Integrative Analysis of Genome, 3D Genome,and Transcriptome Alterations of Clinical Lung Cancer Samples

Genomic studies of cancer cell alterations, such as mutations, copy number variations (CNVs), and translocations, greatly promote our understanding of the genesis and development of cancers. However, the 3D genome architecture of cancers remains less studied due to the complexity of cancer genomes and technical difficulties. To explore the 3D genome structure in clinical lung cancer, we performed Hi-C experiments using paired normal and tumor cells harvested from patients with lung cancer, combining with RNA sequenceing analysis. We demonstrated the feasibility of studying 3D genome of clinical lung cancer samples with a small number of cells (1 × 10⁴), compared the genome architecture between clinical samples and cell lines of lung cancer, and identified conserved and changed spatial chromatin structures between normal and cancer samples. We also showed that Hi-C data can be used to infer CNVs and point mutations in cancer. By integrating those different types of cancer alterations, we showed significant associations between CNVs, 3D genome, and gene expression. We propose that 3D genome mediates the effects of cancer genomic alterations on gene expression through altering regulatory chromatin structures. Our study highlights the importance of analyzing 3D genomes of clinical cancer samples in addition to cancer cell lines and provides an integrative genomic analysis pipeline for future larger-scale studies in lung cancer and other cancers.     

Circulating Tumor DNA as Biomarkers for Cancer Detection

Detection of circulating tumor DNAs (ctDNAs) in cancer patients is an important component of cancer precision medicine ctDNAs. Compared to the traditional physical and biochemical methods, blood-based ctDNA detection offers a non-invasive and easily accessible way for cancer diagnosis, prognostic determination, and guidance for treatment. While studies on this topic are currently underway, clinical translation of ctDNA detection in various types of cancers has been attracting much attention, due to the great potential of ctDNA as blood-based biomarkers for early diagnosis and treatment of cancers. ctDNAs are detected and tracked primarily based on tumor-related genetic and epigenetic alterations. In this article, we reviewed the available studies on ctDNA detection and described the representative methods. We also discussed the current understanding of ctDNAs in cancer patients and their availability as potential biomarkers for clinical purposes. Considering the progress made and challenges involved in accurate detection of specific cell-free nucleic acids, ctDNAs hold promise to serve as biomarkers for cancer patients, and further validation is needed prior to their broad clinical use.     

Circulating tumor cells in the early detection of human cancers

Cancer is a severe disease with high morbidity and mortality globally. Thus, early detection is emerging as an important topic in modern oncology. Although the strategies for early detection have developed rapidly in recent decades, they remain challenging due to the subtle symptoms in the initial stage of the primary tumor. Currently, tumor biomarkers, imaging, and specific screening tests are widely used in various cancer types; however, each method has limitations. The harms are even overweight against the benefits in some cases. Therefore, early detection approaches should be improved urgently. Liquid biopsy, for now, is a convenient and non-invasive way compared to the traditional tissue biopsy in screening and early diagnosis. Circulating tumor cells (CTCs) are vital in liquid biopsy and play a central role in tumor dissemination and metastases. They have promising potential as cancer biomarkers in early detection. This review updates the knowledge of the biology of CTC; it also highlights the CTC enrichment technologies and their applications in the early detection of several human cancers.     

Detection of Circulating Tumor Cells and Circulating Tumor Microemboli in Gastric Cancer

PURPOSE: Gastric cancer studies indicated a potential correlation between circulating tumor cells (CTCs) in peripheral blood and tumor relapse/metastasis. The prevalence and significance of circulating tumor microemboli (CTM) in gastric cancer remain unknown. We investigated the prevalence and prognostic value of CTCs and CTM for progression-free survival (PFS) and overall survival (OS) in gastric cancer patients. METHODS:Eighty-one gastric cancer patients consented to provide 5 ml of peripheral blood before systematic therapy. CTCs and CTM were isolated using isolation by size of epithelial tumor cells and characterized by cytopathologists. For 41 stage IV gastric cancer patients, CTM was investigated as a potential biomarker to predict prognosis. RESULTS:CTCs were detected in 51 patients; the average count was 1.81. In clinical stage I, II, III, and IV patients, the average CTC counts were 1.40, 0.67, 1.24, and 2.71, respectively. CTM were detected in 3 of 33 clinical stage I to IIIb patients, at an average of 0.12 (0-2). CTM were detected in 13 of 53 clinical stage IIIc to IV patients, at an average of 1.26 (0-22). In stage IV patients, CTM positivity correlated with the CA125 level. PFS and OS in CTM-positive patients were significantly lower than in CTM-negative patients (P < .001). CTM positivity was an independent factor for determining the PFS (P = .016) and OS (P = .003) of stage IV patients in multivariate analysis. Using markers of the epithelial-mesenchymal transition, single CTCs were divided into three phenotypes including epithelial CTCs, biphenotypic epithelial/mesenchymal CTCs, and mesenchymal CTCs. For CTM, CK?/Vimentin+/CD45? and CK+/Vimentin+/CD45? phenotypes were observed, but the CK+/Vimentin?/CD45? CTM phenotype was not. CA125 was detected in gastric cancer cell lines BGC823 and MGC803. CONCLUSIONS: In stage IV patients, CTM positivity was correlated with serum CA125 level. CTM were an independent predictor of shorter PFS and OS in stage IV patients. Thus, CTM detection may be a useful tool to predict prognosis in stage IV patients.     

微流控芯片技术在循环肿瘤细胞分离中的研究进展

循环肿瘤细胞(circulating tumor cells,CTCs)是指从原发肿瘤或转移灶脱落、发生上皮-间质转化进入患者外周血血液循环的恶性肿瘤细胞.CTCs在肿瘤研究和临床诊断上的作用逐渐得到认可,外周血中CTCs存在与否以及数量多少不但可以用于肿瘤的早期诊断,还可以用于评估肿瘤预后、监测肿瘤的转移和复发.微流控芯片作为一个高通量、小型化的细胞实验平台,已被应用于CTCs的分选当中.本文综述了用于CTCs捕获的微流控芯片系统的最新研究进展,着重介绍各类芯片的捕获原理、芯片结构和捕获效率,最后对微流控芯片技术在CTCs分选中的应用前景进行了展望.     

Sensitive and Specific Biomimetic Lipid Coated Microfluidics to Isolate Viable Circulating Tumor Cells and Microemboli for Cancer Detection

Here we presented a simple and effective membrane mimetic microfluidic device with antibody conjugated supported lipid bilayer (SLB) “smart coating” to capture viable circulating tumor cells (CTCs) and circulating tumor microemboli (CTM) directly from whole blood of all stage clinical cancer patients. The non-covalently bound SLB was able to promote dynamic clustering of lipid-tethered antibodies to CTC antigens and minimized non-specific blood cells retention through its non-fouling nature. A gentle flow further flushed away loosely-bound blood cells to achieve high purity of CTCs, and a stream of air foam injected disintegrate the SLB assemblies to release intact and viable CTCs from the chip. Human blood spiked cancer cell line test showed the ~95% overall efficiency to recover both CTCs and CTMs. Live/dead assay showed that at least 86% of recovered cells maintain viability. By using 2 mL of peripheral blood, the CTCs and CTMs counts of 63 healthy and colorectal cancer donors were positively correlated with the cancer progression. In summary, a simple and effective strategy utilizing biomimetic principle was developed to retrieve viable CTCs for enumeration, molecular analysis, as well as ex vivo culture over weeks. Due to the high sensitivity and specificity, it is the first time to show the high detection rates and quantity of CTCs in non-metastatic cancer patients. This work offers the values in both early cancer detection and prognosis of CTC and provides an accurate non-invasive strategy for routine clinical investigation on CTCs.     

Programmed Cell Death–Ligand 1 Overexpression in Thyroid Cancer

Objective: Programmed cell death–ligand 1 (PD-L1) expression on tumor tissue has been associated with favorable response to anti–programmed cell death–receptor 1/PD-L1 therapy in many human cancers. Studies have reported that PD-L1 is also expressed in thyroid cancer. The objective of this paper is to introduce the potential predictive and therapeutic values of PD-L1 in thyroid cancer.Methods: A literature search was conducted in the PubMed database using the terms “PD-L1,” “B7-H1,” and “thyroid cancer.” PD-L1 positivity was determined by immunohistochemical assay.Results: The frequency of PD-L1 positivity in different studies ranged from 6.1 to 82.5% in papillary thyroid cancer (PTC) patients and 22.2 to 81.2% in anaplastic thyroid cancer (ATC) patients. PD-L1 positivity rate was higher in ATC than in PTC within the same studies, and its expression intensity was significantly higher in tumor tissue than in the corresponding nontumor thyroid tissues. Moreover, PD-L1 expression was positively associated with the aggressiveness and recurrence of thyroid cancers and negatively associated with the differentiation status and outcomes. PD-L1 checkpoint pathway blockade may emerge as a promising therapeutic target in the treatment of thyroid cancers.Conclusion: PD-L1 is a potential biomarker to predict the recurrence and prognosis of thyroid cancers. It is also a novel immunotherapy target for optimizing the management landscape of radioiodine-refractory and ATCs.Abbreviations: ATC = anaplastic thyroid cancer; DTC = differentiated thyroid cancer; IHC = immunohistochemical; OS = overall survival; PD-1 = programmed cell death–receptor 1; PD-L1 = programmed cell death–ligand 1; PD-L2 = programmed cell death–ligand 2; PTC = papillary thyroid cancer; TNM = tumor-node-metastasis; Treg = regulatory T cell     

Classification of Circulating Tumor Cells by Epithelial-Mesenchymal Transition Markers

In cancer, epithelial-mesenchymal transition (EMT) is associated with metastasis. Characterizing EMT phenotypes in circulating tumor cells (CTCs) has been challenging because epithelial marker-based methods have typically been used for the isolation and detection of CTCs from blood samples. The aim of this study was to use the optimized CanPatrol CTC enrichment technique to classify CTCs using EMT markers in different types of cancers. The first step of this technique was to isolate CTCs via a filter-based method; then, an RNA in situ hybridization (RNA-ISH) method based on the branched DNA signal amplification technology was used to classify the CTCs according to EMT markers. Our results indicated that the efficiency of tumor cell recovery with this technique was at least 80%. When compared with the non-optimized method, the new method was more sensitive and more CTCs were detected in the 5-ml blood samples. To further validate the new method, 164 blood samples from patients with liver, nasopharyngeal, breast, colon, gastric cancer, or non-small-cell lung cancer (NSCLC) were collected for CTC isolation and characterization. CTCs were detected in 107(65%) of 164 blood samples, and three CTC subpopulations were identified using EMT markers, including epithelial CTCs, biophenotypic epithelial/mesenchymal CTCs, and mesenchymal CTCs. Compared with the earlier stages of cancer, mesenchymal CTCs were more commonly found in patients in the metastatic stages of the disease in different types of cancers. Circulating tumor microemboli (CTM) with a mesenchymal phenotype were also detected in the metastatic stages of cancer. Classifying CTCs by EMT markers helps to identify the more aggressive CTC subpopulation and provides useful evidence for determining an appropriate clinical approach. This method is suitable for a broad range of carcinomas.     

Prognostic impact of circulating tumor cells in patients with ampullary cancer

Circulating tumor cells (CTCs) are an important topic of investigation for both basic and clinical cancer research. In this prospective study, we evaluated the clinical role of CTCs in ampullary cancer. We analyzed blood samples from 62 consecutively diagnosed patients with ampullary adenocarcinoma and 24 healthy controls for their CTC content. Combined data from immunostaining of CD45, 4′,6‐diamidino‐2‐phenylindole (DAPI), and fluorescence in situ hybridization with a chromosome 8 centromere (CEP8) probe were used to identify CTCs; cells that were CD45‐/DAPI+/CEP8>2 were considered CTCs. The Cox proportional hazards model was used to assess the relationship between CTCs, clinical characteristics, and patient outcomes. We detected ≥2 CTCs/3.2 ml whole blood in 43 of 62 patients (69.4%), as well as ≥5 CTCs/3.2 ml in 16 of these patients (25.8%). A CTC cutoff value of 2 cells/3.2 ml achieved 69.4% sensitivity and 95.8% specificity as a diagnostic tool; CTCs were associated with tumor burden. CTC levels ≥3/3.2 ml (hazard ratio [HR]: 2.5, 95% confidence interval [CI]: (1.2–5.2), p = 0.014) and ≥5/3.2 ml (HR: 3.5, 95% CI: 1.7–7.3, p < 0.001) were both associated with shorter disease‐free survival. Moreover, ≥3 CTCs/3.2 ml (HR: 2.7, 95% CI: 1.2–6.3, p = 0.019) and ≥5 CTCs/3.2 ml (HR: 3.8, 95% CI: 1.8–8.5, p < 0.001) were predictive of shorter overall survival. CTC assessment may help identify patients with ampullary cancer who are at high risk of an unfavorable outcome.     

2-(4-Chlorophenyl)-2-oxoethyl 4-benzamidobenzoate derivatives,a novel class of SENP1 inhibitors: Virtual screening,synthesis and biological evaluation

Prostate cancer is one of the most prevalent types of malignant cancers in men and has a high mortality rate among all male cancers. Previous studies have demonstrated that Sentrin/SUMO-specific protease 1 (SENP1) plays an important role in the occurrence and development of prostate cancer, and has been identified as a novel drug target for development of small molecule drugs against prostate cancer. In this paper, we used virtual screening and docking to identify compound J5 as a novel lead compound inhibiting SENP1, from SPECS library. We further investigated the SAR (structure–activity relationship) of the benzoate substituent of compound J5, and discovered compounds 8d and 8e as better small molecule inhibitors of SENP1. Both compounds are the high potent SENP1 small molecule inhibitors discovered up to date, and further lead optimization may lead to a series of novel anti-SENP1 agents. Further SAR studies are in process and will be reported in due course.     

Molecular Biomarker Analyses Using Circulating Tumor Cells

Background

Evaluation of cancer biomarkers from blood could significantly enable biomarker assessment by providing a relatively non-invasive source of representative tumor material. Circulating Tumor Cells (CTCs) isolated from blood of metastatic cancer patients hold significant promise in this regard.

Methodology/Principal Findings

Using spiked tumor-cells we evaluated CTC capture on different CTC technology platforms, including CellSearch® and two biochip platforms, and used the isolated CTCs to develop and optimize assays for molecular characterization of CTCs. We report similar performance for the various platforms tested in capturing CTCs, and find that capture efficiency is dependent on the level of EpCAM expression. We demonstrate that captured CTCs are amenable to biomarker analyses such as HER2 status, qRT-PCR for breast cancer subtype markers, KRAS mutation detection, and EGFR staining by immunofluorescence (IF). We quantify cell surface expression of EGFR in metastatic lung cancer patient samples. In addition, we determined HER2 status by IF and FISH in CTCs from metastatic breast cancer patients. In the majority of patients (89%) we found concordance with HER2 status from patient tumor tissue, though in a subset of patients (11%), HER2 status in CTCs differed from that observed in the primary tumor. Surprisingly, we found CTC counts to be higher in ER+ patients in comparison to HER2+ and triple negative patients, which could be explained by low EpCAM expression and a more mesenchymal phenotype of tumors belonging to the basal-like molecular subtype of breast cancer.

Conclusions/Significance

Our data suggests that molecular characterization from captured CTCs is possible and can potentially provide real-time information on biomarker status. In this regard, CTCs hold significant promise as a source of tumor material to facilitate clinical biomarker evaluation. However, limitations exist from a purely EpCAM based capture system and addition of antibodies to mesenchymal markers could further improve CTC capture efficiency to enable routine biomarker analysis from CTCs.     

Forward and reverse mutations in stages of cancer development

Background

Massive occurrences of interstitial loss of heterozygosity (LOH) likely resulting from gene conversions were found by us in different cancers as a type of single-nucleotide variations (SNVs), comparable in abundance to the commonly investigated gain of heterozygosity (GOH) type of SNVs, raising the question of the relationships between these two opposing types of cancer mutations.

Methods

In the present study, SNVs in 12 tetra sample and 17 trio sample sets from four cancer types along with copy number variations (CNVs) were analyzed by AluScan sequencing, comparing tumor with white blood cells as well as tissues vicinal to the tumor. Four published “nontumor”-tumor metastasis trios and 246 pan-cancer pairs analyzed by whole-genome sequencing (WGS) and 67 trios by whole-exome sequencing (WES) were also examined.

Results

Widespread GOHs enriched with CG-to-TG changes and associated with nearby CNVs and LOHs enriched with TG-to-CG changes were observed. Occurrences of GOH were 1.9-fold higher than LOH in “nontumor” tissues more than 2 cm away from the tumors, and a majority of these GOHs and LOHs were reversed in “paratumor” tissues within 2 cm of the tumors, forming forward-reverse mutation cycles where the revertant LOHs displayed strong lineage effects that pointed to a sequential instead of parallel development from “nontumor” to “paratumor” and onto tumor cells, which was also supported by the relative frequencies of 26 distinct classes of CNVs between these three types of cell populations.

Conclusions

These findings suggest that developing cancer cells undergo sequential changes that enable the “nontumor” cells to acquire a wide range of forward mutations including ones that are essential for oncogenicity, followed by revertant mutations in the “paratumor” cells to avoid growth retardation by excessive mutation load. Such utilization of forward-reverse mutation cycles as an adaptive mechanism was also observed in cultured HeLa cells upon successive replatings. An understanding of forward-reverse mutation cycles in cancer development could provide a genomic basis for improved early diagnosis, staging, and treatment of cancers.

     

A label-free microfluidic chip for the highly selective isolation of single and cluster CTCs from breast cancer patients

BackgroundCirculating tumor cells (CTCs) existing in peripheral blood can be used to predict the prognosis and survival of cancer patients. The study was designed to detect circulating tumor cells and circulating tumor single cell genes by applying microfluidic chip technology. It was used to explore the clinical application value in breast cancer.MethodsWe have developed a size-based CTCs sorting microfluidic chip, which contains a hexagonal array and a micro-pipe channel array to isolate and confirm both single CTCs and CTCs clusters. The sorting performance of the as-fabricated chip was tested by analyzing the clinical samples collected from 129 breast cancer patients and 50 healthy persons.ResultsIn this study, the chip can detect different immunophenotypes of CTCs in breast cancer patients. It was found that the new microfluidic device had high sensitivity (73.6%) and specificity (82.0%) in detecting CTCs. By detecting the blood samples of 129 breast cancer patients and 50 healthy blood donors, it was found that the number of CTCs was not associated with clinical factors such as age, gender, pathological type, and tumor size of breast cancer patients (P > 0.05), but was associated with TNM staging of breast cancer, with or without metastasis (P < 0.005). There was a statistically significant difference in the number of CTCs between luminal A (ER+/PR+/HER2-) and HER-2+ (ER-/PR-/HER2+) (P < 0.05). The best cut-off level distinguished by CTC between the breast cancer patients and the healthy persons was 3.5 cells/mL, with 0.845 for AUC-ROC, 0.790–0.901 for 95% CI, 73.6% for sensitivity, and 82% for specificity (P = 0.000). The combination of CTC, CEA, CA125 and CA153 can provide more effective breast cancer screening.ConclusionsThe CTCs analysis method presented here doesn''t rely on the specific antibody, such as anti-EpCAM, which would avoid the missed inspection caused by antibody-relied methods and offer more comprehensive biological information for clinical breast cancer diagnosis and treatment.     

Unique Characteristics and Outcomes of Patients Diagnosed With Both Primary Thyroid and Primary Renal Cell Carcinoma

Objective: Patients with multiple primary malignancies may exhibit unique clinical characteristics that suggest a common predisposition or lead to different disease management. Given the association of primary thyroid (TC) and renal cell carcinoma (RCC), we characterized the clinicopathologic features of patients treated for both malignancies (TC/RCC).Methods: TC/RCC patients were identified through the institutional tumor registry and using data compiled by retrospective chart review. To compare with broader institutional and national cohorts, we examined patients admitted with TC or RCC institution-wide and reviewed the National Cancer Institute's Surveillance, Epidemiology, and End Results (SEER) program for these cancers.Results: Overall, 51% of patients developed TC before RCC, 27% developed RCC before TC, and 22% were diagnosed within 1 year of each other. The mean age at TC diagnosis was 52 ± 15 (18–77), which was significantly older than institutional TC patients (45 ± 16.5 years, P≤.0001), and the mean age at RCC diagnosis was 59 ± 12 (32–79). The TC/RCC cohort had a balanced sex distribution (51% female) compared with the institutional TC group (67% female, P = .0003) and the institutional RCC group (31% female, P<.0001). Similar age and sex ratio differences were seen when compared with SEER cohorts. In the TC/RCC cohort, 43% of patients developed other cancers (52% of females, 33% of males; P = .04); among the females, 45% developed breast cancer.Conclusion: Individuals who develop both TC and RCC may represent a unique subset of cancer patients. Further prospective research is warranted to explore the unanticipated association with breast cancer in female patients and to investigate a possible common pathogenesis underlying these malignancies.Abbreviations: RCC = renal cell carcinoma SEER = Surveillance, Epidemiology, and End Results SPC = second primary cancer SPTC = subsequent primary thyroid cancer TC = thyroid cancer VHL = von Hippel-Lindau     

Mutational Analysis of Circulating Tumor Cells from Colorectal Cancer Patients and Correlation with Primary Tumor Tissue

Circulating tumor cells (CTCs) provide a non-invasive accessible source of tumor material from patients with cancer. The cellular heterogeneity within CTC populations is of great clinical importance regarding the increasing number of adjuvant treatment options for patients with metastatic carcinomas, in order to eliminate residual disease. Moreover, the molecular profiling of these rare cells might lead to insight on disease progression and therapeutic strategies than simple CTCs counting. In the present study we investigated the feasibility to detect KRAS, BRAF, CD133 and Plastin3 (PLS3) mutations in an enriched CTCs cell suspension from patients with colorectal cancer, with the hypothesis that these genes` mutations are of great importance regarding the generation of CTCs subpopulations. Subsequently, we compared CTCs mutational status with that of the corresponding primary tumor, in order to access the possibility of tumor cells characterization without biopsy. CTCs were detected and isolated from blood drawn from 52 colorectal cancer (CRC) patients using a quantum-dot-labelled magnetic immunoassay method. Mutations were detected by PCR-RFLP or allele-specific PCR and confirmed by direct sequencing. In 52 patients, discordance between primary tumor and CTCs was 5.77% for KRAS, 3.85% for BRAF, 11.54% for CD133 rs3130, 7.69% for CD133 rs2286455 and 11.54% for PLS3 rs6643869 mutations. Our results support that DNA mutational analysis of CTCs may enable non-invasive, specific biomarker diagnostics and expand the scope of personalized medicine for cancer patients.     

基于微纳技术的循环肿瘤细胞免疫检测新方法北大核心CSCD

本研究旨在探索一种高灵敏度、高特异性检测循环肿瘤细胞(circulating tumor cells, CTCs)的免疫检测新方法,以尽早地检出结直肠癌,提高该疾病的检出率。首先制备含有线性微柱结构的微芯片,通过在其表面孵育氧化石墨烯-链霉亲和素(graphite oxide-streptavidin, GO-SA)及偶联广谱一抗(antibody1, Ab₁),即上皮特异性黏附分子(epithelial cell adhesion molecule, EpCAM)单克隆抗体以捕获CTCs。运用羧基化多壁碳纳米管(carboxylated multi-walled carbon nanotubes, MWCNTs-COOH)与结直肠癌相关抗体,即特异性二抗(antibody 2, Ab₂)偶联制备抗体复合物。在捕获CTCs的微芯片上孵育该抗体复合物,构建以Ab₁-CTCs-Ab₂为主体的超级三明治结构,通过电化学工作站检测并验证其高灵敏度和高特异性。结果发现,在免疫传感器的构建中结合应用微纳技术,极大地提高了CTCs的检测灵敏度和特异性。本研究验证了该免疫传感器应用于临床血样检测的可行性,并通过该免疫传感器对结直肠癌患者外周血中CTCs进行检测和计数。结果表明,基于微纳技术的超级三明治式免疫传感器为CTCs的检测提供了新的途径,对临床工作中的疾病诊断及病情实时监控方面均具有潜在的应用价值。     

Clinical significance of circulating tumor cells after chemotherapy in unresectable pancreatic ductal adenocarcinoma

Circulating tumor cells (CTCs) have emerged as liquid biopsy biomarker providing non-invasive assessment of cancer progression and biology. We investigated whether longitudinal analysis of CTCs could monitor disease progression, response to chemotherapy, and survival in patients with unresectable pancreatic ductal adenocarcinoma (PDAC). A total of 52 patients with PDAC were prospectively enrolled in this study. Peripheral blood samples were serially collected at the time of diagnosis and after chemotherapy with clinical assessments. CTCs were isolated through a centrifugal microfluidic disc, enumerated with immunostaining against Epithelial cell adhesion molecule (EpCAM), Cytokeratin (CK), Plectin-1 and CD45, and identified by an automated imaging system. One or more CTCs were detected in 84.62% patients with unresectable PDAC at the time of diagnosis. CTC numbers were not statistically different across tumor sizes, location and metastatic sites. The absolute number of CTCs after chemotherapy was inversely related to overall survival (OS), and the decreased number of CTCs after chemotherapy was significantly associated with longer OS in patients with PDAC. Identifying CTCs and monitoring CTC changes after chemotherapy could be a useful prognostic marker for survival in patients with unresectable PDACs.     

Clinical Validation of an Ultra High-Throughput Spiral Microfluidics for the Detection and Enrichment of Viable Circulating Tumor Cells

Background

Circulating tumor cells (CTCs) are cancer cells that can be isolated via liquid biopsy from blood and can be phenotypically and genetically characterized to provide critical information for guiding cancer treatment. Current analysis of CTCs is hindered by the throughput, selectivity and specificity of devices or assays used in CTC detection and isolation.

Methodology/Principal Findings

Here, we enriched and characterized putative CTCs from blood samples of patients with both advanced stage metastatic breast and lung cancers using a novel multiplexed spiral microfluidic chip. This system detected putative CTCs under high sensitivity (100%, n = 56) (Breast cancer samples: 12–1275 CTCs/ml; Lung cancer samples: 10–1535 CTCs/ml) rapidly from clinically relevant blood volumes (7.5 ml under 5 min). Blood samples were completely separated into plasma, CTCs and PBMCs components and each fraction were characterized with immunophenotyping (Pan-cytokeratin/CD45, CD44/CD24, EpCAM), fluorescence in-situ hybridization (FISH) (EML4-ALK) or targeted somatic mutation analysis. We used an ultra-sensitive mass spectrometry based system to highlight the presence of an EGFR-activating mutation in both isolated CTCs and plasma cell-free DNA (cf-DNA), and demonstrate concordance with the original tumor-biopsy samples.

Conclusions/Significance

We have clinically validated our multiplexed microfluidic chip for the ultra high-throughput, low-cost and label-free enrichment of CTCs. Retrieved cells were unlabeled and viable, enabling potential propagation and real-time downstream analysis using next generation sequencing (NGS) or proteomic analysis.     

A novel metformin derivative,HL010183, inhibits proliferation and invasion of triple-negative breast cancer cells

Mounting evidence suggests that metformin (N,N-dimethylbiguanide), a widely prescribed drug for the treatment of type II diabetes, exerts an anti-tumor effect on several cancers including breast cancer. Breast cancer has been estimated as one of the most commonly diagnosed types of cancer among women. In particular, triple-negative breast cancers are associated with poor prognosis and metastatic growth. In the present study, we synthesized a novel metformin derivative 5 (HL010183) and metformin salts, 9a, 9b, and 9c (metformin gamma-aminobutyric acid (GABA) salt, metformin pregabalin salt and metformin gabapentin salt), which exerted more potent inhibitory effects on the proliferation and invasiveness of Hs578T triple-negative breast carcinoma cells than metformin. Importantly, 5 showed approximately 100-fold more potent effects compared to metformin. In a triple-negative breast cancer xenograft model, 5 showed a comparable degree of inhibitory effect on in vivo tumor growth at the 100 mg/kg dose to that of metformin at 500 mg/kg. Our results clearly demonstrate that 5 exerts a potent anti-tumor effect both in vitro and in vivo, paving the way for a strategy for treatment of triple-negative breast cancer.