Integration of ζ-deficient CARs into the CD3ζ gene conveys potent cytotoxicity in T and NK cells.

ABSTRACT: Chimeric antigen receptor (CAR)-redirected immune cells hold significant therapeutic potential for oncology, autoimmune diseases, transplant medicine, and infections. All approved CAR-T therapies rely on personalized manufacturing using undirected viral gene transfer, which results in nonphysiological regulation of CAR-signaling and limits their accessibility due to logistical challenges, high costs and biosafety requirements. Random gene transfer modalities pose a risk of malignant transformation by insertional mutagenesis. Here, we propose a novel approach utilizing CRISPR-Cas gene editing to redirect T cells and natural killer (NK) cells with CARs. By transferring shorter, truncated CAR-transgenes lacking a main activation domain into the human CD3ζ (CD247) gene, functional CAR fusion-genes are generated that exploit the endogenous CD3ζ gene as the CAR's activation domain. Repurposing this T/NK-cell lineage gene facilitated physiological regulation of CAR expression and redirection of various immune cell types, including conventional T cells, TCRγ/δ T cells, regulatory T cells, and NK cells. In T cells, CD3ζ in-frame fusion eliminated TCR surface expression, reducing the risk of graft-versus-host disease in allogeneic off-the-shelf settings. CD3ζ-CD19-CAR-T cells exhibited comparable leukemia control to TCRα chain constant (TRAC)-replaced and lentivirus-transduced CAR-T cells in vivo. Tuning of CD3ζ-CAR-expression levels significantly improved the in vivo efficacy. Notably, CD3ζ gene editing enabled redirection of NK cells without impairing their canonical functions. Thus, CD3ζ gene editing is a promising platform for the development of allogeneic off-the-shelf cell therapies using redirected killer lymphocytes.

A combined computational and functional approach identifies IGF2BP2 as a driver of chemoresistance in a wide array of pre-clinical models of colorectal cancer.

AIM: Chemoresistance is a major cause of treatment failure in colorectal cancer (CRC) therapy. In this study, the impact of the IGF2BP family of RNA-binding proteins on CRC chemoresistance was investigated using in silico, in vitro, and in vivo approaches. METHODS: Gene expression data from a well-characterized cohort and publicly available cross-linking immunoprecipitation sequencing (CLIP-Seq) data were collected. Resistance to chemotherapeutics was assessed in patient-derived xenografts (PDXs) and patient-derived organoids (PDOs). Functional studies were performed in 2D and 3D cell culture models, including proliferation, spheroid growth, and mitochondrial respiration analyses. RESULTS: We identified IGF2BP2 as the most abundant IGF2BP in primary and metastastatic CRC, correlating with tumor stage in patient samples and tumor growth in PDXs. IGF2BP2 expression in primary tumor tissue was significantly associated with resistance to selumetinib, gefitinib, and regorafenib in PDOs and to 5-fluorouracil and oxaliplatin in PDX in vivo. IGF2BP2 knockout (KO) HCT116 cells were more susceptible to regorafenib in 2D and to oxaliplatin, selumitinib, and nintedanib in 3D cell culture. Further, a bioinformatic analysis using CLIP data suggested stabilization of target transcripts in primary and metastatic tumors. Measurement of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) revealed a decreased basal OCR and an increase in glycolytic ATP production rate in IGF2BP2 KO. In addition, real-time reverse transcriptase polymerase chain reaction (qPCR) analysis confirmed decreased expression of genes of the respiratory chain complex I, complex IV, and the outer mitochondrial membrane in IGF2BP2 KO cells. CONCLUSIONS: IGF2BP2 correlates with CRC tumor growth in vivo and promotes chemoresistance by altering mitochondrial respiratory chain metabolism. As a druggable target, IGF2BP2 could be used in future CRC therapy to overcome CRC chemoresistance.

Decreased eukaryotic initiation factors expression upon temozolomide treatment-potential novel implications for eIFs in glioma therapy.

PURPOSE: Since glioma therapy is currently still limited until today, new treatment options for this heterogeneous group of tumours are of great interest. Eukaryotic initiation factors (eIFs) are altered in various cancer entities, including gliomas. The purpose of our study was to evaluate the potential of eIFs as novel targets in glioma treatment. METHODS: We evaluated eIF protein expression and regulation in 22 glioblastoma patient-derived xenografts (GBM PDX) after treatment with established cytostatics and with regards to mutation profile analyses of GBM PDX. RESULTS: We observed decreased expression of several eIFs upon temozolomide (TMZ) treatment independent from the phosphatidylinositol 3-kinase (PI3K)/ AKT/ mammalian target of the rapamycin (mTOR) signalling pathway. These effects of TMZ treatment were not present in TMZ-resistant PDX. Combination therapy of regorafenib and TMZ re- established the eIF/AKT/mTOR axis. CONCLUSION: Our study provides novel insights into chemotherapeutic effects on eIF regulation in gliomas and suggests that eIFs are interesting candidates for future research to improve glioma therapy.

Calcium-binding protein S100P is a new target gene of MACC1, drives colorectal cancer metastasis and serves as a prognostic biomarker.

BACKGROUND: The metastasis inducing gene MACC1 is a prognostic and predictive biomarker for metastasis in several cancers. Its mechanism of inducing metastasis includes the transcriptional control of other cancer-related target genes. Here, we investigate the interplay with the metastasis driver S100P in CRC progression. METHODS: MACC1-dependent S100P expression was analysed by qRT-PCR. The binding of MACC1 to the S100P promoter was determined by ChIP. Alterations in cell proliferation and motility were determined by functional in vitro assays. In vivo metastasis after intrasplenic transplantation was assessed by bioluminescence imaging and evaluation of tumour growth and liver metastasis. The prognostic value of S100P was determined in CRC patients by ROC-based Kaplan-Meier analyses. RESULTS: Expression of S100P and MACC1 correlated positively in CRC cells and colorectal tumours. MACC1 was found binding to the S100P promoter and induces its expression. The overexpression of S100P increased proliferation, migration and invasion in vitro and significantly induced liver metastasis in vivo. S100P expression was significantly elevated in metachronously metastasising CRC and was associated with shorter metastasis-free survival. CONCLUSIONS: We identified S100P as a transcriptional target gene of MACC1. Expression of S100P increases the metastatic potential of CRC cells in vitro and in vivo, and serves as a prognostic biomarker for metastasis-free survival of CRC patients, emphasising novel therapeutic interventions targeting S100P.

Heterogeneous pathway activation and drug response modelled in colorectal-tumor-derived 3D cultures.

Organoid cultures derived from colorectal cancer (CRC) samples are increasingly used as preclinical models for studying tumor biology and the effects of targeted therapies under conditions capturing in vitro the genetic make-up of heterogeneous and even individual neoplasms. While 3D cultures are initiated from surgical specimens comprising multiple cell populations, the impact of tumor heterogeneity on drug effects in organoid cultures has not been addressed systematically. Here we have used a cohort of well-characterized CRC organoids to study the influence of tumor heterogeneity on the activity of the KRAS/MAPK-signaling pathway and the consequences of treatment by inhibitors targeting EGFR and downstream effectors. MAPK signaling, analyzed by targeted proteomics, shows unexpected heterogeneity irrespective of RAS mutations and is associated with variable responses to EGFR inhibition. In addition, we obtained evidence for intratumoral heterogeneity in drug response among parallel "sibling" 3D cultures established from a single KRAS-mutant CRC. Our results imply that separate testing of drug effects in multiple subpopulations may help to elucidate molecular correlates of tumor heterogeneity and to improve therapy response prediction in patients.

Inhibiting phosphoglycerate dehydrogenase counteracts chemotherapeutic efficacy against MYCN-amplified neuroblastoma.

Here we sought metabolic alterations specifically associated with MYCN amplification as nodes to indirectly target the MYCN oncogene. Liquid chromatography-mass spectrometry-based proteomics identified seven proteins consistently correlated with MYCN in proteomes from 49 neuroblastoma biopsies and 13 cell lines. Among these was phosphoglycerate dehydrogenase (PHGDH), the rate-limiting enzyme in de novo serine synthesis. MYCN associated with two regions in the PHGDH promoter, supporting transcriptional PHGDH regulation by MYCN. Pulsed stable isotope-resolved metabolomics utilizing 13 C-glucose labeling demonstrated higher de novo serine synthesis in MYCN-amplified cells compared to cells with diploid MYCN. An independence of MYCN-amplified cells from exogenous serine and glycine was demonstrated by serine and glycine starvation, which attenuated nucleotide pools and proliferation only in cells with diploid MYCN but did not diminish these endpoints in MYCN-amplified cells. Proliferation was attenuated in MYCN-amplified cells by CRISPR/Cas9-mediated PHGDH knockout or treatment with PHGDH small molecule inhibitors without affecting cell viability. PHGDH inhibitors administered as single-agent therapy to NOG mice harboring patient-derived MYCN-amplified neuroblastoma xenografts slowed tumor growth. However, combining a PHGDH inhibitor with the standard-of-care chemotherapy drug, cisplatin, revealed antagonism of chemotherapy efficacy in vivo. Emergence of chemotherapy resistance was confirmed in the genetic PHGDH knockout model in vitro. Altogether, PHGDH knockout or inhibition by small molecules consistently slows proliferation, but stops short of killing the cells, which then establish resistance to classical chemotherapy. Although PHGDH inhibition with small molecules has produced encouraging results in other preclinical cancer models, this approach has limited attractiveness for patients with neuroblastoma.

Feasibility of precise and reliable glucose quantification in human whole blood samples by 1 tesla benchtop NMR.

The standard procedure for blood glucose measurements is enzymatic testing. This method is cheap, but requires small samples of open blood with direct contact to the test medium. In principle, NMR provides non-contact analysis of body fluids, but high-field spectrometers are expensive and cannot be easily utilized under clinical conditions. Low-field NMR systems with permanent magnets are becoming increasingly smaller and more affordable. The studies presented here aim at exploring the capabilities of low-field NMR for measuring glucose concentrations in whole blood. For this purpose, a modern 1 T benchtop NMR spectrometer was used. Challenges arise from broad spectral lines, the glucose peak locations close to the water signal, low SNR and the interference with signals from other blood components. Whole blood as a sample comprises even more boundary conditions: crucial for reliable results are avoiding the separation of plasma and cells by gravitation and reliable reference values. First, the accuracy of glucose levels measured by NMR was tested using aqueous glucose solutions and commercially available bovine plasma. Then, 117 blood samples from oral glucose tolerance testing were measured with minimal preparation by simple pulse-acquire NMR experiments. The analysis itself is the key to achieve high precision, so several approaches were investigated: peak integration, orthogonal projection to latent structure analysis and support vector machine regression. Correlations between results from the NMR spectra and the routine laboratory automated analyzer revealed an RMSE of 7.90 mg/dL for the best model. 91.5% of the model output lies within the limits of the German Medical Association guidelines, which require the glucose measurement to be within 11% of the reference method. It is concluded that spectral quantification of glucose in whole blood samples by high-quality NMR spectrometers operating at 1 T is feasible with sufficient accuracy.

A phosphoarray platform is capable of personalizing kinase inhibitor therapy in head and neck cancers.

Tyrosine kinase inhibitors are effective treatments for cancers. Knowing the specific kinase mutants that drive the underlying cancers predict therapeutic response to these inhibitors. Thus, the current protocol for personalized cancer therapy involves genotyping tumors in search of various driver mutations and subsequently individualizing the tyrosine kinase inhibitor to the patients whose tumors express the corresponding driver mutant. While this approach works when known driver mutations are found, its limitation is the dependence on driver mutations as predictors for response. To complement the genotype approach, we hypothesize that a phosphoarray platform is equally capable of personalizing kinase inhibitor therapy. We selected head and neck squamous cell carcinoma as the cancer model to test our hypothesis. Using the receptor tyrosine kinase phosphoarray, we identified the phosphorylation profiles of 49 different tyrosine kinase receptors in five different head and neck cancer cell lines. Based on these results, we tested the cell line response to the corresponding kinase inhibitor therapy. We found that this phosphoarray accurately informed the kinase inhibitor response profile of the cell lines. Next, we determined the phosphorylation profiles of 39 head and neck cancer patient derived xenografts. We found that absent phosphorylated EGFR signal predicted primary resistance to cetuximab treatment in the xenografts without phosphorylated ErbB2. Meanwhile, absent ErbB2 signaling in the xenografts with phosphorylated EGFR is associated with a higher likelihood of response to cetuximab. In summary, the phosphoarray technology has the potential to become a new diagnostic platform for personalized cancer therapy.

Basal subtype is predictive for response to cetuximab treatment in patient-derived xenografts of squamous cell head and neck cancer.

Cetuximab is the single targeted therapy approved for the treatment of head and neck cancer (HNSCC). Predictive biomarkers have not been established and patient stratification based on molecular tumor profiles has not been possible. Since EGFR pathway activation is pronounced in basal subtype, we hypothesized this activation could be a predictive signature for an EGFR directed treatment. From our patient-derived xenograft platform of HNSCC, 28 models were subjected to Affymetrix gene expression studies on HG U133+ 2.0. Based on the expression of 821 genes, the subtype of each of the 28 models was determined by integrating gene expression profiles through centroid-clustering with previously published gene expression data by Keck et al. The models were treated in groups of 5-6 animals with docetaxel, cetuximab, everolimus, cis- or carboplatin and 5-fluorouracil. Response was evaluated by comparing tumor volume at treatment initiation and after 3 weeks of treatment (RTV). Tumors distributed over the 3 signature-defined subtypes: 5 mesenchymal/inflamed phenotype (MS), 15 basal type (BA), 8 classical type (CL). Cluster analysis revealed a strong correlation between response to cetuximab and the basal subtype. RTV MS 3.32 vs. BA 0.78 (MS vs. BA, unpaired t-test, p 0.0002). Cetuximab responders were distributed as following: 1/5 in MS, 5/8 in CL and 13/15 in the BA group. Activity of classical chemotherapies did not differ between the subtypes. In conclusion basal subtype was associated with response to EGFR directed therapy in head and neck squamous cell cancer patient-derived xenografts.

Combination of a multimode antenna and TIAMO for traveling-wave imaging at 9.4 Tesla.

PURPOSE: To investigate the performance of a multimode antenna combined with time-interleaved acquisition of modes (TIAMO) for improved (1)H image homogeneity as compared to conventional traveling-wave imaging in the human brain at 9.4 Tesla (T). METHODS: An adjustable three-port antenna was built to stimulate the propagation of three basic waveguide modes within a 9.4 T scanner bore. For TIAMO, two time-interleaved acquisitions using different linear combinations of these modes were optimized to achieve a homogeneous rooted sum-of-squares combination of their B1+ patterns ( B1,RSS+). The antenna's transmit and receive performance, as well as local specific absorption rate, were analyzed using experiments and numerical simulations. RESULTS: The optimized TIAMO B1,RSS+ combination was superior to radiofrequency shimming. Across the entire brain, it improved the homogeneity of the excitation field by a factor of two and its maximum-to-minimum ratio by almost a factor of five as compared to the circularly polarized mode. The two-fold increase in "virtual" receive channels enhanced the parallel imaging performance and enabled the use of higher acceleration factors. CONCLUSION: Despite the limited number of channels, a remote three-port antenna combined with TIAMO represents an easily implementable setup to achieve void-free (1)H images from the entire brain at 9.4 T, which can be used for anatomical localization and B0 shimming.

Triple-quantum-filtered sodium imaging at 9.4 Tesla.

PURPOSE: Efficient acquisition of triple-quantum-filtered (TQF) sodium images at ultra-high field (UHF) strength. METHODS: A three-pulse preparation and a stack of double-spirals were used for the acquisition of TQF images at 9.4 Tesla. The flip angles of the TQ preparation were smoothly reduced toward the edge of k-space along the partition-encoding direction. In doing so, the specific absorption rate could be reduced while preserving the maximal signal intensity for the partitions most relevant for image contrast in the center of k-space. Simulations, phantom and in vivo measurements were used to demonstrate the usefulness of the proposed method. RESULTS: A higher sensitivity (∼ 20%) was achieved compared to the standard acquisition without flip angle apodization. Signals from free sodium ions were successfully suppressed irrespective of the amount of apodization used. B0 corrected TQF images with a nominal resolution of 5 × 5 × 5 mm(3) and an acceptable signal-to-noise ratio could be acquired in vivo within 21 min. CONCLUSION: Conventional TQF in combination with flip angle apodization permits to exploit more efficiently the increased sensitivity available at 9.4T.

Three-layered radio frequency coil arrangement for sodium MRI of the human brain at 9.4 Tesla.

PURPOSE: A multinuclei imaging setup with the capability to acquire both sodium ((23) Na) and proton ((1) H) signals at 9.4 Tesla is presented. The main objective was to optimize coil performance at the (23) Na frequency while still having the ability to acquire satisfactory (1) H images. METHODS: The setup consisted of a combination of three radio frequency (RF) coils arranged in three layers: the innermost layer was a 27-channel (23) Na receive helmet which was surrounded by a four-channel (23) Na transceiver array. The outer layer consisted of a four-channel (1) H dipole array for B0 shimming and anatomical localization. Transmit and receive performance of the (23) Na arrays was compared to a single-tuned (23) Na birdcage resonator. RESULTS: While the transmit efficiency of the (23) Na transceiver array was comparable to the birdcage, the (23) Na receive array provided substantial signal-to-noise ratio (SNR) gain near the surface and comparable SNR in the center. The utility of this customized setup was demonstrated by (23) Na images of excellent quality. CONCLUSION: High SNR, efficient transmit excitation and B0 shimming capability can be achieved for (23) Na MRI at 9.4T using novel coil combination. This RF configuration is easily adaptable to other multinuclei applications at ultra high field (≥ 7T).

Safety testing and operational procedures for self-developed radiofrequency coils.

The development of novel radiofrequency (RF) coils for human ultrahigh-field (≥7 T), non-proton and body applications is an active field of research in many MR groups. Any RF coil must meet the strict requirements for safe application on humans with respect to mechanical and electrical safety, as well as the specific absorption rate (SAR) limits. For this purpose, regulations such as the International Electrotechnical Commission (IEC) standard for medical electrical equipment, vendor-suggested test specifications for third party coils and custom-developed test procedures exist. However, for higher frequencies and shorter wavelengths in ultrahigh-field MR, the RF fields may become extremely inhomogeneous in biological tissue and the risk of localized areas with elevated power deposition increases, which is usually not considered by existing safety testing and operational procedures. In addition, important aspects, such as risk analysis and comprehensive electrical performance and safety tests, are often neglected. In this article, we describe the guidelines used in our institution for electrical and mechanical safety tests, SAR simulation and verification, risk analysis and operational procedures, including coil documentation, user training and regular quality assurance testing, which help to recognize and eliminate safety issues during coil design and operation. Although the procedure is generally applicable to all field strengths, specific requirements with regard to SAR-related safety and electrical performance at ultrahigh-field are considered. The protocol describes an internal procedure and does not reflect consensus among a large number of research groups, but rather aims to stimulate further discussion related to minimum coil safety standards. Furthermore, it may help other research groups to establish their own procedures. Copyright © 2015 John Wiley & Sons, Ltd.

Volumetric imaging with homogenised excitation and static field at 9.4 T.

OBJECTIVES: To overcome the challenges of B0 and RF excitation inhomogeneity at ultra-high field MRI, a workflow for volumetric B0 and flip-angle homogenisation was implemented on a human 9.4 T scanner. MATERIALS AND METHODS: Imaging was performed with a 9.4 T human MR scanner (Siemens Medical Solutions, Erlangen, Germany) using a 16-channel parallel transmission system. B0- and B1-mapping were done using a dual-echo GRE and transmit phase-encoded DREAM, respectively. B0 shims and a small-tip-angle-approximation kT-points pulse were calculated with an off-line routine and applied to acquire T1- and T 2 (*) -weighted images with MPRAGE and 3D EPI, respectively. RESULTS: Over six in vivo acquisitions, the B0-distribution in a region-of-interest defined by a brain mask was reduced down to a full-width-half-maximum of 0.10 ± 0.01 ppm (39 ± 2 Hz). Utilising the kT-points pulses, the normalised RMSE of the excitation was decreased from CP-mode's 30.5 ± 0.9 to 9.2 ± 0.7 % with all B 1 (+)  voids eliminated. The SNR inhomogeneities and contrast variations in the T1- and T 2 (*) -weighted volumetric images were greatly reduced which led to successful tissue segmentation of the T1-weighted image. CONCLUSION: A 15-minute B0- and flip-angle homogenisation workflow, including the B0- and B1-map acquisitions, was successfully implemented and enabled us to reduce intensity and contrast variations as well as echo-planar image distortions in 9.4 T images.

Predictive In Vivo Models for Oncology.

Experimental oncology research and preclinical drug development both substantially require specific, clinically relevant in vitro and in vivo tumor models. The increasing knowledge about the heterogeneity of cancer requested a substantial restructuring of the test systems for the different stages of development. To be able to cope with the complexity of the disease, larger panels of patient-derived tumor models have to be implemented and extensively characterized. Together with individual genetically engineered tumor models and supported by core functions for expression profiling and data analysis, an integrated discovery process has been generated for predictive and personalized drug development.Improved "humanized" mouse models should help to overcome current limitations given by xenogeneic barrier between humans and mice. Establishment of a functional human immune system and a corresponding human microenvironment in laboratory animals will strongly support further research.Drug discovery, systems biology, and translational research are moving closer together to address all the new hallmarks of cancer, increase the success rate of drug development, and increase the predictive value of preclinical models.

[School shootings by adult outsiders ­ psychiatric aspects and risk markers]. / Amokläufe an Schulen durch Außenstehende - Psychiatrische Auffälligkeiten und Risikomarker.

Objective: Three cases of rampage killings at German schools by adult outsiders were identified and analyzed. The cases took place between 1913 and 1983. To what extent do psychiatric aspects play a role and are there risk factors that can be identified und used for prevention? Method: For the identification of risk factors the warning behavior typology was utilized which covers eight behavioral factors. Results: Severe mental problems were found in all three cases. The factors of the warning behavior typology were present in different relevance: Pathway (100 %), Fixation (100 %), Identification (33 %), novel aggression (33 %), energy burst (33 %), Leakage (66 %), last resort (66 %), directly communicated threat (0 %). Conclusions: The prevention of such cases seems to be most promising installing a regional and interdisciplinary threat management model. The field of threat management offers a scientific frame with evidence based tools and methods.

A comprehensively characterized large panel of head and neck cancer patient-derived xenografts identifies the mTOR inhibitor everolimus as potential new treatment option.

Patient-derived xenograft (PDX) models have shown to reflect original patient tumors better than any other preclinical model. We embarked in a study establishing a large panel of head and neck squamous cell carcinomas PDX for biomarker analysis and evaluation of established and novel compounds. Out of 115 transplanted specimens 52 models were established of which 29 were characterized for response to docetaxel, cetuximab, methotrexate, carboplatin, 5-fluorouracil and everolimus. Further, tumors were subjected to sequencing analysis and gene expression profiling of selected mTOR pathway members. Most frequent response was observed for docetaxel and cetuximab. Responses to carboplatin, 5-fluorouracil and methotrexate were moderate. Everolimus revealed activity in the majority of PDX. Mutational profiling and gene expression analysis did not reveal a predictive biomarker for everolimus even though by trend RPS6KB1 mRNA expression was associated with response. In conclusion we demonstrate a comprehensively characterized panel of head and neck cancer PDX models, which represent a valuable and renewable tissue resource for evaluation of novel compounds and associated biomarkers.

High-resolution quantitative sodium imaging at 9.4 Tesla.

PURPOSE: Investigation of the feasibility to perform high-resolution quantitative sodium imaging at 9.4 Tesla (T). METHODS: A proton patch antenna was combined with a sodium birdcage coil to provide a proton signal without compromising the efficiency of the X-nucleus coil. Sodium density weighted images with a nominal resolution of 1 × 1 × 5 mm(3) were acquired within 30 min with an ultrashort echo time sequence. The methods used for signal calibration as well as for B0, B1, and off-resonance correction were verified on a phantom and five healthy volunteers. RESULTS: An actual voxel volume of roughly 40 µL could be achieved at 9.4T, while maintaining an acceptable signal-to-noise ratio (8 for brain tissue and 35 for cerebrospinal fluid). The measured mean sodium concentrations for gray and white matter were 36 ± 2 and 31 ± 1 mmol/L of wet tissue, which are comparable to values previously reported in the literature. CONCLUSION: The reduction of partial volume effects is essential for accurate measurement of the sodium concentration in the human brain. Ultrahigh field imaging is a viable tool to achieve this goal due to its increased sensitivity.

The Concept of Identification in Threat Assessment.

Identification is one of eight warning behaviors--superordinate patterns of accelerating risk--that are theorized to correlate with targeted violence, and have some empirical validation. It is characterized by one or more of five characteristics: pseudo-commando behavior, evidence of a warrior mentality, a close association with weapons or other military or law enforcement paraphernalia, wanting to imitate and often surmount previous attackers or assassins, or believing oneself to be an agent to advance a particular cause or belief system. The authors briefly explore the history of the psychology of identification, its current usage, and its application to threat assessment. Four cases are used to illustrate identification as both a process and a product, and a likely motive for targeted violence in some subjects. Its operational relevance for threat assessment is suggested.