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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 -
Genetics and Molecular Research : GMR Nov 2012Different treatment outcomes and prognoses in patients with breast cancer can be observed with similar clinical predictors; this is because the biology of breast cancer...
Different treatment outcomes and prognoses in patients with breast cancer can be observed with similar clinical predictors; this is because the biology of breast cancer is complex and heterogenous, involving multiple unknown contributing factors. We looked for plasma human mammaglobin (hMAM) mRNA by RT-PCR in 82 Korean patients with breast cancer to determine if there is an association between the presence of plasma hMAM mRNA in these patients and known prognostic factors. The prognostic usefulness of detection of plasma hMAM mRNA expression in these patients was also evaluated by determining overall survival and event-free survival. A significant difference was observed in the rate of positivity of plasma hMAM mRNA between the early stages of cancer (stages I-II, 23.4%) and advanced stages (stages III-IV, 82.9%). The expression rates of estrogen receptor, progesterone receptor, and HER-2/neu in the breast tissue of these patients, by immunohistochemistry, were 69.5, 75.6, and 20.7%, respectively. In the univariate analysis, plasma hMAM expression was significantly correlated with high histological and nuclear grades, nodal metastasis, and negative estrogen receptor and progesterone receptor status. Patients negative for plasma hMAM mRNA had significantly higher rates of event-free survival compared to the patients positive for plasma hMAM mRNA. However, no significant association with overall survival was observed for expression of plasma hMAM mRNA (P = 0.16). Qualitative detection of plasma hMAM mRNA appears to be associated with unfavorable prognostic factors and lower rates of event-free survival in patients with breast cancer.
Topics: Adult; Aged; Aged, 80 and over; Breast Neoplasms; Disease-Free Survival; Female; Gene Expression Regulation, Neoplastic; Humans; MCF-7 Cells; Mammaglobin A; Middle Aged; RNA, Messenger
PubMed: 23212340
DOI: 10.4238/2012.November.28.2 -
Histopathology Jan 2013In addition to oestrogen and progesterone receptors, gross cystic disease fluid protein-15 (GCDFP-15) and mammaglobin A (MAM) are the most common markers used to...
AIMS
In addition to oestrogen and progesterone receptors, gross cystic disease fluid protein-15 (GCDFP-15) and mammaglobin A (MAM) are the most common markers used to identify breast origin by immunohistochemistry. GCDFP-15 expression has been reported in approximately 60% of breast carcinomas and MAM expression in approximately 80%. Data on their expression in triple-negative breast cancer (TNBC) are very limited. The aim of this study was to examine the expression of these markers in TNBC to determine their utility in pathological diagnosis.
METHODS AND RESULTS
We studied the immunohistochemical (IHC) expression of GCDFP-15 and MAM in 63 primary and 118 metastatic TNBCs. GCDFP-15 staining was present in 14% of primary and 21% of metastatic TNBCs. MAM staining was present in 25% of primary and 41% of metastatic TNBCs. The frequency of expression of GCDFP-15 and/or MAM was 30% in primary and 43% in metastatic TNBCs, and many positive tumours had only focal staining.
CONCLUSIONS
Staining for GCDFP-15 and/or MAM in triple-negative carcinomas helps to confirm breast origin, but most tumours in this subgroup of breast carcinomas lack expression of either marker.
Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Breast Neoplasms; Carrier Proteins; Female; Glycoproteins; Humans; Immunohistochemistry; Mammaglobin A; Membrane Transport Proteins; Middle Aged; Receptor, ErbB-2; Receptors, Estrogen; Receptors, Progesterone
PubMed: 22963676
DOI: 10.1111/j.1365-2559.2012.04344.x -
PloS One 2012Adoptive T cell therapy has proven to be beneficial in a number of tumor systems by targeting the relevant tumor antigen. The tumor antigen targeted in our model is...
Adoptive T cell therapy has proven to be beneficial in a number of tumor systems by targeting the relevant tumor antigen. The tumor antigen targeted in our model is Mammaglobin-A, expressed by approximately 80% of human breast tumors. Here we evaluated the use of adoptively transferred Mammaglobin-A specific CD8 T cells in combination with low dose irradiation to induce breast tumor rejection and prevent relapse. We show Mammaglobin-A specific CD8 T cells generated by DNA vaccination with all epitopes (Mammaglobin-A2.1, A2.2, A2.4 and A2.6) and full-length DNA in vivo resulted in heterogeneous T cell populations consisting of both effector and central memory CD8 T cell subsets. Adoptive transfer of spleen cells from all Mammaglobin-A2 immunized mice into tumor-bearing SCID/beige mice induced tumor regression but this anti-tumor response was not sustained long-term. Additionally, we demonstrate that only the adoptive transfer of Mammaglobin-A2 specific CD8 T cells in combination with a single low dose of irradiation prevents tumors from recurring. More importantly we show that this single dose of irradiation results in the down regulation of the macrophage scavenger receptor 1 on dendritic cells within the tumor and reduces lipid uptake by tumor resident dendritic cells potentially enabling the dendritic cells to present tumor antigen more efficiently and aid in tumor clearance. These data reveal the potential for adoptive transfer combined with a single low dose of total body irradiation as a suitable therapy for the treatment of established breast tumors and the prevention of tumor recurrence.
Topics: Adoptive Transfer; Animals; Breast Neoplasms; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Cytotoxicity, Immunologic; Dendritic Cells; Epitopes, T-Lymphocyte; Female; Humans; Immunologic Memory; Interferon-gamma; Interleukin-7 Receptor alpha Subunit; L-Selectin; Lipid Metabolism; Mammaglobin A; Mice; Mice, SCID; Mice, Transgenic; Scavenger Receptors, Class A; Tumor Necrosis Factor Receptor Superfamily, Member 7; Tumor Necrosis Factor-alpha; Vaccines, DNA; Whole-Body Irradiation
PubMed: 22911764
DOI: 10.1371/journal.pone.0041240 -
Breast Cancer Research and Treatment Feb 2013Mammaglobin-A (Mam-A) is a 10 kDa secretory protein that is overexpressed in 80 % of primary and metastatic human breast cancers. Previous studies from our laboratory...
Mammaglobin-A (Mam-A) is a 10 kDa secretory protein that is overexpressed in 80 % of primary and metastatic human breast cancers. Previous studies from our laboratory demonstrated that Mam-A cDNA vaccine can induce Mam-A-specific CD8 T cell responses and mediate regression of human breast cancer xenografts in NOD/SCID mice. In this article, we present our results on a phase I clinical trial of a Mam-A cDNA vaccination in breast cancer patients with stage-IV metastatic disease, including the impact of vaccination on the expression of the inducible co-stimulator molecule (ICOS) on CD4 T cells. Specimens from seven patients with stage-IV metastatic cancer were available for these analyses. Patients were vaccinated with a Mam-A cDNA vaccine on days 0, 28, and 56, and immune responses were assessed at serial time points following vaccination. At 6 months following the first vaccination, flow cytometric analysis demonstrated a significant increase in the frequency of CD4+ICOS(hi) T cells from 5 ± 2 % pre-vaccination to 23 ± 4 % (p < 0.001), with a concomitant decrease in the frequency of CD4+FoxP3+ T cells (regulatory T cells [Treg]) from 19 ± 6 to 10 ± 5 % (p < 0.05). ELISpot analysis of CD4+ICOS(hi) sorted T cells demonstrated that following vaccination the cytokines produced by Mam-A-specific T cells switched from IL-10 (78 ± 21 spm pre-vaccination to 32 ± 14 spm 5 months post-vaccine p < 0.001) to IFN-γ (12 ± 6 spm pre-vaccination to 124 ± 31 spm 5 months post-vaccine p < 0.001). The ratio of CD4+ICOS(hi) T cells to CD4+FoxP3+ T cells increased from 0.37 ± 0.12 before vaccination to 2.3 ± 0.72 (p = 0.021) following vaccination. Further, these activated CD4+ICOS(hi) T cells induced preferential lysis of human breast cancer cells expressing Mam-A protein. We conclude that Mam-A cDNA vaccination is associated with specific expansion and activation of CD4+ICOS(hi) T cells, with a concomitant decrease in Treg frequency. These encouraging results strongly suggest that Mam-A cDNA vaccination can induce antitumor immunity in breast cancer patients.
Topics: Breast Neoplasms; CD4-Positive T-Lymphocytes; Cancer Vaccines; DNA, Complementary; Female; Forkhead Transcription Factors; Humans; Inducible T-Cell Co-Stimulator Protein; Interferon-gamma; Interleukin-10; Lymphocyte Count; Mammaglobin A; T-Lymphocytes, Cytotoxic; Vaccines, DNA
PubMed: 22678162
DOI: 10.1007/s10549-012-2110-9 -
The International Journal of Biological... Jul 2012human mammaglobin (hMAM) expression has been reported in pleural effusions (PE). The aim of this study was to assess the clinical relevance of hMAM mRNA in PE from...
BACKGROUND
human mammaglobin (hMAM) expression has been reported in pleural effusions (PE). The aim of this study was to assess the clinical relevance of hMAM mRNA in PE from patients who underwent thoracoscopy.
MATERIAL AND METHODS
A total of 288 patients with PE were studied, 155 of which were diagnosed with malignant and 133 with non-malignant diseases by thoracoscopy. Cells from PE were analyzed by nested hMAM RT-PCR. Statistical analyses were performed to evaluate the diagnostic performance parameters (DPP), the association between hMAM expression and benign or malignant status and the relative risk of cancer for patients with negative thoracoscopy showing hMAM positivity.
RESULTS
hMAM mRNA was found in 68/288 (23.6%) PE samples of which 51 were from the 155 patients diagnosed with malignant diseases and 17 were from the 133 patients diagnosed with non-malignant diseases. A significant correlation between hMAM expression and malignancy was found (OR=3.04) and the DPP were as follows: sensitivity=32.9%, specificity=87.2%, accuracy=58.0%, positive predictive value=75.0% and negative predictive value=52.7%. Among the patients with negative thoracoscopy (n=133), 5/17 (29.4%) hMAM-positive patients had or developed a tumor during the 18-month follow up period, as compared to 10/116 (8.6%) hMAM-negative patients (relative risk of 4.6 for developing a malignancy).
CONCLUSION
These findings suggest a possible application of hMAM RT-PCR detection in PE as to identify a false-negative thoracoscopy in non-specific pleuritis.
Topics: Adult; Aged; Aged, 80 and over; Female; Humans; Male; Mammaglobin A; Middle Aged; Pilot Projects; Pleural Effusion, Malignant; RNA, Messenger; Reverse Transcriptase Polymerase Chain Reaction
PubMed: 22653740
DOI: 10.5301/JBM.2012.9305 -
BMC Cancer May 2012To investigate the prognostic significance of disseminated tumor cells (DTCs) in bone marrow (BM) from non-metastatic breast cancer patients before and after surgery.
BACKGROUND
To investigate the prognostic significance of disseminated tumor cells (DTCs) in bone marrow (BM) from non-metastatic breast cancer patients before and after surgery.
METHODS
Patients with non-metastatic breast cancer were consecutively recruited to this project during the years 1998-2000. Real-time RT-PCR quantification of a DTC multimarker panel consisting of cytokeratin 19, mammaglobin A and TWIST1 mRNA was performed in BM samples obtained from 154 patients three weeks (BM2) and/or six months after surgery (BM3). The results were compared to previously published data from pre-operative BM analyses for the same patients.
RESULTS
DTCs were identified in post-operative BM samples (BM2 and/or BM3) from 23 (15%) of the 154 patients investigated. During a median follow-up of 98 months, 10 (44%) of these patients experienced systemic relapse as compared to 16 (12%) of 131 DTC-negative patients. Kaplan-Meier estimates of systemic recurrence-free- and breast-cancer specific survival demonstrated significantly shorter survival for patients with persistent DTCs in BM after surgery (p≤0.001). By multivariate Cox regression analyses, persistent DTCs after surgery was an independent predictor of both systemic recurrence-free- (HR = 5.4, p < 0.001) and breast-cancer specific survival (HR = 5.3, p < 0.001). Furthermore, the prognostic value of DTCs in BM was similar for pre- and post surgery samples. However, patients with DTCs both before and after surgery (BM1 and BM2/3) had a particularly poor prognosis (systemic recurrence-free survival: HR = 7.2, p < 0.0001 and breast-cancer specific survival: HR = 8.0, p < 0.0001).
CONCLUSIONS
Detection of persistent DTCs in BM samples obtained after surgery identified non-metastatic breast cancer patients at high risk for systemic relapse, and with reduced breast-cancer specific survival. Furthermore, patients with positive DTC status both before and after surgery had a particularly poor prognosis.
Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Bone Marrow; Breast Neoplasms; Female; Humans; Keratin-19; Mammaglobin A; Middle Aged; Neoplasm Staging; Nuclear Proteins; Prognosis; Time Factors; Twist-Related Protein 1
PubMed: 22640166
DOI: 10.1186/1471-2407-12-190 -
Human Immunology Jan 2012Human breast cancer-associated antigen, mammaglobin-A (Mam-A), potentially offers a novel therapeutic target as a breast cancer vaccine. In this study, we define the...
Human breast cancer-associated antigen, mammaglobin-A (Mam-A), potentially offers a novel therapeutic target as a breast cancer vaccine. In this study, we define the CD8(+) cytotoxic T lymphocyte (CTL) response to Mam-A-derived candidate epitopes presented in the context of HLA-A24 (A*2402). HLA-A24 has a frequency of 72% in Japanese, 27% in Asian Indian, and 18% in Caucasian populations. Using a human leukocyte antigen (HLA)-binding prediction algorithm we identified 7 HLA-A24-restricted Mam-A-derived candidate epitopes (MAA24.1-7). Membrane stabilization studies with TAP-deficient T2 cells transfected with HLA-A2402 (T2.A24) indicated that MAA24.2 (CYAGSGCPL) and MAA24.4 (ETLSNVEVF) have the highest HLA-A24 binding affinity. Further, 2 CD8(+) CTL cell lines generated in vitro against T2.A24 cells individually loaded with Mam-A-derived candidate epitopes demonstrated significant cytotoxic activity against MAA24.2 and MAA24.4. In addition, the same CD8(+) CTL lines lysed the HLA-A24(+)/Mam-A(+) stable transfected human breast cancer cell lines AU565 and MDA-MB-361. However, these CTLs had no cytotoxicity against HLA-A24(-)/Mam-A(+) and HLA-A24(+)/Mam-A(-) breast cancer cell lines. In summary, our results define HLA-A24-restricted, Mam-A-derived, CD8(+) CTL epitopes that can potentially be employed for Mam-A-based breast cancer vaccine therapy to breast cancer patients with HLA-A24 phenotype.
Topics: Amino Acid Sequence; Binding, Competitive; Breast Neoplasms; CD8 Antigens; Cancer Vaccines; Cell Line, Tumor; Cells, Cultured; Cytotoxicity, Immunologic; Epitope Mapping; Epitopes, T-Lymphocyte; Female; HLA-A24 Antigen; Humans; Mammaglobin A; Protein Binding; T-Lymphocytes, Cytotoxic; Transfection
PubMed: 22074997
DOI: 10.1016/j.humimm.2011.10.017 -
Clinical Cancer Research : An Official... Nov 2011To investigate the associations between baseline and posttreatment circulating tumor cell (CTC) gene expression and outcome of patients enrolled in four North Central...
Cytokeratin-19 and mammaglobin gene expression in circulating tumor cells from metastatic breast cancer patients enrolled in North Central Cancer Treatment Group trials, N0234/336/436/437.
PURPOSE
To investigate the associations between baseline and posttreatment circulating tumor cell (CTC) gene expression and outcome of patients enrolled in four North Central Cancer Treatment Group metastatic breast cancer (MBC) trials in which specimens were shipped (at 4°C) from community-based sites to a reference laboratory (Mayo Clinic, Rochester, MN).
EXPERIMENTAL DESIGN
Blood was collected at treating sites from MBC patients before (baseline), during, and at the end of treatment with erlotinib + gemcitabine (N0234), sorafenib (N0336), irinotecan + cetuximab (N0436), or paclitaxel-poliglumex + capecitabine (N0437). CTCs from 10 mL of EDTA blood were enriched with CD45 depletion, 24 to 30 hours postblood collection. Reverse transcription/quantitative PCR was used to determine cytokeratin-19 (CK19) and mammaglobin (MGB1) mRNA levels in CTCs from up to 13 (N0234), 16 (N0336), 18 (N0436), and 39 (N0437) patients. The gene expressions were normalized to β(2)-microglobulin and calibrated to healthy blood using the 2(-ΔΔCq) algorithm; positivity was defined as 2 or more.
RESULTS
CK19+mRNA cells were detected in 56% to 75% and MGB1+mRNA cells in 23% to 38% of 86 patients at baseline. CK19+mRNA cells were detected in 30% to 67% and MGB1+mRNA cells in 14% to 64% of 110 postbaseline serial samples. The presence of baseline CK19+mRNA cells (P = 0.01) but not MGB1+mRNA cells (P = 0.14) was significantly associated with shorter overall survival. A decrease in MGB1+mRNA levels (baseline-week 8) seemed to be associated with clinical response (P = 0.05).
CONCLUSIONS
CTC gene expression analysis conducted by a reference laboratory is feasible when blood is collected from treating sites and processed 24 to 30 hours postcollection. The presence of baseline CK19+mRNA CTCs was associated with poor prognosis; a decrease in MGB1+mRNA CTCs may help predict response to therapy of MBC patients.
Topics: Adult; Aged; Breast Neoplasms; Female; Gene Expression; Humans; Keratin-19; Mammaglobin A; Middle Aged; Neoplasm Metastasis; Neoplastic Cells, Circulating; Prognosis; RNA, Messenger; Treatment Outcome
PubMed: 21976532
DOI: 10.1158/1078-0432.CCR-11-0981