Fixation Artifacts Research Articles

The morphology of internal elastic lamina corrugations in arteries under physiological conditions

Background: In elastic and resistance arteries, an elastin-rich membrane, the Internal Elastic Lamina (IEL), separates the tunica intima from the underlying tunica media. The IEL often appears wrinkled or corrugated in histological images. These corrugations are sometimes ascribed to vessel contraction ex vivo, and to fixation artifacts, and therefore regarded as not physiologically relevant. We examine whether the IEL remains corrugated even under physiological conditions.Methods: The diameters of carotid arteries of anesthetized pigs were measured by ultrasound. The arteries were then excised, inflated within a conical sleeve, fixed, and imaged by confocal microscopy. The conical sleeve allows fixing each artery across a wide range of diameters which bracket its ultrasound diameter. Thus the study was designed to quantify how corrugations change with diameter for a single artery, and test whether corrugations exist when the fixed artery matches the ultrasound diameter.Results: At diameters below the ultrasound diameter (i.e. when the artery was constricted as compared to ultrasound conditions), the IEL corrugations were found to decrease significantly with increasing diameter, but not fully flatten at the ultrasound diameter. The contour length of the IEL was found to be roughly 10% larger than the circumference of the artery measured by ultrasound. The physiological diameter is likely to be even smaller than the ultrasound diameter since ultrasound was conducted with the animal under general anesthesia which leads to vasodilation, suggesting a higher level of corrugation under physiological conditions. For arterial cross sections constricted below the ultrasound diameter, the IEL contour length decreased roughly with the square root of the diameter.Conclusion: The primary conclusions of this study are: a) the IEL is corrugated when the artery is constricted and flattens as the artery diameter increases, b) the IEL is corrugated under physiological conditions and has a contour length at least 10% more than the physiological arterial diameter, and c) the IEL despite being relatively stiffer than the surrounding arterial layers, does not behave like an inextensible membrane.

The advent of preventive high-resolution structural histopathology by artificial-intelligence-powered cryogenic electron tomography.

Advances in cryogenic electron microscopy (cryoEM) single particle analysis have revolutionized structural biology by facilitating the in vitro determination of atomic- and near-atomic-resolution structures for fully hydrated macromolecular complexes exhibiting compositional and conformational heterogeneity across a wide range of sizes. Cryogenic electron tomography (cryoET) and subtomogram averaging are rapidly progressing toward delivering similar insights for macromolecular complexes in situ, without requiring tags or harsh biochemical purification. Furthermore, cryoET enables the visualization of cellular and tissue phenotypes directly at molecular, nanometric resolution without chemical fixation or staining artifacts. This forward-looking review covers recent developments in cryoEM/ET and related technologies such as cryogenic focused ion beam milling scanning electron microscopy and correlative light microscopy, increasingly enhanced and supported by artificial intelligence algorithms. Their potential application to emerging concepts is discussed, primarily the prospect of complementing medical histopathology analysis. Machine learning solutions are poised to address current challenges posed by "big data" in cryoET of tissues, cells, and macromolecules, offering the promise of enabling novel, quantitative insights into disease processes, which may translate into the clinic and lead to improved diagnostics and targeted therapeutics.

Imaging Assessment of Peripapillary Vessel Diameters in Postmortem Eyes.

Proof of concept of ex vivo retinal vessel diameter measurements in human postmortem eyes. En face near-infrared (IR) images and optical coherence tomography (OCT) of the optic nerve head (ONH) were captured ex vivo with a Heidelberg Engineering Spectralis (Spectralis, version 7.0.4, Image Capture Module, version 1.2.4, Heidelberg Heidelberg, Germany) device, using a custom-made eye chamber holding and positioning the eyes during the image process. Thirty-two formaldehyde-fixated eyes of 16 patients were imaged. In the IR images, two independent graders measured retinal vessel diameters at the intersection of a drawn circle centered on the ONH with diameters of 2.0 mm and 3.4 mm, respectively. The anatomically corresponding measurements between both graders were statistically analyzed using a Wilcoxon signed-rank test. A total of 246 matched measurements of both graders were analyzed across all 32 imaged eyes. Statistically significant differences between the graders were found for arterioles at 2 mm from the ONH. The other measurements did not show statistically significant intergrader differences. The mean values for arteriole diameters were 72.2 µm at 2.0 mm and 61.5 µm at 3.4 mm for grader 1, and 66.4 µm at 2.0 mm and 63.2 µm at 3.4 mm for grader 2. The mean diameter for venules were 75.5 µm at 2.0 mm and 79.3 µm at 3.4 mm for grader 1, and 67.4 µm at 2 mm and 79.1 µm at 3.4 mm for grader 2. To the best of our knowledge, this is the first study to present IR image-based retinal vessel diameters in ex vivo postmortem eyes. Retinal IR/OCT imaging is possible, and measurements are reproducible in formaldehyde-fixated human eyes. Fixation artefacts result in lower image quality, and this can impose challenges in correctly detecting, classifying, and measuring retinal vessels.

Inherent Fluorescence Demonstrates Immunotropic Properties for Novel Janus Kinase 3 Inhibitors.

There is a general question in small molecule pharmacology about how apparent compound concentrations in blood, plasma, and organs actually relate to actual amounts at the target site of a compound. In this study, we used inherently fluorescent JAK3 ligands and their macrolide conjugates to investigate the relationship between physical properties, apparent bulk concentration, and organ and subcellular distribution. In vitro uptake into immune cells suggested that much of the substance was associated with granules or organelles. Samples from murine pharmacokinetic studies were analyzed by both conventional mass spectrometry and cryofluorescence microscopy methods to show the distribution of a compound within organs and cells without artifacts of fixation. These observations confirm the uptake of granules observed in vitro. Data from macrolides carrying either a coumarin fluorophore or a JAK3 inhibitor were similar, suggesting that the distribution is directed by the properties of the larger macrolide. These data show a propensity for azalide macrolides to concentrate in the lung and gut epithelia and suggest that the plasma- or whole-blood-derived estimates of drug levels almost certainly underestimate concentrations of macrolides in the mucous membranes. Thus, their apparent efficacy at sub-bacteriostatic doses may reflect their higher levels in barrier layers.

A cross-comparative analysis of in vivo versus ex vivo MRI indices in a mouse model of concussion

BackgroundWe present a cross-sectional, case-matched, and pair-wise comparison of structural magnetic resonance imaging (MRI), diffusion tensor imaging (DTI), and neurite orientation dispersion and density imaging (NODDI) measures in vivo and ex vivo in a mouse model of concussion, thus aiming to establish the concordance of structural and diffusion imaging findings in living brain and after fixation. MethodsWe allocated 28 male mice aged 3–4 months to sham injury and concussion (CON) groups. CON mice had received a single concussive impact on day 0 and underwent MRI at day 2 (n = 9) or 7 (n = 10) post-impact, and sham control mice likewise underwent imaging at day 2 (n = 5) or 7 (n = 4). Immediately after the final scanning, we collected the perfusion-fixed brains, which were stored for imaging ex vivo 6–12 months later. We then compared the structural imaging, DTI, and NODDI results between different methods. ResultsIn vivo to ex vivo structural and DTI/NODDI findings were in notably poor agreement regarding the effects of concussion on structural integrity of the brain.Comparison with existing methods:ex vivo imaging was frequently done to study the effects of diseases and treatments, but our results showed that ex vivo and in vivo imaging can detect completely opposite and contradictory results. This is also the first study that compares in vivo and ex vivo NODDI. ConclusionOur findings call for caution in extrapolating translational capabilities obtained ex vivo to physiological measurements in vivo. The divergent findings may reflect fixation artefacts and the contribution of the glymphatic system changes.

Visualizing Protein Localizations in Fixed Cells: Caveats and the Underlying Mechanisms.

Fluorescence microscopy techniques have been widely adopted in biology for their ability to visualize the structure and dynamics of a wide range of cellular and subcellular processes. The specificity and sensitivity that these techniques afford have made them primary tools in the characterization of protein localizations within cells. Many of the fluorescence microscopy techniques require cells to be fixed via chemical or alternative methods before being imaged. However, some fixation methods have been found to induce the redistribution of particular proteins in the cell, resulting in artifacts in the characterization of protein localizations and functions under physiological conditions. Here, we review the ability of commonly used cell fixation methods to faithfully preserve the localizations of proteins that bind to chromatin, undergo liquid-liquid phase separation (LLPS), and are involved in the formation of various membrane-bound organelles. We also review the mechanisms underlying various fixation artifacts and discuss potential alternative fixation methods to minimize the artifacts while investigating different proteins and cellular structures. Overall, fixed-cell fluorescence microscopy is a very powerful tool in biomedical research; however, each experiment demands the careful selection of an appropriate fixation method to avoid potential artifacts and may benefit from live-cell imaging validation.

Fixation can change the appearance of phase separation in living cells.

Fixing cells with paraformaldehyde (PFA) is an essential step in numerous biological techniques as it is thought to preserve a snapshot of biomolecular transactions in living cells. Fixed-cell imaging techniques such as immunofluorescence have been widely used to detect liquid-liquid phase separation (LLPS) in vivo. Here, we compared images, before and after fixation, of cells expressing intrinsically disordered proteins that are able to undergo LLPS. Surprisingly, we found that PFA fixation can both enhance and diminish putative LLPS behaviors. For specific proteins, fixation can even cause their droplet-like puncta to artificially appear in cells that do not have any detectable puncta in the live condition. Fixing cells in the presence of glycine, a molecule that modulates fixation rates, can reverse the fixation effect from enhancing to diminishing LLPS appearance. We further established a kinetic model of fixation in the context of dynamic protein-protein interactions. Simulations based on the model suggest that protein localization in fixed cells depends on an intricate balance of protein-protein interaction dynamics, the overall rate of fixation, and notably, the difference between fixation rates of different proteins. Consistent with simulations, live-cell single-molecule imaging experiments showed that a fast overall rate of fixation relative to protein-protein interaction dynamics can minimize fixation artifacts. Our work reveals that PFA fixation changes the appearance of LLPS from living cells, presents a caveat in studying LLPS using fixation-based methods, and suggests a mechanism underlying the fixation artifact.

Electron tomography of prolamellar bodies and their transformation into grana thylakoids in cryofixed Arabidopsis cotyledons.

The para-crystalline structures of prolamellar bodies (PLBs) and light-induced etioplast-to-chloroplast transformation have been investigated via electron microscopy. However, such studies suffer from chemical fixation artifacts and limited volumes of 3D reconstruction. Here, we examined Arabidopsis thaliana cotyledon cells by electron tomography (ET) to visualize etioplasts and their conversion into chloroplasts. We employed scanning transmission ET to image large volumes and high-pressure freezing to improve sample preservation. PLB tubules were arranged in a zinc blende-type lattice-like carbon atoms in diamonds. Within 2 h after illumination, the lattice collapsed from the PLB exterior and the disorganized tubules merged to form thylakoid sheets (pre-granal thylakoids), which folded and overlapped with each other to create grana stacks. Since the nascent pre-granal thylakoids contained curved membranes in their tips, we examined the expression and localization of CURT1 (CURVATURE THYLAKOID1) proteins. CURT1A transcripts were most abundant in de-etiolating cotyledon samples, and CURT1A was concentrated at the PLB periphery. In curt1a etioplasts, PLB-associated thylakoids were swollen and failed to form grana stacks. In contrast, PLBs had cracks in their lattices in curt1c etioplasts. Our data provide evidence that CURT1A is required for pre-granal thylakoid assembly from PLB tubules during de-etiolation, while CURT1C contributes to cubic crystal growth in the dark.

Confocal Fluorescence Microscopy Investigation for the Existence of Subdomains within Protein Storage Vacuoles in Soybean Cotyledons.

In legumes, the seed storage proteins accumulate within specialized organelles called protein storage vacuoles (PSVs). In several plant species, PSVs are differentiated into subdomains that accumulate different kinds of proteins. Even though the existence of subdomains is common in cereals and legumes, it has not been reported in soybean PSVs. The two most abundant seed proteins of soybean, 7S and 11S globulins, have different temporal accumulation patterns and exhibit considerable solubility differences that could result in differential accretion of these proteins within the PSVs. Here, we employed confocal fluorescent microscopy to examine the presence or absence of subdomains within the soybean PSVs. Eosin-stained sections of FAA-fixed paraffin embedded soybean seeds, when viewed by confocal fluorescence microscopy, revealed the presence of intricate subdomains within the PSVs. However, fluorescence immunolabeling studies demonstrated that the 7S and 11S globulins were evenly distributed within the PSVs and failed to corroborate the existence of subdomains within the PSVs. Similarly, confocal scanning microscopy examination of free-hand, vibratome and cryostat sections also failed to demonstrate the existence of subdomains within PSVs. The subdomains, which were prominently seen in PSVs of FAA-fixed soybean seeds, were not observed when the seeds were fixed either in glutaraldehyde/paraformaldehyde or glutaraldehyde. Our studies demonstrate that the apparent subdomains observed in FAA-fixed seeds may be a fixation artifact.

Perfusion with 10% neutral-buffered formalin is equivalent to 4% paraformaldehyde for histopathology and immunohistochemistry in a mouse model of experimental autoimmune encephalomyelitis.

Intravascular (IV) perfusion of tissue fixative is commonly used in the field of neuroscience as the central nervous system tissues are exquisitely sensitive to handling and fixation artifacts which can affect downstream microscopic analysis. Both 10% neutral-buffered formalin (NBF) and 4% paraformaldehyde (PFA) are used, although IV perfusion with PFA is most commonly referenced. The study objective was to compare the severity of handling and fixation artifacts, semiquantitative scores of inflammatory and neurodegenerative changes, and quantitative immunohistochemistry following terminal IV perfusion of mice with either 10% NBF or 4% PFA in a model of experimental autoimmune encephalitis (EAE). The study included 24 mice; 12 were control animals not immunized and an additional 12 were immunized with PLP139-151 subcutaneously, harvested at day 20, and fixed in the same fashion. Equal numbers (4 per group) were perfused with 10% NBF or 4% PFA, and 4 were immersion-fixed in 10% NBF. NBF-perfused mice had less severe dark neuron artifact than PFA-perfused mice (P < .001). Immersion-fixed animals had significantly higher scores for oligodendrocyte halos, dark neuron artifact, and perivascular clefts than perfusion-fixed animals. Histopathology scores in EAE mice for inflammation, demyelination, and necrosis did not differ among fixation methods. Also, no significant differences in quantitative immunohistochemistry for CD3 and Iba-1 were observed in immunized animals regardless of the method of fixation. These findings indicate that IV perfusion of mice with 10% NBF and 4% PFA are similar and adequate fixation techniques in this model.

A OneStep Solution to Fix and Stain Cells for Correlative Live and Fixed Microscopy.

Correlating the location of subcellular structures with dynamic cellular behaviors is difficult when working with organisms that lack the molecular genetic tools needed for expressing fluorescent protein fusions. Here, we describe a protocol for fixing, permeabilizing, and staining cells in a single step while imaging on a microscope. In contrast to traditional, multi‐step fixing and staining protocols that take hours, the protocol outlined here achieves satisfactory staining within minutes. This approach takes advantage of well‐characterized small molecules that stain specific subcellular structures, including nuclei, mitochondria, and actin networks. Direct visualization of the entire process allows for rapid optimization of cell fixation and staining, as well as straightforward identification of fixation artifacts. Moreover, live imaging prior to fixation reveals the dynamic history of cellular features, making it particularly useful for model systems without the capacity for expressing fluorescent protein fusions. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Fixing, permeabilizing, and staining mammalian cells in one step on the microscope

Formalin-Fixed and Paraffin-Embedded Samples for Next Generation Sequencing: Problems and Solutions.

Over the years, increasing information has been asked of the pathologist: we have moved from a purely morphological diagnosis to biomolecular and genetic studies, which have made it possible to implement the use of molecular targeted therapies, such as anti-epidermal growth factor receptor (EGFR) molecules in EGFR-mutated lung cancer, for example. Today, next generation sequencing (NGS) has changed the approach to neoplasms, to the extent that, in a short time, it has gained a place of absolute importance and diagnostic, prognostic and therapeutic utility. In this scenario, formaldehyde-fixed and paraffin-embedded (FFPE) biological tissue samples are a source of clinical and molecular information. However, problems can arise in the genetic material (DNA and RNA) for use in NGS due to fixation, and work is being devoted to possible strategies to reduce its effects. In this paper, we discuss the applications of FFPE tissue samples in the execution of NGS, we focus on the problems arising with the use of this type of material for nucleic acid extraction and, finally, we consider the most useful strategies to prevent and reduce single nucleotide polymorphisms (SNV) and other fixation artifacts.

Existence of giant mitochondria-containing sheet structures lacking cristae and matrix in the etiolated cotyledon of Arabidopsis thaliana

Mitochondria are essential organelles involved in the production and supply of energy in eukaryotic cells. Recently, the use of serial section scanning electron microscopy (S3EM) has allowed accurate three-dimensional (3D) reconstructed images of even complex organelle structures. Using this method, ultrathin sections of etiolated cotyledons were observed 4 days after germination of Arabidopsis thaliana in the dark, and giant mitochondria were found. To exclude the possibility of chemical fixation artifacts, this study confirmed the presence of giant mitochondria in high-pressure frozen samples. The 3D reconstructed giant mitochondria had a complex structure that included not only the elongated region but also the flattened shape of a disk. It contained the characteristic sheet structure, and the sheet lacked cristae and matrix but consisted of outer and inner membranes. Whether this phenomenon could be observed in living cells was investigated using the transformant with mitochondrial matrix expressing green fluorescent protein. Small globular mitochondria observed in light-treated samples were also represented in etiolated cotyledons. Although no giant mitochondria were observed in light-treated samples, they were found in the dark 3 days after germination and rapidly increased in number on the fourth day. Therefore, giant mitochondria were observed only in dark samples. These findings were supported by electron microscopy results.

Assessment of Intravascular Perfusion with 10% Neutral Buffered Formalin Versus 4% Paraformaldehyde for Histopathology and Immunohistochemistry in a Mouse Model of Experimental Autoimmune Encephalitis

Intravascular (IV) perfusion of tissue fixative is a technique that is commonly utilized in the field of neuroscience as central nervous system tissues are exquisitely sensitive to handling and fixation artifacts which can affect downstream morphologic readouts. In the literature, IV perfusion with 4% paraformaldehyde (PFA) is most referenced, although 10% neutral buffered formalin (NBF) is more commonly used for immersion fixation in both clinical and experimental pathology laboratories and has also been used in perfusion fixation. Unlike 4% PFA, 10% NBF is stable at room temperature and is not light sensitive. The study objective was to compare the frequency and severity of handling and fixation artifacts (including tissue tears, folds, oligodendrocyte halos, perivascular clefts, vacuolation, and dark neuron artifacts), semi-quantitative inflammation and necrosis scores, and quantitative immunohistochemistry following terminal IV perfusion of mice with 10% NBF or 4% PFA in a model of experimental autoimmune encephalitis (EAE). We hypothesized that no differences would be observed between the two fixatives after IV perfusion. SJL mice were immunized with 50µg of PLP139–151 subcutaneously and at day 20 mice were harvested for tissue collection. A total of 24 mice were used; 12 were control animals not immunized (4 perfused with 10% NBF, 4 perfused with 4% PFA and 4 were not perfused, but brain and spinal cord were collected fresh and then immersion fixed in 10% NBF). An additional 12 were immunized and fixed in the same fashion. Histologically, spinal cord sections from PFA perfused mice had less severe tissue folds than those from NBF perfused mice (P<0.01). NBF perfused mice had less severe dark neuron artifact than PFA perfused mice (P<0.001). Immersion fixed animals had significantly higher scores for oligodendrocyte halos, dark neuron artifact, and perivascular clefts than perfusion fixed animals. No significant differences were noted in EAE histopathology scores for inflammation, demyelination, or necrosis in immunized animals regardless of the method of fixation. Finally, no significant differences in quantitative immunohistochemistry for CD3 and Iba-1 were observed regardless of method of fixation, although scores for EAE animals were significantly higher than controls. These findings indicate that whole body perfusion of mice with 10% NBF and 4% PFA are similar and adequate fixation techniques for the study of neuropathology, especially in an EAE model.

Norbormide-Based Probes and Their Application for Mitochondrial Imaging in Drosophila Melanogaster.

Fluorescent live imaging on Drosophila melanogaster is a microscopy technique in rapid expansion. The growing number of probes available to detect cellular components and the relatively easy genetic manipulation of fruit fly make this model one of the most used for in vivo analysis of several physiological and/or pathological processes. Here we describe the chemical synthesis of two norbormide-derived BODIPY-conjugated fluorescent probes (NRBMC009 and NRBZLW0047). Moreover, we describe the larval dissection method, and subsequent live imaging acquisition. Both probes are able to label mitochondria in different Drosophila larval tissues, which allows for the characterization of mitochondrial morphological alterations by using a simple and quick method that avoids the fixation artefacts that often occur in immunofluorescence studies.

Clear Cell Morphology in a Salivary Gland Tumor - Clearly a Morphological Dilemma

Primary salivary gland tumors with clear cell morphology comprise an uncommonly encountered subgroup of salivary gland neoplasm. Clear cell morphology is a known feature in both benign and malignant salivary gland tumors. Oncocytomas are uncommon tumors of the salivary gland. They have an abundance of mitochondria, which is manifested as granular eosinophilic cytoplasm by light microscopy with presence of cytoplasmic glycogen. Fixation artifact can impart cytoplasmic clearing and oncocytomas with a predominance of clear cytoplasm are labeled Clear cell oncocytomas. Accurate diagnosis of salivary gland tumors can be challenging because of the many diagnostic entities, extensive morphologic overlap, and the rarity of most tumor types. Here we report a case of Clear cell salivary oncoctyoma which was confirmed by Immunohistochemical markers by excluding morphological differential of Clear cell myoepithelioma.

Evaluation and comparative analysis of minimal formalin containing compound fixatives

Introduction: Fixation is the crucial step in the practice of diagnostic pathology. Formalin has been used as a general fixative for more than a century. The IARC (International Agency for Research on Cancer) reported that there is a causal association between formalin exposure and leukemia and nasopharyngeal carcinoma. Hence this study was done to find an alternative fixative to minimize formalin exposure in histopathology laboratory. Materials and Methods: 100 specimens were fixed in 3 different minimal formalin containing compound fixatives composed of ethanol, glycerin, formalin (EFG fixatives)and hypotonic saline at fixation time of 10, 8 and 7 hours. The processed specimens were analyzed for fixation artifacts, staining characteristics and cytoarchitectural features and compared with conventional formalin fixation. Results: Cytoarchitectural features were well preserved as conventional formalin fixation in both EFG I and EFG II at 8 and 10 hours of fixation, and with inadequate fixation characters at 7 hours. EFG II was found to evaporate less formaldehyde vapor than 10% buffered formalin and fixation time reduced upto 8 hours. EFG III resulted in poor preservation of tissues in all three fixation times. Conclusion: As formaldehyde is a group 1 human carcinogen, it should be replaced by less toxic fixatives in histopathology laboratories. The present study highlights that minimal formalin containing EFG fixatives can be easily prepared in the laboratory and their effectiveness in routine histopathology examination is comparable with conventional formalin fixation. Keywords: Compound fixatives, Architecture, Fixation, Formalin toxicity, Histopathology.

7 Tesla MRI Followed by Histological 3D Reconstructions in Whole-Brain Specimens.

Post mortem magnetic resonance imaging (MRI) studies on the human brain are of great interest for the validation of in vivo MRI. It facilitates a link between functional and anatomical information available from MRI in vivo and neuroanatomical knowledge available from histology/immunocytochemistry. However, linking in vivo and post mortem MRI to microscopy techniques poses substantial challenges. Fixation artifacts and tissue deformation of extracted brains, as well as co registration of 2D histology to 3D MRI volumes complicate direct comparison between modalities. Moreover, post mortem brain tissue does not have the same physical properties as in vivo tissue, and therefore MRI approaches need to be adjusted accordingly. Here, we present a pipeline in which whole-brain human post mortem in situ MRI is combined with subsequent tissue processing of the whole human brain, providing a 3-dimensional reconstruction via blockface imaging. To this end, we adapted tissue processing procedures to allow both post mortem MRI and subsequent histological and immunocytochemical processing. For MRI, tissue was packed in a susceptibility matched solution, tailored to fit the dimensions of the MRI coil. Additionally, MRI sequence parameters were adjusted to accommodate T1 and T2∗ shortening, and scan time was extended, thereby benefiting the signal-to-noise-ratio that can be achieved using extensive averaging without motion artifacts. After MRI, the brain was extracted from the skull and subsequently cut while performing optimized blockface imaging, thereby allowing three-dimensional reconstructions. Tissues were processed for Nissl and silver staining, and co-registered with the blockface images. The combination of these techniques allows direct comparisons across modalities.

Robust assessment of tumor mutational burden in cytological specimens from lung cancer patients

ObjectivesTumor mutational burden (TMB) has emerged as a promising predictive biomarker for immune checkpoint inhibitor therapy. While the feasibility of TMB analysis on formalin-fixed paraffin-embedded (FFPE) samples has been thoroughly evaluated, only limited analyses have been performed on cytological samples, and no dedicated study has investigated concordance of TMB between different sample types. Here, we assessed TMB on matched histological and cytological samples from lung cancer patients and evaluated the accuracy of TMB estimation in these sample types. Materials and MethodsWe analyzed mutations and resulting TMB in FFPE samples and matched ethanol-fixed cytological smears (n = 12 matched pairs) by using a targeted next-generation sequencing assay (Oncomine™ Tumor Mutational Load). Two different variant allele frequency (VAF) thresholds were used to estimate TMB (VAF = 5% or 10%). ResultsAt 5% VAF threshold, 73% (107/147) of mutations were concordantly detected in matched histological and cytological samples. Discordant variants were mainly unique to FFPE samples (34/40 discordant variants) and mostly C:G > T:A transitions with low allelic frequency, likely indicating formalin fixation artifacts. Increasing the VAF threshold to 10% clearly increased the number of concordantly detected mutations in matched histological and cytological samples to 96% (100/106 mutations), and drastically reduced the number of FFPE-only mutations (from 34 to 4 mutations). In contrast, cytological samples showed consistent mutation count and TMB values at both VAF thresholds. Using FFPE samples, 2 out of 12 patients were classified as TMB-high at VAF cutoff of 5% but TMB-low at 10%, whereas cytological specimens allowed consistent patient classification independently from VAF cutoff. ConclusionOur results show that cytological smears provide more consistent TMB values due to high DNA quality and lack of formalin-fixation induced artifacts. Therefore, cytological samples should be the preferred sample type for robust TMB estimation.

Abstract 6495: Development of robust and reproducible controls to support clinical implementation of quantitative immunofluorescence assays for pharmacodynamic biomarkers

Abstract Robust pharmacodynamic (PD) assays are valuable to confirm putative drug mechanisms and downstream effects in support of early phase clinical trials. Evolving methods to achieve consistent performance across patients, clinical trials, and laboratories is critical for the success of clinical pharmacodynamic measurements. We have developed integral quality control tools to support clinical implementation of quantitative multiplex immunofluorescence assays (qmIFAs) that include xenograft tissue reference calibrators. These reference slides help to provide confirmation that the pharmacodynamic assay is reporting biomarker levels within designated target ranges with expected subcellular localization, helping to control for run-to-run issues with the assay procedures, reagents, or equipment; however, significant challenges are faced in producing these key reagents. Compared to conventional cell pellets, xenograft controls require more time, expense and specialized facilities; however, the tumor tissues are much more representative of the heterogeneity found in clinical biopsies. The challenges associated with xenograft controls include tissue and biomarker variability attributed to necrotic areas, fixation artifacts, and variable drug distribution. During our qualification process, xenografts undergo multiple rounds of selection and analysis to identify specimens that exhibit good tissue quality, adequate regions of interest (ROI), and appropriate target signal levels to support the intended biomarker measurements. Slide lots are produced, qualified, and stored in a manner that ensures adequate lot sizes, robust and reproducible slide to slide performance, and appropriate shelf life. Xenograft tissue reference standards representing a range of basal or drug-induced biomarker expression in appropriate models have been developed for several informative PD markers including Epithelial Mesenchymal Transition (EMT) markers such as E-Cadherin, Vimentin, and Beta-Catenin; DNA damage response markers γH2AX, pNBS1 and Rad51; apoptosis induction markers for colocalized γH2AX/cleaved Caspase 3; and the cell cycle markers pY15cdk and pHH3 among others and have been used as important tools to support ongoing clinical pharmacodynamic analyses of research biopsies at the NCI and other institutions (PMIDs 25964244, 29682208, 30792217, 31732654). In summary, generalized methods and workflows that will be made public have been developed by the NCI to optimize the production of qualified reference slide lots of sufficient reproducibility, stability, and quantity to support ongoing NCI-supported clinical trial pharmacodynamic assays for several key biomarkers. Funded by NCI Contract No HHSN261200800001E. Citation Format: Gabriel J. Benton, Katherine V. Ferry-Galow, Victor E. Lonsberry, Manisha Mohandoss, Howard Stotler, Debbie Trail, Kyle Georgius, Melinda Hollingshead, Deborah Wilsker, Robert Kinders, James H. Doroshow, Ralph E. Parchment. Development of robust and reproducible controls to support clinical implementation of quantitative immunofluorescence assays for pharmacodynamic biomarkers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6495.