Statistical validation of the mammaglobin-nested RT-PCR assay for tumor cell detection in blood of breast cancer patients.

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.     

The diagnostic value of RNA oncomarkers in evaluation of malignant breast tumors

The levels of the RNA oncomarkers, telomerase (hTERT), cytokeratin-19 (CK-19), and mammaglobin (MAM) have been investigated in capillary blood of female patients with mammary ductal carcinoma. The study revealed overexpression of all three factors in patients with this pathology. This overexpression was not found in healthy donors and female patients with mammary fibroadenoma. Levels of the RNA oncomarkers return to the normal level within 10 days after successful tumor resection. These results have been used for the development of diagnostic kits, which may be applicable for differential diagnostics, screening and postoperation monitoring of patients with malignant breast tumors.     

Particularities of blood lymphocyte response to irradiation in vitro in breast cancer patients

DNA breaks and their repair efficiency were analyzed in irradiated in vitro lymphocytes (at doses 1 Gy, gamma-radiation of 60Co, dose rate 1 Gy/min) isolated from peripheral blood of 41 untreated patients with breast cancer and 25 healthy donors using the DNA comet assay under non-denaturing conditions (mainly double-strand DNA breaks (DSB), as well as apoptotic cell death using the DNA halo assay. To estimate the expression of bystander effect, the cells were incubated in a culture medium obtained from lymphocytes irradiated in vitro at doses 1 Gy. The average DSB level in blood lymphocytes of breast cancer patients was shown to be significantly higher (p < 0.05) compared with that in control donors. In general, the following effects were observed in irradiated in vitro lymphocytes of cancer patients: (1) increased sensitivity to y-radiation-induced DNA DSBs compared with lymphocytes from healthy donors, (2) reduced repair efficiency of these damages. Incubation of irradiated blood lymphocytes in a medium from irradiated cells led to an increased relative number of DNA DSBs and an elevated fraction of cells dying through apoptotic pathway both in blood lymphocytes from cancer patients and control donors. However, these non-targeted effects were more expressed for the blood lymphocytes of breast cancer patients.     

食管癌根治性放疗前后外周血CK19 mRNA变化的临床意义

目的：研究检测食管癌根治性放疗前后外周血中循环肿瘤细胞（circulating tumor cells,CTC）标记细胞角蛋白19（Cytokeratin19,CK19）mRNA的变化及其临床意义。方法：收集72例食管癌患者根治性放疗前后外周血,应用巢式RT-PCR检测CK19 mRNA的表达,分析其与放疗疗效及两年无进展生存（progerssion-free survival,PFS）的关系。结果：放疗前后CK19 mRNA阳性者分别占44.4%（32/72）与30.6%（22/72）,差别无统计学意义（P=0.085）。放疗前32例表达阳性者,放疗后其中12例转阴（37.5%）,20例持续阳性（62.5%）;40例阴性者,2例转为阳性（5.0%）,38例持续阴性（81.2%）。并且,放疗前后CK19 mRNA变化与放疗疗效相关,放疗后CK19 mRNA阳性提示2年无进展生存期较差。结论：食管癌根治性前后外周血CK19 mRNA变化对于判断放疗疗效及预后具有重要意义。     

CXCL12 rs1801157 polymorphism and expression in peripheral blood from breast cancer patients

The role of chemokines has been extensively analyzed both in cancer risk and tumor progression. Among different cytokines, CXCR4 and its ligand CXCL12 have been recently subjected to a closer examination. The single-nucleotide polymorphism (SNP) rs1801157 (previously known as CXCL12-A/SDF1-3'A) in the CXCL12 gene and the relative expression of mRNA CXCL12 in peripheral blood were assessed in breast cancer patients, since the chemokine CXCL12 and its receptor CXCR4 regulate leukocyte trafficking and many essential biological processes, including tumor growth, angiogenesis and metastasis of different types of tumors. Genotyping was performed by PCR-RFLP (polymerase chain reaction followed by restriction fragment length polymorphism) using MspI restriction enzyme and the expression analyses by quantitative RT-PCR. No difference in GG genotype and allele A carrier frequencies were observed between breast cancer patients and healthy blood donors and nor when CXCL12 mRNA expression was assessed among patients with different tumor stages. However a significant difference was observed when CXCL12 mRNA relative expression was analyzed in breast cancer patients in accordance to the presence or absence of the CXCL12 rs1801157 allele A. Allele A breast cancer patients presented a mRNA CXCL12 expression about 2.1-fold smaller than GG breast cancer patients. Estrogen positive patients presenting CXCL12 allele A presented a significantly lower expression of CXCL12 in peripheral blood (p=0.039) than GG hormone positive patients. Our findings demonstrated that allele A is associated with low expression of CXCL12 in the peripheral blood from ER-positive breast cancer patients, which suggests implications on breast cancer clinical outcome.     

Phosphorylation of FAK, PI-3K, and impaired actin organization in CK-positive micrometastatic breast cancer cells

Several markers have been used to detect circulating tumor cells (CTC) in the peripheral blood of patients with breast cancer. However, analysis of activated signaling kinases in CTC implicated in cellular transformation, migration, and survival has not been addressed so far. In the present study, we focused on the phenotypic profile of micrometastatic cells in peripheral blood mononuclear cells (PBMC) preparations from 45 breast cancer patients. PBMC cytospins from 28 cytokeratin (CK)-positive and 17 CK-negative samples were assessed for the expression of phosphorylated FAK (p-FAK), phosphorylated PI-3 kinase (p-PI-3K), and HER2 using confocal laser scanning microscopy. The expression of p-FAK was documented in all 28 CK-positive samples, while all 17 CK-negative samples were tested negative for p-FAK. Immunomagnetic separation using EpCAM antibody fully confirmed these findings, implying a sound correlation for the co-expression of the two molecules. Interestingly, 15 of 28 CK- and p-FAK-positive samples also expressed the HER2 oncoprotein. p-PI-3K was documented in 15 of 17 CK- and p-FAK-positive samples. Immunoblot analysis of micrometastatic cells in co-culture with PBMC confirmed the specific expression of both p-FAK and p-PI-3K. Finally, impaired actin organization was apparent in CK- and p-FAK/p-PI-3K-positive samples, comparable to that observed in MCF-7 human breast cancer cells. Our findings provide strong evidence that micrometastatic cells express activated signaling kinases, which may regulate migration mechanisms, supporting the presumption of their malignant and metastatic nature.     

Identification of a cyclin B1-derived CTL epitope eliciting spontaneous responses in both cancer patients and healthy donors

With the aim to identify cyclin B1-derived peptides with high affinity for HLA-A2, we used three in silico prediction algorithms to screen the protein sequence for possible HLA-A2 binders. One peptide scored highest in all three algorithms, and the high HLA-A2-binding affinity of this peptide was verified in an HLA stabilization assay. By stimulation with peptide-loaded dendritic cells a CTL clone was established, which was able to kill two breast cancer cell lines in an HLA-A2-dependent and peptide-specific manner, demonstrating presentation of the peptide on the surface of cancer cells. Furthermore, blood from cancer patients and healthy donors was screened for spontaneous T-cell reactivity against the peptide in IFN-γ ELISPOT assays. Patients with breast cancer, malignant melanoma, or renal cell carcinoma hosted powerful and high-frequency T-cell responses against the peptide. In addition, when blood from healthy donors was tested, similar responses were observed. Ultimately, serum from cancer patients and healthy donors was analyzed for anti-cyclin B1 antibodies. Humoral responses against cyclin B1 were frequently detected in both cancer patients and healthy donors. In conclusion, a high-affinity cyclin B1-derived HLA-A2-restricted CTL epitope was identified, which was presented on the cell surface of cancer cells, and elicited spontaneous T-cell responses in cancer patients and healthy donors.     

Human conjunctival epithelial precursor cells and their progeny in 3D organotypic culture

We report on an in vitro organ culture method to investigate human conjunctival epithelial basal precursor cells and their progeny within a more natural three-dimensional microenvironment. Conjunctival fragments were cultured on gelatin sponges in medium with 10% FBS. The conjunctival phenotype of the epithelium was confirmed by the expression and distribution of a panel of markers (p63, CK-13/CK-10, CK-19, Ki-67, PAS for goblet cells, CD45 for infiltrating interlamellar leukocytes and nestin for mesenchymal and ocular epithelial precursor cells). After 7 days, the epithelium had exfoliated its superficial layers (mostly CK-19( )positive cells and all goblets), maintaining only 1-2 layers of basal/parabasal cells, p63, CK-13/CK-10 and nestin positive cells, firmly attached to the specimen. After 14 days, a new multilayered epithelium was formed, consisting of p63, CK-13/CK-10, nestin positive cells and in the high-zone CK-19 positive cells with new goblets. Additionally, we found interlamellar leukocytes which had probably migrated from capillaries that continued to be well maintained in the subepithelial stroma. Cells dispersed from conjunctival epithelium and co-cultured with feeder post-mitotic NIH3T3 fibroblasts formed mosaics displaying a basal epithelial phenotype. These cells expressed CD133 as revealed by RT-PCR. These organ cultures provide new opportunities to investigate epithelial reconstitution of the conjunctival surface and changes that may have occurred to their stem/precursor cells during adaptation to varying conditions in vitro.     

Phenotypic and Genetic Characterization of Circulating Tumor Cells by Combining Immunomagnetic Selection and FICTION Techniques

The presence of circulating tumor cells (CTCs) in breast cancer patients has been proven to have clinical relevance. Cytogenetic characterization of these cells could have crucial relevance for targeted cancer therapies. We developed a method that combines an immunomagnetic selection of CTCs from peripheral blood with the fluorescence immunophenotyping and interphase cytogenetics as a tool for investigation of neoplasm (FICTION) technique. Briefly, peripheral blood (10 ml) from healthy donors was spiked with a predetermined number of human breast cancer cells. Nucleated cells were separated by double density gradient centrifugation of blood samples. Tumor cells (TCs) were immunomagnetically isolated with an anti-cytokeratin antibody and placed onto slides for FICTION analysis. For immunophenotyping and genetic characterization of TCs, a mixture of primary monoclonal anti-pancytokeratin antibodies was used, followed by fluorescent secondary antibodies, and finally hybridized with a TOP2A/HER-2/CEP17 multicolor probe. Our results show that TCs can be efficiently isolated from peripheral blood and characterized by FICTION. Because genetic amplification of TOP2A and ErbB2 (HER-2) in breast cancer correlates with response to anthracyclines and herceptin therapies, respectively, this novel methodology could be useful for a better classification of patients according to the genetic alterations of CTCs and for the application of targeted therapies. (J Histochem Cytochem 56:667–675, 2008)     

Tube-Like Structures with Co-Expression of D2-40 and CD34: Newly Formed Vasculatures?

Background: A great number of in vitro and in vivo studies have suggested that many pathways or factors can stimulate angiogenesis and lymphangiogenesis, which facilitate tumor progression and metastasis. However, the morphological and immunohistochemical profile of newly formed vasculatures has not been elucidated, making it difficult to differentiate them from the pre-existing ones, and to identify their unique molecular profiles for diagnosis and therapeutic interventions.Experimental findings: As cytokeratin (CK)-19 is a well-recognized stem cell marker and CK-19-positive cells are frequently detected in the peripheral blood of patients with metastatic cancer, our recent studies have assessed the involvement of CK-19 in the formation of new vasculatures in primary colorectal cancer (CRC) tissues. Our studies showed that a subset of lymph node-positive cases harbored some isolated normal epithelial structures with distinct CK-19 immunostaining within an otherwise CK-19-negative background. These structures are exclusively located within or adjacent to lymphoid follicles and are often surrounded by tube-like structures expressing lymphatic endothelial marker D2-40. Similar structures are more frequently seen at the junctions between pre-invasive and invasive CRC with the following features: (1). they consist of a single layer of endothelial cells that express both D2-40 and CD34, (2). their endothelial walls are often incomplete with disseminated cells protruding into the adjacent stroma, and (3). they are exclusively associated with disseminated CK-19-positive cellsHypothesis: Based on these findings, we propose that these tube-like structures represent newly formed vasculatures, which are derived by the convergence of aberrant lymphocyte infiltration and tumor stem cells. Because of their close physical proximity, tumor stem cells within the epithelial and stromal components contribute equally and coordinately to the morphogenesis of new vasculatures, which constitutes the basis for the unique morphologic and immunohistochemical features of newly formed vasculatures. Our hypothesis appears to be applicable to all epithelium-derived cancers.     

Therapy of human tumors in NOD/SCID mice with patient-derived reactivated memory T cells from bone marrow

In an analysis of 84 primary-operated breast cancer patients and 11 healthy donors, we found that the bone marrow of most patients contained memory T cells with specificity for tumor-associated antigens. Patients' bone marrow and peripheral blood contained CD8+ T cells that specifically bound HLA/peptide tetramers. In short-term culture with autologous dendritic cells pre-pulsed with tumor lysates, patients' memory T cells from bone marrow (but not peripheral blood) could be specifically reactivated to interferon-gamma-producing and cytotoxic effector cells. A single transfer of restimulated bone-marrow T cells into NOD/SCID mice caused regression of autologous tumor xenotransplants associated with infiltration by human T cells and tumor-cell apoptosis and necrosis. T cells from peripheral blood showed much lower anti-tumor reactivity. Our findings reveal an innate, specific recognition of breast cancer antigens and point to a possible novel cancer therapy using patients' bone-marrow-derived memory T cells.     

Basal, oxidative and alkylative DNA damage, DNA repair efficacy and mutagen sensitivity in breast cancer

Impaired DNA repair may fuel up malignant transformation of breast cells due to the accumulation of spontaneous mutations in target genes and increasing susceptibility to exogenous carcinogens. Moreover, the effectiveness of DNA repair may contribute to failure of chemotherapy and resistance of breast cancer cells to drugs and radiation. The breast cancer susceptibility genes BRCA1 and BRCA2 are involved in DNA repair. To evaluate further the role of DNA repair in breast cancer we determined: (1) the kinetics of removal of DNA damage induced by hydrogen peroxide and the anticancer drug doxorubicin, and (2) the level of basal, oxidative and alkylative DNA damage before and during/after chemotherapy in the peripheral blood lymphocytes of breast cancer patients and healthy individuals. The level of DNA damage and the kinetics of DNA repair were evaluated by alkaline single cell gel electrophoresis (comet assay). Oxidative and alkylative DNA damage were assayed with the use of DNA repair enzymes endonuclease III (Endo III) and formamidopyrimidine-DNA glycosylase (Fpg), recognizing oxidized DNA bases and 3-methyladenine-DNA glycosylase II (AlkA) recognizing alkylated bases. We observed slower kinetics of DNA repair after treatment with hydrogen peroxide and doxorubicin in lymphocytes of breast cancer patients compared to control individuals. The level of basal, oxidative and alkylative DNA damage was higher in breast cancer patients than in the control and the difference was more pronounced when patients after chemotherapy were engaged, but usually the level of DNA damage in these patients was too high to be measured with our system. Our results indicate that peripheral blood lymphocytes of breast cancer patients have more damaged DNA and display decreased DNA repair efficacy. Therefore, these features can be considered as risk markers for breast cancer, but the question whether they are the cause or a consequence of the illness remains open. Nevertheless, our results suggest that research on the mutagen sensitivity and efficacy of DNA repair could impact the development of new diagnostic and screening strategies as well as indicate new targets to prevent and cure cancer. Moreover, the comet assay may be applied to evaluate the suitability of a particular mode of chemotherapy to a particular cancer patient.     

DNA repair capacity measured by high throughput alkaline comet assays in EBV-transformed cell lines and peripheral blood cells from cancer patients and healthy volunteers

We collected peripheral blood (PB) from 556 patients with various types of cancer who had undergone radiotherapy and from 81 healthy volunteers. We exposed whole PB and Epstein-Barr virus-transformed lymphoblastoid cell lines (EBLs) derived from the PB mononucleocytes to X-irradiation (5 Gy). Using the alkaline comet assay, we measured the immediate DNA damage and, at 15 min, the % residual damage. In PB, the immediate damage was similar in patients and healthy volunteers while the % residual damage (mean+/-S.D.) was significantly higher in patients with breast (54.3+/-A23.9), cervical (54.7+/-A23.9), head/neck (56.8+/-A24.4), lung (60.1+/-23.5), or esophageal cancers (59.5+/-A33.7) than in healthy donors (42.9+/-19.6) (P<0.05). We did not observe such differences in the EBV-transformed cell lines. Thus, radiation sensitivity of fresh PB cells measured by the alkaline comet assay was related to cancer status.     

Lack of dendritic cell mobilization into the peripheral blood of cancer patients following standard- or high-dose chemotherapy plus granulocyte-colony stimulating factor

BACKGROUND: Dendritic cells (DC), the most specialized antigen-presenting cells, can be detected in the peripheral blood (PB) and divided into two subsets of populations, DC1 and DC2, endowed with different functions. The aim of this study was to evaluate the effect on DC release and on their subsets of three regimens utilized to mobilize CD34+ cells into the PB in cancer patients and in normal CD34+ cell donors. PATIENTS AND METHODS: The mobilizing sequences were: standard-dose epirubicin+taxol+granulocyte-colony-stimulating factor (G-CSF; 15 patients with advanced breast cancer), high-dose cyclophosphamide (CTX)+G-CSF (10 patients with breast cancer patients and 7 with non-Hodgkin's lymphoma, NHL), and G-CSF alone (5 normal donors of CD34+ cells for allogeneic transplantation). Comparative data were obtained from the steady-state PB of 20 healthy volunteers. For flow cytometric analysis, DC were gated as negative for specific lineage markers (CD3, CD11b, CD14, CD16, CD56, CD19, CD20, CD34) and positive for HLA-DR. The DC1 and DC2 subsets were defined as CD11c and CDw123 positive, respectively. RESULTS: The percentages of DC at baseline and the time of CD34+ cell peak were: 0.48 and 0.51 for standard-dose chemotherapy (CT); 0.55 and 0.63 for breast cancer after high-dose CTX+G-CSF; 0.53 and 0.71 for NHL after high-dose CTX+G-CSF; and 0.51 and 0.54 for normal donors of CD34+ cells after G-CSF alone (all p=n.s.).Mean DC1/DC2 ratios in each study group at the time of CD34+ cell peak were 0.10, 0.12, and 0.18, respectively. Finally, in the group of healthy volunteers, the percentage of circulating DC was 0.95 and the mean DC1/DC2 ratio was 1.28. CONCLUSION: To our knowledge, this is the first report that demonstrates that both standard-dose or high-dose CT, when utilized together with G-CSF, do not induce DC mobilization into the PB, whereas a reversed DC1/DC2 ratio is observed. Furthermore, a lack of significant DC mobilization after G-CSF alone was also seen, in contrast to what was previously observed by others. These data should be taken in account when evaluating clinical correlations between DC number and CPC engraftment in both the transplantation setting, when monitoring the effects on the immune system of combinations of new drugs and/or cytokines, and when high numbers of DC are required for both experimental and clinical applications.     

Regulatory T cells increase in breast cancer and in stage IV breast cancer

Expression levels of VEGF and Her-2, levels of T-regulatory (Treg) cells, levels of CD3+ cells, and ratios of Th (CD4+ T cells)/Tr (Treg) cells were compared between stage I, II, III, and IV breast cancer patients (n?=?120) prior to chemotherapy and healthy women (n?=?30). Cells from peripheral blood were counted by flow cytometry, Her-2 and VEGF expression was detected by pathological examination, and Her-2 was detected by FISH. Breast cancer patients had more Treg cells and a lower ratio of Th/Tr cells than the healthy women. Stage IV breast cancer patients had more Treg cells and a lower ratio of Th/Tr cells than stage I, II, or III breast cancer patients. Patients positive for VEGF had a lower ratio of Th/Tr cells compared with patients negative for VEGF, and those positive for both VEGF and Her-2 also had a lower ratio of Th/Tr cells compared with patients not positive for both VEGF and Her-2. The decreased Th/Tr cells ratio indicates impaired immune function, suggesting that the stage IV breast cancer and the Her-2/VEGF-positive breast cancer patients have lower immune function.     

MN/CA9: a potential gene marker for detection of malignant cells in effusions.

Many cancers cause malignant effusions. The presence of malignant cells in effusions has implications in diagnosis, tumour staging and prognosis. The detection of malignant cells currently presents a challenge for cytopathologists. New adjunctive methods are needed. Although the effusions provide excellent materials for molecular assay, the available molecular markers are extremely limited, which hinders its clinical application. MN/CA9 has proved to be a valuable marker in many cancers such as lung, breast, colon, kidney, etc. The present study was to evaluate MN/CA9 as a new molecular marker for the detection of cancer cells in pleural effusions. Seventy-one pleural effusions including 59 malignant effusions from patients with cancer, and 12 patients with benign diseases as a control, were subjected to RT-PCR for detection of MN/CA9 gene expression. MN/CA9 gene expression was detected in 53/59 (89.8%) pleural effusions from cancer patients (15/16 for breast cancers, 10/11 for lung cancers, 4/4 for ovary cancers, 2/3 for colon-rectal cancers, 5/6 for cancers of unknown site, 7/8 for mesothelioma and 10/11 for other cancers). Furthermore, MN/CA9 was positive in 13/18 (72.2%) of cytologically negative effusions of cancer patients. MN/CA9 was detected in only 1/12 (8.3%) effusions from the control patients (p < 0.01). The sensitivity and specificity of MN/CA9 gene expression were, respectively, 89.8% and 91.7%. Our preliminary results suggest that MN/CA9 could be a potential marker for the detection of malignant cells in effusions. A large-scale study is needed to confirm these results.     

MN/CA9: a potential gene marker for detection of malignant cells in effusions

Many cancers cause malignant effusions. The presence of malignant cells in effusions has implications in diagnosis, tumour staging and prognosis. The detection of malignant cells currently presents a challenge for cytopathologists. New adjunctive methods are needed. Although the effusions provide excellent materials for molecular assay, the available molecular markers are extremely limited, which hinders its clinical application. MN/CA9 has proved to be a valuable marker in many cancers such as lung, breast, colon, kidney, etc. The present study was to evaluate MN/CA9 as a new molecular marker for the detection of cancer cells in pleural effusions. Seventy-one pleural effusions including 59 malignant effusions from patients with cancer, and 12 patients with benign diseases as a control, were subjected to RT-PCR for detection of MN/CA9 gene expression. MN/CA9 gene expression was detected in 53/59 (89.8%) pleural effusions from cancer patients (15/16 for breast cancers, 10/11 for lung cancers, 4/4 for ovary cancers, 2/3 for colon–rectal cancers, 5/6 for cancers of unknown site, 7/8 for mesothelioma and 10/11 for other cancers). Furthermore, MN/CA9 was positive in 13/18 (72.2%) of cytologically negative effusions of cancer patients. MN/CA9 was detected in only 1/12 (8.3%) effusions from the control patients (p<0.01). The sensitivity and specificity of MN/CA9 gene expression were, respectively, 89.8% and 91.7%. Our preliminary results suggest that MN/CA9 could be a potential marker for the detection of malignant cells in effusions. A large-scale study is needed to confirm these results.