Spatial interactions modulate tumor growth and immune infiltration.

Direct observation of immune cell trafficking patterns and tumor-immune interactions is unlikely in human tumors with currently available technology, but computational simulations based on clinical data can provide insight to test hypotheses. It is hypothesized that patterns of collagen formation evolve as a mechanism of immune escape, but the exact nature of the interaction between immune cells and collagen is poorly understood. Spatial data quantifying the degree of collagen fiber alignment in squamous cell carcinomas indicates that late stage disease is associated with highly aligned fibers. Here, we introduce a computational modeling framework (called Lenia) to discriminate between two hypotheses: immune cell migration that moves 1) parallel or 2) perpendicular to collagen fiber orientation. The modeling recapitulates immune-ECM interactions where collagen patterns provide immune protection, leading to an emergent inverse relationship between disease stage and immune coverage. We also illustrate the capabilities of Lenia to model the evolution of tumor progression and immune predation. Lenia provides a flexible framework for considering a spectrum of local (cell-scale) to global (tumor-scale) dynamics by defining a kernel cell-cell interaction function that governs tumor growth dynamics under immune predation with immune cell migration. Mathematical modeling provides important mechanistic insights into cell interactions. Short-range interaction kernels provide a mechanism for tumor cell survival under conditions with strong Allee effects, while asymmetric tumor-immune interaction kernels lead to poor immune response. Thus, the length scale of tumor-immune interactions drives tumor growth and infiltration.

Identification of a Biomarker Panel from Genome-Wide Methylation to Detect Early HPV-Associated Oropharyngeal Cancer.

As oropharyngeal cancer (OPC) associated with human papillomavirus (HPV) increases in men, the need for a screening test to diagnose OPC early is crucial. This study agnostically identified differentially methylated CpG sites to identify additional biomarkers to improve screening for early OPC.DNA was extracted from oral gargles of 89 early cases and 108 frequency matched healthy controls, and processed for genome-wide methylation using the Illumina Infinium MethylationEPIC BeadChip. Selected sites were combined with our prior methylation data in the EPB41L3 gene (CpG sites 438, 427, and 425) and oral HPV16 and HPV18 status were considered as binary variables (positive/negative). Lasso regression identified CpG sites strongly associated with early OPC. ROC curves with AUC were generated. The panel was validated utilizing bootstrap resampling.Machine learning analyses identified 14 markers that are significantly associated with early OPC, including one EPB41L3 CpG site (438) and oral HPV16 status. A final model was trained on all available samples using the discovered panel and was able to predict early OPC compared with controls with an AUC of 0.970 on the training set. In the bootstrap validation sets, the average AUC was 0.935, indicating adequate internal validity.Our data suggest that this panel can detect OPC early, however external validation of this panel is needed. Further refinement of a panel of biomarkers to diagnose OPC earlier is urgently needed to prevent complex treatment of OPC and associated comorbidities, while reducing risk of recurrence. PREVENTION RELEVANCE: This study identified biomarkers using genome-wide methylation to create a panel capable of discerning early oropharyngeal cancer (OPC) from those without OPC. Such a biomarker panel would be an effective tool to detect OPC early and prevent complications of treatment associated with later diagnosis.

Poor oral health influences head and neck cancer patient survival: an International Head and Neck Cancer Epidemiology Consortium pooled analysis.

BACKGROUND: Poor oral health has been identified as a prognostic factor potentially affecting the survival of patients with head and neck squamous cell carcinoma. However, evidence to date supporting this association has emanated from studies based on single cohorts with small-to-modest sample sizes. METHODS: Pooled analysis of 2449 head and neck squamous cell carcinoma participants from 4 studies of the International Head and Neck Cancer Epidemiology Consortium included data on periodontal disease, tooth brushing frequency, mouthwash use, numbers of natural teeth, and dental visits over the 10 years prior to diagnosis. Multivariable generalized linear regression models were used and adjusted for age, sex, race, geographic region, tumor site, tumor-node-metastasis stage, treatment modality, education, and smoking to estimate risk ratios (RR) of associations between measures of oral health and overall survival. RESULTS: Remaining natural teeth (10-19 teeth: RR = 0.81, 95% confidence interval [CI] = 0.69 to 0.95; ≥20 teeth: RR = 0.88, 95% CI = 0.78 to 0.99) and frequent dental visits (>5 visits: RR = 0.77, 95% CI = 0.66 to 0.91) were associated with better overall survival. The inverse association with natural teeth was most pronounced among patients with hypopharyngeal and/or laryngeal, and not otherwise specified head and neck squamous cell carcinoma. The association with dental visits was most pronounced among patients with oropharyngeal head and neck squamous cell carcinoma. Patient-reported gingival bleeding, tooth brushing, and report of ever use of mouthwash were not associated with overall survival. CONCLUSIONS: Good oral health as defined by maintenance of the natural dentition and frequent dental visits appears to be associated with improved overall survival among head and neck squamous cell carcinoma patients.

Genetically engineered mouse models of head and neck cancers.

The head and neck region is one of the anatomic sites commonly afflicted by cancer, with ~1.5 million new diagnoses reported worldwide in 2020 alone. Remarkable progress has been made in understanding the underlying disease mechanisms, personalizing care based on each tumor's individual molecular characteristics, and even therapeutically exploiting the inherent vulnerabilities of these neoplasms. In this regard, genetically engineered mouse models (GEMMs) have played an instrumental role. While progress in the development of GEMMs has been slower than in other major cancer types, several GEMMs are now available that recapitulate most of the heterogeneous characteristics of head and neck cancers such as the tumor microenvironment. Different approaches have been employed in GEMM development and implementation, though each can generally recapitulate only certain disease aspects. As a result, appropriate model selection is essential for addressing specific research questions. In this review, we present an overview of all currently available head and neck cancer GEMMs, encompassing models for head and neck squamous cell carcinoma, nasopharyngeal carcinoma, and salivary and thyroid gland carcinomas.

Immune-based classification of HPV-associated oropharyngeal cancer with implications for biomarker-driven treatment de-intensification.

BACKGROUND: There is significant interest in treatment de-escalation for human papillomavirus-associated (HPV+) oropharyngeal squamous cell carcinoma (OPSCC) patients given the generally favourable prognosis. However, 15-30% of patients recur after primary treatment, reflecting a need for improved risk-stratification tools. We sought to develop a molecular test to risk stratify HPV+ OPSCC patients. METHODS: We created an immune score (UWO3) associated with survival outcomes in six independent cohorts comprising 906 patients, including blinded retrospective and prospective external validations. Two aggressive radiation de-escalation cohorts were used to assess the ability of UWO3 to identify patients who recur. Multivariate Cox models were used to assess the associations between the UWO3 immune class and outcomes. FINDINGS: A three-gene immune score classified patients into three immune classes (immune rich, mixed, or immune desert) and was strongly associated with disease-free survival in six datasets, including large retrospective and prospective datasets. Pooled analysis demonstrated that the immune rich group had superior disease-free survival compared to the immune desert (HR = 9.0, 95% CI: 3.2-25.5, P = 3.6 × 10-5) and mixed (HR = 6.4, 95% CI: 2.2-18.7, P = 0.006) groups after adjusting for age, sex, smoking status, and AJCC8 clinical stage. Finally, UWO3 was able to identify patients from two small treatment de-escalation cohorts who remain disease-free after aggressive de-escalation to 30 Gy radiation. INTERPRETATION: With additional prospective validation, the UWO3 score could enable biomarker-driven clinical decision-making for patients with HPV+ OPSCC based on robust outcome prediction across six independent cohorts. Prospective de-escalation and intensification clinical trials are currently being planned. FUNDING: CIHR, European Union, and the NIH.

Tumor Cell Extrinsic Synaptogyrin 3 Expression as a Diagnostic and Prognostic Biomarker in Head and Neck Cancer.

Over 70% of oropharyngeal head and neck squamous cell carcinoma (HNSC) cases in the United States are positive for human papillomavirus (HPV) yet biomarkers for stratifying oropharyngeal head and neck squamous cell carcinoma (HNSC) patient risk are limited. We used immunogenomics to identify differentially expressed genes in immune cells of HPV(+) and HPV(-) squamous carcinomas. Candidate genes were tested in clinical specimens using both quantitative RT-PCR and IHC and validated by IHC using the Carolina Head and Neck Cancer Study (CHANCE) tissue microarray of HNSC cases. We performed multiplex immunofluorescent staining to confirm expression within the immune cells of HPV(+) tumors, receiver operating characteristic (ROC) curve analyses, and assessed survival outcomes. The neuronal gene Synaptogyrin-3 (SYNGR3) is robustly expressed in immune cells of HPV(+) squamous cancers. Multiplex immunostaining and single cell RNA-seq analyses confirmed SYNGR3 expression in T cells, but also unexpectedly in B cells of HPV(+) tumors. ROC curve analyses revealed that combining SYNGR3 and p16 provides more sensitivity and specificity for HPV detection compared to p16 IHC alone. SYNGR3-high HNSC patients have significantly better prognosis with five-year OS and DSS rates of 60% and 71%, respectively. Moreover, combining p16 localization and SYNGR3 expression can further risk stratify HPV(+) patients such that high cytoplasmic, low nuclear p16 do significantly worse (Hazard Ratio, 8.6; P = 0.032) compared to patients with high cytoplasmic, high nuclear p16. SYNGR3 expression in T and B cells is associated with HPV status and enhanced survival outcomes of HNSC patients.

Lysyl hydroxylase 2 mediated collagen post-translational modifications and functional outcomes.

Lysyl hydroxylase 2 (LH2) is a member of LH family that catalyzes the hydroxylation of lysine (Lys) residues on collagen, and this particular isozyme has been implicated in various diseases. While its function as a telopeptidyl LH is generally accepted, several fundamental questions remain unanswered: 1. Does LH2 catalyze the hydroxylation of all telopeptidyl Lys residues of collagen? 2. Is LH2 involved in the helical Lys hydroxylation? 3. What are the functional consequences when LH2 is completely absent? To answer these questions, we generated LH2-null MC3T3 cells (LH2KO), and extensively characterized the type I collagen phenotypes in comparison with controls. Cross-link analysis demonstrated that the hydroxylysine-aldehyde (Hylald)-derived cross-links were completely absent from LH2KO collagen with concomitant increases in the Lysald-derived cross-links. Mass spectrometric analysis revealed that, in LH2KO type I collagen, telopeptidyl Lys hydroxylation was completely abolished at all sites while helical Lys hydroxylation was slightly diminished in a site-specific manner. Moreover, di-glycosylated Hyl was diminished at the expense of mono-glycosylated Hyl. LH2KO collagen was highly soluble and digestible, fibril diameters were diminished, and mineralization impaired when compared to controls. Together, these data underscore the critical role of LH2-catalyzed collagen modifications in collagen stability, organization and mineralization in MC3T3 cells.

Multiplexed bioluminescence microscopy via phasor analysis.

Bioluminescence imaging with luciferase-luciferin pairs is a well-established technique for visualizing biological processes across tissues and whole organisms. Applications at the microscale, by contrast, have been hindered by a lack of detection platforms and easily resolved probes. We addressed this limitation by combining bioluminescence with phasor analysis, a method commonly used to distinguish spectrally similar fluorophores. We built a camera-based microscope equipped with special optical filters to directly assign phasor locations to unique luciferase-luciferin pairs. Six bioluminescent reporters were easily resolved in live cells, and the readouts were quantitative and instantaneous. Multiplexed imaging was also performed over extended time periods. Bioluminescent phasor further provided direct measures of resonance energy transfer in single cells, setting the stage for dynamic measures of cellular and molecular features. The merger of bioluminescence with phasor analysis fills a long-standing void in imaging capabilities, and will bolster future efforts to visualize biological events in real time and over multiple length scales.

Activation of the CREB Coactivator CRTC2 by Aberrant Mitogen Signaling promotes oncogenic functions in HPV16 positive head and neck cancer.

Head and neck squamous cell carcinoma (HNSCC) is the 6th most common cancer worldwide and incidence rates are continuing to rise globally. Patients often present with locally advanced disease and a staggering 50% chance of relapse following treatment. Aberrant activation of adaptive response signaling pathways, such as the cAMP/PKA pathway, induce an array of genes associated with known cancer pathways that promote tumorigenesis and drug resistance. We identified the cAMP Regulated Transcription Coactivator 2 (CRTC2) to be overexpressed and constitutively activated in HNSCCs and this confers poor prognosis. CRTCs are regulated through their subcellular localization and we show that CRTC2 is exclusively nuclear in HPV(+) HNSCC, thus constitutively active, due to non-canonical Mitogen-Activated Kinase Kinase 1 (MEKK1)-mediated activation via a MEKK1-p38 signaling axis. Loss-of-function and pharmacologic inhibition experiments decreased CRTC2/CREB transcriptional activity by reducing nuclear CRTC2 via nuclear import inhibition and/or by eviction of CRTC2 from the nucleus. This shift in localization was associated with decreased proliferation, migration, and invasion. Our results suggest that small molecules that inhibit nuclear CRTC2 and p38 activity may provide therapeutic benefit to patients with HPV(+) HNSCC.

Lysyl hydroxylase 2-induced collagen cross-link switching promotes metastasis in head and neck squamous cell carcinomas.

Head and neck squamous cell carcinoma (HNSCC) is the 6th most common cancer worldwide and incidence rates are continuing to rise globally. HNSCC patient prognosis is closely related to the occurrence of tumor metastases, and collagen within the tumor microenvironment (TME) plays a key role in this process. Lysyl hydroxylase 2 (LH2), encoded by the Procollagen-Lysine,2-Oxoglutarate 5-Dioxygenase 2 (PLOD2) gene, catalyzes hydroxylation of telopeptidyl lysine (Lys) residues of fibrillar collagens which then undergo subsequent modifications to form stable intermolecular cross-links that change the biomechanical properties (i.e. quality) of the TME. While LH2-catalyzed collagen modification has been implicated in driving tumor progression and metastasis in diverse cancers, little is known about its role in HNSCC progression. Thus, using gain- and loss-of-function studies, we examined the effects of LH2 expression levels on collagen cross-linking and cell behavior in vitro and in vivo using a tractable bioluminescent imaging-based orthotopic xenograft model. We found that LH2 overexpression dramatically increases HNSCC cell migratory and invasive abilities in vitro and that LH2-driven changes in collagen cross-linking robustly induces metastasis in vivo. Specifically, the amount of LH2-mediated collagen cross-links increased significantly with PLOD2 overexpression, without affecting the total quantity of collagen cross-links. Conversely, LH2 knockdown significantly blunted HNSCC cells invasive capacity in vitro and metastatic potential in vivo. Thus, regardless of the total "quantity" of collagen crosslinks, it is the "quality" of these cross-links that is the key driver of HNSCC tumor metastatic dissemination. These data implicate LH2 as a key regulator of HNSCC tumor invasion and metastasis by modulating collagen cross-link quality and suggest that therapeutic strategies targeting LH2-mediated collagen cross-linking in the TME may be effective in controlling tumor progression and improving disease outcomes.

Application of bioluminescence resonance energy transfer-based cell tracking approach in bone tissue engineering.

Bioluminescent imaging (BLI) has emerged as a popular in vivo tracking modality in bone regeneration studies stemming from its clear advantages: non-invasive, real-time, and inexpensive. We recently adopted bioluminescence resonance energy transfer (BRET) principle to improve BLI cell tracking and generated the brightest bioluminescent signal known to date, which thus enables more sensitive real-time cell tracking at deep tissue level. In the present study, we brought BRET-based cell tracking strategy into the field of bone tissue engineering for the first time. We labeled rat mesenchymal stem cells (rMSCs) with our in-house BRET-based GpNLuc reporter and evaluated the cell tracking efficacy both in vitro and in vivo. In scaffold-free spheroid 3D culture system, using BRET-based GpNLuc labeling resulted in significantly better correlation to cell numbers than a fluorescence based approach. In scaffold-based 3D culture system, GpNLuc-rMSCs displayed robust bioluminescence signals with minimal background noise. Furthermore, a tight correlation between BLI signal and cell number highlighted the robust reliability of using BRET-based BLI. In calvarial critical sized defect model, robust signal and the consistency in cell survival evaluation collectively supported BRET-based GpNLuc labeling as a reliable approach for non-invasively tracking MSC. In summary, BRET-based GpNLuc labeling is a robust, reliable, and inexpensive real-time cell tracking method, which offers a promising direction for the technological innovation of BLI and even non-invasive tracking systems, in the field of bone tissue engineering.

CRTC1/MAML2 directs a PGC-1α-IGF-1 circuit that confers vulnerability to PPARγ inhibition.

Mucoepidermoid carcinoma (MEC) is a life-threatening salivary gland cancer that is driven primarily by a transcriptional coactivator fusion composed of cyclic AMP-regulated transcriptional coactivator 1 (CRTC1) and mastermind-like 2 (MAML2). The mechanisms by which the chimeric CRTC1/MAML2 (C1/M2) oncoprotein rewires gene expression programs that promote tumorigenesis remain poorly understood. Here, we show that C1/M2 induces transcriptional activation of the non-canonical peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) splice variant PGC-1α4, which regulates peroxisome proliferator-activated receptor gamma (PPARγ)-mediated insulin-like growth factor 1 (IGF-1) expression. This mitogenic transcriptional circuitry is consistent across cell lines and primary tumors. C1/M2-positive tumors exhibit IGF-1 pathway activation, and small-molecule drug screens reveal that tumor cells harboring the fusion gene are selectively sensitive to IGF-1 receptor (IGF-1R) inhibition. Furthermore, this dependence on autocrine regulation of IGF-1 transcription renders MEC cells susceptible to PPARγ inhibition with inverse agonists. These results yield insights into the aberrant coregulatory functions of C1/M2 and identify a specific vulnerability that can be exploited for precision therapy.

Synergistic efficacy of combined EGFR and HDAC inhibitors overcomes tolerance to EGFR monotherapy in salivary mucoepidermoid carcinoma.

OBJECTIVES: Mucoepidermoid carcinoma (MEC) is the most common type of salivary gland malignancy. Advanced or high-grade MECs are refractory to chemotherapy, often leading to tumor recurrence/metastasis and abysmal ~35% 5-year survival. Causal links have been established between Epithelial Growth Factor Receptor (EGFR) activation and poor outcome. Herein we investigated the therapeutic efficacy of EGFR inhibition against MEC using in vitro pre-clinical models. MATERIALS AND METHODS: Five human MEC cell lines were used in cell viability, cytotoxicity, apoptosis, cell cycle, 2D-clonogenicity, and 3D-spheroid formation assays following treatment with Erlotinib (EGFR inhibitor), SAHA (Histone Deacetylase inhibitor; HDAC) and CUDC-101 (dual EGFR-HDAC inhibitor). Effects on MEC cancer stem cells were evaluated using flow cytometry. Gene expression and pathway regulation were evaluated via qPCR and Western blot, respectively. RESULTS: MEC cells enter a quiescent, non-proliferative yet rapidly reversible drug tolerant state upon EGFR inhibition. Despite robust suppression of MEC cell proliferation, no discernable apoptosis is detected. Combination of EGFR and HDAC inhibitors exhibits synergistic effects, exerting ~5-fold more potent cell cytotoxicity compared to HDAC or EGFR monotherapy. CUDC-101, a single molecule with dual EGFR-HDAC inhibitor moieties, exerts irreversible and potent cytotoxic activity against MEC cells and blunts MEC cancer stem-cell tumorigenicity. CONCLUSION: MEC cells are intrinsically tolerant to EGFR inhibition. Combining EGFR and HDAC inhibitors exerts synergistic and potent cytotoxic effects, suggesting that EGFR inhibitors still hold significant promise against MEC. Future studies are needed to assess the applicability and efficacy of dual EGFR-HDAC inhibitors for the clinical management of MEC.

Impact of Tumor Site and Adjuvant Radiotherapy on Survival of Patients with Adenoid Cystic Carcinoma: A SEER Database Analysis.

Adenoid cystic carcinoma (ACC) is a rare salivary gland tumor, displaying aggressive behavior with frequent recurrence and metastasis. Little information exists regarding the impact of clinicopathological parameters and adjuvant radiotherapy (aRT) on ACC disease specific (DSS) and overall survival (OS). We extracted demographic, treatment, and survival information of 1439 patients with major or minor intraoral salivary gland ACC from the Surveillance, Epidemiology, and End Results (SEER) database. The associations between tumor characteristics and aRT with OS and DSS were estimated using hazard ratios (HR) and 95% confidence intervals (CI). Submandibular gland ACCs had the worst prognosis (adjusted DSS HR = 1.48; 95% CI = 0.99-2.20, compared to parotid), and this difference was more pronounced among patients with advanced-stage tumors (adjusted DSS HR = 1.93; 95% CI = 1.13-3.30). aRT was associated with increased overall survival only among stage III submandibular ACC patients (HR = 0.64; 95% CI = 0.42-0.98) and had no benefit in any other group. In conclusion, submandibular gland ACC carries a worse prognosis than other gland subsites and may benefit from aRT. The different outcomes between submandibular gland and other major or minor gland ACCs warrant further mechanistic investigation.

Engineered BRET-Based Biologic Light Sources Enable Spatiotemporal Control over Diverse Optogenetic Systems.

Light-inducible optogenetic systems offer precise spatiotemporal control over a myriad of biologic processes. Unfortunately, current systems are inherently limited by their dependence on external light sources for their activation. Further, the utility of laser/LED-based illumination strategies are often constrained by the need for invasive surgical procedures to deliver such devices and local heat production, photobleaching and phototoxicity that compromises cell and tissue viability. To overcome these limitations, we developed a novel BRET-activated optogenetics (BEACON) system that employs biologic light to control optogenetic tools. BEACON is driven by self-illuminating bioluminescent-fluorescent proteins that generate "spectrally tuned" biologic light via bioluminescence resonance energy transfer (BRET). Notably, BEACON robustly activates a variety of commonly used optogenetic systems in a spatially restricted fashion, and at physiologically relevant time scales, to levels that are achieved by conventional laser/LED light sources.

An Immunocompetent Mouse Model of HPV16(+) Head and Neck Squamous Cell Carcinoma.

The incidence of human papilloma virus (HPV)-associated head and neck squamous cell carcinoma (HNSCC) is increasing and implicated in more than 60% of all oropharyngeal carcinomas (OPSCCs). Although whole-genome, transcriptome, and proteome analyses have identified altered signaling pathways in HPV-induced HNSCCs, additional tools are needed to investigate the unique pathobiology of OPSCC. Herein, bioinformatics analyses of human HPV(+) HNSCCs revealed that all tumors express full-length E6 and identified molecular subtypes based on relative E6 and E7 expression levels. To recapitulate the levels, stoichiometric ratios, and anatomic location of E6/E7 expression, we generated a genetically engineered mouse model whereby balanced expression of E6/E7 is directed to the oropharyngeal epithelium. The addition of a mutant PIK3CAE545K allele leads to the rapid development of pre-malignant lesions marked by immune cell accumulation, and a subset of these lesions progress to OPSCC. This mouse provides a faithful immunocompetent model for testing treatments and investigating mechanisms of immunosuppression.

Resveratrol Targets Urokinase-Type Plasminogen Activator Receptor Expression to Overcome Cetuximab-Resistance in Oral Squamous Cell Carcinoma.

Drug resistance to anti-cancer agents is a major concern regarding the successful treatment of malignant tumors. Recent studies have suggested that acquired resistance to anti-epidermal growth factor receptor (EGFR) therapies such as cetuximab are in part caused by genetic alterations in patients with oral squamous cell carcinoma (OSCC). However, the molecular mechanisms employed by other complementary pathways that govern resistance remain unclear. In the current study, we performed gene expression profiling combined with extensive molecular validation to explore alternative mechanisms driving cetuximab-resistance in OSCC cells. Among the genes identified, we discovered that a urokinase-type plasminogen activator receptor (uPAR)/integrin ß1/Src/FAK signal circuit converges to regulate ERK1/2 phosphorylation and this pathway drives cetuximab-resistance in the absence of EGFR overexpression or acquired EGFR activating mutations. Notably, the polyphenolic phytoalexin resveratrol, inhibited uPAR expression and consequently the signaling molecules ERK1/2 downstream of EGFR thus revealing additive effects on promoting OSCC cetuximab-sensitivity in vitro and in vivo. The current findings indicate that uPAR expression plays a critical role in acquired cetuximab resistance of OSCC and that combination therapy with resveratrol may provide an attractive means for treating these patients.

A Bioluminescence Resonance Energy Transfer-Based Approach for Determining Antibody-Receptor Occupancy In Vivo.

Elucidating receptor occupancy (RO) of monoclonal antibodies (mAbs) is a crucial step in characterizing the therapeutic efficacy of mAbs. However, the in vivo assessment of RO, particularly within peripheral tissues, is greatly limited by current technologies. In the present study, we developed a bioluminescence resonance energy transfer (BRET)-based system that leverages the large signal:noise ratio and stringent energy donor-acceptor distance dependency to measure antibody RO in a highly selective and temporal fashion. This versatile and minimally invasive system enables longitudinal monitoring of the in vivo antibody-receptor engagement over several days. As a proof of principle, we quantified cetuximab-epidermal growth factor receptor binding kinetics using this system and assessed cetuximab RO in a tumor xenograft model. Incomplete ROs were observed, even at a supratherapeutic dose of 50 mg/kg, indicating that fractional target accessibility is achieved. The BRET-based imaging approach enables quantification of antibody in vivo RO and provides critical information required to optimize therapeutic mAb efficacy.

Identification of NSDHL mutations associated with CHILD syndrome in oral verruciform xanthoma.

OBJECTIVE: The aim of this study was to perform a systematic analysis of the nicotinamide adenine dinucleotide phosphate (NAD[P])-dependent steroid dehydrogenase-like (NSDHL) gene in cases of oral verruciform xanthoma (VX) and to test for the presence of mutations associated with congenital hemidysplasia with ichthyosiform nevus and limb defects (CHILD) syndrome. STUDY DESIGN: DNA was extracted from archived paraffin-embedded tissue of oral VX and control cases. Polymerase chain reaction (PCR) was then used to screen exons 4 and 6 of the NSDHL gene for the presence of 4 known germline mutations associated with CHILD syndrome and 1 somatic mutation previously identified in VX lesions with no known association with CHILD syndrome. RESULTS: Of the 16 oral VX tissue samples, 8 (50%) had known missense mutations associated with CHILD syndrome. Furthermore, 2 of these 8 tissue samples also had an additional missense mutation previously identified in cutaneous VX lesions. No mutations of exons 4 and 6 were found in the 5 negative control tissue samples. CONCLUSIONS: NSDHL gene mutations associated with CHILD syndrome are common in sporadic oral VX cases, suggesting that these mutations confer a greater risk for the development of epithelial barrier defects that promote recurrent oral VX lesions and the potential for direct germline transmission of oral VX susceptibility.

Adaptive Transcriptional Responses by CRTC Coactivators in Cancer.

Adaptive stress signaling networks directly influence tumor development and progression. These pathways mediate responses that allow cancer cells to cope with both tumor cell-intrinsic and cell-extrinsic insults and develop acquired resistance to therapeutic interventions. This is mediated in part by constant oncogenic rewiring at the transcriptional level by integration of extracellular cues that promote cell survival and malignant transformation. The cAMP-regulated transcriptional coactivators (CRTCs) are a newly discovered family of intracellular signaling integrators that serve as the conduit to the basic transcriptional machinery to regulate a host of adaptive response genes. Thus, somatic alterations that lead to CRTC activation are emerging as key driver events in the development and progression of many tumor subtypes.