María J. Serrano

Biomarkers characterization of circulating tumour cells in breast cancer patients

IntroductionIncreasing evidence supports the view that the detection of circulating tumor cells (CTCs) predicts outcomes of nonmetastatic breast cancer patients. CTCs differ genetically from the primary tumor and may contribute to variations in prognosis and response to therapy. As we start to understand more about the biology of CTCs, we can begin to address how best to treat this form of disease.MethodsNinety-eight nonmetastatic breast cancer patients were included in this study. CTCs were isolated by immunomagnetic techniques using magnetic beads labelled with a multi-CK-specific antibody (CK3-11D5) and CTC detection through immunocytochemical methods. Estrogen receptor, progesterone receptor and epidermal growth factor receptor (EGFR) were evaluated by immunofluorescence experiments and HER2 and TOP2A by fluorescence in situ hybridization. We aimed to characterize this set of biomarkers in CTCs and correlate it with clinical-pathological characteristics.ResultsBaseline detection rate was 46.9% ≥ 1 CTC/30 ml threshold. CTC-positive cells were more frequent in HER2-negative tumors (p = 0.046). In patients younger than 50 years old, HER2-amplified and G1-G2 tumors had a higher possibility of being nondetectable CTCs. Heterogeneous expression of hormonal receptors (HRs) in samples from the same patients was found. Discordances between HR expression, HER2 and TOP2A status in CTCs and their primary tumor were found in the sequential blood samples. Less that 35% of patients switched their CTC status after receiving chemotherapy. EGFR-positive CTCs were associated with Luminal tumors (p = 0.03).ConclusionsThis is the largest exploratory CTC biomarker analysis in nonmetastatic BC patients. Our study suggests that CTC biomarkers profiles might be useful as a surrogate marker for therapeutic selection and monitoring since heterogeneity of the biomarker distribution in CTCs and the lack of correlation with the primary tumor biomarker status were found. Further exploration of the association between EGFR-positive CTCs and Luminal tumors is warranted.

Detection of circulating tumor cells in the context of treatment: prognostic value in breast cancer.

Circulating tumor cells (CTCs) in patients with breast cancer can be regarded as the pre-stadium of clinically manifest distant metastases. Here we present results on CTCs determination in peripheral blood (PB) of breast cancer patients in the context of treatment. Ninety-two patients were enrolled onto a prospective, unicenter study, and 71 of those are the focus of our analyses. CTC assessment was performed by isolating cytokeratin-positive (CK) cells by immunomagnetic techniques, with further identification by immunocytochemical methods. CTCs were detected in 47 (66%) patients: 35 with primary breast cancer, and 12 with metastatic disease. Five (14.3%) of those patients with primary cancer and CTCs showed first disease progression or died. Eleven (91.6%) of those patients with metastatic disease and CTCs before chemotherapy, died, During chemotherapy, >6 CTCs was correlated with a worse prognostic of disease in patients with metastatic disease (p=0.05). Four weeks after chemotherapy, 59 patients underwent a follow-up assessment. CTCs were detected in 54.2% of those patients. CTCs levels, and not the presence of CTCs alone, was associated with progression free of disease (p=0.052) and showed borderline significance with overall survival (p= 0.071). The differential prognostic and overall survival showed between patients with and without elevated CTCs before and at the end of chemotherapy, is of special interest in patients without clinical evidence of metastasis.

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.

EGFR mutations in circulating tumour DNA

www.thelancet.com/oncology Vol 13 October 2012 971 predictive markers is important to enable selection of subgroups likely to benefi t from a particular drug. The investigators assayed 32 plasma proteins associated with the reported targets of regorafenib. Obtained at baseline and 15 days after start of treatment, protein concentrations were tested for association with response and outcome. Identifi cation of soluble plasma biomarkers is particularly interesting because it could circumvent the potential pitfalls associated with biomarker development based on single biopsy samples of genetically heterogeneous primary tumours. Plasma concentrations of cytokeratin fragments (CK18M30) associated with apoptosis increased more in patients with at least 40% maximum tumour shrinkage compared with those with less than 40% tumour shrinkage in Eisen and colleagues’ study. Furthermore, baseline concentrations of soluble TIE1 and TIMP2, which suppresses endothelial cell proliferation, were higher in patients with 40% or more tumour shrinkage than in those with less than 40%. The biomarker analyses included only 28 patients, and therefore should be interpreted with caution. The researchers suggest further investigation of regorafenib in subpopulations of patients with renal-cell carcinoma who have upregulated angiopoietin or FGF pathways. However, defi nition of distinct subgroups is diffi cult because there are few clinical trials where biomarker assessment is used effi ciently. Randomised phase 2 biomarker trials with small sample sizes could provide recommendations for phase 3 trials such as identifi cation of populations for enrolment that have specifi c biomarkers. The study of Eisen and colleagues is a reminder of the challenges created by increasing treatment options at a time when the incorporation of biomarker candidates into novel trials will be key to maximise therapeutic benefi ts for patient subgroups and personalisation of treatment.

Relevance of molecular characterization of circulating tumor cells in breast cancer in the era of targeted therapies

Development in circulating tumor cells (CTCs) technologies represents a valuable tool for the better understanding of tumor biology. The clinical relevance of CTCs as a prognostic factor is well established both in metastatic and early-stage breast cancer patients. The eradication or decrease of CTCs following treatment is associated with improved clinical outcomes. Because of the availability of novel cancer treatments that specifically target tumor cells underlying signaling pathways, molecular characterization of CTCs has strong potential to translate into personalized treatments. A handful of studies have explored relevant markers such as the estrogen and progesterone receptor, HER2 and EGF receptor. However, there is not a single validation of a molecular marker in CTCs that provides prognostic information or predicts response to cancer therapies. This review describes the latest results on the characterization of breast cancer CTCs with a focus on CTC biology and implications in clinical practice.

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.

Circulating cancer cells in division in an early breast cancer patient

A 63-year-old woman came for management of a second breast cancer (BC). Ten years earlier, she had a lumpectomy and axillary lymph node dissection for invasive ductal carcinoma (pT1cN0M0). The tumor cells expressed estrogen receptor (ER) and progesterone receptor (PR) and did not overexpress human epidermal growth factor receptor 2 (ERBB2). After discussion of the risks and the benefits of adjuvant therapy, the patient declined further treatment. In July 2009, a follow-up mammography revealed an architectural distortion in the upper outer quadrant. Specimens from a stereotactically guided core biopsy revealed an invasive lobular carcinoma. Lumpectomy and sentinel lymph node biopsy was carried out. This patient’s tumor was pT1bN0M0, ER positive, PR positive, and ERBB2 negative. Adjuvant treatment with aromatase inhibitors was recommended and she elected to proceed with letrozole. The patient was treated with radiation therapy to the right breast. She discontinued the adjuvant treatment due to clinically relevant musculoskeletal symptoms in March 2010. Approximately 20 months after the diagnosis of the second tumor, she had no evidence of recurrent BC. From March 2009 to September 2010, as a part of a protocol approved by the institutional review board, 98 nonmetastatic BC patients donated 10 ml of blood for the analysis of circulating tumor cells (CTCs). Samples were processed according to the protocol established for our group [1]. In brief, CTCs were captured from the peripheral blood by pan-cytokeratin (CK) 7/8 antibody-bearing ferrofluid and subsequently MACs MS columns were used for cell immunoselection. CTCs were identified by an immunocytochemical method and visualized under a direct light microscope to perform the cytomorphologic and immunophenotypic evaluation of CTC [2] (Figure 1—Legend). In this patient, positive CTCs were detected in the blood samples obtainedbefore the initiationof endocrine therapy andon 3 and 12months followup visit. Themost unexpected observation was the finding of cytomorphological features suggesting that a CTC was undergoing cell division among the five CK-positive cells detected in the last sample. Using an antibody that recognizes phosphorylated Histone 3 on Ser 10 (phosphor-H3) that is involved in the mitotic process [3], we confirmed our suspicion. There was a fluorescent signal of phospho-H3 in the separating chromosomes in one CTC that was previously identified as a CK-positive CTC (Figure 1—Legend). To the best of our knowledge, this is the first report of a CTC in division in the peripheral blood of a BC patient. Although CTCs are not usually considered in making decisions about adjuvant treatment in early BC patients, the detection of CTC in division raises an issue in the management of these patients. A possible role of the cessation of endocrine therapy and the detection of a mitotic CTC may not be ruled out; however, no reports of a similar nature are available regarding this phenomenon. Another explanation for this finding lies in the role of therapy resistance and minimal residual disease is thought to result from drug-induced molecular changes in CTCs, reflecting clonal selection during treatment [4]. CTCs have been detected in BC patients many years after diagnosis who are clinically disease free, suggesting that tumor cells are in a state of dynamic dormancy. Clinical dormancy is reflected by relapses at distant organs, following the primary cancer diagnosis. However, the mechanism of outgrowth and replacement of dormant tumor cells over time remain in question. Although we recognize that a follow-up of 20 months is a rather short time and we cannot rule out that the CTC came from the first breast cancer; we propose that the dormant CTCs turnover may be consistent with a model of homeostasis involving CTCs with self-renewing properties. There is not much individual information about the features of CTCs that can predict whether or not patients will develop a late recurrence or should expect the induction of CTCs death [5]. Further characterization of such precursors of metastasis may provide powerful translational tools to improve the clinical outcome of BC patients.

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.