Francisco G. Ortega

Circulating Tumor Cells Identify Early Recurrence in Patients with Non-Small Cell Lung Cancer Undergoing Radical Resection

Background Surgery is the treatment of choice for patients with non-small cell lung cancer (NSCLC) stages I-IIIA. However, more than 20% of these patients develop recurrence and die due to their disease. The release of tumor cells into peripheral blood (CTCs) is one of the main causes of recurrence of cancer. The objectives of this study are to identify the prognostic value of the presence and characterization of CTCs in peripheral blood in patients undergoing radical resection for NSCLC. Patients and Methods 56 patients who underwent radical surgery for previously untreated NSCLC were enrolled in this prospective study. Peripheral blood samples for CTC analysis were obtained before and one month after surgery. In addition CTCs were phenotypically characterized by epidermal growth factor receptor (EGFR) expression. Results 51.8% of the patients evaluated were positive with the presence of CTCs at baseline. A decrease in the detection rate of CTCs was observed in these patients one month after surgery (32.1%) (p = 0.035). The mean number of CTCs was 3.16 per 10 ml (range 0–84) preoperatively and 0.66 (range 0–3) in postoperative determination. EGFR expression was found in 89.7% of the patients at baseline and in 38.9% patients one month after surgery. The presence of CTCs after surgery was significantly associated with early recurrence (p = 0.018) and a shorter disease free survival (DFS) (p = .008). In multivariate analysis CTC presence after surgery (HR = 5.750, 95% CI: 1.50–21.946, p = 0.010) and N status (HR = 0.296, 95% CI: 0.091–0.961, p = 0.043) were independent prognostic factors for DFS. Conclusion CTCs can be detected and characterized in patients undergoing radical resection for non-small cell lung cancer. Their presence might be used to identify patients with increased risk of early recurrence.

Study of antitumor activity in breast cell lines using silver nanoparticles produced by yeast

In the present article, we describe a study of antitumor activity in breast cell lines using silver nanoparticles (Ag NPs) synthesized by a microbiological method. These Ag NPs were tested for their antitumor activity against MCF7 and T47D cancer cells and MCF10-A normal breast cell line. We analyzed cell viability, apoptosis induction, and endocytosis activity of those cell lines and we observed that the effects of the biosynthesized Ag NPs were directly related with the endocytosis activity. Moreover, Ag NPs had higher inhibition efficacy in tumor lines than in normal lines of breast cells, which is due to the higher endocytic activity of tumor cells compared to normal cells. In this way, we demonstrate that biosynthesized Ag NPs can be an alternative for the treatment of tumors.

miRNA in situ hybridization in circulating tumor cells - MishCTC

Circulating tumor cells (CTCs) must be phenotypically and genetically characterized before they can be utilized in clinical applications. Here, we present the first protocol for the detection of miRNAs in CTCs using in situ hybridization (ISH) combined with immunomagnetic selection based on cytokeratin (CK) expression and immunocytochemistry. Locked-Nucleic Acid (LNA) probes associated with an enzyme-labeled fluorescence (ELF) signal amplification approach were used to detect miRNA-21 in CTCs. This protocol was optimized using both epithelial tumor (MDA-MB468) and epithelial non-tumor (MCF-10A) cell lines, and miRNA-21 was selected as the target miRNA because of its known role as an onco-miRNA. Hematopoietic cells do not express miRNA-21; thus, miRNA-21 is an ideal marker for detecting CTCs. Peripheral blood samples were taken from 25 cancer patients and these samples were analyzed using our developed protocol. Of the 25 samples, 11 contained CTCs. For all 11 CTC-positive samples, the isolated CTCs expressed both CK and miRNA-21. Finally, the protocol was applied to monitor miRNA-21 expression in epithelial to mesenchymal transition (EMT)-induced MCF-7 cells, an epithelial tumor cell line. CK expression was lost in these cells, whereas miRNA-21 was still expressed, suggesting that miRNA-21 might be a good marker for detecting CTCs with an EMT phenotype.

Prognostic factor analysis of circulating tumor cells in peripheral blood of patients with peritoneal carcinomatosis of colon cancer origin treated with cytoreductive surgery plus an intraoperative hyperthermic intraperitoneal chemotherapy procedure (CRS + HIPEC)

PURPOSE Complete cytoreductive surgery (CRS) with hyperthermic intraperitoneal chemotherapy (HIPEC) has changed the therapeutic landscape, improving overall survival in patients with peritoneal carcinomatosis with a colonic origin. The main limitation of this aggressive locoregional procedure, however, is extra-abdominal or distant spread. The objective of this study was to identify the prognostic value of circulating tumor cells (CTCs) in patients with peritoneal carcinomatosis of colonic origin undergoing CRS + HIPEC. PATIENTS AND METHODS Fourteen patients diagnosed with peritoneal carcinomatosis from colon cancer and suitable for potentially curative treatment with CRS + HIPEC were included in this study. CTCs were isolated from the peripheral blood by immunomagnetic techniques by the use of a multi-cytokeratin-specific antibody and detected via immunocytochemical methods. The phenotypic characterization of EGFR on CTCs was analyzed by immunofluorescence. RESULTS At baseline, 50% of the patients were positive for CTCs, with a mean value of 5.5 CTCs per 10 mL of peripheral blood. After surgery, 28.57% of the patients presented CTCs, with a mean value of 6.75 CTCs per 10 mL. A positive correlation was found between the presence of CTC-negative, epidermal growth factor receptor-positive at baseline and the patients who had symptoms of intestinal obstruction (21.4%). In addition, the presence of CTCs identified patients with distant dissemination and was also significantly correlated with progression-free survival (P = .0024). CONCLUSION The detection and characterization of CTCs are good prognostic and predictive markers in patients with peritoneal carcinomatosis resulting from colon cancer. These analyses could be used as a new tool to identify subpopulations of patients who could benefit from CRS + HIPEC treatment.

Integrated bio-affinity nano-platform into a microfluidic immunosensor based on monoclonal bispecific trifunctional antibodies for the electrochemical determination of epithelial cancer biomarker

BACKGROUND The epithelial cell adhesion molecule (EpCAM) is a biomarker that is highly overexpressed on the surface of epithelial carcinoma cells. In this study, silver nanoparticles covered with polyvinyl alcohol (AgNPs-PVA) were synthesized, characterized and used in a microfluidic immunosensor based on the use of anti-EpCAM recombinant antibodies as a trapping agent. METHODS The concentration of trapped EpCAM is then electrochemically quantified by HRP-conjugated anti-EpCAM-antibody. HRP reacted with its enzymatic substrate in a redox process which resulted in the appearance of a current whose magnitude (at a working voltage as low as -0.10V) is directly proportional to the concentration of EpCAM. RESULTS Under optimized conditions, the detection limits for the microfluidic immunosensor and a commercial ELISA were 0.8 and 13.9pg/L, respectively. The within-assay and between-assay coefficients of variation are below 6.5% for the proposed method. The immunosensor was validated by analyzing patient samples, and a good correlation with a commercial ELISA was obtained. CONCLUSIONS The good analytical performance is attributed to the efficient immobilization of the anti-EpCAM recombinant antibodies on the AgNPs-PVA, and its high specificity for EpCAM. This microfluidic immunosensor is intended for use in diagnosis and prognosis of epithelial cancer, to monitor the disease, and to assess therapeutic efficacy.

CK-coated magnetic-based beads as a tool to isolate circulating tumor cells (CTCs) in human tumors

Circulating tumor cells (CTCs) can be detected in the blood of many cancer patients and play a key role in metastasis. In addition, after the development of technologies with the necessary sensitivity and reproducibility, the diagnostic potential of these cells is being actively explored. Recently, the U.S. Food and Drug Administration has approved the CellSearch(®) System, based on magnetic beads coated with epithelial cell-adhesion molecule (EpCAM) antibody. Despite its usefulness, this system can miss CTCs that lose epithelial antigens due to the epithelial-mesenchymal transition and, in the case of advanced NSCLC, CTCs positivity can be demonstrated only in 30-50% of patients. In an effort to overcome these drawbacks, new methods are being developed. In this study, we have evaluated CK-coated beads as a system to isolate CTCs from lung cancer patients in the clinical setting, and have evaluated if they can be a useful source of material for genetic testing. We were able to identify CTCs in 17 of the 30 patients included in the study (57%), with a range of 1 to 7 cells. In two of them, we found only CTCs with an EMT pattern. CTC positivity seemed to correlate with the clinical history of the malignancy. CTCs could be detected in more than 80% of stage III-IV lung cancer patients at presentation or in blood samples taken immediately after surgery. The percentage dropped to 13% in patients responding to chemotherapy or TKIs, raising again to 57% after tumor progression. Finally, we tested the CTCs isolated from 8 patients for EGFR and k-ras mutations, but gene amplification was successful only in the 3 patients with 4 or more CTCs.

EGFR detection in extracellular vesicles of breast cancer patients through immunosensor based on silica-chitosan nanoplatform

In the present work, we designed a microfluidic electrochemical immunosensor with enough sensibility and precision to quantify epithermal growth factor receptor (EGFR) in plasma extracellular vesicles (EVs) of plasma from breast cancer patients. The sensor employs SiNPs coated with chitosan (SiNPs-CH) as reactions platform, based on the covalently immobilization of monoclonal anti-EGFR on SiNPs-CH retained in the central channel (CC) of the microfluidic device. The synthetized SiNPs-CH were characterized by UV-visible spectroscopy (UV-visible), energy dispersive spectrometry (EDS), Nanoparticle Tracking Analysis (NTA) and transmission electron microscopy (TEM). EGFR was quantified by a direct sandwich immunoassay measuring through a horseradish peroxidase (HRP)-conjugated anti-EGFR. The enzymatic product (benzoquinone) was detected by reduction at - 100 mV on a sputtering gold electrode. The measured current was directly proportional to the level of EGFR in human serum samples. The linear range was from 0 ng mL-1 to 50 ng mL-1. The detection limit was 1.37 pg mL-1, and the within- and between-assay coefficients of variation were below 6.25%. Finally, plasma samples from 30 early breast cancer patients and 20 healthy donor were analyzed by the novel method. EGFR levels in EVs (EVs-EGFR) were significantly higher than in the healthy control group (p = 0.002) and also, more sensitivity and specificity than normal serum markers like CEA and CA15.3 has been observed. EVs-EGFR concentration correlates with EGFR tumor status (p = 0.0003) as well as it correlate with the tumor size and pathological grade. To conclude, plasma EVs are suitable for proteomic characterization of cancer disease, as long as the employed method has sufficient sensitivity, like the case of immune-electrochemical nanosensors with incremented reaction surface.

肿瘤患者循环肿瘤细胞(CTCs)的分离工具：细胞角蛋白包被磁珠

多种癌症患者的外周血中均可检测到循环肿瘤细胞(circulating tumourcells，CTCs)，它在肿瘤 转移过程中发挥关键作用。此外，伴随CTCs检测技术的逐步发展，其敏感度和可重复性逐步 提高，进而可充分发挥其在肿瘤诊断中的潜能。最近，美国食品和药物管理局(food and drug administration，FDA)已批准CellSearch®检测系统用于检测外周血CTCs，该系统采用的是上皮细胞 粘附分子(EpCAM)抗体包被的磁珠。尽管它能有效检测出CTCs，但同样可因为上皮—间质转化 中上皮抗原的丢失而错失CTCs，数据显示只有30%~50%的晚期NSCLC外周血CTCs检测阳性。为 了克服这些缺点，研究人员正探索新的检测方法。本研究建立了细胞角蛋白(CK)包被的磁珠检测 系统来分离肺癌临床病例外周血中的CTCs，并评估该系统所分离的CTCs是否可作为基因检测的 有效材料来源。纳入研究的30例患者中，17例检测到CTCs (57%)，细胞数从1~7个，其中2例患者 中，仅存在EMT形式的CTCs。CTCs检测阳性率与恶性肿瘤的临床病史相关。在III-IV期肺癌患者 首诊时或术后血标本中，超过80%可检测到CTCs。对化疗或TKIs治疗有效的患者，其检测阳性率 下降到13%，肿瘤进展后又上升至57%。最后，我们对其中8例患者的CTCs进行EGFR和K-ras突变 检测，但只有3例CTCs≥4个的患者基因扩增成功。