Histone H3.3G34-Mutant Interneuron Progenitors Co-opt PDGFRA for Gliomagenesis.

Histone H3.3 glycine 34 to arginine/valine (G34R/V) mutations drive deadly gliomas and show exquisite regional and temporal specificity, suggesting a developmental context permissive to their effects. Here we show that 50% of G34R/V tumors (n = 95) bear activating PDGFRA mutations that display strong selection pressure at recurrence. Although considered gliomas, G34R/V tumors actually arise in GSX2/DLX-expressing interneuron progenitors, where G34R/V mutations impair neuronal differentiation. The lineage of origin may facilitate PDGFRA co-option through a chromatin loop connecting PDGFRA to GSX2 regulatory elements, promoting PDGFRA overexpression and mutation. At the single-cell level, G34R/V tumors harbor dual neuronal/astroglial identity and lack oligodendroglial programs, actively repressed by GSX2/DLX-mediated cell fate specification. G34R/V may become dispensable for tumor maintenance, whereas mutant-PDGFRA is potently oncogenic. Collectively, our results open novel research avenues in deadly tumors. G34R/V gliomas are neuronal malignancies where interneuron progenitors are stalled in differentiation by G34R/V mutations and malignant gliogenesis is promoted by co-option of a potentially targetable pathway, PDGFRA signaling.

Mechanisms and therapeutic implications of hypermutation in gliomas.

A high tumour mutational burden (hypermutation) is observed in some gliomas1-5; however, the mechanisms by which hypermutation develops and whether it predicts the response to immunotherapy are poorly understood. Here we comprehensively analyse the molecular determinants of mutational burden and signatures in 10,294 gliomas. We delineate two main pathways to hypermutation: a de novo pathway associated with constitutional defects in DNA polymerase and mismatch repair (MMR) genes, and a more common post-treatment pathway, associated with acquired resistance driven by MMR defects in chemotherapy-sensitive gliomas that recur after treatment with the chemotherapy drug temozolomide. Experimentally, the mutational signature of post-treatment hypermutated gliomas was recapitulated by temozolomide-induced damage in cells with MMR deficiency. MMR-deficient gliomas were characterized by a lack of prominent T cell infiltrates, extensive intratumoral heterogeneity, poor patient survival and a low rate of response to PD-1 blockade. Moreover, although bulk analyses did not detect microsatellite instability in MMR-deficient gliomas, single-cell whole-genome sequencing analysis of post-treatment hypermutated glioma cells identified microsatellite mutations. These results show that chemotherapy can drive the acquisition of hypermutated populations without promoting a response to PD-1 blockade and supports the diagnostic use of mutational burden and signatures in cancer.

Aberrant DNA repair reveals a vulnerability in histone H3.3-mutant brain tumors.

Pediatric high-grade gliomas (pHGG) are devastating and incurable brain tumors with recurrent mutations in histone H3.3. These mutations promote oncogenesis by dysregulating gene expression through alterations of histone modifications. We identify aberrant DNA repair as an independent mechanism, which fosters genome instability in H3.3 mutant pHGG, and opens new therapeutic options. The two most frequent H3.3 mutations in pHGG, K27M and G34R, drive aberrant repair of replication-associated damage by non-homologous end joining (NHEJ). Aberrant NHEJ is mediated by the DNA repair enzyme polynucleotide kinase 3'-phosphatase (PNKP), which shows increased association with mutant H3.3 at damaged replication forks. PNKP sustains the proliferation of cells bearing H3.3 mutations, thus conferring a molecular vulnerability, specific to mutant cells, with potential for therapeutic targeting.

Digital holographic microscopy as a tool to study the thermal shape fluctuations of lipid vesicles.

The bending elasticity modulus of lipid membranes is obtained by applying for the first time, to the best of our knowledge, a novel experimental technique based on digital holographic microscopy. The fluctuations of the radius with time were extracted by tracking and measuring the optical thickness at the vesicle poles. The temporal autocorrelation function of the vesicle diameter computed for each of the studied vesicles was then fitted with the theoretical expression to deduce the membrane's tension and bending constant. For the bending elasticity modulus of SOPC bilayers, the value of (0.93 ± 0.03) × 10(-12) erg was obtained. This result is in accordance with values previously obtained by means of other conventional methods for the same type of lipid membrane in the presence of sugar molecules in aqueous medium. The obtained results encourage the future development of the digital holographic microscopy as a technique suitable for the measurement of the bending elasticity of lipid membranes.

Quantitative assessment of noise reduction with partial spatial coherence illumination in digital holographic microscopy.

Improving image quality in digital holographic microscopy is achievable by using partial spatial coherence (PSC) illumination instead of fully coherent illumination. This Letter presents simple theoretical models to quantitatively assess the reduction of noise as a function of both the spatial coherence of the illumination and the defocus distance of the noise source. The first developed model states that the effect of the PSC can be studied by discretizing the field of view in the plane of the noise source. The second model, following a continuous approach, corroborates the discrete model and extends it. Experimental results confirm theoretical expectations.

Fast numerical autofocus of multispectral complex fields in digital holographic microscopy with a criterion based on the phase in the Fourier domain.

The knowledge of the complex amplitude of optical fields, that is, both quantitative phase and intensity, enables numeric reconstruction along the optical axis. Nonetheless, a criterion is required for autofocusing. This Letter presents a robust and rapid refocusing criterion suitable for color interferometric digital holographic microscopy, and, more generally, for applications where complex amplitude is known for at least two different wavelengths. This criterion uses the phase in the Fourier domain, which is compared among wavelengths. It is applicable whatever the nature of the observed object: opaque, refractive, or both mixed. The method is validated with simulated and experimental holograms.

Complex Drop Impact Morphology.

The aim of this work is to understand the changes in the observed phenomena during particle-laden drop impact. The impact of millimeter-size drops was investigated onto hydrophilic (glass) and hydrophobic (polycarbonate) substrates. The drops were dispersions of water and spherical and nearly iso-dense hydrophobic particles with diameters of 200 and 500 µm. The impact was studied by side and bottom view images in the range 150 ≤ We ≤ 750 and 7100 ≤ Re ≤ 16400. The particles suppressed the appearance of singular jetting and drop partial rebound but promoted splashing, receding breakup, and rupture. The drops with 200 µm particles spread in two phases: fast and slow, caused by inertial and capillary forces, respectively. Also, the increase in volume fraction of 200 µm particle led to a linear decrease in the maximum spreading factor caused by the inertia force on both hydrophilic and hydrophobic substrates. The explanation of this reduction was argued to be the result of energy dissipation through frictional losses between particles and the substrate.

High throughput holographic imaging-in-flow for the analysis of a wide plankton size range.

We developed a Digital Holographic Microscope (DHM) working with a partial coherent source specifically adapted to perform high throughput recording of holograms of plankton organisms in-flow, in a size range of 3 µm-300 µm, which is of importance for this kind of applications. This wide size range is achieved with the same flow cell and with the same microscope magnification. The DHM configuration combines a high magnification with a large field of view and provides high-resolution intensity and quantitative phase images refocusing on high sample flow rate. Specific algorithms were developed to detect and extract automatically the particles and organisms present in the samples in order to build holograms of each one that are used for holographic refocusing and quantitative phase contrast imaging. Experimental results are shown and discussed.

Color imaging-in-flow by digital holographic microscopy with permanent defect and aberration corrections.

Color imaging-in-flow of particles is performed using red-green-blue (RGB) digital holographic microscopy (DHM), whose sources are partially coherent. RGB DHM provides intensity and quantitative phase images in the three color channels, which is valuable for observing small objects in numerous fields. In-flow investigation on a large depth of field is made possible by the refocusing capability of DHM and has many potential applications. A method is also developed to automatically correct the color balance and compensate both intensity and phase defects and aberrations, providing high-quality imaging. Experimental results show color in-flow analysis of microplankton and confirm the efficiency of the correction method.

Refocusing based on amplitude analysis in color digital holographic microscopy.

A refocusing criterion adapted to red-green-blue (RGB) digital holographic microscopy is established. It is applicable for both amplitude and phase objects. This color criterion is based on a monochromatic criterion, using the integrated modulus amplitude. Simulated RGB holograms show the value of having color information, even for colorless samples; in addition, the position of the focus plane along the optical axis is determined more accurately. Simulations take into account both the numerical apertures of lenses and noise during the holographic process. We also implement an algorithm exponentially reducing the computation time required for detecting the focus plane. The method is validated on experimental holograms.

Refocus criterion for both phase and amplitude objects in digital holographic microscopy.

For digital holographic microscopy applications, we modify the focus criterion based on the integration of the amplitude modulus to make possible its use regardless of the phase or amplitude nature of the objects under test. When applied on holographic data, the original criterion gives, at the focus plane, a minimum or a maximum, for amplitude or phase objects. The criterion we propose here operates on high-pass filtered complex amplitudes. It is shown that the proposed criterion gives a minimum for both types of objects when the focus plane is reached. Experimental results on real samples and simulations are provided, illustrating the efficiency and the potential of the method.

Comparison of germline and somatic structural variants in cancers reveal systematic differences in variant generating and selection processes.

Although several recent studies have characterized structural variants (SVs) in germline and cancer genomes, the features of SVs in these different contexts have not been directly compared. We examined similarities and differences between 2 million germline and 115 thousand tumor SVs from a cohort of 963 patients from The Cancer Genome Atlas (TCGA). We found significant differences in features related to their genomic sequences and localization that suggest differences between SV-generating processes and selective pressures. For example, we found that transposon-mediated processes shape germline much more than somatic SVs, while somatic SVs more frequently show features characteristic of chromoanagenesis. These differences were extensive enough to enable us to develop a classifier - "the great GaTSV" - that accurately distinguishes between germline and cancer SVs in tumor samples that lack a matched normal sample.

Separation of overlapped particles in digital holographic microscopy.

In this paper, we present a procedure to separate aggregates of overlapped particles in digital holograms, based on a focus plane analysis applied to each particle. The method can be applied either on phase or on amplitude objects, according that each object has a border in one focus plane. Numerical simulations are performed to quantify the robustness of the process by increasing the overlapping areas between the particles. The separation algorithm is successfully demonstrated experimentally on different types of aggregates.

Automated three-dimensional detection and classification of living organisms using digital holographic microscopy with partial spatial coherent source: application to the monitoring of drinking water resources.

In this paper, we investigate the use of a digital holographic microscope working with partially coherent spatial illumination for an automated detection and classification of living organisms. A robust automatic method based on the computation of propagating matrices is proposed to detect the 3D position of organisms. We apply this procedure to the evaluation of drinking water resources by developing a classification process to identify parasitic protozoan Giardia lamblia cysts among two other similar organisms. By selecting textural features from the quantitative optical phase instead of morphological ones, a robust classifier is built to propose a new method for the unambiguous detection of Giardia lamblia cyst that present a critical contamination risk.

Digital holographic interferometry with CO2 lasers and diffuse illumination applied to large space reflector metrology [Invited].

Digital holographic interferometry in the long-wave infrared domain has been developed by combining a CO(2) laser and a microbolometer array. The long wavelength allows large deformation measurements, which are of interest in the case of large space reflectors undergoing thermal changes when in orbit. We review holography at such wavelengths and present some specific aspects related to this spectral range on our measurements. For the design of our digital holographic interferometer, we studied the possibility of illuminating specular objects by a reflective diffuser. We discuss the development of the interferometer and the results obtained on a representative space reflector, first in the laboratory and then during vacuum cryogenic test.

ALK Amplification and Rearrangements Are Recurrent Targetable Events in Congenital and Adult Glioblastoma.

PURPOSE: Anaplastic lymphoma kinase (ALK) aberrations have been identified in pediatric-type infant gliomas, but their occurrence across age groups, functional effects, and treatment response has not been broadly established. EXPERIMENTAL DESIGN: We performed a comprehensive analysis of ALK expression and genomic aberrations in both newly generated and retrospective data from 371 glioblastomas (156 adult, 205 infant/pediatric, and 10 congenital) with in vitro and in vivo validation of aberrations. RESULTS: ALK aberrations at the protein or genomic level were detected in 12% of gliomas (45/371) in a wide age range (0-80 years). Recurrent as well as novel ALK fusions (LRRFIP1-ALK, DCTN1-ALK, PRKD3-ALK) were present in 50% (5/10) of congenital/infant, 1.4% (3/205) of pediatric, and 1.9% (3/156) of adult GBMs. ALK fusions were present as the only candidate driver in congenital/infant GBMs and were sometimes focally amplified. In contrast, adult ALK fusions co-occurred with other oncogenic drivers. No activating ALK mutations were identified in any age group. Novel and recurrent ALK rearrangements promoted STAT3 and ERK1/2 pathways and transformation in vitro and in vivo. ALK-fused GBM cellular and mouse models were responsive to ALK inhibitors, including in patient cells derived from a congenital GBM. Relevant to the treatment of infant gliomas, we showed that ALK protein appears minimally expressed in the forebrain at perinatal stages, and no gross effects on perinatal brain development were seen in pregnant mice treated with the ALK inhibitor ceritinib. CONCLUSIONS: These findings support use of brain-penetrant ALK inhibitors in clinical trials across infant, pediatric, and adult GBMs. See related commentary by Mack and Bertrand, p. 2567.

Structural variations in cancer and the 3D genome.

Structural variations (SVs) affect more of the cancer genome than any other type of somatic genetic alteration but difficulties in detecting and interpreting them have limited our understanding. Clinical cancer sequencing also increasingly aims to detect SVs, leading to a widespread necessity to interpret their biological and clinical relevance. Recently, analyses of large whole-genome sequencing data sets revealed features that impact rates of SVs across the genome in different cancers. A striking feature has been the extent to which, in both their generation and their influence on the selective fitness of cancer cells, SVs are more specific to individual cancer types than other genetic alterations such as single-nucleotide variants. This Perspective discusses how the folding of the 3D genome, and differences in its folding across cell types, affect observed SV rates in different cancer types as well as how SVs can impact cancer cell fitness.

Shear induced diffusion of platelets revisited.

The transport of platelets in blood is commonly assumed to obey an advection-diffusion equation with a diffusion constant given by the so-called Zydney-Colton theory. Here we reconsider this hypothesis based on experimental observations and numerical simulations including a fully resolved suspension of red blood cells and platelets subject to a shear. We observe that the transport of platelets perpendicular to the flow can be characterized by a non-trivial distribution of velocities with and exponential decreasing bulk, followed by a power law tail. We conclude that such distribution of velocities leads to diffusion of platelets about two orders of magnitude higher than predicted by Zydney-Colton theory. We tested this distribution with a minimal stochastic model of platelets deposition to cover space and time scales similar to our experimental results, and confirm that the exponential-powerlaw distribution of velocities results in a coefficient of diffusion significantly larger than predicted by the Zydney-Colton theory.

Three-dimensional analysis of blood platelet spreading using digital holographic microscopy: a statistical study of the differential effect of coatings in healthy volunteers and dialyzed patients.

In cardiovascular disorders, the study of thrombocytes, commonly known as platelets, is highly important since they are involved in blood clotting, essential in hemostasis, and they can in pathological situations affect the blood circulation. In this paper, single deposited platelets are measured using interferometric digital holographic microscopy. We have shown that the average optical height of platelets is significantly lower in healthy volunteers than in dialyzed patients, meaning a better spreading. It demonstrates the great interest for assessing this parameter in any patients, and therefore the high potential of analyzing single spread platelets using digital holographic microscopy in fundamental research as well as a diagnostic tool in routine laboratories, for usual blood tests.

Therapeutic targeting of ATR in alveolar rhabdomyosarcoma.

Despite advances in multi-modal treatment approaches, clinical outcomes of patients suffering from PAX3-FOXO1 fusion oncogene-expressing alveolar rhabdomyosarcoma (ARMS) remain dismal. Here we show that PAX3-FOXO1-expressing ARMS cells are sensitive to pharmacological ataxia telangiectasia and Rad3 related protein (ATR) inhibition. Expression of PAX3-FOXO1 in muscle progenitor cells is not only sufficient to increase sensitivity to ATR inhibition, but PAX3-FOXO1-expressing rhabdomyosarcoma cells also exhibit increased sensitivity to structurally diverse inhibitors of ATR. Mechanistically, ATR inhibition leads to replication stress exacerbation, decreased BRCA1 phosphorylation and reduced homologous recombination-mediated DNA repair pathway activity. Consequently, ATR inhibitor treatment increases sensitivity of ARMS cells to PARP1 inhibition in vitro, and combined treatment with ATR and PARP1 inhibitors induces complete regression of primary patient-derived ARMS xenografts in vivo. Lastly, a genome-wide CRISPR activation screen (CRISPRa) in combination with transcriptional analyses of ATR inhibitor resistant ARMS cells identifies the RAS-MAPK pathway and its targets, the FOS gene family, as inducers of resistance to ATR inhibition. Our findings provide a rationale for upcoming biomarker-driven clinical trials of ATR inhibitors in patients suffering from ARMS.