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Journal of Translational Medicine Jun 2015Locally advanced HER2-overexpressing breast cancer (BC) patients achieve a high rate of pathological complete responses (pCR) after neoadjuvant chemotherapy (NC). The...
Improved Natural Killer cell activity and retained anti-tumor CD8(+) T cell responses contribute to the induction of a pathological complete response in HER2-positive breast cancer patients undergoing neoadjuvant chemotherapy.
BACKGROUND
Locally advanced HER2-overexpressing breast cancer (BC) patients achieve a high rate of pathological complete responses (pCR) after neoadjuvant chemotherapy (NC). The apparently unaltered immune proficiency of these patients together with the immune-modulating activities of NC drugs suggest a potential contribution of host immunity in mediating clinical responses. We thus performed an extensive immunomonitoring in locally advanced BC patients undergoing NC to identify immunological correlates of pCR induction.
METHODS
The immune profile of 40 HER2-positive and 38 HER2-negative BC patients was characterized at diagnosis and throughout NC (Paclitaxel and Trastuzumab, or Docetaxel and Epirubicin, respectively). The percentages of circulating immune cell subsets including T and B lymphocytes, Natural Killer (NK) cells, regulatory T cells, T helper 17 lymphocytes, were quantified by multiparametric flow cytometry. NK cells functional activity was evaluated through the analysis of NF-kB nuclear translocation by Multispectral flow cytometry, and with the in vitro monitoring of Trastuzumab-mediated antibody-dependent cell cytotoxicity (ADCC). CD8(+) T cell responses against six different tumor-associated antigens (TAA) were characterized by IFN-γ ELISPOT and IFN-γ/IL-2 DualSpot assays.
RESULTS
After NC, HER2-positive patients showed a significant increase in the number of NK cells and regulatory T cells irrespective of the pathological response, whereas patients undergoing a pCR disclosed higher percentages of T helper 17 cells. Notably, a significant increase in the number of activated NK cells was observed only in HER2-positive patients achieving a pCR. Characterization of anti-tumor T cell responses highlighted sustained levels of CD8(+) T cells specific for survivin and mammaglobin-A throughout NC in patients undergoing a pCR in both arms. Moreover, HER2-positive patients achieving a pCR were characterized by a multi-epitopic and polyfunctional anti-tumor T cell response, markedly reduced in case of partial response.
CONCLUSIONS
These results indicate that maintenance of functional T cell responses against selected antigens and improvement of NK cell proficiency during NC are probably critical requirements for pCR induction, especially in HER2-positive BC patients. Trail registration:
TRIAL REGISTRATION NUMBER
NCT02307227, registered on ClinicalTrials.gov ( http://www.clinicaltrials.gov , November 26, 2014).
Topics: Adaptive Immunity; Adult; Aged; Aged, 80 and over; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; CD8-Positive T-Lymphocytes; Female; Humans; Immunity, Innate; Immunophenotyping; Killer Cells, Natural; Middle Aged; NF-kappa B; Neoadjuvant Therapy; Paclitaxel; Receptor, ErbB-2; Remission Induction; Trastuzumab; Treatment Outcome
PubMed: 26116238
DOI: 10.1186/s12967-015-0567-0 -
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 -
European Journal of Medical Research Sep 2009The diagnostic tools to predict the prognosis in patients suffering from breast cancer (BC) need further improvements. New technological achievements like the gene...
BACKGROUND
The diagnostic tools to predict the prognosis in patients suffering from breast cancer (BC) need further improvements. New technological achievements like the gene profiling of circulating tumour cells (CTC) could help identify new prognostic markers in the clinical setting. Furthermore, gene expression patterns of CTC might provide important informations on the mechanisms of tumour cell metastasation.
MATERIALS AND METHODS
We performed realtime-PCR and multiplex-PCR analyses following immunomagnetic separation of CTC. Peripheral blood (PB) samples of 63 patients with breast cancer of various stages were analyzed and compared to a control group of 14 healthy individuals. After reverse-transcription, we performed multiplex PCR using primers for the genes ga733.3, muc-1 and c-erbB2. Mammaglobin1, spdef and c-erbB2 were analyzed applying realtime-PCR.
RESULTS
ga733.2 overexpression was found in 12.7% of breast cancer cases, muc-1 in 15.9%, mgb1 in 9.1% and spdef in 12.1%. In this study, c-erbB2 did not show any significant correlation to BC, possibly due to a highly ambient expression. Besides single gene analyses, gene profiles were additionally evaluated. Highly significant correlations to BC were found in single gene analyses of ga733.2 and muc-1 and in gene profile analyses of ga733.3*muc-1 and GA7 ga733.3*muc-1*mgb1*spdef.
CONCLUSION
Our study reveals that the single genes ga733.3, muc-1 and the gene profiles ga733.3*muc-1 and ga733.3*3muc-1*mgb1*spdef can serve as markers for the detection of CTC in BC. The multigene analyses found highly positive levels in BC patients. Our study indicates that not single gene analyses but subtle patterns of multiple genes lead to rising accuracy and low loss of specificity in detection of breast cancer cases.
Topics: Adult; Aged; Breast Neoplasms; Female; Gene Expression Profiling; Humans; Immunohistochemistry; Mammaglobin A; Middle Aged; Mucin-1; Neoplasm Proteins; Neoplastic Cells, Circulating; Polymerase Chain Reaction; Receptor, ErbB-2; Uteroglobin
PubMed: 19748849
DOI: 10.1186/2047-783x-14-10-426 -
BMC Cancer May 2002Immunomagnetic enrichment followed by RT-PCR (immunobead RT-PCR) is an efficient methodology to identify disseminated carcinoma cells in the blood and bone marrow. The...
BACKGROUND
Immunomagnetic enrichment followed by RT-PCR (immunobead RT-PCR) is an efficient methodology to identify disseminated carcinoma cells in the blood and bone marrow. The RT-PCR assays must be both specific for the tumor cells and sufficiently sensitive to enable detection of single tumor cells. We have developed a method to test RT-PCR assays for any cancer. This has been investigated using a panel of RT-PCR markers suitable for the detection of breast cancer cells.
METHODS
In the assay, a single cell line-derived tumor cell is added to 100 peripheral blood mononuclear cells (PBMNCs) after which mRNA is isolated and reverse transcribed for RT-PCR analysis. PBMNCs without added tumor cells are used as specificity controls. The previously studied markers epidermal growth factor receptor (EGFR), mammaglobin 1 (MGB1), epithelial cell adhesion molecule (EpCAM/TACSTD1), mucin 1 (MUC1), carcinoembryonic antigen (CEA) were tested. Two new epithelial-specific markers ELF3 and EphB4 were also tested.
RESULTS
MUC1 was unsuitable as strong amplification was detected in 100 cell PBMNC controls. Expression of ELF3, EphB4, EpCAM, EGFR, CEA and MGB1 was found to be both specific for the tumor cell, as demonstrated by the absence of a signal in most 100 cell PBMNC controls, and sensitive enough to detect a single tumor cell in 100 PBMNCs using a single round of RT-PCR.
CONCLUSIONS
ELF3, EphB4, EpCAM, EGFR, CEA and MGB1 are appropriate RT-PCR markers for use in a marker panel to detect disseminated breast cancer cells after immunomagnetic enrichment.
Topics: Antigens, Neoplasm; Biomarkers, Tumor; Breast Neoplasms; Carcinoembryonic Antigen; Cell Adhesion Molecules; DNA-Binding Proteins; Ephrin-B2; Epithelial Cell Adhesion Molecule; ErbB Receptors; Genes, erbB-1; Humans; Immunomagnetic Separation; Leukocytes, Mononuclear; Mammaglobin A; Membrane Proteins; Neoplasm Proteins; Nucleic Acid Amplification Techniques; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-ets; RNA, Neoplasm; Reverse Transcriptase Polymerase Chain Reaction; Transcription Factors; Tumor Cells, Cultured; Uteroglobin
PubMed: 12031094
DOI: 10.1186/1471-2407-2-14 -
Cancer Mar 2006Methods for identifying sentinel lymph nodes (SNs) and their clinical significance have been established. Recent advances in molecular immunology have enabled the...
BACKGROUND
Methods for identifying sentinel lymph nodes (SNs) and their clinical significance have been established. Recent advances in molecular immunology have enabled the analysis of precise immune responses. The objective of the current study was to clarify the dendritic cell (DC) maturation, T-helper type 1 (Th-1) and Th-2 responses, and regulatory T-cell responses of SNs in patients with breast carcinoma.
METHODS
SNs and non-SNs were identified by radioguided and blue dye-guided methods in 70 consecutive patients with clinically lymph node negative (N0) breast carcinoma. Lymphocytes were collected from SNs and non-SNs and were subjected to flow cytometric analysis (FCM) using antibodies of CD83-fluorescein isothiocyanate (FITC), CD80-phycoerythrin (PE), CD86-PE, CD40-PE, human leukemic D-related antigen (HLA-DR)-FITC, CD4-FITC, and CD25-PE. Total RNA was extracted from SNs and non-SNs, and the expression of CD83, interleukin 12p40 (IL-12p40), interferon gamma (IFN-gamma), IL-4, IL-10, and Foxp3 was evaluated by using quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. The immunologic status of SNs was analyzed further with regard to micrometastases, which were identified as negative microscopically but positive according to an RT-PCR analysis that was specific for mammaglobin.
RESULTS
SNs were detectable in 70 of 71 consecutive patients (98.6%) with clinically N0 breast carcinoma. Fourteen of 70 patients (20.0%) had positive metastasis in SNs. When SNs were compared with non-SNs in 56 metastasis-negative patients, FCM revealed that HLA-DR-positive, CD80-positive, CD86-positive, and CD40-positive cell populations were decreased significantly in SNs. RT-PCR analysis demonstrated that, among 44 patients with metastasis-negative SNs, the expression levels of CD83 and IFN-gamma mRNA were significantly lower in SNs compared with non-SNs. Immunologic parameters also were compared between 44 metastasis-negative SNs and 14 metastasis-positive SNs. The metastasis-positive SNs demonstrated significantly higher expression of CD83, IL-12p40, IFN-gamma, IL-10, and Foxp3 mRNA than the metastasis-negative SNs. Correction of micrometastasis detected by mammaglobin enhanced these differences consistently.
CONCLUSIONS
In patients with breast carcinoma, cellular immune responses, from DC maturation to Th-1 responses, appeared to be less active in SNs compared with non-SNs before metastasis developed. Once metastasis was established in SNs, DC maturation was triggered and was followed by the up-regulation of Th-1 responses, which may reflect antigen-specific immune responses in SNs. Unlike DC maturation and Th-1 responses after metastasis in SNs, up-regulation of Th-2 and regulatory T-cell responses developed in parallel.
Topics: Adult; Aged; Antigens, CD; Biomarkers, Tumor; Breast Neoplasms; Dendritic Cells; Female; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; Lymphatic Metastasis; Mammaglobin A; Middle Aged; Neoplasm Proteins; RNA, Messenger; RNA, Neoplasm; Reverse Transcriptase Polymerase Chain Reaction; Sentinel Lymph Node Biopsy; T-Lymphocytes, Regulatory; Th1 Cells; Th2 Cells; Uteroglobin
PubMed: 16475148
DOI: 10.1002/cncr.21729 -
British Journal of Cancer May 2005In cancer patients, the ability to detect disseminated tumour cells in peripheral blood or bone marrow could improve prognosis and consent both early detection of...
Effect of different cytokines on mammaglobin and maspin gene expression in normal leukocytes: possible relevance to the assays for the detection of micrometastatic breast cancer.
In cancer patients, the ability to detect disseminated tumour cells in peripheral blood or bone marrow could improve prognosis and consent both early detection of metastatic disease and monitoring of the efficacy of systemic therapy. These objectives remain elusive mainly due to the lack of specific genetic markers for solid tumours. The use of surrogate tissue-specific markers can reduce the specificity of the assays and give rise to a clinically unacceptable false-positive rate. Mammaglobin (MAM) and maspin are two putative breast tissue-specific markers frequently used for detection of occult tumour cells in the peripheral blood, bone marrow and lymph nodes of breast cancer patients. In this study, it was evaluated whether MAM and maspin gene expression may be induced in the normal blood and bone marrow cells exposed to a panel of cytokines, including chemotactic factors (C5a, interleukin (IL)-8), LPS, proinflammatory cytokines (TNF-alpha, IL-1beta) and growth factors (IL-3, granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor). The experimental data show that all cytokines included in the panel, except for IL-8, were able to induce maspin expression; on the contrary, MAM gene was never induced. These results suggest that MAM is more specific than maspin and that the possible interference of cytokines should be taken into account in interpreting molecular assays for detection of isolated tumour cells.
Topics: Biomarkers, Tumor; Breast Neoplasms; Cytokines; False Positive Reactions; Gene Expression Regulation; Genes, Tumor Suppressor; Humans; Mammaglobin A; Neoplasm Metastasis; Neoplasm Proteins; Neoplastic Cells, Circulating; Reverse Transcriptase Polymerase Chain Reaction; Sensitivity and Specificity; Serpins; Tumor Cells, Cultured; Uteroglobin
PubMed: 15841077
DOI: 10.1038/sj.bjc.6602563 -
BMC Cancer Feb 2008We sought to examine the detection rate of cancer cells in peripheral blood (PBL) and in bone marrow (BM) using an established 7-gene marker panel and evaluated whether...
BACKGROUND
We sought to examine the detection rate of cancer cells in peripheral blood (PBL) and in bone marrow (BM) using an established 7-gene marker panel and evaluated whether there were any definable associations of any individual gene with traditional predictors of prognosis.
METHODS
Patients with T1-T3 primary breast cancer were enrolled into a prospective, multi-institutional cohort study. In this interim analysis 215 PBL and 177 BM samples were analyzed by multimarker, real-time RT-PCR analysis designed to detect circulating and disseminated breast cancer cells.
RESULTS
At a threshold of three standard deviations from the mean expression level of normal controls, 63% (136/215) of PBL and 11% (19/177) of BM samples were positive for at least one cancer-associated marker. Marker positivity in PBL demonstrated a statistically significant association with grade II-III (vs. grade I; p = 0.0083). Overexpression of the mammaglobin (mam) gene alone had a statistically significant association with high tumor grade (p = 0.0315), and showed a trend towards ER-negative tumors and a high risk category. There was no association between marker positivity in PBL and the pathologic (H&E) and/or molecular (RT-PCR) status of the axillary lymph nodes (ALN).
CONCLUSION
This study suggests that molecular detection of circulating cancer cells in PBL detected by RT-PCR is associated with high tumor grade and specifically that overexpression of the mam gene in PBL may be a poor prognostic indicator. There was no statistically significant association between overexpression of cancer-associated genes in PBL and ALN status, supporting the concept of two potentially separate metastatic pathways.
Topics: Adult; Aged; Aged, 80 and over; Bone Marrow Cells; Breast Neoplasms; Cohort Studies; Female; Humans; Lymph Nodes; Mammaglobin A; Middle Aged; Neoplasm Proteins; Predictive Value of Tests; Prognosis; Prospective Studies; RNA, Messenger; Uteroglobin
PubMed: 18289390
DOI: 10.1186/1471-2407-8-55 -
The American Journal of Surgical... Jul 2013Acinic cell carcinoma (ACC) is a low-grade salivary gland malignancy characterized by serous acinar differentiation. Most ACCs arise in the parotid gland, but ACCs have...
Acinic cell carcinoma (ACC) is a low-grade salivary gland malignancy characterized by serous acinar differentiation. Most ACCs arise in the parotid gland, but ACCs have been reported to originate in nonparotid salivary glands where serous acini are less abundant. Given the recent discovery of mammary analog secretory carcinoma (MASC)-a salivary malignancy that histologically mimics ACC-a retrospective reevaluation of nonparotid ACCs is warranted. The surgical pathology archives of The Johns Hopkins Hospital were searched for all ACCs arising outside of the parotid gland. For each case, the histologic slides were reviewed; immunohistochemical analysis (mammaglobin, S100 protein) was performed; and confirmatory ETV6 breakapart fluorescence in situ hybridization assay was completed. Demographic and clinical data were obtained from the medical records. Fourteen extraparotid tumors diagnosed as ACC were identified. Eleven of 14 (79%) tumors harbored the ETV6 translocation (oral cavity=9 of 11; submandibular gland=2 of 2). The translocation-positive tumors occurred in 7 women and 4 men ranging in age from 20 to 86 years (mean, 56 y) and usually presented as painless masses. Immunohistochemistry for mammaglobin and S100 was positive in all 11 translocation-positive tumors but negative in the 3 translocation-negative tumors. Histologically, the translocation-positive tumors exhibited uniform cells with vacuolated cytoplasm, microcystic/cystic and papillary architecture, and intraluminal secretions; however, the presence of basophilic cytoplasmic granules was conspicuously absent. Basophilic cytoplasmic granules, indicative of true serous acinar differentiation, were present in the 3 translocation-negative tumors. Of the translocation-positive tumors, only 1 locally recurred, and none metastasized. Most alleged ACCs of nonparotid origin actually represent misclassified MASCs. The impact of diagnostic error is mitigated by the low-grade nature of MASC that, like ACCs, do not appear to be clinically aggressive.
Topics: Acinar Cells; Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Breast Neoplasms; Carcinoma, Acinar Cell; Female; Humans; Male; Mammaglobin A; Middle Aged; Mouth Neoplasms; Retrospective Studies; S100 Proteins; Salivary Gland Neoplasms; Submandibular Gland Neoplasms; Translocation, Genetic; Young Adult
PubMed: 23681074
DOI: 10.1097/PAS.0b013e3182841554 -
Acta Otorhinolaryngologica Italica :... Jun 2019
Topics: Adolescent; Adult; Aged; Aquaporin 5; Caveolin 1; Chronic Disease; Cyclooxygenase 2; Cytokines; Epithelial Cells; Gene Expression; Gene Expression Profiling; Humans; Lactoferrin; Mammaglobin A; Middle Aged; Mucin 5AC; Nasal Polyps; Retrospective Studies; Rhinitis; Sinusitis; Transforming Growth Factor beta1; Tumor Necrosis Factor-alpha; Young Adult
PubMed: 31131836
DOI: 10.14639/0392-100X-2361 -
Molecular Therapy : the Journal of the... Oct 2004Expression of secretoglobin family 2A member 2 (SCGB2A2, also known as mammaglobin-1) has been detected in a high percentage of primary and metastatic breast tumors, to...
The human SCGB2A2 (mammaglobin-1) promoter/enhancer in a helper-dependent adenovirus vector directs high levels of transgene expression in mammary carcinoma cells but not in normal nonmammary cells.
Expression of secretoglobin family 2A member 2 (SCGB2A2, also known as mammaglobin-1) has been detected in a high percentage of primary and metastatic breast tumors, to a lesser extent in normal breast, but not in other normal tissues. Plasmid transfection studies in our lab and others, however, were unable to identify the genetic elements regulating this specificity. Here we demonstrate that a 25-kb DNA fragment derived from the human SCGB2A2 gene upstream of the protein coding sequence was highly active and preferentially expressed in breast cancer cells when introduced via a helper-dependent adenoviral (HDAd) vector. HDAd delivery was selected for its high cloning capacity, its high efficiency of gene transfer, and the absence of cis-acting viral sequences that can potentially interfere with specificity of the inserted promoters. A series of vectors with deletions in the 25-kb fragment was constructed to identify important regulatory regions of the SCGB2A2 promoter. We have determined that elements controlling the specificity of expression reside within the first 345 bp upstream of the coding sequence. In addition, we identified a strong enhancer several kilobases upstream of this minimal promoter. We suggest that the SCGB2A2 promoter/enhancer should be particularly advantageous for gene therapy protocols involving oncolytic viruses or toxic gene transfer via adenovectors to mammary tumors.
Topics: Adenoviridae; Animals; Base Pairing; Breast Neoplasms; Carcinoma; Cell Line, Tumor; Enhancer Elements, Genetic; Female; Gene Expression Regulation, Neoplastic; Genes, Reporter; Genetic Therapy; Genetic Vectors; Helper Viruses; Humans; Luciferases; Mammaglobin A; Mammary Neoplasms, Experimental; Mice; Mice, Transgenic; Molecular Sequence Data; Neoplasm Proteins; Promoter Regions, Genetic; Sequence Deletion; Uteroglobin
PubMed: 15451460
DOI: 10.1016/j.ymthe.2004.06.849