Androgen Receptor Splice Variant 7 in Asian Patients With Metastatic Castration-Resistant Prostate Cancer.

PURPOSE: Androgen receptor splice variant 7 (ARV-7) is a resistance mechanism to hormonal therapy in metastatic castrate-resistant prostate cancer (mCRPC). It has been associated with poor outcomes. On progression to castrate resistance, ARV-7 positivity has been identified in global populations at an incidence of 17.8%-28.8%. Here, we characterize the incidence of ARV-7 positivity in Asian patients with mCRPC in a prospective fashion and evaluate its implications on treatment outcomes. METHODS: Patients with mCRPC from multiple centers in Southeast and East Asia were enrolled in a prospective manner before initiation of androgen receptor signaling inhibitors or docetaxel. ARV-7 status was evaluated at baseline with three commercially available assays: AdnaTest Prostate Cancer platform, Clearbridge method, and IBN method. Clinical outcomes at progression were assessed. The primary end point of this study was prevalence of ARV-7 positivity; secondary end points were incidence of ARV-7 positivity, prostate specific antigen (PSA) response rate, PSA progression-free survival (PFS), and overall survival (OS). RESULTS: A total of 102 patients with a median age of 72 years at enrollment participated. Overall, an incidence of ARV-7 positivity of between 14.3% and 33.7% in Asian patients with mCRPC was demonstrated depending on the assay used. Patients found to have ARV-7 positivity at enrollment had a numerically worse PSA PFS compared with ARV-7 negative patients. CONCLUSION: In this study, the incidence of ARV-7 positivity in Asian patients with mCRPC was shown to be similar to the global population. Patients with ARV-7 positivity appear to have more aggressive disease with numerically worse PSA PFS and OS. Further prospective studies are needed to fully characterize the relationship that ARV-7 positivity has on prognosis of Asian patients with mCRPC.

Metered-Dose Inhaler Spacer with Integrated Spirometer for Home-Based Asthma Monitoring and Drug Uptake.

This work introduces Spiromni, a single device incorporating three different pressurised metered-dose inhaler (pMDI) accessories: a pMDI spacer, an electronic monitoring device (EMD), and a spirometer. While there are devices made to individually address the issues of technique, adherence and monitoring, respectively, for asthma patients as laid out in the Global Initiative for Asthma's (GINA) global strategy for asthma management and prevention, Spiromni was designed to address all three issues using a single, combination device. Spiromni addresses the key challenge of measuring both inhalation and exhalation profiles, which are different by an order of magnitude. Moreover, the innovative design prevents exhalation from entering the spacer chamber and prevents medication loss during inhalation using umbrella valves without a loss in flow velocity. Apart from recording the peak exhalation flow rate, data from the sensors allow us to extract other key lung volume and capacities measures similar to a medical pulmonary function test. We believe this low-cost portable multi-functional device will benefit both asthma patients and clinicians in the management of the disease.

A Sensorised Glove to Detect Scratching for Patients with Atopic Dermatitis.

In this work, a lightweight compliant glove that detects scratching using data from microtubular stretchable sensors on each finger and an inertial measurement unit (IMU) on the palm through a machine learning model is presented: the SensorIsed Glove for Monitoring Atopic Dermatitis (SIGMA). SIGMA provides the user and clinicians with a quantifiable way of assaying scratch as a proxy to itch. With the quantitative information detailing scratching frequency and duration, the clinicians would be able to better classify the severity of itch and scratching caused by atopic dermatitis (AD) more objectively to optimise treatment for the patients, as opposed to the current subjective methods of assessments that are currently in use in hospitals and research settings. The validation data demonstrated an accuracy of 83% of the scratch prediction algorithm, while a separate 30 min validation trial had an accuracy of 99% in a controlled environment. In a pilot study with children (n = 6), SIGMA accurately detected 94.4% of scratching when the glove was donned. We believe that this simple device will empower dermatologists to more effectively measure and quantify itching and scratching in AD, and guide personalised treatment decisions.

Marker-free characterization of full-length transcriptomes of single live circulating tumor cells.

The identification and characterization of circulating tumor cells (CTCs) are important for gaining insights into the biology of metastatic cancers, monitoring disease progression, and medical management of the disease. The limiting factor in the enrichment of purified CTC populations is their sparse availability, heterogeneity, and altered phenotypes relative to the primary tumor. Intensive research both at the technical and molecular fronts led to the development of assays that ease CTC detection and identification from peripheral blood. Most CTC detection methods based on single-cell RNA sequencing (scRNA-seq) use a mix of size selection, marker-based white blood cell (WBC) depletion, and antibodies targeting tumor-associated antigens. However, the majority of these methods either miss out on atypical CTCs or suffer from WBC contamination. We present unCTC, an R package for unbiased identification and characterization of CTCs from single-cell transcriptomic data. unCTC features many standard and novel computational and statistical modules for various analyses. These include a novel method of scRNA-seq clustering, named deep dictionary learning using k-means clustering cost (DDLK), expression-based copy number variation (CNV) inference, and combinatorial, marker-based verification of the malignant phenotypes. DDLK enables robust segregation of CTCs and WBCs in the pathway space, as opposed to the gene expression space. We validated the utility of unCTC on scRNA-seq profiles of breast CTCs from six patients, captured and profiled using an integrated ClearCell FX and Polaris workflow that works by the principles of size-based separation of CTCs and marker-based WBC depletion.

Wearable Soft Microtube Sensors for Quantitative Home-Based Erectile Dysfunction Monitoring.

Quantifiable erectile dysfunction (ED) diagnosis involves the monitoring of rigidity and tumescence of the penile shaft during nocturnal penile tumescence (NPT). In this work, we introduce Erectile Dysfunction SENsor (EDSEN), a home-based wearable device for quantitative penile health monitoring based on stretchable microtubular sensing technology. Two types of sensors, the T- and R-sensors, are developed to effectively measure penile tumescence and rigidity, respectively. Conical models mimicking penile shaft were fabricated with polydimethylsiloxane (PDMS) material, using different base to curing agent ratios to replicate the different hardness properties of a penile shaft. A theoretical buckling force chart for the different penile models is generated to determine sufficiency criteria for sexual intercourse. An average erect penile length and circumference requires at least a Young's modulus of 179 kPa for optimal buckling force required for satisfactory sexual intercourse. The conical penile models were evaluated using EDSEN. Our results verified that the circumference of a penile shaft can be accurately measured by T-sensor and rigidity using the R-sensor. EDSEN provides a private and quantitative method to detect ED within the comfortable confines of the user's home.

Hybrid double-spiral microfluidic chip for RBC-lysis-free enrichment of rare cells from whole blood.

Drug selection and treatment monitoring via minimally invasive liquid biopsy using circulating tumor cells (CTCs) are expected to be realized in the near future. For clinical applications of CTCs, simple, high-throughput, single-step CTC isolation from whole blood without red blood cell (RBC) lysis and centrifugation remains a crucial challenge. In this study, we developed a novel cancer cell separation chip, "hybrid double-spiral chip", that involves the serial combination of two different Dean flow fractionation (DFF) separation modes of half and full Dean cycles, which is the hybrid DFF separation mode for ultra-high-throughput blood processing at high precision and size-resolution separation. The chip allows fast processing of 5 mL whole blood within 30 min without RBC lysis and centrifugation. RBC and white blood cell (WBC) depletion rates of over 99.9% and 99%, respectively, were achieved. The average recovery rate of spiked A549 cancer cells was 87% with as low as 200 cells in 5 mL blood. The device can achieve serial reduction in the number of cells from approximately 1010 cells of whole blood to 108 cells, and subsequently to an order of 106 cells. The developed method can be combined with measurements of all recovered cells using imaging flow cytometry. As proof of concept, CTCs were successfully enriched and enumerated from the blood of metastatic breast cancer patients (N = 10, 1-69 CTCs per 5 mL) and metastatic prostate cancer patients (N = 10, 1-39 CTCs per 5 mL). We believe that the developed method will be beneficial for automated clinical analysis of rare CTCs from whole blood.

Modular micro-PCR system for the onsite rapid diagnosis of COVID-19.

Effective containment of the COVID-19 pandemic requires rapid and accurate detection of the pathogen. Polymerase chain reaction (PCR) remains the gold standard for COVID-19 confirmation. In this article, we report the performance of a cost-effective modular microfluidic reverse transcription (RT)-PCR and RT-loop mediated isothermal amplification (RT-LAMP) platform, Epidax®, for the point-of-care testing and confirmation of SARS-CoV-2. This platform is versatile and can be reconfigured either for screening using endpoint RT-PCR or RT-LAMP tests or for confirmatory tests using real-time RT-PCR. Epidax® is highly sensitive and detects as little as 1 RNA copy per µL for real-time and endpoint RT-PCR, while using only half of the reagents. We achieved comparable results with those of a commercial platform when detecting SARS-CoV-2 viruses from 81 clinical RNA extracts. Epidax® can also detect SARS-CoV-2 from 44 nasopharyngeal samples without RNA extraction by using a direct RT-PCR assay, which shortens the sample-to-answer time to an hour with minimal user steps. Furthermore, we validated the technology using an RT-LAMP assay on 54 clinical RNA extracts. Overall, our platform provides a sensitive, cost-effective, and accurate diagnostic solution for low-resource settings.

Erratum: Iyer, A., et al. Integrative Analysis and Machine Learning Based Characterization of Single Circulating Tumor Cells. J. Clin. Med. 2020, 9, 1206.

In the published manuscript [...].

Integrative Analysis and Machine Learning based Characterization of Single Circulating Tumor Cells.

We collated publicly available single-cell expression profiles of circulating tumor cells (CTCs) and showed that CTCs across cancers lie on a near-perfect continuum of epithelial to mesenchymal (EMT) transition. Integrative analysis of CTC transcriptomes also highlighted the inverse gene expression pattern between PD-L1 and MHC, which is implicated in cancer immunotherapy. We used the CTCs expression profiles in tandem with publicly available peripheral blood mononuclear cell (PBMC) transcriptomes to train a classifier that accurately recognizes CTCs of diverse phenotype. Further, we used this classifier to validate circulating breast tumor cells captured using a newly developed microfluidic system for label-free enrichment of CTCs.

Prospective Molecular Profiling of Circulating Tumor Cells from Patients with Melanoma Receiving Combinatorial Immunotherapy.

BACKGROUND: Blood molecular profiling of circulating tumor cells (CTCs) can enable monitoring of patients with metastatic melanoma during checkpoint inhibitor immunotherapy (CII) and in combination with targeted therapies. We developed a microfluidics-based CTC platform to explore CTC profiling utility in CII-treated patients with melanoma using a melanoma messenger RNA (mRNA)/DNA biomarker panel. METHODS: Blood samples (n = 213) were collected prospectively from 75 American Joint Committee on Cancer-staged III/IV melanoma patients during CII treatment and those enriched for CTCs. CTC profiling was performed using 5 known melanoma mRNA biomarkers and BRAF V600E DNA mutation. CTC biomarker status associations with clinical outcomes were assessed. RESULTS: CTCs were detected in 88% of blood samples from patients with melanoma. CTC-derived biomarkers and clinical variables analyzed using classification and regression tree analysis revealed that a combination of lactate dehydrogenase, CTC-mRNA biomarkers, and tumor BRAF-mutation status was indicative of clinical outcomes for patients with stage IV melanoma (n = 52). The panel stratified low-risk and high-risk patients, whereby the latter had poor disease-free (P = 0.03) and overall survival (P = 0.02). Incorporation of a DNA biomarker with mRNA profiling increased overall CTC-detection capability by 57% compared to mRNA profiling only. RNA sequencing of isolated CTCs identified significant catenin beta 1 (CTNNB1) overexpression (P <0.01) compared to nondisease donor blood. CTC-CTNNB1 was associated with progressive disease/stable disease compared to complete-responder patient status (P = 0.02). Serial CTC profiling identified subclinical disease in patients who developed progressive disease during treatment/follow-up. CONCLUSIONS: CTC-derived mRNA/DNA biomarkers have utility for monitoring CII, targeted, and combinatorial therapies in metastatic melanoma patients.

Detection and prognostic relevance of circulating tumour cells (CTCs) in Asian breast cancers using a label-free microfluidic platform.

OBJECTIVES: We aimed to study the prevalence of CTCs in breast cancer (BC) patients undergoing neoadjuvant or palliative therapy with a label-free microfluidic platform (ClearCell FX), and its prognostic relevance in metastatic BC (mBC). MATERIALS AND METHODS: Peripheral blood samples were collected from 108 BC patients before starting a new line of treatment ("baseline"), majority of whom had mBC (76/108; 70.4%). CTCs were retrieved by dean flow fractionation that enriched for larger cells, and enumerated using immunofluorescence-based staining. Progression-free survival (PFS) in mBC patients was analysed using Kaplan-Meier method; cox proportional hazard models were used for univariable and multivariable analyses. RESULTS: The detection rate of CTCs before starting a new line of treatment was 75.9% (n = 108; median: 8 CTCs/7.5 ml blood) at a cut off of ≥2 CTCs. PFS was inferior for mBC patients with baseline CTC count ≥5 CTCs/7.5 ml blood vs. those with < 5 CTCs/7.5 ml blood (median PFS: 4.3 vs. 7.0 months; p-value: 0.037). The prognostic relevance of CTCs was most significant in patients with HER2- mBC (median PFS: 4.1 vs. 8.3 months; p-value: 0.032), luminal (HR+HER2-) subtype (median PFS: 4.2 vs. 8.3 months; p-value: 0.048), and patients who had one or more prior treatments (median PFS: 4.2 vs. 7.0 months; p-value: 0.02). On multivariable analysis, baseline CTC level (hazard ratio (HR): 1.84, p-value: 0.02) and pre-treatment status (HR: 1.87, p-value: 0.05) were independent predictors of PFS. CONCLUSIONS: This work demonstrates the prognostic significance of CTCs in mBC detected using a label-free size-based enrichment platform.

Addressing cellular heterogeneity in tumor and circulation for refined prognostication.

Despite pronounced genomic and transcriptomic heterogeneity in non-small-cell lung cancer (NSCLC) not only between tumors, but also within a tumor, validation of clinically relevant gene signatures for prognostication has relied upon single-tissue samples, including 2 commercially available multigene tests (MGTs). Here we report an unanticipated impact of intratumor heterogeneity (ITH) on risk prediction of recurrence in NSCLC, underscoring the need for a better genomic strategy to refine prognostication. By leveraging label-free, inertial-focusing microfluidic approaches in retrieving circulating tumor cells (CTCs) at single-cell resolution, we further identified specific gene signatures with distinct expression profiles in CTCs from patients with differing metastatic potential. Notably, a refined prognostic risk model that reconciles the level of ITH and CTC-derived gene expression data outperformed the initial classifier in predicting recurrence-free survival (RFS). We propose tailored approaches to providing reliable risk estimates while accounting for ITH-driven variance in NSCLC.

ClearCell® FX, a label-free microfluidics technology for enrichment of viable circulating tumor cells.

Circulating tumor cells (CTCs) dissociate from primary tumor into the bloodstream, and carry with them cancer's fingerprints as well as the potential to turn aggressive and metastasize. In order to understand CTCs and develop clinical utility, different methods of enrichment and isolation of CTCs can be used. Here, we report the use of a label-free platform, ClearCell® FX which isolates CTCs by their mechanical features and its advantages. The technology utilizes Dean Flow Fractionation (DFF) principle in a spiral microfluidics system to separate the larger CTCs from smaller blood cells. The gentle and fast workflow allows for a range of downstream assays to be performed on the intact CTCs, particularly studies that examine an epithelial cell adhesion molecular (EpCAM)-independent population. Viable, intact cells are also retrievable for development of culture or in vivo models. © 2018 International Society for Advancement of Cytometry.

A preliminary study for the assessment of PD-L1 and PD-L2 on circulating tumor cells by microfluidic-based chipcytometry.

AIM: Expression of PD-L1 in the tumor is associated with more favorable responses to anti-PD-1 therapy in multiple cancers. However, obtaining tumor biopsies for PD-L1 interrogation is an invasive procedure and challenging to assess repeatedly as the disease progresses. MATERIALS & METHODS: Here we assess an alternative, minimally invasive approach to analyze blood samples for circulating tumor cells (CTCs) that have broken away from the tumor and entered the periphery. Our approach uses sized-based microfluidic CTC enrichment and subsequent characterization with microfluidic-based cytometry (chipcytometry). CONCLUSION: We demonstrate tumor-cell detection and characterization for PD-L1, and other markers, in both spiked and patient samples. This preliminary communication is the first report using chipcytometry for the characterization of CTCs.

Ultra-fast, label-free isolation of circulating tumor cells from blood using spiral microfluidics.

Circulating tumor cells (CTCs) are rare cancer cells that are shed from primary or metastatic tumors into the peripheral blood circulation. Phenotypic and genetic characterization of these rare cells can provide important information to guide cancer staging and treatment, and thus further research into their characteristics and properties is an area of considerable interest. In this protocol, we describe detailed procedures for the production and use of a label-free spiral microfluidic device to allow size-based isolation of viable CTCs using hydrodynamic forces that are present in curvilinear microchannels. This spiral system enables us to achieve ≥ 85% recovery of spiked cells across multiple cancer cell lines and 99.99% depletion of white blood cells in whole blood. The described spiral microfluidic devices can be produced at an extremely low cost using standard microfabrication and soft lithography techniques (2-3 d), and they can be operated using two syringe pumps for lysed blood samples (7.5 ml in 12.5 min for a three-layered multiplexed chip). The fast processing time and the ability to collect CTCs from a large patient blood volume allows this technique to be used experimentally in a broad range of potential genomic and transcriptomic applications.

Micromagnetic resonance relaxometry for rapid label-free malaria diagnosis.

We report a new technique for sensitive, quantitative and rapid detection of Plasmodium spp.-infected red blood cells (RBCs) by means of magnetic resonance relaxometry (MRR). During the intraerythrocytic cycle, malaria parasites metabolize large amounts of cellular hemoglobin and convert it into hemozoin crystallites. We exploit the relatively large paramagnetic susceptibility of these hemozoin particles, which induce substantial changes in the transverse relaxation rate of proton nuclear magnetic resonance of RBCs, to infer the 'parasite load' in blood. Using an inexpensive benchtop 0.5-Tesla MRR system, we show that with minimal sample preparatory steps and without any chemical or immunolabeling, a parasitemia level of fewer than ten parasites per microliter in a volume below 10 µl of whole blood is detected in a few minutes. We demonstrate this method both for cultured Plasmodium falciparum parasites and in vivo with Plasmodium berghei-infected mice.

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.

Single cell kinase signaling assay using pinched flow coupled droplet microfluidics.

Droplet-based microfluidics has shown potential in high throughput single cell assays by encapsulating individual cells in water-in-oil emulsions. Ordering cells in a micro-channel is necessary to encapsulate individual cells into droplets further enhancing the assay efficiency. This is typically limited due to the difficulty of preparing high-density cell solutions and maintaining them without cell aggregation in long channels (>5 cm). In this study, we developed a short pinched flow channel (5 mm) to separate cell aggregates and to form a uniform cell distribution in a droplet-generating platform that encapsulated single cells with >55% encapsulation efficiency beating Poisson encapsulation statistics. Using this platform and commercially available Sox substrates (8-hydroxy-5-(N,N-dimethylsulfonamido)-2-methylquinoline), we have demonstrated a high throughput dynamic single cell signaling assay to measure the activity of receptor tyrosine kinases (RTKs) in lung cancer cells triggered by cell surface ligand binding. The phosphorylation of the substrates resulted in fluorescent emission, showing a sigmoidal increase over a 12 h period. The result exhibited a heterogeneous signaling rate in individual cells and showed various levels of drug resistance when treated with the tyrosine kinase inhibitor, gefitinib.

An ultra-high-throughput spiral microfluidic biochip for the enrichment of circulating tumor cells.

The detection and characterization of rare circulating tumor cells (CTCs) from the blood of cancer patients can potentially provide critical insights into tumor biology and hold great promise for cancer management. The ability to collect a large number of viable CTCs for various downstream assays such as quantitative measurements of specific biomarkers or targeted somatic mutation analysis is increasingly important in medical oncology. Here, we present a simple yet reliable microfluidic device for the ultra-high-throughput, label-free, size-based isolation of CTCs from clinically relevant blood volumes. The fast processing time of the technique (7.5 mL blood in less than 10 min) and the ability to collect more CTCs from larger blood volumes lends itself to a broad range of potential genomic and transcriptomic applications. A critical advantage of this protocol is the ability to return all fractions of blood (i.e., plasma (centrifugation), CTCs and white blood cells (WBCs) (size-based sorting)) that can be utilized for diverse biomarker studies or time-sensitive molecular assays such as RT-PCR. The clinical use of this biochip was demonstrated by detecting CTCs from 100% (10/10) of blood samples collected from patients with advanced-stage metastatic breast and lung cancers. The CTC recovery rate ranged from 20 to 135 CTCs mL(-1) and obtained under high purity (of 1 CTC out of every 30-100 WBCs which gives ∼4 log depletion of WBCs). They were identified with immunofluorescence assays (pan-cytokeratin+/CD45-) and molecular probes such as HER2/neu.

Slanted spiral microfluidics for the ultra-fast, label-free isolation of circulating tumor cells.

The enumeration and characterization of circulating tumor cells (CTCs), found in the peripheral blood of cancer patients, provide a potentially accessible source for cancer diagnosis and prognosis. This work reports on a novel spiral microfluidic device with a trapezoidal cross-section for ultra-fast, label-free enrichment of CTCs from clinically relevant blood volumes. The technique utilizes the inherent Dean vortex flows present in curvilinear microchannels under continuous flow, along with inertial lift forces which focus larger CTCs against the inner wall. Using a trapezoidal cross-section as opposed to a traditional rectangular cross-section, the position of the Dean vortex core can be altered to achieve separation. Smaller hematologic components are trapped in the Dean vortices skewed towards the outer channel walls and eventually removed at the outer outlet, while the larger CTCs equilibrate near the inner channel wall and are collected from the inner outlet. By using a single spiral microchannel with one inlet and two outlets, we have successfully isolated and recovered more than 80% of the tested cancer cell line cells (MCF-7, T24 and MDA-MB-231) spiked in 7.5 mL of blood within 8 min with extremely high purity (400-680 WBCs mL(-1); ~4 log depletion of WBCs). Putative CTCs were detected and isolated from 100% of the patient samples (n = 10) with advanced stage metastatic breast and lung cancer using standard biomarkers (CK, CD45 and DAPI) with the frequencies ranging from 3-125 CTCs mL(-1). We expect this simple and elegant approach can surmount the shortcomings of traditional affinity-based CTC isolation techniques as well as enable fundamental studies on CTCs to guide treatment and enhance patient care.