Tumor Cells In Peripheral Blood Research Articles

Determination of Circulating Tumor Cells in Peripheral Blood By Flow Cytometry

Determination of Circulating Tumor Cells in Peripheral Blood By Flow Cytometry

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)

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. 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. 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). 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.

Notes for developing a molecular test for the full characterization of circulating tumor cells.

The proved association between the circulating tumor cell (CTC) levels and the patients' survival parameters has been growing interest to investigate the molecular profile of these neoplastic cells among which hide out precursors capable of initiating a new distant metastatic lesion. The full characterization of the tumor cells in peripheral blood of cancer patients is expected to be of help for understanding and (prospectively) for counteracting the metastatic process. The major hitch that is hampering the successful gaining of this result is the lack of a consensus onto standard operating procedures (SOPs) for performing what we generally define as the "liquid biopsy". Here we review the more recent acquisitions in the analysis of CTCs and tumor related nucleic acids, looking to the main open questions that are hampering their definitive employ in the routine clinical practice.

Abstract 379: Circulating tumor cells (CTCs) in clinically localized prostate cancer (PCa): searching a prognostic tool

Abstract Background If PCa screening has reduced advanced disease and disease-specific mortality, the tradeoff is an over-diagnosis of cases that would not have caused clinical consequences during a man's lifetime if left untreated. Conversely, a vast numbers of men, which harbor occult extra-prostatic extension, develop recurrence after surgery. Despite it looks like an addressable question by quantifying Circulating Tumor Cells (CTCs), unlike breast cancer, in which prognostic and predictive impact of tumor cells in peripheral blood or bone marrow (Disseminated Tumor Cells, DTCs) was largely provided, their role in localized PCa is far from clear. Methods From July 2011 to August 2013, we quantify the tumor burden at diagnosis in peripheral blood of 153 PCa patients stage T2a-T3b, enrolled at three clinical sites (Roma, Orbassano and Padova). All the patients were candidate to undergo radical prostatectomy because of positive biopsy for cancer. Two labs (Orbassano and Padova) performed the CellSearch assay. In patients enrolled in Roma (Janus trial, a phase II study for the use of zoledronic acid as neoadjuvant treatment of invasive PCa), we evaluated in parallel the DTCs. Results We used data obtained in patients enrolled in Orbassano (n = 50) and Roma (n = 15) as training set, meanwhile we used patients of Padova (n = 88) as validation set. Consistently with previous reports (Kraan, Sleijfer et al. 2011) reporting low inter-test and inter-lab variability, the baseline CTC count in the two sets did not significantly differ (Mann-Whitney Rank Sum Test, p = 0.170), so that they were further analyzed altogether. Overall, we found that 74 out of 153 PC patients (48,4%) had at least 1 CTC per 7.5 ml of peripheral blood; 45 PC (29,4%) patients had > 2 cells and 21 PC patients (13,7%) had > 3 cells. Interestingly, 8 out of 153 PC patients (5,2%) had > 5 cells at the first blood draw, before surgery. When we synchronously detected CTCs and DTCs, we found consistent results in 64% of patients (Wilcoxon signed Rank Test, p = 0.339). Conclusions Even when the PCa is pathologically organ confined at surgery, it was reported 5% to 20% of these patients harbor foci of micro-metastatic disease that will later manifest itself as recurrent disease. Here we show CTCs by CellSearch assay in close to 50% of biopsy-confirmed PCa patients, obtaining evidence of a systemic disease. Prospective studies will be required to investigate whether CTC-status is associated with worst outcome in the M0 setting of PCa, as already provided in early breast cancer patients (Lucci, Hall et al. 2012). Citation Format: Elisabetta Rossi, Antonella Facchinetti, Vittorio Aneloni, Emanuel Zilio, Massimo Dal Bianco, Alice Zoccoli, Daniele Santini, Diletta Garrou, Francesco Porpiglia, Rita Zamarchi. Circulating tumor cells (CTCs) in clinically localized prostate cancer (PCa): searching a prognostic tool. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 379. doi:10.1158/1538-7445.AM2015-379

High-Density Dielectrophoretic Microwell Array for Detection, Capture, and Single-Cell Analysis of Rare Tumor Cells in Peripheral Blood.

Development of a reliable platform and workflow to detect and capture a small number of mutation-bearing circulating tumor cells (CTCs) from a blood sample is necessary for the development of noninvasive cancer diagnosis. In this preclinical study, we aimed to develop a capture system for molecular characterization of single CTCs based on high-density dielectrophoretic microwell array technology. Spike-in experiments using lung cancer cell lines were conducted. The microwell array was used to capture spiked cancer cells, and captured single cells were subjected to whole genome amplification followed by sequencing. A high detection rate (70.2%–90.0%) and excellent linear performance (R2 = 0.8189–0.9999) were noted between the observed and expected numbers of tumor cells. The detection rate was markedly higher than that obtained using the CellSearch system in a blinded manner, suggesting the superior sensitivity of our system in detecting EpCAM− tumor cells. Isolation of single captured tumor cells, followed by detection of EGFR mutations, was achieved using Sanger sequencing. Using a microwell array, we established an efficient and convenient platform for the capture and characterization of single CTCs. The results of a proof-of-principle preclinical study indicated that this platform has potential for the molecular characterization of captured CTCs from patients.

Recent progress of diagnosis and treatment in NK cell neoplasms

Aggressive natural killer cell leukemia (ANKL) is a malignant disorder of mature NK cells that is relatively common in East Asia. It is associated with Epstein-Barr virus in more than 80% of patients. The median survival is not more than three months after diagnosis. In a recent survey of 34 cases with ANKL, the median patient age was 40 years and the age distribution followed a bimodal pattern. Morphologically, the ANKL cells varied from large granular lymphocytes to atypical cells with pleomorphic-like appearances. Clinical characteristics include fever, liver dysfunction, hemophagocytic syndrome, and a rapidly progressive course. In one third of patients, tumor cells in peripheral blood or the bone marrow are below 20% at initial presentation, which might lead to diagnostic delay. L-asparaginase-based chemotherapy plus allogeneic hematopoietic stem cell transplantation (HSCT) is a potential therapeutic option with curative intent.As for extranodal NK/T cell lymphoma, nasal type, HSCT could be anticipated to provide survival benefit or a chance of cure for patients in clinical stage II to IV with complete remission at transplantation. However, the optimal stem cell source, timing of transplantation, and preconditioning regimen require further elucidation.

Heterogeneity of circulating tumor cells in primary breast cancer patients.

e22037 Background: Circulating tumor cells (CTCs) are tumor cells in peripheral blood and represent a heterogeneous population of cells with different phenotypes and biological value. The aim of th...

SELEX aptamer used as a probe to detect circulating tumor cells in peripheral blood of pancreatic cancer patients.

Many studies have shown that the quantity and dynamics of circulating tumor cells (CTCs) in peripheral blood of patients afflicted with solid tumours have great relevance in therapeutic efficacy and prognosis. Different methods based on various strategies have been developed to isolate and identify CTCs, but their efficacy needs to be improved because of the rarity and complexity of CTCs. This study was designed to examine the possibility of using a SELEX aptamer (BC-15) as a probe to identify rare CTCs out of background nucleated cells. Aptamer BC-15 was selected from a random oligonucleotide library screened against human breast cancer tissue. Fluorescence staining showed that BC-15 had a high affinity for nuclei of human cancer cell lines of various origins as well as CTCs isolated from pancreatic cancer patients, whereas its binding capacity for non-tumor breast epithelial cells and leukocytes was almost undetectable. BC-15+/CD45- cells in cancer patient blood were also found to be cytokeratins 18-positive and aneuploid by immunofluorescence staining and fluorescent in situ hybridization, respectively. Finally, the aptamer method was compared with the well-established anti-cytokeratin method using 15 pancreatic cancer patient blood samples, and enumeration indicated no difference between these two methods. Our study establishes a novel way to identify CTCs by using a synthetic aptamer probe. This new approach is comparable with the anti-cytokeratin-based CTC identification method.

Clinical test on circulating tumor cells in peripheral blood of lung cancer patients, based on novel immunomagnetic beads

This paper aims to establish a novel and highly sensitive method to detect circulating tumor cells (CTCs) in the peripheral blood of patients with lung cancer. This therefore enables the discovery of invisible micrometastasis in the early stage of lung cancer, leading to better prognostic assessments of lung cancer and detection of the post-operative tumor recurrence and metastasis, treatment options, and evaluation of curative effects. In this research study, various lung cancer cells were mixed with adult blood samples to simulate blood samples of tumor patients. With novel test methods, CTCs in peripheral blood of lung cancer patients were calculated, after the reaction between the cells obtained from the mix and EpCAM (epithelial cell adhesion molecule) antibodies which were marked by immunomagnetic beads. The results showed that 18 out of 42 (42.9%) lung cancer patients had a positive CTCs, which increased with tumor enlargement or metastasis. CTCs were not detected in a total of 20 blood samples from healthy volunteers. This indicated that the technology of novel immunomagnetic bead-enrichment could effectively separate and identify CTCs in peripheral blood of lung cancer patients, which is of great clinical value for prognostic assessments and treatment guidance of lung cancer.

Clinical Significance of Circulating Tumor Cells in Peripheral Blood of Patients with Esophageal Squamous Cell Carcinoma.

BackgroundEsophageal squamous cell carcinoma is an aggressive gastrointestinal tract cancer. To date, the presence of circulating tumor cells (CTC) has been reported as a prognostic factor in peripheral blood from patients with gastrointestinal cancers.MethodsThe CellSearch system was used to isolate and enumerate CTCs. A total of 90 patients with esophageal squamous cell carcinoma who received chemotherapy or chemoradiotherapy were enrolled. Peripheral blood specimens were collected before and after treatments.ResultsAt baseline analysis, CTCs were detected in 25 patients (27.8 %). Overall survival was significantly shorter in patients with than without CTCs. Follow-up blood specimens were obtained from 71 patients. Partial response, stable disease, and progressive disease after treatment were seen in 32, 12, and 27 patients, respectively. CTC positivity after treatment in the progressive disease group (40.7 %) was significantly higher than that of the partial response group (6.3 %). Patients with a change in CTC status from positive to negative had a good prognosis as well as patients without baseline CTCs.ConclusionsEvaluation of CTCs may be a promising indicator for predicting tumor prognosis and the clinical efficacy of chemotherapy or chemoradiation therapy in patients with esophageal squamous cell carcinoma.Electronic supplementary materialThe online version of this article (doi:10.1245/s10434-015-4392-8) contains supplementary material, which is available to authorized users.

Detection of circulating tumour cells in peripheral blood of patients with malignant pleural mesothelioma.

The independent prognostic value of Circulating Tumour Cells (CTC) level has been demonstrated in several solid tumours. There is currently few data on Malignant Pleural Mesothelioma (MPM) and CTC. We investigated whether the presence of CTC was correlated with prognosis factors and treatment efficacy. MPM patients (pts) were enrolled in a prospective monocentric study. CTC detection was made using the "CellSearch" assay. The correlation between the presence of CTC and worse prognosis factors was assessed using the X(2) test. Comparison of Overall Survival (OS) and Progression Free Survival (PFS) according to CTC detection was performed using the log-rank test. Twenty-seven MPM pts with a median follow-up of 4.2 months were included. CTC were detected in 44% of pts with a median level of 1.5. No significant correlation was observed between the presence of CTC and worse prognosis factors. Moreover, CTC detection was not a significant predictor of OS or PFS (p=0.155 and p=0.32 respectively). CTC were detected in a small cohort of MPM patients. We couldn't demonstrate a significant prognostic value or a difference in OS/PFS between CTC levels. Further analyses, validation studies and detection techniques are needed to establish their real clinical value in MPM.

Occult Breast Lobular Carcinoma with Numerous Circulating Tumor Cells in Peripheral Blood

We experienced a very rare case of occult breast lobular carcinoma with numerous circulating tumor cells in peripheral blood. The diagnosis was very difficult because there were no symptoms of breast cancer and the preceding chief complaints such as general fatigue and weight loss or abnormality of peripheral blood findings were suggestive of a hematological disease. We could make a correct diagnosis of this case by checking the findings of complete blood count and bone marrow biopsy at the same time using immunohistochemistry.

Effects of Herceptin on circulating tumor cells in HER2 positive early breast cancer.

The objective of this study was to determine the changes in peripheral blood circulating tumor cells in HER2-positive early breast cancer before and after Herceptin therapy, and to explore the effects of the HER2 gene and Herceptin on circulating tumor cells. CK19 mRNA expression in peripheral blood was evaluated by qRT-PCR as an index of circulating tumor cells in 15 cases of HER-2-positive breast cancer and 18 cases of HER2-negative breast cancer before, and after chemotherapy as well. Ten cases of HER2-positive breast cancer continued on Herceptin therapy for 3 months after chemotherapy, and their peripheral blood was again drawn and assayed for CK-19 mRNA expression. Preoperatively, all cases of HER2-positive cancer were positive for CK19 mRNA in peripheral blood, but 6 cases of HER2-negative breast cancer were positive (33.3%), where there was a substantial difference between the two groups. After 6 cycles of adjuvant chemotherapy, CK19 positive rates in cases of HER2-positive and -negative breast cancer reduced by 93.3 and 11.1%, respectively, with a significant difference still existing. After 3 months of Herceptin therapy, expression of CK19 mRNA declined considerably in 10 cases of HER2 positive breast cancer (113.66 ± 88.65 vs 63.35 ± 49.27, P = 0.025). HER-2 gene expression closely correlated with circulating tumor cells in peripheral blood of early breast cancer patients. Moreover, Herceptin, a monoclonal antibody for HER2, can reduce the number of circulating tumor cells, which can be an early predictive factor for Herceptin therapy effectiveness against breast cancer.

Location of NLS-RARα protein in peripheral blood tumor cells of patients with acute promyeolic leukemia

Location of NLS-RARα protein in peripheral blood tumor cells of patients with acute promyeolic leukemia

Analysis of the IL-31 pathway in Mycosis fungoides and Sézary syndrome.

IL-31, predominantly produced by CD45RO + CLA + Th2 cells, plays an important pathogenetic role in pruritic skin diseases like atopic dermatitis. As tumor cells in Sézary syndrome (SS) and Mycosis fungoides (MF) possess similar immunophenotypes and the conditions mentioned are often associated with pruritus, the analysis of the IL-31 pathway in MF/SS patients is of interest. Serum samples from the peripheral blood of 23 patients and 17 controls were analyzed for IL-31 abundance and correlated with disease stage and pruritus. Furthermore IL-31-, IL-31 receptor alpha (IL-31Rα)- and Oncostatin M receptor beta (OSMRβ)-mRNA expression was measured in blood tumor cells from SS patients, memory T-cells from controls and lymphoma cell lines. Serum IL-31 levels were low but differed between groups with no or strong pruritus. Expression of IL-31 was detectable at low levels in cell lines, but not in the tumor cells of SS patients. Stimulation with PMA/ionomycin led to indiscriminate expression in peripheral blood tumor cells and control T-cells. IL-2-stimulation resulted in expression only in 9/11 patient samples. IL-31Rα-expression was detectable in 10/10 cell lines, 8/15 peripheral blood samples from SS patients, and 4/10 controls; whereas, OSMRβ mRNA was detectable in 4/10 cell lines, but only one patient and control sample. The results of our analyses regarding serum levels and receptor expression do not suggest a central role of IL-31 in MF/SS pathogenesis. However, the results of IL-2 stimulation as well as the increased IL-31 levels in patients with strong pruritus offer a rationale for therapeutic approach in this subset of patients.

In Vivo Ibrutinib Abrogates BCR-Dependent Adhesion in Tumor Cells of Patients with Chronic Lymphocytic Leukemia

Ibrutinib, now FDA approved for use in both previously treated mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL), induces objective clinical responses and is well tolerated (Advani, JCO 2013; Byrd, NEJM 2013). As with other BCR-directed kinase inhibitors, (such as fostamatinib and idelalisib) ibrutinib leads to a treatment-induced transient lymphocytosis that resolves over the first 2-3 months on therapy in patients with CLL. The initial increase in absolute lymphocyte count (ALC) has been shown to be due to the release of tumor cells from the tissue compartments; specifically the lymph nodes, into the peripheral blood (Herman, Leukemia 2014). In vitro ibrutinib reduces the ability of CLL cells to migrate towards chemokines and prevents adhesion of BCR stimulated CLL cells (Ponader, Blood 2012; de Rooij, Blood 2012). We hypothesized that the initial rise in ALC is due to inhibition of CLL cell adhesion. We tested this ex vivo in patients enrolled on a single agent phase II trial of ibrutinib in CLL (NCT01500733) and investigated the mechanism.In order to evaluate the effect of ibrutinib on CLL cell adhesion in vivo we compared peripheral blood tumor cells collected from patients pre-treatment and on day 28 of treatment. Fluorescently labeled cells were plated on fibronectin-coated plates and allowed to adhere for 1 hour. Adhesion was then evaluated using florescence microscopy and quantified using the ImageJ software. After 28 days on ibrutinib, CLL cells displayed a severely reduced adhesion ability (99% reduction, P<0.001, n=13). Next, we tested the effect of ibrutinib on CLL cell adhesion to a confluent stromal cell layer. Although less dramatic, we found a similar reduction in the ability of cells to adhere to the stromal cell layer in all three patients evaluated. As the initial rise in ALC occurs very rapidly in some patients, we evaluated the ability of cells collected on day 2 of ibrutinib therapy and again found a significant inhibition of cell adhesion to fibronectin (81% reduction, P=0.005, n=10). This suggests that even one dose of ibrutinib is sufficient to disrupt CLL cell adhesion.We next sought to determine if ibrutinib could directly interfere with adhesion by causing cells to detach from fibronectin. We found that after one hour of 1µM ibrutinib treatment in vitro there was a significant reduction in the number of CLL cells that remained attached (33% reduction, P=0.03, n=10). This suggests that ibrutinib can not only prevent CLL cell adhesion but can release adhered cells from the microenvironment, consistent with influx of cells from the lymph node into the peripheral blood within hours of starting treatment.To determine whether the observed defect in cell adhesion is due to changes in the expression of CD29/CD49d (VLA-4 or α4β1 integrin), or CD18/CD11a (LFA-1), we measured the expression of these known regulators of CLL cell adhesion in samples obtained pre-treatment and during treatment. While there was inter-patient variability, overall there was no significant change in expression of CD29 after 28 days on treatment. In contrast albeit minor, we found reductions in both CD49d and CD18 (P<.05). When analyzing cells obtained on day 2, we found no significant changes suggesting that alteration of adhesion molecule expression does not account for the rapid increase in ALC. Integrin α4β1 is activated by PKC downstream of BTK. We thus sought to determine whether stimulation with PMA, an activator of PKC, could restore the ability of CLL cells to adhere to fibronectin. Indeed, PMA stimulation of CLL cells obtained from patients after 28 days on ibrutinib partially restored their ability to adhere to fibronectin, suggesting that reduced α4β1 integrin activity contributes to inhibition of adhesion by ibrutinib.In conclusion, ibrutinib abolished CLL cell adhesion at least in part due to direct inhibition of BTK- and BCR-mediated α4β1 integrin activity. Additionally, ibrutinib not only prevented adhesion but also caused the detachment of adhered cells from the extracellular matrix. Together, this suggests that disruption of cellular adhesion is a major contributor to the observed rise in ALC early on treatment.This work was supported by the Intramural Research Program of NHLBI, NIH. We thank our patients for donating blood samples to make this research possible. DisclosuresWiestner:Pharmacyclics Inc.: Research Funding.

Inhibitory effect of endostar on lymphangiogenesis in non-small cell lung cancer and its effect on circulating tumor cells

背景与目的血管内皮抑素可以抑制肿瘤新生血管的生成，但对肿瘤微淋巴管的形成与发展是否存在抑制效应引起我们关注。本研究旨在探讨重组人血管内皮抑素（recombinant human endostatin injection）对非小细胞肺癌组织中血管内皮生长因子（vascular endothelial growth factor, VEGF）-C、VEGF-D和VEGF受体（VEGFR）-3表达及对外周血循环肿瘤细胞数目的影响。方法荷瘤裸鼠随机分为空白对照组、顺铂组、不同浓度重组人血管内皮抑素组及重组人血管内皮抑素+顺铂组，连续给药2周。1周后检测肿瘤组织中VEGF-C、VEGF-D、VEGFR-3的表达水平和微淋巴管密度。免疫荧光染色诊断和计数循环肿瘤细胞。结果重组人血管内皮抑素组与重组人血管内皮抑素联合顺铂组的表达阳性率、微淋巴管密度均明显低于空白对照组与顺铂组（P < 0.05）；较高浓度的重组人血管内皮抑素联合顺铂组与重组人血管内皮抑素组表达阳性率和微淋巴管密度低于相应较低重组人血管内皮抑素浓度的组别（P < 0.05）。各组微淋巴管密度与VEGF-C、VEGF-D、VEGFR-3表达阳性率存在正相关。重组人血管内皮抑素联合顺铂各组的循环肿瘤细胞数目明显低于单独使用顺铂或重组人血管内皮抑素（P < 0.05）。结论重组人血管内皮抑素可以抑制肿瘤新生淋巴管生成，减少循环肿瘤细胞，作用大小与浓度有关。与顺铂联合使用能够更有效的减少循环肿瘤细胞。

334P - Clinical Significance and the Detection of Circulating Tumor Cells in Peripheral Blood of Patients with Breast Cancer

ABSTRACT Aim: To explore the detection methods of detecting circulating tumor cells (CTCs) in peripheral blood of breast cancer patients using multiparameter flow cytometry (FCM) and analyze its clinical significance. Methods: The breast cancer cell line SKBR-3 was serially diluted and added into the peripheral blood mononuclear cells from healthy donors to evaluate the sensitivity of FCM. Mononuclear cells were isolated from 7.5 mL peripheral venous blood of 62 patients with breast cancer and 20 cases of healthy volunteers using density gradient centrifugation. Superspeed flow cytometry were used to detect expression level of immunofluorescence antibody (EpCAM、CK18 and CD45). The amount of CTCs ≥ 5/7.5 ml in peripheral blood was considered micrometastases positive. Results: The expression of CTCs in 20 healthy volunteers was all negative, while the positive rate was 53.2% in 62 case breast cancer patients, the difference was significant (P 0.05). The positive rate of CTCs gradually increased with positive rate of TNM staging ranks. The positive rate of CTCs was significant different among different breast cancer molecular subtypes (P Conclusions: The sensitivity and specificity of the detection of CTCs using ultra high speed flow cytometry was high. The positive rate CTCs in breast cancer patients was related to its clinical TNM staging, the expression of Ki 67 and HER-2, but unrelated to menopausal status, the expression of ER and PR. Breast cancer molecular subtypes can assess the risk of peripheral blood micrometastases, the positive rate of CTCs was high in HER-2 amplification type and triple-negative type. Disclosure: All authors have declared no conflicts of interest.

Assessing the Presence of Circulating Tumor Cells in Breast Cancer Patients Using Multiplex Reverse Transcription Polymerase Chain Reaction and Specific Primers for Mammaglobin, Pthrp and Ck19

ABSTRACT Aim: The aim of this study was to develop a multiplex reverse transcription polymerase chain reaction (RT-PCR) assay for the detection of circulating tumor cells in peripheral blood of patients with breast cancer. Methods: Peripheral blood samples were collected from 54 breast cancer patients and 20 healthy blood donors. Subsequently, the samples processed for RNA extraction and then were analyzed for the expression of PTHrP, CK19 and Mammaglobin using specific primers and multiplex PCR. Results: The detection rates in breast cancer patients for PTHrP, CK19 and Mammaglobin were 68.5%, 63% and 22.2% and for healthy donors 10%, 0% and 10%., respectively. The statistical analysis revealed that PTHrP, CK19 and their combination could be used for diagnosing breast cancer. The combination of positive detection rates of PTHrP with CK19 correlated with the presence of distant metastasis in patients with breast cancer, especially with bone metastasis. Moreover, positive detection rates of CK19 correlated with high proliferation rate of breast cancer Conclusions: Multiplex-PCR based detection of circulating tumor cells can provide useful information for the disease stage and presence of metastasis in breast cancer patients. Disclosure: All authors have declared no conflicts of interest.

Optimization of rhabdomyosarcoma disseminated disease assessment by flow cytometry.

Rhabdomyosarcoma (RMS) is the most common type of soft tissue sarcoma in children. Circulating tumor cells in peripheral blood or disseminated to bone marrow, a concept commonly referred to as minimal residual disease (MRD), are thought to be key to the prediction of metastasis and treatment efficacy. To date, two MRD markers, MYOD and MYOGENIN, have been tested; however, MRD detection continues to be challenging mainly owing to the closeness of the detection limit and the discordance of both markers in some samples. Therefore, the addition of a third marker could be useful for more accurate MRD assessment. The PAX3 gene is expressed during embryo development in all myogenic precursor cells in the dermomyotome. As RMS cells are thought to originate from these muscle precursor cells, they are expected to be positive for PAX3. In this study, PAX3 expression was characterized in cancer cell lines and tumors, showing wide expression in RMS. Detection sensitivities by quantitative polymerase chain reaction (qPCR) of the previously proposed markers, MYOD and MYOGENIN, were similar to that of PAX3, thereby indicating the feasibility of its detection. Interestingly, the flow cytometry experiments supported the usefulness of this technique in the quantification of MRD in RMS using PAX3 as a marker. These results indicate that flow cytometry, albeit in some cases slightly less sensitive, can be considered a good approach for MRD assessment in RMS and more consistent than qPCR, especially owing to its greater specificity. Furthermore, fluorescence-activated cell sorting permits the recovery of cells, thereby providing material for further characterization of circulating or disseminated cancer cells.