The modified FACS calcein AM retention assay: a high throughput flow cytometer based method to measure cytotoxicity.

Current methods to determine cellular cytotoxicity in vitro are hampered by background signals that are caused by auto-fluorescent target and effector cells and by non-specific cell death. We combined and adjusted existing cell viability assays to develop a method that allows for highly reproducible, accurate, single cell analysis by high throughput FACS, in which non-specific cell death is corrected for. In this assay the number of living, calcein AM labeled cells that are green fluorescent are quantified by adding a fixed number of unlabeled calibration beads to the analysis. Using this modified FACS calcein AM retention method, we found EC50 values to be highly reproducible and considerably lower compared to EC50 values obtained by conventional assays, displaying the high sensitivity of this assay.
Copyright © 2015. Published by Elsevier B.V.

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Whole Brain Radiotherapy and RRx-001: Two Partial Responses in Radioresistant Melanoma Brain Metastases from a Phase I/II Clinical Trial: A TITE-CRM Phase I/II Clinical Trial.

Kim et al. report two patients with melanoma metastases to the brain that responded to treatment with RRx-001 and whole brain radiotherapy (WBRT) without neurologic or systemic toxicity in the context of a phase I/II clinical trial. RRx-001 is an reactive oxygen and reactive nitrogen species (ROS/RNS)-dependent systemically nontoxic hypoxic cell radiosensitizer with vascular normalizing properties under investigation in patients with various solid tumors including those with brain metastases.
Metastatic melanoma to the brain is historically associated with poor outcomes and a median survival of 4 to 5 months. WBRT is a mainstay of treatment for patients with multiple brain metastases, but no significant therapeutic advances for these patients have been described in the literature. To date, candidate radiosensitizing agents have failed to demonstrate a survival benefit in patients with brain metastases, and in particular, no agent has demonstrated improved outcome in patients with metastatic melanoma. Kim et al. report two patients with melanoma metastases to the brain that responded to treatment with novel radiosensitizing agent RRx-001 and WBRT without neurologic or systemic toxicity in the context of a phase I/II clinical trial.
Published by Elsevier Inc.

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Adaptive Biotechnologies and Collaborators to Present 10 Studies Demonstrating the Utility of Immunosequencing in Both Tissue and Blood Samples at 2016 American Association of Cancer Research Annual Meeting

On April 16, 2016 Adaptive Biotechnologies, the leader in combining next generation sequencing (NGS) and expert bioinformatics to profile T- and B-cell receptors of the adaptive immune system, along with its collaborators from institutions around the world, will present data demonstrating how Adaptive’s immunosequencing platform can be used as a novel oncology diagnostic to accurately and reliably quantify the density and clonality of Tumor Infiltrating Lymphocytes (TILs) to assess disease prognosis and response to therapy (Press release, Adaptive Biotechnologies, APR 16, 2016, View Source [SID:1234511008]). Two oral presentations and eight posters will be presented at the American Association for Cancer Research (AACR) (Free AACR Whitepaper) Annual Meeting, April 16-20, 2016, in New Orleans, Louisiana.

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Notably, many of the data to be presented explore the relevance of the immune repertoire in the blood as well as in the tumor tissue. The potential to identify blood-based immune molecular biomarkers of response to the growing class of immunomodulatory drugs may offer clinicians a more accessible sample source for widespread benefit to patients across many tumor types.

Select data presentations of interest include:

Dr. Robert Prins, et al. from UCLA will present data showing that in patients with glioblastoma undergoing immunotherapy, immunosequencing identified a potential biomarker of response and overall survival (Abstract 767).
Dr. Padmanee Sharma, et al. from MD Anderson Cancer Center will present data from a prostate cancer patient cohort receiving ipilumumab and androgen deprivation therapy correlating immune profiling data with adverse events which led to discovery of a potential predictive biomarker of patients who are at risk of grade 3 toxicities. (Abstract 1402).
Dr. Rebecca Gardner, et al. from Seattle Children’s Research Institute will present data on children with acute lymphoblastic leukemia (ALL) treated with a CART-19 therapy showing that high-throughput sequencing of patient samples had significantly greater sensitivity in detecting minimal residual disease (MRD) compared with samples analyzed by flow cytometry. Furthermore, in these patients, monitoring disease status by peripheral blood sampling is often more informative and far less invasive than bone marrow sampling. (Abstract 4893).
"These data being presented at AACR (Free AACR Whitepaper) demonstrate the utility of immunosequencing in both tissue and blood samples as a critical component in guiding clinicians’ approach to managing and treating cancer," said Harlan Robins, Chief Scientific Officer and Co-Founder of Adaptive Biotechnologies. "Ultimately incorporating immunosequencing into a potential blood-based biomarker for use in the clinic may help enhance the diagnosis, prognosis and monitoring of disease in cancer patients."

Representatives from Adaptive Biotechnologies will be exhibiting at AACR (Free AACR Whitepaper) booth #2530 to answer questions about their proprietary, transformative immunosequencing technology.

Oral Presentations:
Abstract #847: Molecular characterization of breast tumor T-cell infiltration in exome datasets
Date and Time: Sunday, Apr 17, 2016, 4:35 PM – 4:50 PM
Location: Room 243, Morial Convention Center
Presenter: Ricardo Armisen, Universidad de Chile, Santiago, Chile

Abstract #4362: T cell repertoire diversification is associated with immune related toxicities following immune checkpoint inhibition in metastatic cancer patients
Date and Time: Tuesday, Apr 19, 2016, 3:50 PM – 4:05 PM
Location: New Orleans Theater C, Morial Convention Center
Presenter: Lawrence Fong, University of California, San Francisco

Posters:

Abstract #767: TCR sequencing can identify and track tumor-specific T cell populations and is a predictive biomarker of response to DC vaccination in glioblastoma patients
Date and Time: Sunday, Apr 17, 2016, 1:00 PM – 5:00 PM
Location: Section 33, Poster Board #26
Author: Robert Prins, et al., University of California, Los Angeles

Abstract #1402: Exploratory biomarkers that predict for clinical outcomes in a Phase II trial with ipilimumab plus finite androgen deprivation therapy for metastatic non-castrate prostate cancer
Date and Time: Monday, Apr 18, 2016, 8:00 AM -12:00 PM
Location: Section 22, Poster Board #3
Author: Padmanee Sharma, et al., The University of Texas MD Anderson Cancer Center

Abstract #1405: Vesigenurtacel-L stimulates tumor infiltration of unique polyclonal T cell clones in non-muscle invasive bladder cancer patients
Date and Time: Monday, Apr 18, 2016, 8:00 AM -12:00 PM
Location: Section 22, Poster Board #6
Author: Melissa Price, et al., Heat Biologics, Inc., Durham, NC

Abstract #2392: Genomic and immune heterogeneity in synchronous melanoma metastases is associated with differential tumor growth and response to therapy
Date and Time: Monday, Apr 18, 2016, 1:00 PM -5:00 PM
Location: Section 28, Poster Board #22
Author: Jennifer A. Wargo, et al., MD Anderson Cancer Center, Houston, TX

Abstract #4159: Characterization of the T-cell receptor (TCR) repertoire in extensive disease small cell lung cancer (ED SCLC)
Date and Time: Tuesday, Apr 19, 2016, 1:00 PM – 5:00 PM
Location: Section 31, Poster board #26
Author: Cory Batenchuk, et al., Bristol-Myers Squibb, NJ

Abstract #4903: Multimodal therapy with a potent vaccine, metronomic cyclophosphamide and anti-PD-1 enhances immunotherapy of advanced tumors by increasing activation and clonal expansion of tumor infiltrating T cells
Date and Time: Wednesday, Apr 20, 2016, 7:30 AM – 11:00 AM
Location: Section 23, Poster board #14
Author: Genevieve Weir, et al., Immunovaccine, Inc., Halifax, NS, Canada

Abstract #4893: Molecular detection of ALL in the peripheral blood is more sensitive than flow cytometric analysis of the bone marrow in patients with treatment-related hypocellularity
Date and Time: Wednesday, Apr 20, 2016, 8:00 AM -12:00 PM
Location: Section 23, Poster board #4
Author: Rebecca Gardner, et al., Seattle Children’s Research Institute, Seattle, WA

Abstract #4897: First evidence of changes in the TCRβ repertoire from a cutaneous melanoma patient immunized with the CSF-470 vaccine.
Date and Time: Wednesday, Apr 20, 2016, 8:00 AM -12:00 PM
Location: Section 23, Poster board #8
Author: José Mordoh, et al., Centro de Investigaciones Oncológicas-Fundación Cáncer, Ciudad Autónoma de Buenos Aires, Argentina

About the immunoSEQ Platform
Adaptive’s immunoSEQ Platform helps researchers make discoveries in areas such as oncology, autoimmune disorders, infectious diseases and basic immunology. The immunoSEQ Assays can identify millions of T- and B-cell receptors from a single sample in exquisite detail. Offered as a Service or Kit, immunoSEQ Assays provide quantitative, reproducible sequencing results along with access to powerful, easy-to-use analysis tools. The immunoSEQ Assays are for research use only and are not for use in diagnostic procedures.

About Minimal Residual Disease
Minimal residual disease (MRD) refers to cancer cells that may remain in the body of a person with lymphoid cancer after treatment. These cells are present at levels undetectable by traditional microscopic examination (also called morphologic examination) of blood, bone marrow or a lymph node biopsy. Sensitive molecular technologies, such as the next-generation sequencing utilized by Adaptive’s clonoSEQ MRD Test, are needed for reliable detection of very low levels of MRD. Learn more at knowMRD.com.

About the clonoSEQ Process
Adaptive’s clonoSEQ Process enables physicians to utilize sequencing-based minimal residual disease (MRD) detection as an aid to clinical decision making for patients with lymphoid cancers (blood cancers). With its ability to detect cancer cells at a level as low as one per one million white blood cells, the clonoSEQ MRD Test is one to two orders of magnitude more sensitive than the other methods of MRD detection, such as ASO-PCR and flow cytometry.

Functional Characterization of D9, a Novel Deazaneplanocin A (DZNep) Analog, in Targeting Acute Myeloid Leukemia (AML).

Aberrant epigenetic events contribute to tumorigenesis of all human cancers. Significant efforts are underway in developing new generation of epigenetic cancer therapeutics. Although clinical trials for agents targeting DNA hypermethylation and histone deacetylation have yielded promising results, developing agents that target histone methylation remains to be in the early stage. We and others have previously reported that 3-Deazaneplanocin A (DZNep) is a histone methylation inhibitor that has a wide range of anticancer effects in various human cancers. Here, focusing on acute myeloid leukemia (AML) as a model, we reported a less toxic analog of DZNep, named D9, which is shown to be efficacious in AML cell lines and patient-derived samples in vitro, as well as AML tumorigenesis in vivo. Gene expression analysis in a panel of AML cell lines treated with D9 identified a set of genes that is associated with D9 sensitivity and implicated in multiple oncogenic signaling pathways. Moreover, we show that D9 is able to deplete the leukemia stem cells (LSC) and abolish chemotherapy-induced LSC enrichment, leading to dramatic elimination of AML cell survival. Thus, D9 appears to be a robust epigenetic compound that may constitute a potential for AML therapy.

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Bioorthogonal Turn-On Probe Based on Aggregation-Induced Emission Characteristics for Cancer Cell Imaging and Ablation.

Bioorthogonal turn-on probes have been widely utilized in visualizing various biological processes. Most of the currently available bioorthogonal turn-on probes are blue or green emissive fluorophores with azide or tetrazine as functional groups. Herein, we present an alternative strategy of designing bioorthogonal turn-on probes based on red-emissive fluorogens with aggregation-induced emission characteristics (AIEgens). The probe is water soluble and non-fluorescent due to the dissipation of energy through free molecular motion of the AIEgen, but the fluorescence is immediately turned on upon click reaction with azide-functionalized glycans on cancer cell surface. The fluorescence turn-on is ascribed to the restriction of molecular motion of AIEgen, which populates the radiative decay channel. Moreover, the AIEgen can generate reactive oxygen species (ROS) upon visible light (λ=400-700 nm) irradiation, demonstrating its dual role as an imaging and phototherapeutic agent.
© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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