Rare expression of target antigens for immunotherapy on disseminated tumor cells in breast cancer patients without overt metastases

Occult disseminated tumor cells are the major cause of relapse in patients with primary operable breast cancer but detection and characterization of these few cells is difficult. Applying immunohistochemistry, an immunomagnetic enrichment technique (IET) and immunocytochemistry (IC), we studied 58 breast cancer patients without overt metastases for the frequency of cytokeratin-positive (CK+) bone marrow (BM) cells coexpressing the epithelial adhesion molecule 17-1A (EpCAM) and c-erbB-2 and analyzed the primary tumor for these antigens as a strategy for additional immunotherapy. The primary tumors were analyzed for the target antigens by a pathologist. Dissemination of CK+ cells was studied in 4-6 x 10(6) BM cells by IC alone. For characterization of CK+ cells, 10-15 x 10(6) BM cells were incubated with microbeads coupled to antibodies detecting the target antigens, labelled cells were separated on selection columns and the positively (BM cells carrying the target antigen) and negatively (BM cells without target antigen) selected fractions were stained for CK+ cells. The effectiveness of these methods was confirmed in cell culture models. 17-1A was detected in all primary tumors and c-erbB-2 overexpression (2+, 3+) was found in 25/58 tissue samples. In total, analyzing 15-20 x 10(6) BM cells in each patient, the detection rate for CK+ cells in the BM was 69% (40/58 patients). Interestingly, analysis of the positive and negative enrichment fractions showed that the 17-1A antigen was coexpressed on CK+ cells in only 6 patients and c-erbB-2/CK+ cells were found in only one patient. Although 17-1A and c-erbB-2 were frequently detected in the primary tumor, these antigens were rarely expressed on CK+ BM cells. Whether the applied IET is not able to detect low amounts of these target antigens has to be clarified. Nevertheless, applying cell-cycle independent protocols in clinical trials requires careful elucidation of those patients who might benefit from these therapies.     

Sensitive fluorescent in situ hybridisation method for the characterisation of breast cancer cells in bone marrow aspirates.

AIM: The presence of malignant cells in the blood and bone marrow of patients with cancer at the time of surgery may be indicative of early relapse. In addition to their numbers, the phenotypes of the micrometastatic cells might be essential in determining whether overt metastases will develop. This study aimed to establish a sensitive method for the detection and characterisation of malignant cells present in bone marrow. METHODS: In spiking experiments, SKBR3 cells were mixed with mononuclear cells in known proportions to mimic bone marrow samples with micrometastatic cells. Tumour cells were extracted using SAM-M450 Dynabeads coupled to the MOC-31 anti-epithelial antibody, and were further analysed for amplification of erbB2 and int2 by fluorescent in situ hybridisation (FISH). erbB2 and int2 copy numbers were also determined in 15 primary breast cancers, and bone marrow samples from patients with amplification were analysed for micrometastatic cells by immunomagnetic enrichment and FISH. RESULTS: In model experiments, cells with amplification could be detected in bead selected fractions when ratios of tumour cells (SKBR3) to mononuclear cells were as low as 10:10(7). Among the tumour samples, eight showed increased copy numbers of erbB2 and/or int2, and three of these patients had detectable numbers of tumour cells in their bone marrow: 4000, 540, and 26 tumour cells/10(7) mononuclear cells, respectively. The patient with 540 tumour cells/10(7) mononuclear cells showed high level amplification of erbB2 and suffered from a particularly aggressive disease, whereas the patient with 4000 tumour cells/10(7) mononuclear cells had favourable disease progression. CONCLUSION: These results demonstrate the feasibility and advantage of combining immunomagnetic selection and FISH characterisation of cancer cells in bone marrow samples. It is possible that molecular characterisation of such cells could provide prognostically valuable information.     

Sensitive fluorescent in situ hybridisation method for the characterisation of breast cancer cells in bone marrow aspirates.

AIM: The presence of malignant cells in the blood and bone marrow of patients with cancer at the time of surgery may be indicative of early relapse. In addition to their numbers, the phenotypes of the micrometastatic cells might be essential in determining whether overt metastases will develop. This study aimed to establish a sensitive method for the detection and characterisation of malignant cells present in bone marrow. METHODS: In spiking experiments, SKBR3 cells were mixed with mononuclear cells in known proportions to mimic bone marrow samples with micrometastatic cells. Tumour cells were extracted using SAM-M450 Dynabeads coupled to the MOC-31 anti-epithelial antibody, and were further analysed for amplification of erbB2 and int2 by fluorescent in situ hybridisation (FISH). erbB2 and int2 copy numbers were also determined in 15 primary breast cancers, and bone marrow samples from patients with amplification were analysed for micrometastatic cells by immunomagnetic enrichment and FISH. RESULTS: In model experiments, cells with amplification could be detected in bead selected fractions when ratios of tumour cells (SKBR3) to mononuclear cells were as low as 10:10(7). Among the tumour samples, eight showed increased copy numbers of erbB2 and/or int2, and three of these patients had detectable numbers of tumour cells in their bone marrow: 4000, 540, and 26 tumour cells/10(7) mononuclear cells, respectively. The patient with 540 tumour cells/10(7) mononuclear cells showed high level amplification of erbB2 and suffered from a particularly aggressive disease, whereas the patient with 4000 tumour cells/10(7) mononuclear cells had favourable disease progression. CONCLUSION: These results demonstrate the feasibility and advantage of combining immunomagnetic selection and FISH characterisation of cancer cells in bone marrow samples. It is possible that molecular characterisation of such cells could provide prognostically valuable information.     

Chemokine receptor CXCR4 expression in breast cancer as a potential predictive marker of isolated tumor cells in bone marrow

Interactions between the CXCR4 chemokine receptor in breast cancer cells and the ligand CXCL12/SDF-1α are thought to play an important role in breast cancer metastases. In this pilot study, CXCR4 expression along with other biomarkers including HER2-neu and EGFR, were measured in primary tumor samples of patients with operable breast cancer to test whether any of these biomarkers alone and in combination could indicate breast cancer with high likelihood of metastasizing to bone marrow. Cytokeratin (CK) positive cells in bone marrow were identified by flow-cytometry following enrichment with CK 7/8 antibody-coupled magnetic beads. Primary tumors (n = 18) were stained with specific antibodies for CXCR4, HER2-neu, EGFR, and PCNA using an indirect avidin–biotin horseradish peroxidase method. The majority of the patients had T2/T3 tumors (72%), or lymph node involvement (67%) as pathologic characteristics that were more indicative of high-risk breast cancer. High CXCR4 cytoplasmic expression was found in 7 of 18 patients (39%), whereas 6 of 18 patients (33%) were found to have CK positivity in bone marrow. The median number of CK⁺ cells was 236 (range, 20–847) per 5 × 10⁴ enriched BM cells. The presence of CK⁺ cells in bone marrow was found to be associated with increased expression of CXCR4 alone or in addition to EGFR and/or HER2-neu expression (P = 0.013, P = 0.005, and P = 0.025, respectively) in primary tumors. Furthermore, three patients with high CK positivity (>236 CK⁺ per 5 × 10⁴ enriched bone marrow cells) in bone marrow exclusively expressed high levels of CXCR4 with EGFR/HER2-neu (P = 0.001). Our data suggest that high CXCR4 expression in breast cancer may be a potential marker in predicting isolated tumor cells in bone marrow. CXCR4 coexpression with EGFR/HER2-neu might further predict a particular subset of patients with high CK positivity in bone marrow.     

Detection of bone marrow micrometastasis

The detection of metastasizing single tumor cells has so far been difficult. Using a monoclonal antibody (MAb A45-B/B3) recognizing human cytokeratin, we identified immunocytochemically single tumor cells and micrometastases in patients (n = 24) with nonsmall cell lung cancer at the time of surgery of the primary tumor. The cytokeratin-positive cells (1-14/5 x 10(5) cells) in the bone marrow samples of 9 (9/24) patients were found. We also found a garland-like cluster, which consists of seven cancer cells and two closely connected tumor cells from one bone marrow sample. These results indicate that this technique can be used as a early diagnostic technique of bone marrow micrometastasis in the patient with the nonsmall cell lung cancer.     

免疫磁性活化细胞分选方法优化及纯化后细胞的生物学特性检测

目的:改良常规免疫磁性活化细胞分选(MACS)的分离纯化方法,提高分选后细胞的纯度并确保细胞仍具有良好活性及功能。方法:以干细胞抗原-1(Sca-1)标记的Sca-1+造血干/祖细胞(Sca-1+HSC/HPC)为例,分别用改良后和常规MACS法分选出小鼠骨髓细胞Sca-1+细胞,流式细胞术检测两种分选方法获得Sca-1+细胞纯度;计算阳性细胞回收率;台酚蓝染色检测分选细胞存活百分率;CCK-8检测细胞增殖,混合造血祖细胞(CFU-Mix)体外培养评价分选Sca-1+细胞分化能力。结果:改良MACS法分选获得的Sca-1+细胞纯度达93%以上(常规MACS法仅为87%),阳性细胞的回收率可达73%(常规法仅为62.3%);细胞存活率、细胞增殖和CFU-Mix集落形成能力两种分选方法无明显差别。结论:改良法能明显提高细胞分选纯度及回收率,细胞活性和功能保持良好,值得各种细胞免疫磁性分选参考采用。     

Detection of metastatic colon cancer cells in sentinel nodes by flow cytometry

In colon cancer the presence of metastases in the regional lymph nodes is an important prognostic factor. Currently, examination is based on histological and immunohistochemical evaluations of lymph node sections. However, these methods are time-consuming, labour intensive and micro-metastases might be missed. The sentinel node technique may be used to identify the direct tumour draining lymph node. In this study the possible use of flow cytometry for detection of sentinel node metastases in patients with colon cancer has been investigated.

Peripheral blood mononuclear cells (PBMC) with the addition of DLD-1 colon cancer cells were stained intracellularly for cytokeratin 20 (CK20), and for the cell surface markers epithelial cell adhesion molecule (EpCAM) and carbohydrate antigen 19-9 (CA19-9). CK20 positive colon cancer cells were reliably detected with as few as 0.037% events. However, PBMCs from both colon cancer patients and from healthy individuals contained CK20 positive cells. Staining for EpCAM resulted in an almost complete recovery of tumour cells, and as few as 0.037% added cells were detected. Low intra-assay variability was determined for EpCAM (CV 8.8) and CA19-9 (CV 9.7) stainings. The results from staining for CK20 or for EpCAM and CA19-9 concorded, but the degree to which respective antigens were expressed varied. The use of flow cytometry for detection of metastatic colon cancer cells was verified in fourteen sentinel nodes specimens from patients with colon cancer.

Herein we have explored the potential of flow cytometry to become a fast, sensitive and reliable method for detection of lymphatic metastases in patients with colon cancer using direct fluorophore-conjugated antibodies against multiple surface antigens. The method seems feasible and further testing is warranted.     

Keratinocyte growth factor improves repair in the injured tracheal epithelium

Keratinocyte growth factor (KGF) is a critical growth factor in lung development and is a protective agent after lung injury, although the exact mechanisms of this protective effect have not yet been elucidated. Our laboratory has shown that circulating epithelial progenitor cells can traffic to the airway and that they appear to be derived from the bone marrow. On this basis, we hypothesized that KGF and its putative receptor (KGFR) would be important to these cells. We showed that the KGFR, which is found almost exclusively on epithelial cells, was present on cells in the bone marrow and circulation of mice that identified a subpopulation of cytokeratin 5+ circulating epithelial progenitor cells (CEPC). In addition, the KGFR co-localized with a population of cytokeratin 5+ basal cells in the repairing proximal airway. Systemic administration of KGF resulted in a significant increase in mobilization of cytokeratin 5+ CEPC at 6 h after injection. Administration of KGF to mouse recipients of heterotopic syngeneic tracheal transplants resulted in protection and more rapid repair of the tracheal epithelium, with an increase in the number of CEPC in the epithelium of the airway, and this effect was abrogated by blocking CEPC with anti-CXCL12 antibodies. KGF therefore appears to be an important growth factor for local resident progenitor epithelial cell repair and for mobilization and enhanced engraftment of CEPC to the injured proximal airway epithelium.     

免疫磁珠分选系统在分离大鼠骨髓干细胞群中的应用

采用免疫磁珠分选系统(MACS)分离大鼠骨髓Thy-1.1 干细胞群。收集大鼠胫骨和股骨的骨髓细胞,Percoll密度梯度离心分离单个核细胞,Thy1.1单抗标记,间接MACS分离纯化Thy-1.1 干细胞群,流式细胞仪检测其纯度和分选前后CD34 百分比,台盼兰拒染法检测细胞活力,计算回收率。结果表明分选后的Thy-1.1 细胞纯度达94.2%,回收率64.7%;分选后细胞活力为99.6%,与分选前99.8%无明显差异;分选前CD34 百分比为1.2%,与分选后1.3%无差异。MACS能有效分选大鼠骨髓Thy-1.1 干细胞群,所得细胞纯度高,细胞活力保持好。大鼠Thy-1.1抗原与CD34分子无明显相关性。     

Immunohistochemical identification of six cytokeratin-defined subsets of the rat thymic epithelial cells

Rat thymic epithelial cells (TEC) have been studied by a panel of monoclonal antibodies specific for single cytokeratin polypeptides or cytokeratin pairs. Using various combinations of single and double immunostainings 6 TEC subsets (CK types) were identified, each characterized by different cytokeratin expression. Subcapsular/perivascular TEC (TEC-CK type 1) share cytokeratins 7, 8, 19 with a subset of medullary TEC, while cortical TEC were reactive with anti-CK 8 and anti-CK 18 mAbs only (TEC-CK type 2). Additional 4 subsets were identified in the medulla; TEC-CK type 3 (CK 8 + 18 + 19 +), TEC-CK type 4 (CK 8 + 10 + 18 + 19 +), TEC-CK Type 5 (CK 8 + 10 + 10 +) and TEC-CK type 6 positive only with CK 8 of all above cytokeratins. This study extends the concept of TEC heterogeneity and might also be useful to further understanding of TEC origin, development and functions.     

Immunomagnetic enrichment of disseminated tumor cells in bone marrow and blood of breast cancer patients by the Thomsen-Friedenreich-Antigen

Purpose The presence of disseminated tumor cells in the bone marrow (DTC-BM) of breast cancer patients has shown independent prognostic impact. Immunomagnetic enrichment of such cells is an approach to increase the number of detected cells with limited sample volume, especially for circulating tumor cells (CTCs) in blood. The Thomsen-Friedenreich (TF) antigen (CD 176) is a specific oncofetal carbohydrate epitope (Galβ1-3GalNAcα-O) expressed on the surface of various carcinomas. Own studies demonstrated a nearly complete TF expression on DTC-BM, indicating its suitability as marker for immunomagnetic enrichment. Methods BM samples of 65 and peripheral blood samples of 11 breast cancer patients were examined immunocytochemically by staining with the anti-Cytokeratin antibody A45-B/B3 before and after immunomagnetic enrichment. Enrichment was done by incubation with the primary antibody TF 2 (IgM), followed by secondary magnetically labelled rat-anti mouse IgM. Cytospin slides were screened manually by bright-field microscopy. Results 15/65 pts (23%) showed DTC-BM in primary screening with a median of 2/2 mio cells (range 1–10). By enrichment, a median of 23.3 mio cells (0.8–218) could be analysed, increasing positivity to 72% (47/65 pts) with a med. of 4 DTCs (1–105, P < .0001). Blood from 1/11 pts before and 5/11 (45%) after enrichment showed CTCs (med. 2, 1–20), at a med. of 12.4 mio (2.6–38.5) cells analysed. Comparing BM and blood of the same patients after enrichment, 5 were positive in both compartments, 4 showed DTC-BM without presence of CTCs. Conclusion The positive immunomagnetic enrichment technique with TF-antibodies enables to analyse larger sample volumes and increase tumor cell detection rate. This could allow monitoring and characterisation of CTCs as targets for therapies.     

The relationship between micrometastases in the bone marrow, histopathologic features of the primary tumor in breast cancer and prognosis

The pathologic features of the primary tumors in 285 patients with breast cancer at the time of initial presentation, and with no clinical evidence of distant metastases, have been analyzed. The results have been compared with the detection of tumor cells in the bone marrow by use of an immunocytochemical method using antisera raised against the epithelial membrane antigen (EMA). The authors found EMA-positive cells (i.e., tumor cells) in the bone marrow of 77 (27%) patients and a significant association between the presence of such EMA-positive cells in the bone marrow and tumor size (P = 0.006) and peritumoral vascular invasion (P = less than 0.001). A possible relationship with estrogen receptor negativity (P = 0.06) also was noted.     

Circulating Breast Cancer Cells Are Frequently Apoptotic

Automatic search for cytokeratin/mucin-1 double immunofluorescence was performed to detect and characterize circulating epithelial tumor cells in patients with advanced breast cancer. The peripheral blood samples in 8 of 19 patients (42.1%) presented with cytokeratin-positive and epithelial-type mucin-positive (CK(+)/MUC1(+)) tumor cells. Detailed microscopic analysis, however, suggested that the majority of the double immunopositive cells was apoptotic according to an "inclusion type" cytokeratin staining pattern and nuclear condensation. Furthermore, apoptosis-related DNA strand breaks could be demonstrated by applying the TdT-uridine nick end labeling assay in these cells. In 3 of 8 positive samples all of the CK(+)/MUC1(+) cells displayed apoptotic features. We conclude that apoptotic cells significantly contribute to the circulating tumor cell fraction in breast cancer patients. As the predictive value of such cells for the outcome of the disease is unclear, they should be considered separately when analyzing tumor cell dissemination.     

体外诱导骨髓间充质干细胞向胆管上皮样细胞分化

背景：肝外胆管和胆囊上皮细胞的分离、纯化相对比较容易,但是胆管上皮细胞在体外易失去增殖能力,难以提供基础研究所需的细胞量,限制了胆管修复等基础研究的进程。虽然骨髓间充质干细胞可以转化为肝细胞,但是尚无体外培养骨髓间充质干细胞分化为胆管上皮细胞的报道。 目的：探讨体外诱导骨髓间充质干细胞分化为胆管上皮细胞的可行性。 方法：采取全骨髓贴壁筛选法体外分离并纯化大鼠骨髓间充质干细胞后,在第3代骨髓间充质干细胞培养基中加入肝细胞生长因子和表皮生长因子,倒置显微镜下观察骨髓间充质干细胞的形态学变化,免疫荧光检测不同时间CK19的表达情况。 结果与结论：在肝细胞生长因子和表皮生长因子的诱导下,骨髓间充质干细胞由梭形逐渐变为多边形、三角形;免疫荧光检查显示诱导第4周细胞膜开始表达CK19,诱导第6周CK19表达率明显提高。结果表明在两种细胞因子联合诱导下骨髓间充质干细胞能够转化为胆管上皮样细胞,从而为骨髓间充质干细胞修复损伤胆管提供了一个新思路。     

改良IME-FICC 法检测鼻咽癌患者外周血循环肿瘤细胞及其临床意义分析

目的: 探索改良免疫磁珠富集联合免疫荧光细胞化学技术(IME-FICC)检测鼻咽癌患者外周血循环肿瘤细胞(CTCs)的临床意义。方法: 取76例初治鼻咽癌患者外周血,分离单个核细胞,用与磁珠共价结合的上皮细胞黏附分子(EpCAM)抗体富集外周血中表达EpCAM的肿瘤细胞,再采用宽波段滤板检测细胞角蛋白(CK)8/18阳性的肿瘤细胞。中位随访25个月后,对包括循环肿瘤细胞在内的预后因子做统计分析。结果: 20 例正常人的外周血中未检测到CK8/18,63例鼻咽癌患者的外周血检测到CK8/18,阳性率为82.9%(63/76,P<0.01)。复发患者治疗前检测到的外周血CK8/18⁺ CTC中位数明显高于不复发患者(P<0.01),两组中位病毒壳蛋白抗原(VCA)-IgA滴度无显著差异(P>0.05)。外周血CK8/18⁺CTCs个数在3以上,无复发生存率逐渐下降。VCA-IgA滴度不能预测生存。结论: 外周血中CK8/18⁺CTCs是初治鼻咽癌患者的预后不良因素。     

骨髓间充质干细胞体外向子宫内膜上皮细胞的分化

BACKGROUND: Previous studies have confirmed that as an exogenous origin of endometrial epithelial cells, bone marrow stem cells are involved in the functional reconstruction of the injured endometrium. OBJECTIVE:To discuss whether bone marrow mesenchymal stem cells can differentiate into endometrial epithelial cells in vitro. METHODS:Passage 2 bone marrow mesenchymal stem cells and endometrial stromal cells were collected and subjected to different treatments: bone marrow mesenchymal stem cells were cultured alone in the DMEM/F12 medium containing 2% fetal bovine serum or in the DMEM/F12 medium containing 2% fetal bovine serum, 10^-7 mol/L β-estradiol and 10 μg/L epidermal growth factor as groups 1 and 2, respectively; bone marrow mesenchymal stem cells co-cultured with endometrial stromal cells by Transwell chamber were cultured in the DMEM/F12 medium containing 2% fetal bovine serum or in the DMEM/F12 medium containing 2% fetal bovine serum, 10^-7 mol/L β-estradiol and 10 μg/L epidermal growth factor as groups 3 and 4, respectively. After 5 days culture, bone marrow mesenchymal stem cells at the bottom of the culture plate were collected to detect expressions of epithelial cell markers, such as CK7, CK18, CK1 and EMA by RT-PCR technology, and to determine keratin expression using immunofluorescence assay. Besides, expressions of these epithelial cell markers in the group 3 were detected using RT-PCR at 1, 3, 5 and 7 days of culture, respectively. RESULTS AND CONCLUSION:mRNA expression of CK7 showed a significant successive rise in the groups 1, 2, 3, 4 and it was highest in the group 4. mRNA expressions of CK18 and CKl9 in the later three groups had no significant differences, but all significantly higher than those in the group 1. Compared with the groups 1 and 3, mRNA expression of EMA in the groups 2, 4 were significantly higher, but no significant differences existed between groups. Expression of keratin was strongly positive in the group 4, weakly positive in the group 3 and negative in the groups 1, 2, respectively. Furthermore, with the increase of culture time, mRNA expression of CK7 exhibited a constant increase, which was significantly higher at 5 and 7 days than at 1 and 3 days; but there were no significant differences in expressions of CKl9, CKl8 and EMA at different time points. These results show that bone marrow mesenchymal stem cells can differentiate into endometrial epithelial cells in vitro under certain conditions. Moreover, it can remarkably promote the endometrial differentiation under the combined effects of some exogenous and endogenous factors.