Jinfukang regulates integrin/Src pathway and anoikis mediating circulating lung cancer cells migration.

ETHNOPHARMACOLOGICAL RELEVANCE: Metastasis is the main cause of death in lung cancer patients. Circulating tumor cells (CTCs) may be an important target of metastasis intervention. Previous studies have shown that Jinfukang could prevent the recurrence and metastasis of lung cancer, and we have established a circulating lung tumor cell line CTC-TJH-01. However, whether Jinfukang inhibition of lung cancer metastasis is related to CTCs is still unknown. AIM OF THE STUDY: To further explore the mechanism of Jinfukang in anti-metastasis of lung cancer from the perspective of intervention of CTCs. MATERIALS AND METHODS: CTC-TJH-01 and H1975 cells were treated with Jinfukang. Cell viability was detected by CCK8, and the cell apoptosis was detected by flow cytometry. Transwell was used to detected cell migration and invasion. Cell anoikis was detected by anoikis detection kit. Protein expression was analysis by Western blot. RESULTS: Jinfukang could inhibit the proliferation, migration and invasion of CTC-TJH-01 and H1975 cells. Besides, Jinfukang could also induce anoikis in CTC-TJH-01 and H1975 cells. Analysis of the mRNA expression profile showed ECM-receptor interaction and focal adhesion were regulated by Jinfukang. Moreover, it was also find that Jinfukang significantly inhibited integrin/Src pathway in CTC-TJH-01 and H1975 cells. When suppress the expression of integrin with ATN-161, it could promote Jinfukang to inhibit migration and induce anoikis in CTC-TJH-01 and H1975 cells. CONCLUSIONS: Our results indicate that the migration and invasion of CTCs are inhibited by Jinfukang, and the mechanism may involve the suppression of integrin/Src axis to induce anoikis. These data suggest that Jinfukang exerts anti-metastatic effects in lung cancer may through anoikis.

Circulating tumor-cell-targeting Au-nanocage-mediated bimodal phototherapeutic properties enriched by magnetic nanocores.

Metastasis resulting from circulating tumor cells (CTCs) is associated with 90% of all cancer mortality. To disrupt cancer dissemination, therapeutic targeting of CTCs by extracorporeal photodynamic therapy (PDT) has emerged; however, it still remains impractical due to its limited therapeutic window. Herein, we developed a photosensitive and magnetic targeted core-satellite nanomedicine (TCSN) to augment the light-induced damage to the targeted cells. The magnetic nanocore (MNC) with multiple iron oxide nanoparticles stabilized using thiolated polyvinyl alcohol can magnetize the CTCs to achieve magnetic enrichment under a magnetic field. Multiple gold nanocage (AuNC) satellites were conjugated on the MNC to facilitate bimodal photothermal therapy and PDT. Adjusting the thiol content in the MNC allows manipulating the AuNC density on TCSNs, which has been found to demonstrate a density-dependent bimodal phototherapeutic effect under laser irradiation at 808 and 940 nm. Moreover, with the immobilization of anti-epithelial cell adhesion molecule (anti-EpCAM), TCSN exhibited an enhanced affinity toward EpCAM-expressing 4T1 cells. We demonstrate that TCSN-labeled 4T1 cells can be isolated and photo-eradicated in a microfluidic channel with a dynamic flow. Our studies showed that TCSN with the complementary properties of MNC and AuNCs can largely augment the therapeutic window by magnetic enrichment and bimodal phototherapy, serving as an advanced extracorporeal strategy to remove CTCs.

Advances in the Characterization of Circulating Tumor Cells in Metastatic Breast Cancer: Single Cell Analyses and Interactions, and Patient-Derived Models for Drug Testing.

Metastasis is the major cause of breast cancer death worldwide. In metastatic breast cancer, circulating tumor cells (CTCs) can be captured from patient blood samples sequentially over time and thereby serve as surrogates to assess the biology of surviving cancer cells that may still persist in solitary or multiple metastatic sites following treatment. CTCs may thus function as potential real-time decision-making guides for selecting appropriate therapies during the course of disease or for the development and testing of new treatments. The heterogeneous nature of CTCs warrants the use of single cell platforms to better inform our understanding of these cancer cells. Current techniques for single cell analyses and techniques for investigating interactions between cancer and immune cells are discussed. In addition, methodologies for growing patient-derived CTCs in vitro or propagating them in vivo to facilitate CTC drug testing are reviewed. We advocate the use of CTCs in appropriate microenvironments to appraise the effectiveness of cancer chemotherapies, immunotherapies, and for the development of new cancer treatments, fundamental to personalizing and improving the clinical management of metastatic breast cancer.

Proteomics analysis of tumor exosomes reveals vital pathways of Jinfukang inhibiting circulating tumor cells metastasis in lung cancer.

ETHNOPHARMACOLOGICAL RELEVANCE: Jinfukang has long been used for the clinical treatment of lung cancer. Previous studies have shown that Jinfukang can induce the apoptosis of circulating tumor cells by intervening ROS-mediated DNA damage pathway. However, whether Jinfukang can inhibit the metastasis of circulating tumor cells and its mechanism are still unclear. AIM OF THE STUDY: To further investigate the mechanism of Jinfukang in anti-metastasis of lung cancer from the perspective of intervention of tumor exosomes. MATERIALS AND METHODS: The invadopodia formation was determined with immunofluorescence. Invasion and migration were detected using the Transwell assay. Ultracentrifugation was used to isolate exosomes. Exosomes were characterized by electron microscopy, nanoparticle tracking analysis and immunoblotting, and the protein profile was evaluated by proteomic analysis. The molecular functions, biological processes and signaling pathway enrichment analysis were performed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). Key differentially expressed proteins were verified by Western blot. RESULTS: Jinfukang can inhibit the expression of MMP14, cortactin, Tks5 and the formation of invadopodia of CTC-TJH-01 cells. Furthermore, Jinfukang can significantly inhibit the invasion and migration of CTC-TJH-01 cells. The diameter of exosomes extracted from the CTC-TJH-01 cells treated by Jinfukang was 30-100 nm, and the exosomal markers CD63, CD81 and TSG101 were expressed. We identified 680 deferentially expressed proteins. Gene oncology analysis indicated that exosomes were mostly derived from plasma membrane and mainly involved in protein localization and intracellular signaling. The ingenuity pathway analysis showed that the EGF pathway was significantly inhibited, whereas the GP6 signaling pathway was significantly activated. We also confirmed that Jinfukang inhibited the expression of EGF pathway-related proteins in CTC-TJH-01 cells. Besides, when EGF was used to activate EGF signaling pathway, the inhibition of Jinfukang on CTC cell metastasis was reversed. CONCLUSION: Jinfukang inhibits the metastasis of CTC-TJH-01 cells through the EGF pathway.

Dendrimer-Au Nanoparticle Network Covered Alumina Membrane for Ion Rectification and Enhanced Bioanalysis.

Ion transport in an artificial asymmetric nanoporous membrane, which is similar to biological ion channels, can be used for biosensing. Here, a dendrimer-Au nanoparticle network (DAN) is in situ assembled on a nanoporous anodic aluminum oxide (AAO) surface, forming a DAN/AAO hybrid membrane. Benefiting from the high surface area and anion selectivity of DAN, the prepared DAN/AAO hybrid presents selective ion transport. Under a bias potential, a diode-like current-potential (I-V) response is observed. The obtained ionic current rectification (ICR) property can be tuned by the ion valence and pH value of the electrolyte. The rectified ionic current endows the as-prepared DAN/AAO hybrid with the ability of enhanced bioanalysis. Sensitive capture and detection of circulating tumor cells (CTCs) with a detection limit of 80 cells mL-1 as well as excellent reusability can be achieved.

Insulin enhancement of the antitumor activity of chemotherapeutic agents in colorectal cancer is linked with downregulating PIK3CA and GRB2.

The present state of cancer chemotherapy is unsatisfactory. New anticancer drugs that marginally improve the survival of patients continue to be developed at an unsustainably high cost. The study aimed to elucidate the effects of insulin (INS), an inexpensive drug with a convincing safety profile, on the susceptibility of colon cancer to chemotherapeutic agents: 5-fluorouracil (FU), oxaliplatin (OXA), irinotecan (IRI), cyclophosphamide (CPA) and docetaxel (DOC). To examine the effects of insulin on cell viability and apoptosis, we performed an in vitro analysis on colon cancer cell lines Caco-2 and SW480. To verify the results, we performed in vivo analysis on mice bearing MC38 colon tumors. To assess the underlying mechanism of the therapy, we examined the mRNA expression of pathways related to the signaling downstream of insulin receptors (INSR). Moreover, we performed Western blotting to confirm expression patterns derived from the genetic analysis. For the quantification of circulating tumor cells in the peripheral blood, we used the maintrac method. The results of our study show that insulin-pretreated colon cancer cells are significantly more susceptible to commonly used chemotherapeutics. The apoptosis ratio was also enhanced when INS was administered complementary to the examined drugs. The in vivo study showed that the combination of INS and FU resulted in significant inhibition of tumor growth and reduction of the number of circulating tumor cells. This combination caused a significant downregulation of the key signaling substrates downstream of INSR. The results indicate that the downregulation of PIK3CA (phosphatidylinositol 3-kinase catalytic subunit alpha), which plays a critical role in cell signaling and GRB2 (growth factor receptor-bound protein 2), a regulator of cell proliferation and differentiation may be responsible for the sensitizing effect of INS. These findings were confirmed at protein levels by Western blotting. In conclusion, these results suggest that INS might be potentially applied to clinical use to enhance the therapeutic effectiveness of chemotherapeutic drugs. The findings may become a platform for the future development of new and inexpensive strategies for the clinical chemotherapy of tumors.

Jingfukang induces anti-cancer activity through oxidative stress-mediated DNA damage in circulating human lung cancer cells.

BACKGROUND: Metastasis is the main cause of lung cancer death. As a seed of metastasis, circulating tumor cells are an important target for metastasis intervention. The traditional Chinese medicine, Jinfukang, has been clinically available for the treatment of non-small cell lung cancer (NSCLC). In this study, we investigated the action and underlying mechanisms of Jinfukang against circulating lung tumor cells. METHODS: The cell counting kit-8 (CCK-8), colony formation and cell cycle assays were used to study the cell proliferation ability. Flow cytometry was used to detect the apoptosis and the expression level of ROS and Caspase-3. Comet and TUNEL assays were used to detect DNA damage. DNA damage related pathway protein was detected by western blot. RESULTS: Jinfukang significantly inhibits the proliferation of CTC-TJH-01 cells by inducing G1 phase arrest and inhibits their colony formation in a dose-dependent manner. Moreover, Jinfukang induces apoptosis in CTC-TJH-01 cells through the ROS-mediated ATM/ATR-p53 pathway and DNA damage. CONCLUSIONS: Our findings suggest that Jinfukang may be a potential drug for lung cancer metastasis.

Flow Cytometric Detection of Circulating Osteosarcoma Cells in Dogs.

Osteosarcoma (OSA) is a malignant tumor of middle-aged dogs and adolescent humans. The clinical outcome of OSA has not improved over more than three decades, and dogs typically succumb to metastatic disease within 6 months despite tumor resection through limb amputation and adjuvant chemotherapy. Therefore, undetectable tumor cells with potential to form metastases are present at diagnosis. An assay to identify canine immortalized and primary OSA cells through flow cytometric detection of intracellular collagen 1 (Col I) and osteocalcin was optimized, and applied to blood samples from tumor-bearing dogs for detection of circulating tumor cells (CTCs). Spiking variable number of OSA cells into normal dog blood recovered 50-60% of Col I positive cells with high forward and variable side light scatter. An algorithm to exclude nonviable, doublet, and autofluorescent cells was applied to sequential blood samples from three dogs obtained prior to and after limb amputation, and at approximately, triweekly intervals over 121, 142, and 183 days of chemotherapy, respectively. Dogs had >100 CTC/106 leukocytes prior to amputation, variably frequent CTC during chemotherapy, and an increase up to 4,000 CTC/106 leukocytes within 4 weeks before overt metastases or death. Sorted CTCs were morphologically similar to direct tumor aspirates and positive for Col I. Although preliminary, findings suggest that CTCs are frequent in canine OSA, more numerous than carcinoma CTC in humans, and that an increase in CTC frequency may herald clinical deterioration. This assay may enable enumeration and isolation of OSA CTC for prognostic and functional studies, respectively. © 2019 International Society for Advancement of Cytometry.

Apoptotic and Nonapoptotic Activities of Pterostilbene against Cancer.

Cancer is a major cause of death. The outcomes of current therapeutic strategies against cancer often ironically lead to even increased mortality due to the subsequent drug resistance and to metastatic recurrence. Alternative medicines are thus urgently needed. Cumulative evidence has pointed out that pterostilbene (trans-3,5-dimethoxy-4-hydroxystilbene, PS) has excellent pharmacological benefits for the prevention and treatment for various types of cancer in their different stages of progression by evoking apoptotic or nonapoptotic anti-cancer activities. In this review article, we first update current knowledge regarding tumor progression toward accomplishment of metastasis. Subsequently, we review current literature regarding the anti-cancer activities of PS. Finally, we provide future perspectives to clinically utilize PS as novel cancer therapeutic remedies. We, therefore, conclude and propose that PS is one ideal alternative medicine to be administered in the diet as a nutritional supplement.

The effect of selected food phytochemicals on breast cancer metastasis based on in vivo capture of circulating tumor cells.

The number of circulating tumor cells (CTCs) in a mouse tumor model fed with different dietary factors was first evaluated by in vivo CTC capture at different time points of cancer progression. The results showed that the number of CTCs reflected the degree of cancer progression. A new assessment method of dietary factor effects on cancer metastasis was established.

Circulating tumor markers: harmonizing the yin and yang of CTCs and ctDNA for precision medicine.

Current trajectory of clinical care is heading in the direction of personalized medicine. In an ideal scenario, clinicians can obtain extensive diagnostic and prognostic information via minimally-invasive assays. Information available in the peripheral blood has the potential to bring us closer to this goal. In this review we highlight the contributions of circulating tumor cells and circulating tumor DNA and RNA (ctDNA/ctRNA) towards cancer therapeutic field. We discuss clinical relevance, summarize available and upcoming technologies, and hypothesize how future care could be impacted by a combined study.

Surface Functionalized Graphene Biosensor on Sapphire for Cancer Cell Detection.

Graphene has several unique physical, optical and electrical properties such as a two-dimensional (2D) planar structure, high optical transparency and high carrier mobility at room temperature. These make graphene interesting for electrical biosensing. Using a catalyst-free chemical vapor deposition (CVD) method, graphene film is grown on a sapphire substrate. There is a single or a few sheets as confirmed by Raman spectroscopy and atomic force microscopy (AFM). Electrical graphene biosensors are fabricated to detect large-sized biological analytes such as cancer cells. Human colorectal carcinoma cells are sensed by the resistance change of an active bio-functionalized graphene device as the cells are captured by the immobilized antibody surface. The functionalized sensors show an increase in resistance as large as ~20% of the baseline with a small number of adhered cells. This study suggests that the bio-functionalized electrical graphene sensors on sapphire, which is a highly transparent material, can potentially detect circulating tumor cells (CTCs) and monitor cellular electrical behavior while being compatible with fluorescence-based optical-detection bioassays.

Heterogeneous estrogen receptor expression in circulating tumor cells suggests diverse mechanisms of fulvestrant resistance.

Fulvestrant is a dose dependent selective estrogen receptor (ER) down-regulator (SERD) used in ER-positive metastatic breast cancer (MBC). Nearly all patients develop resistance. We performed molecular analysis of circulating tumor cells (CTC) to gain insight into fulvestrant resistance. Preclinical studies were performed with cultured breast cancer cells spiked into human blood and analyzed on the CellSearch(®) system. Clinical data are limited to a subset of patients with ER-positive MBC from a previously reported pilot trial whose disease was progressing on fulvestrant (N = 7) or aromatase inhibitors (AIs) (N = 10). CTCs were enumerated and phenotyped for ER and B-cell lymphoma (BCL2) using the CellSearch(®) CXC kit. In preclinical modeling, tamoxifen and AIs resulted in stabilized ER expression, whereas fulvestrant eliminated it. Five of seven patients progressing on fulvestrant had ≥5CTC/7.5 ml WB. Two of these five, treated with 500 mg/month fulvestrant, had no detectable CTC-expression of ER and BCL2 (an ER regulated gene). Three patients had heterogeneous CTC-ER and BCL2 expression indicating incomplete degradation of the ER target by fulvestrant. Two of these patients received 250 mg/month whereas the third patient received 500 mg/month fulvestrant. Her cancer harbored a mutation (Y537S) in the estrogen receptor alpha gene (ESR1). All seven ER positive patients progressing on AIs had heterogeneous CTC-ER expression. These results suggest heterogeneous mechanisms of resistance to fulvestrant, including insufficient dosage, ESR1 mutation, or conversion to dependence on non-ER pathways. CTC enumeration, phenotyping, and genotyping might identify patients who would benefit from fulvestrant dose escalation versus switching to alternative therapies.

Polymer microfilters with nanostructured surfaces for the culture of circulating cancer cells.

There is a critical need to improve the accuracy of drug screening and testing through the development of in vitro culture systems that more effectively mimic the in vivo environment. Surface topographical features on the nanoscale level, in short nanotopography, effect the cell growth patterns, and hence affect cell function in culture. We report the preliminary results on the fabrication, and subsequent cellular growth, of nanoscale surface topography on polymer microfilters using cell lines as a precursor to circulating tumor cells (CTCs). To create various nanoscale features on the microfilter surface, we used reactive ion etching (RIE) with and without an etching mask. An anodized aluminum oxide (AAO) membrane fabricated directly on the polymer surface served as an etching mask. Polymer filters with a variety of modified surfaces were used to compare the effects on the culture of cancer cell lines in blank culture wells, with untreated microfilters or with RIE-treated microfilters. We then report the differences of cell shape, phenotype and growth patterns of bladder and glioblastoma cancer cell lines after isolation on the various types of material modifications. Our data suggest that RIE modified polymer filters can isolate model cell lines while retaining ell viability, and that the RIE filter modification allows T24 monolayering cells to proliferate as a structured cluster.

Evaluation of epithelial-mesenchymal transitioned circulating tumor cells in patients with resectable gastric cancer: Relevance to therapy response.

AIM: To evaluate the epithelial-to-mesenchymal transition (EMT) of circulating tumor cells (CTCs) in gastric cancer patients. METHODS: We detected tumor cells for expression of four epithelial (E(+)) transcripts (keratins 8, 18, and 19 and epithelial cell adhesion molecule) and two mesenchymal (M(+)) transcripts (Vimentin and Twist) by a quantifiable, dual-colorimetric RNA-in situ hybridization assay. Between July 2014 and October 2014, 44 patients with gastric cancer were recruited for CTC evaluation. Blood samples were obtained from selected patients during the treatment course [before surgery, after surgery and at the 6(th) cycle of XELOX based chemotherapy (about 6 mo postoperatively)]. RESULTS: We found the EMT phenomenon in which there were a few biphenotypic E(+)/M(+) cells in primary human gastric cancer specimens. Of the 44 patients, the presence of CTCs was reported in 35 (79.5%) patients at baseline. Five types of cells including from exclusively E(+) CTCs to intermediate CTCs and exclusively M(+) CTCs were identified (4 patients with M(+) CTCs and 10 patients with M(+) or M(+) > E(+) CTCs). Further, a chemotherapy patient having progressive disease showed a proportional increase of mesenchymal CTCs in the post-treatment blood specimens. We used NCI-N87 cells to analyze the linearity and sensitivity of CanPatrol(TM) system and the correlation coefficient (R(2)) was 0.999. CONCLUSION: The findings suggest that the EMT phenomenon was both in a few cells of primary tumors and abundantly in CTCs from the blood of gastric cancer patients, which might be used to monitor therapy response.

Prognostic value of disseminated tumor cells in the bone marrow of patients with operable primary breast cancer: a long-term follow-up study.

BACKGROUND: Detection of disseminated tumor cells (DTC) in primary breast cancer (BC) patients' bone marrow (BM) seems to be a surrogate marker of tumor spread and an independent prognostic factor for disease-free and overall survival. METHODS: Here we present the largest single-center cohort of patients (n = 1378) with the longest observation time (median 82.0 months). Immunocytochemical staining was performed using murine monoclonal antibody 2E11 with the avidin-biotin complex technique. RESULTS: At primary surgery, 49 % of patients showed MUC-1 positive cells inside their BM. Patients without BM DTC had significantly more often T1-tumors (P = 0.007) with less often affected axillary lymph nodes (P < 0.001). We observed a significantly higher incidence of distant metastases in DTC positive patients (P < 0.001). This leads to a reduced disease-free survival (P < 0.0001). Furthermore, in DTC positive patients there was a higher mortality rate and, accordingly, a reduced overall survival (P < 0.0001). CONCLUSIONS: Due to the presence of BM DTC, patients with a clinically poorer outcome can be identified at primary surgery. We therefore suggest that DTC analysis can be used as a prognostic factor and monitoring tool in clinical trials. Future study concepts relating to DTC should aim at identification of BC patients who may profit from adjuvant systemic therapy.

Cytokeratin-19 and mammaglobin gene expression in circulating tumor cells from metastatic breast cancer patients enrolled in North Central Cancer Treatment Group trials, N0234/336/436/437.

PURPOSE: To investigate the associations between baseline and posttreatment circulating tumor cell (CTC) gene expression and outcome of patients enrolled in four North Central Cancer Treatment Group metastatic breast cancer (MBC) trials in which specimens were shipped (at 4°C) from community-based sites to a reference laboratory (Mayo Clinic, Rochester, MN). EXPERIMENTAL DESIGN: Blood was collected at treating sites from MBC patients before (baseline), during, and at the end of treatment with erlotinib + gemcitabine (N0234), sorafenib (N0336), irinotecan + cetuximab (N0436), or paclitaxel-poliglumex + capecitabine (N0437). CTCs from 10 mL of EDTA blood were enriched with CD45 depletion, 24 to 30 hours postblood collection. Reverse transcription/quantitative PCR was used to determine cytokeratin-19 (CK19) and mammaglobin (MGB1) mRNA levels in CTCs from up to 13 (N0234), 16 (N0336), 18 (N0436), and 39 (N0437) patients. The gene expressions were normalized to ß(2)-microglobulin and calibrated to healthy blood using the 2(-ΔΔCq) algorithm; positivity was defined as 2 or more. RESULTS: CK19+mRNA cells were detected in 56% to 75% and MGB1+mRNA cells in 23% to 38% of 86 patients at baseline. CK19+mRNA cells were detected in 30% to 67% and MGB1+mRNA cells in 14% to 64% of 110 postbaseline serial samples. The presence of baseline CK19+mRNA cells (P = 0.01) but not MGB1+mRNA cells (P = 0.14) was significantly associated with shorter overall survival. A decrease in MGB1+mRNA levels (baseline-week 8) seemed to be associated with clinical response (P = 0.05). CONCLUSIONS: CTC gene expression analysis conducted by a reference laboratory is feasible when blood is collected from treating sites and processed 24 to 30 hours postcollection. The presence of baseline CK19+mRNA CTCs was associated with poor prognosis; a decrease in MGB1+mRNA CTCs may help predict response to therapy of MBC patients.

[The prognostic value of tumor cells blood circulation after liver surgery for cancer lesions].

The study aimed to prove the prognostic meaning of micrometastases blood circulation during liver resections for cancer lesions. 33 patients took part in the study. Circulating micrometastases were detected in blood using immunocytological method with pancytoceratine antibodies KL-1 and CAM 5.2. The majority of patients had colon cancer liver metastases (72,7%). Blood was sampled once in 8 patients, the rest 25 patients had double sampling: before and after liver mobilization. Patients with multiple liver metastases demonstrated tumor cells circulation more often. Of 58 tests, 25 were positive for tumor cells. 3-year survival in those patients was 45,7 ± 13,1%, 5-year survival was 24,4 ± 13,3%. Survival rates for patients with no circulating tumor cells detected were significantly higher.

Detection and HER2 expression of circulating tumor cells: prospective monitoring in breast cancer patients treated in the neoadjuvant GeparQuattro trial.

PURPOSE: This study was aimed at detecting and characterizing circulating tumor cells (CTC) before and after neoadjuvant therapy (NT) in the peripheral blood of patients with breast cancer. EXPERIMENTAL DESIGN: The clinical trial GeparQuattro incorporated NT approaches (epirubicin/cyclophosphamide prior to randomization to docetaxel alone, docetaxel in combination with capecitabine, or docetaxel followed by capecitabine) and additional trastuzumab treatment for patients with HER2-positive tumors. We used the Food and Drug Administration-approved CellSearch system for CTC detection and evaluation of HER2 expression and developed HER2 immunoscoring for CTC. RESULTS: We detected > or =1 CTC/7.5 mL in 46 of 213 patients (21.6%) before NT and in 22 of 207 patients (10.6%) after NT (P = 0.002). Twenty (15.0%) initially CTC-positive cases were CTC-negative after NT, whereas 11 (8.3%) cases were CTC-positive after NT, although no CTC could be found before NT. CTC detection did not correlate with primary tumor characteristics. Furthermore, there was no association between tumor response to NT and CTC detection. HER2-overexpressing CTC were observed in 14 of 58 CTC-positive patients (24.1%), including 8 patients with HER2-negative primary tumors and 3 patients after trastuzumab treatment. CTC scored HER2-negative or weakly HER2-positive before or after NT were present in 11 of 21 patients with HER2-positive primary tumors. HER2 overexpression on CTC was restricted to ductal carcinomas and associated with high tumor stage (P = 0.002). CONCLUSION: CTC number was low in patients with primary breast cancer. The decrease in CTC incidence during treatment was not correlated with standard clinical characteristics and primary tumor response. Information on the HER2 status of CTC might be helpful for stratification and monitoring of HER2-directed therapies.

Enhancing detection of circulating tumor cells with activating KRAS oncogene in patients with colorectal cancer by weighted chemiluminescent membrane array method.

BACKGROUND: In 2008, the National Comprehensive Cancer Network suggested conducting a KRAS mutations test in metastatic colorectal cancer (mCRC) patients prior to administering therapy that uses anti-epidermal growth factor receptor (EGFR) monoclonal antibody. However, tests of KRAS mutations have been limited when traditional molecular techniques, such as polymerase chain reaction (PCR) combined direct sequencing, are used to obtain and analyze patients' cancer tissues. If the primary tumor or metastatic tissues of patients with mCRC is unavailable, then such analysis will not be feasible. Our laboratory has successfully established a colorimetric membrane array analysis platform that could detect activating KRAS mutations from the peripheral blood of patients with various malignancies. METHODS: The current research aims to improve the above-mentioned technique not only by using chemiluminescence detection to replace color development, but also to add scores weighted according to the relevance of each gene to activating KRAS mutations. RESULTS: Our results show that the described weighted chemiluminescent membrane array (WCHMA) can detect circulating tumor cells (CTCs) harboring activating KRAS mutations in the peripheral blood in CRC. The sensitivity, specificity, and accuracy were 90.2, 94.9, and 93.5%, respectively, and the detection limitation was three colon tumor cells per millimeter of blood. The current study would significantly improve the detection sensitivity and accuracy over that of our previously designed membrane array method. CONCLUSIONS: These findings also highlight the need to prompt further prospective studies on more cases of CRC to further establish the clinical relevance of activating KRAS mutation detection from peripheral blood in anti- EGFR-based chemotherapy that uses activating KRAS detection chips and the WCHMA analysis method.