Pan-tumor validation of a NGS fraction-based MSI analysis as a predictor of response to Pembrolizumab.

Microsatellite instability high (MSI-H) and mismatch repair deficient (dMMR) tumor status have been demonstrated to predict patient response to immunotherapies. We developed and validated a next-generation sequencing (NGS)-based companion diagnostic (CDx) to detect MSI-H solid tumors via a comprehensive genomic profiling (CGP) assay, FoundationOne®CDx (F1CDx). To determine MSI status, F1CDx calculates the fraction of unstable microsatellite loci across >2000 loci using a fraction-based (FB) analysis. Across solid tumor types, F1CDx demonstrated a high analytical concordance with both PCR (n = 264) and IHC (n = 279) with an overall percent agreement (OPA) of 97.7% and 97.8%, respectively. As part of a retrospective bridging clinical study from KEYNOTE-158 Cohort K and KEYNOTE-164, patients with MSI-H tumors as determined by F1CDx demonstrated an objective response rate (ORR) of 43.0% to pembrolizumab. In real-world cancer patients from a deidentified clinicogenomic database, F1CDx was at least equivalent in assessing clinical outcome following immunotherapy compared with MMR IHC. Demonstrated analytical and clinical performance of F1CDx led to the pan-tumor FDA approval in 2022 of F1CDx to identify MSI-H solid tumor patients for treatment with pembrolizumab. F1CDx is an accurate, reliable, and FDA-approved method for the identification of MSI-H tumors for treatment with pembrolizumab.

Clinical and analytical validation of FoundationOne®CDx, a comprehensive genomic profiling assay for solid tumors.

FoundationOne®CDx (F1CDx) is a United States (US) Food and Drug Administration (FDA)-approved companion diagnostic test to identify patients who may benefit from treatment in accordance with the approved therapeutic product labeling for 28 drug therapies. F1CDx utilizes next-generation sequencing (NGS)-based comprehensive genomic profiling (CGP) technology to examine 324 cancer genes in solid tumors. F1CDx reports known and likely pathogenic short variants (SVs), copy number alterations (CNAs), and select rearrangements, as well as complex biomarkers including tumor mutational burden (TMB) and microsatellite instability (MSI), in addition to genomic loss of heterozygosity (gLOH) in ovarian cancer. CGP services can reduce the complexity of biomarker testing, enabling precision medicine to improve treatment decision-making and outcomes for cancer patients, but only if test results are reliable, accurate, and validated clinically and analytically to the highest standard available. The analyses presented herein demonstrate the extensive analytical and clinical validation supporting the F1CDx initial and subsequent FDA approvals to ensure high sensitivity, specificity, and reliability of the data reported. The analytical validation included several in-depth evaluations of F1CDx assay performance including limit of detection (LoD), limit of blank (LoB), precision, and orthogonal concordance for SVs (including base substitutions [SUBs] and insertions/deletions [INDELs]), CNAs (including amplifications and homozygous deletions), genomic rearrangements, and select complex biomarkers. The assay validation of >30,000 test results comprises a considerable and increasing body of evidence that supports the clinical utility of F1CDx to match patients with solid tumors to targeted therapies or immunotherapies based on their tumor's genomic alterations and biomarkers. F1CDx meets the clinical needs of providers and patients to receive guideline-based biomarker testing, helping them keep pace with a rapidly evolving field of medicine.

Conductive polymer-based nanoparticles for laser-mediated photothermal ablation of cancer: synthesis, characterization, and in vitro evaluation.

Laser-mediated photothermal ablation of cancer cells aided by photothermal agents is a promising strategy for localized, externally controlled cancer treatment. We report the synthesis, characterization, and in vitro evaluation of conductive polymeric nanoparticles (CPNPs) of poly(diethyl-4,4'-{[2,5-bis(2,3-dihydrothieno[3,4-b][1,4]dioxin-5-yl)-1,4-phenylene] bis(oxy)}dibutanoate) (P1) and poly(3,4-ethylenedioxythiophene) (PEDOT) stabilized with 4-dodecylbenzenesulfonic acid and poly(4-styrenesulfonic acid-co-maleic acid) as photothermal ablation agents. The nanoparticles were prepared by oxidative-emulsion polymerization, yielding stable aqueous suspensions of spherical particles of <100 nm diameter as determined by dynamic light scattering and electron microscopy. Both types of nanoparticles show strong absorption of light in the near infrared region, with absorption peaks at 780 nm for P1 and 750 nm for PEDOT, as well as high photothermal conversion efficiencies (~50%), that is higher than commercially available gold-based photothermal ablation agents. The nanoparticles show significant photostability as determined by their ability to achieve consistent temperatures and to maintain their morphology upon repeated cycles of laser irradiation. In vitro studies in MDA-MB-231 breast cancer cells demonstrate the cytocompatibility of the CPNPs and their ability to mediate complete cancer cell ablation upon irradiation with an 808-nm laser, thereby establishing the potential of these systems as agents for laser-induced photothermal therapy.

Electroactive Polymer Nanoparticles Exhibiting Photothermal Properties.

A method for the synthesis of electroactive polymers is demonstrated, starting with the synthesis of extended conjugation monomers using a three-step process that finishes with Negishi coupling. Negishi coupling is a cross-coupling process in which a chemical precursor is first lithiated, followed by transmetallation with ZnCl2. The resultant organozinc compound can be coupled to a dibrominated aromatic precursor to give the conjugated monomer. Polymer films can be prepared via electropolymerization of the monomer and characterized using cyclic voltammetry and ultraviolet-visible-near infrared (UV-Vis-NIR) spectroscopy. Nanoparticles (NPs) are prepared via emulsion polymerization of the monomer using a two-surfactant system to yield an aqueous dispersion of the polymer NPs. The NPs are characterized using dynamic light scattering, electron microscopy, and UV-Vis-NIR-spectroscopy. Cytocompatibility of NPs is investigated using the cell viability assay. Finally, the NP suspensions are irradiated with a NIR laser to determine their effectiveness as potential materials for photothermal therapy (PTT).

Nanoparticle-mediated photothermal therapy: a comparative study of heating for different particle types.

INTRODUCTION: Near-infrared (NIR) absorbing plasmonic nanoparticles enhance photothermal therapy of tumors. In this procedure, systemically delivered gold nanoparticles preferentially accumulate at the tumor site and when irradiated using laser light, produce localized heat sufficient to damage tumor cells. Gold nanoshells and nanorods have been widely studied for this purpose, and while both exhibit strong NIR absorption, their overall absorption and scattering properties differ widely due to their geometry. In this paper, we compared the photothermal response of both nanoparticle types including the heat generation and photothermal efficiency. METHODS: Tissue simulating phantoms, with varying concentrations of gold nanoparticles, were irradiated with a near-infrared diode laser while concurrently monitoring the surface temperature with an infrared camera. We calculated nanoshell and nanorod optical properties using the Mie solution and the discrete dipole approximation, respectively. In addition, we measured the heat generation of nanoshells and nanorods at the same optical density to determine the photothermal transduction efficiency for both nanoparticle types. RESULTS: We found that the gold nanoshells produced more heat than gold nanorods at equivalent number densities (# of nanoparticles/ml), whereas the nanorods generated more heat than nanoshells at equivalent extinction values at the irradiance wavelength. To reach an equivalent heat generation, we found that it was necessary to have ∼36× more nanorods than nanoshells. However, the gold nanorods were found to have two times the photothermal transduction efficiency than the gold nanoshells. CONCLUSION: For the nanoparticles tested, the nanoshells generated more heat, per nanoparticle, than nanorods, primarily due to their overall larger geometric cross-section. Conversely, we found that the gold nanorods had a higher photothermal efficiency than the gold nanoshells. In conclusion, the ideal choice of plasmonic nanoparticle requires not only per particle efficiency, but also the in vivo particle targeting ability under study.

Copper selenide nanocrystals for photothermal therapy.

Ligand-stabilized copper selenide (Cu(2-x)Se) nanocrystals, approximately 16 nm in diameter, were synthesized by a colloidal hot injection method and coated with amphiphilic polymer. The nanocrystals readily disperse in water and exhibit strong near-infrared (NIR) optical absorption with a high molar extinction coefficient of 7.7 × 10(7) cm(-1) M(-1) at 980 nm. When excited with 800 nm light, the Cu(2-x)Se nanocrystals produce significant photothermal heating with a photothermal transduction efficiency of 22%, comparable to nanorods and nanoshells of gold (Au). In vitro photothermal heating of Cu(2-x)Se nanocrystals in the presence of human colorectal cancer cell (HCT-116) led to cell destruction after 5 min of laser irradiation at 33 W/cm(2), demonstrating the viabilitiy of Cu(2-x)Se nanocrystals for photothermal therapy applications.

Temperature-induced unfolding of epidermal growth factor (EGF): insight from molecular dynamics simulation.

Thermal disruption of protein structure and function is a potentially powerful therapeutic vehicle. With the emerging nanoparticle-targeting and femtosecond laser technology, it is possible to deliver heating locally to specific molecules. It is therefore important to understand how fast a protein can unfold or lose its function at high temperatures, such as near the water boiling point. In this study, the thermal damage of EGF was investigated by combining the replica exchange (136 replicas) and conventional molecular dynamics simulations. The REMD simulation was employed to rigorously explore the free-energy landscape of EGF unfolding. Interestingly, besides the native and unfolded states, we also observed a distinct molten globule (MG) state that retained substantial amount of native contacts. Based on the understanding that which the unfolding of EGF is a three-state process, we have examined the unfolding kinetics of EGF (N-->MG and MG-->D) with multiple 20-ns conventional MD simulations. The Arrhenius prefactors and activation energy barriers determined from the simulation are within the range of previously studied proteins. In contrast to the thermal damage of cells and tissues which take place on the time scale of seconds to hours at relatively low temperatures, the denaturation of proteins occur in nanoseconds when the temperature of heat bath approaches the boiling point.

Microcontact printing of quantum dot bioconjugate arrays for localized capture and detection of biomolecules.

The nanometer size scale of quantum dots (QDs) along with their unique luminescent properties offers great potential as photostable, color-metrically addressable nanoparticle platforms for high-throughput detection and identification of proteins. Here we apply microcontact printing for assembling quantum dot nanoparticle arrays with retained biomolecular capture functionality onto glass surfaces. This method allows the creation of addressable QD arrays on macroscopic glass surfaces. Using fluorescence and AFM imaging, we find that microcontact-printed QDs self-assemble predominantly as monolayers with highly resolved definition. Microcontact-printed streptavidin-conjugated red QDs exhibit retained adsorption onto silane-treated glass and exhibit functionality as demonstrated by the capture of discrete groups of biotin-conjugated red QDs by printed streptavidin-green QD bioconjugates that is at the detection limit of a few discrete protein binding events. These results indicate that microcontact printing of QD bioconjugate arrays serves as a simple technique that allows localized spatial capture and sensitive detection of proteins. This technique may be useful for development of future fluorescent QD-based systems aimed at the parallel capture and detection of trace concentrations of protein.