Circulating biomarkers predictive of tumor response to cancer immunotherapy

Introduction: The advent of checkpoint blockade immunotherapy has revolutionized cancer treatment, but clinical response to immunotherapies is highly heterogeneous among individual patients and between cancer types. This represents a challenge to oncologists when choosing specific immunotherapies for personalized medicine. Thus, biomarkers that can predict tumor responsiveness to immunotherapies before and during treatment are invaluable. Areas covered: We review the latest advances in 'liquid biopsy' biomarkers for noninvasive prediction and in-treatment monitoring of tumor response to immunotherapy, focusing primarily on melanoma and non-small cell lung cancer. We concentrate on high-quality studies published within the last five years on checkpoint blockade immunotherapies, and highlight significant breakthroughs, identify key areas for improvement, and provide recommendations for how these diagnostic tools can be translated into clinical practice. Expert opinion: The first biomarkers proposed to predict tumor response to immunotherapy were based on PD1/PDL1 expression, but their predictive value is limited to specific cancers or patient populations. Recent advances in single-cell molecular profiling of circulating tumor cells and host cells using next-generation sequencing has dramatically expanded the pool of potentially useful predictive biomarkers. As immunotherapy moves toward personalized medicine, a composite panel of both genomic and proteomic biomarkers will have enormous utility in therapeutic decision-making

Characterization of high-Q optical microcavities using confocal microscopy

Confocal microscopy was initially developed to image complex circuits and material defects. Previous imaging studies yielded only qualitative data about the location and number of defects. In the present study, this noninvasive method is used to obtain quantitative information about the Q factor of an optical resonant cavity. Because the intensity of the fluorescent signal measures the number of defects in the resonant cavity, this signal is a measure of the number of surface scattering defects, one of the dominant loss mechanisms in optical microcavities. The Q of the cavities was also determined using conventional linewidth measurements. Based upon a quantitative comparative analysis of these two techniques, it is shown that the Q can be determined without a linewidth measurement, allowing for a noninvasive characterization technique

Counting White Blood Cells from a Blood Smear Using Fourier Ptychographic Microscopy

White blood cell (WBC) count is a valuable metric for assisting with diagnosis or prognosis of various diseases such as coronary heart disease, type 2 diabetes, or infection. Counting WBCs can be done either manually or automatically. Automatic methods are capable of counting a large number of cells to give a statistically more accurate reading of the WBC count of a sample, but the specialized equipment tends to be expensive. Manual methods are inexpensive since they only involve a conventional light microscope setup. However, it is more laborious and error-prone because the small field-of-view (FOV) of the microscope necessitates mechanical scanning of a specimen for counting an adequate number of WBCs. Here, we investigate the use of Fourier ptychographic microscopy (FPM) to bypass these issues of the manual methods. With a 2x objective, FPM can provide a FOV of 120 mm^2 with enhanced resolution comparable to that of a 20x objective, which is adequate for non-differentially counting WBCs in just one FOV. A specialist was able to count the WBCs in FPM images with 100% accuracy compared to the count as determined from conventional microscope images. An automatic counting algorithm was also developed to identify WBCs from FPM’s captured images with 95% accuracy, paving the way for a cost-effective WBC counting setup with the advantages of both the automatic and manual counting methods

Chemical patterning for the highly specific and programmed assembly of nanostructures

We have developed a new chemical patterning technique based on standard lithography-based processes to assemble nanostructures on surfaces with extraordinarily high selectivity. This patterning process is used to create patterns of aminosilane molecular layers surrounded by highly inert poly (ethylene glycol) (PEG) molecules. While the aminosilane regions facilitate nanostructure assembly, the PEG coating prevents adsorption of molecules and nanostructures, thereby priming the semiconductor substrate for the highly localized and programmed assembly of nanostructures. We demonstrate the power and versatility of this manufacturing process by building multilayered structures of gold nanoparticles attached to molecules of DNA onto the aminosilane patterns, with zero nanocrystal adsorption onto the surrounding PEG regions. The highly specific surface chemistry developed here can be used in conjunction with standard microfabrication and emerging nanofabrication technology to seamlessly integrate various nanostructures with semiconductor electronics

Circulating biomarkers predictive of tumor response to cancer immunotherapy

Introduction: The advent of checkpoint blockade immunotherapy has revolutionized cancer treatment, but clinical response to immunotherapies is highly heterogeneous among individual patients and between cancer types. This represents a challenge to oncologists when choosing specific immunotherapies for personalized medicine. Thus, biomarkers that can predict tumor responsiveness to immunotherapies before and during treatment are invaluable. Areas covered: We review the latest advances in 'liquid biopsy' biomarkers for noninvasive prediction and in-treatment monitoring of tumor response to immunotherapy, focusing primarily on melanoma and non-small cell lung cancer. We concentrate on high-quality studies published within the last five years on checkpoint blockade immunotherapies, and highlight significant breakthroughs, identify key areas for improvement, and provide recommendations for how these diagnostic tools can be translated into clinical practice. Expert opinion: The first biomarkers proposed to predict tumor response to immunotherapy were based on PD1/PDL1 expression, but their predictive value is limited to specific cancers or patient populations. Recent advances in single-cell molecular profiling of circulating tumor cells and host cells using next-generation sequencing has dramatically expanded the pool of potentially useful predictive biomarkers. As immunotherapy moves toward personalized medicine, a composite panel of both genomic and proteomic biomarkers will have enormous utility in therapeutic decision-making

Tunability and Noise Dependence in Differentiation Dynamics

The dynamic process of differentiation depends on the architecture, quantitative parameters, and noise of underlying genetic circuits. However, it remains unclear how these elements combine to control cellular behavior. We analyzed the probabilistic and transient differentiation of Bacillus subtilis cells into the state of competence. A few key parameters independently tuned the frequency of initiation and the duration of competence episodes and allowed the circuit to access different dynamic regimes, including oscillation. Altering circuit architecture showed that the duration of competence events can be made more precise. We used an experimental method to reduce global cellular noise and showed that noise levels are correlated with frequency of differentiation events. Together, the data reveal a noise-dependent circuit that is remarkably resilient and tunable in terms of its dynamic behavior

Label-Free, Single-Molecule Detection with Optical Microcavities

Current single-molecule detection techniques require labeling the target molecule. We report a highly specific and sensitive optical sensor based on an ultrahigh quality (Q) factor (Q > 10^8) whispering-gallery microcavity. The silica surface is functionalized to bind the target molecule; binding is detected by a resonant wavelength shift. Single-molecule detection is confirmed by observation of single-molecule binding events that shift the resonant frequency, as well as by the statistics for these shifts over many binding events. These shifts result from a thermo-optic mechanism. Additionally, label-free, single-molecule detection of interleukin-2 was demonstrated in serum. These experiments demonstrate a dynamic range of 10^(12) in concentration, establishing the microcavity as a sensitive and versatile detector

Evaluation of PD-L1 expression on vortex-isolated circulating tumor cells in metastatic lung cancer.

Metastatic non-small cell lung cancer (NSCLC) is a highly fatal and immunogenic malignancy. Although the immune system is known to recognize these tumor cells, one mechanism by which NSCLC can evade the immune system is via overexpression of programmed cell death ligand 1 (PD-L1). Recent clinical trials of PD-1 and PD-L1 inhibitors have returned promising clinical responses. Important for personalizing therapy, patients with higher intensity staining for PD-L1 on tumor biopsies responded better. Thus, there has been interest in using PD-L1 tumor expression as a criterion for patient selection. Currently available methods of screening involve invasive tumor biopsy, followed by histological grading of PD-L1 levels. Biopsies have a high risk of complications, and only allow sampling from limited tumor sections, which may not reflect overall tumor heterogeneity. Circulating tumor cell (CTC) PD-L1 levels could aid in screening patients, and could supplement tissue PD-L1 biopsy results by testing PD-L1 expression from disseminated tumor sites. Towards establishing CTCs as a screening tool, we developed a protocol to isolate CTCs at high purity and immunostain for PD-L1. Monitoring of PD-L1 expression on CTCs could be an additional biomarker for precision medicine that may help in determining response to immunotherapies

Simplified three-dimensional tissue clearing and incorporation of colorimetric phenotyping.

Tissue clearing methods promise to provide exquisite three-dimensional imaging information; however, there is a need for simplified methods for lower resource settings and for non-fluorescence based phenotyping to enable light microscopic imaging modalities. Here we describe the simplified CLARITY method (SCM) for tissue clearing that preserves epitopes of interest. We imaged the resulting tissues using light sheet microscopy to generate rapid 3D reconstructions of entire tissues and organs. In addition, to enable clearing and 3D tissue imaging with light microscopy methods, we developed a colorimetric, non-fluorescent method for specifically labeling cleared tissues based on horseradish peroxidase conversion of diaminobenzidine to a colored insoluble product. The methods we describe here are portable and can be accomplished at low cost, and can allow light microscopic imaging of cleared tissues, thus enabling tissue clearing and imaging in a wide variety of settings