer (49 of 65, 75.4%), but not in patients with other epithelial cancers, benign breast disease, or healthy individuals. No significant differences in the expression level and positive detection rate of BCSG1, CK19, and c-erbB2 mRNA were observed between breast cancer and other epithelial cancers. Furthermore, the expression level and positive detection rate of h-MAM mRNA in PB were significantly correlated to the breast cancer pathologic stage (p=0.012 and p=0.015, respectively). Chemotherapy, radiotherapy, or total tumor resection (after 7 days of treatment) resulted in a significant decrease in the expression level of h-MAM mRNA in PB compared to the levels prior to the treatment (p<0.001). Importantly, an increase in h-MAM mRNA expression was detected in patients immediately after surgery, as well as 3 days post-surgery. These results indicate that the quantitative analysis of h-MAM mRNA is a useful tool for detecting CTCs in breast cancer patients, and can have a potential diagnostic utility in early micrometastasis, clinical evaluation of cancer treatment efficacy, and post-treatment monitoring of breast cancer patients.
Topics: Adolescent; Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Breast Neoplasms; Female; Gene Expression; Humans; Mammaglobin A; Middle Aged; Neoplastic Cells, Circulating; Prognosis; RNA, Messenger; Young Adult
PubMed: 24706379
DOI: 10.5301/jbm.5000065 -
BioTechniques Dec 2001A stochastic model was developed to validate the results obtained with the mammaglobin-nested RT-PCR assay for tumor cell detection in peripheral blood of breast cancer...
A stochastic model was developed to validate the results obtained with the mammaglobin-nested RT-PCR assay for tumor cell detection in peripheral blood of breast cancer patients. Since the assay consists of four PCR setups per peripheral blood sample, the probabilities for receiving 0, 1, 2, 3, or 4 positive setups were calculated. In this model, samples with just 500 mammaglobin mRNA molecules are highly probable to result in at least three positive setups, whereas lower quantities shift the probabilities towards one or two positive setups. In the clinical trial, samples with one or two mammaglobin positive setups were detected in 6/143 (4%) patients with benign lesions of the breast, in 41/310 (13%) breast cancer patients with no evidence of disease and in 39/157 (25%) breast cancer patients with metastatic disease. On the contrary, no sample from patients with benign lesions of the breast resulted in three or four positive setups, but 5/310 (2%) breast cancer patients with no evidence of disease and 46/157 (29%) with metastatic disease. These results correspond with the model: an increased number of tumor cells in peripheral blood lead to a higher amount of mammaglobin mRNA molecules, and these samples may result in at least three positive setups. Samples with three orfour positive setups were mainly derived from breast cancer patients with metastatic disease and only occasionally from patients with no evidence of disease. On account of these results, samples with at least three positive setups are of prognostic value and regarded as tumor cell positive.
Topics: Breast Neoplasms; DNA, Complementary; Female; Humans; Mammaglobin A; Models, Statistical; Neoplasm Proteins; Neoplastic Cells, Circulating; Polymerase Chain Reaction; Reproducibility of Results; Reverse Transcriptase Polymerase Chain Reaction; Stochastic Processes; Uteroglobin
PubMed: 11768665
DOI: 10.2144/01316md03 -
The Journal of Investigative Dermatology Aug 2003
Topics: Humans; Mammaglobin A; Neoplasm Proteins; Sweat Gland Neoplasms; Sweat Glands; Uteroglobin
PubMed: 12880439
DOI: 10.1046/j.1523-1747.2003.12374.x -
Cancer Science Nov 2010( 2010; 101: 2501)
( 2010; 101: 2501)
Topics: Biomarkers, Tumor; Breast; Breast Neoplasms; Female; Humans; Lymphatic Metastasis; Mammaglobin A; Neoplasm Proteins; Reverse Transcriptase Polymerase Chain Reaction; Sentinel Lymph Node Biopsy; Uteroglobin
PubMed: 20950374
DOI: 10.1111/j.1349-7006.2010.01715.x -
Thorax Mar 2006
Topics: Gene Expression; Humans; Mammaglobin A; Mesothelioma; Neoplasm Proteins; Pleural Effusion; Pleural Neoplasms; Uteroglobin
PubMed: 16517588
DOI: 10.1136/thx.2005.049270 -
The Journal of Molecular Diagnostics :... May 2004The risk of developing second primary cancers is increased in patients with breast cancer. The lung is one of the major target organs, and therefore a differential...
The risk of developing second primary cancers is increased in patients with breast cancer. The lung is one of the major target organs, and therefore a differential diagnosis between primary and metastatic cancers is required for the treatment of lung tumors in patients with a history of breast cancer. However, biopsy specimens frequently result in small, fragmented tissues containing only a few, degenerated cancer cells. We attempted to find a useful marker for differential diagnosis, using the online SAGE database. We selected three molecules, small breast epithelial mucin (SBEM), prostate epithelium-specific Ets transcription factor (PDEF), and mammaglobin (MGB1), as potential markers for breast cancer. SBEM and PDEF proved of no use for practical differential diagnosis because they are expressed in the normal bronchus. In contrast, expression of MGB1 was detected in all 22 primary breast cancers, but not in 22 normal lung tissues. Furthermore, all 12 metastatic breast cancers examined demonstrated positive MGB1 transcripts, whereas one of 48 primary lung adenocarcinomas expressed MGB1. This suggests that MGB1 can serve as a differential molecular marker. In practice, prospective examination, using the nine cases with a history of breast cancer, confirmed the usefulness of MGB1 in differential diagnosis.
Topics: Adenocarcinoma; Aged; Aged, 80 and over; Biomarkers, Tumor; Breast Neoplasms; Bronchi; Diagnosis, Differential; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Lung; Lung Neoplasms; Lymphatic Metastasis; Mammaglobin A; Mucins; Neoplasm Proteins; Prospective Studies; Proto-Oncogene Proteins c-ets; Transcription Factors; Uteroglobin
PubMed: 15096563
DOI: 10.1016/S1525-1578(10)60495-3 -
Oncoimmunology Feb 2013The major objectives of tumor vaccination are to induce the regression of established tumors and to favor the establishment of long-lasting tumor-specific immunity,...
The major objectives of tumor vaccination are to induce the regression of established tumors and to favor the establishment of long-lasting tumor-specific immunity, capable of protecting the host from relapse. Immunotherapeutic strategies such as the administration of tumor-associated antigenic peptides offer one means to boost preexisting antitumor CD8 T cell immunity. Our recent work reveals that established breast tumors are rejected and tumor recurrence prevented when low-dose irradiation is combined with the adoptive transfer of Mammaglobin A epitope-specific CD8 T cells.
PubMed: 23525138
DOI: 10.4161/onci.22731