Machine Learning of Three-Dimensional Protein Structures to Predict the Functional Impacts of Genome Variation.

Research in the human genome sciences generates a substantial amount of genetic data for hundreds of thousands of individuals, which concomitantly increases the number of variants of unknown significance (VUS). Bioinformatic analyses can successfully reveal rare variants and variants with clear associations with disease-related phenotypes. These studies have had a significant impact on how clinical genetic screens are interpreted and how patients are stratified for treatment. There are few, if any, computational methods for variants comparable to biological activity predictions. To address this gap, we developed a machine learning method that uses protein three-dimensional structures from AlphaFold to predict how a variant will influence changes to a gene's downstream biological pathways. We trained state-of-the-art machine learning classifiers to predict which protein regions will most likely impact transcriptional activities of two proto-oncogenes, nuclear factor erythroid 2 (NFE2L2)-related factor 2 (NRF2) and c-Myc. We have identified classifiers that attain accuracies higher than 80%, which have allowed us to identify a set of key protein regions that lead to significant perturbations in c-Myc or NRF2 transcriptional pathway activities.

Clinical and genomic characterization of chemoradiation-resistant HPV-positive oropharyngeal squamous cell carcinoma.

Introduction: Most patients with HPV-positive oropharyngeal squamous cell carcinoma (OPSCC) have an excellent response to chemoradiation, and trials are now investigating de-escalated treatment. However, up to 25% of patients with HPV-positive OPSCC will experience recurrence, and up to 5% will even progress through primary treatment. Currently, there are no molecular markers to identify patients with poor prognosis who would be harmed by de-escalation. Herein we report the clinical and genomic characteristics of persistent HPV-positive OPSCC after definitive platinum-based chemoradiation therapy. Methods: Patients with HPV-positive OPSCC treated with curative intent platinum-based chemoradiation between 2007 and 2017 at two institutions and with a persistent locoregional disease were included. We evaluated clinical characteristics, including smoking status, age, stage, treatment, and overall survival. A subset of five patients had tissue available for targeted exome DNA sequencing and RNA sequencing. Genomic analysis was compared to a previously published cohort of 47 treatment-responsive HPV+ OPSCC tumors after batch correction. Mutational landscape, pathway activation, and OncoGPS tumor states were employed to characterize these tumors. Results: Ten patients met the inclusion criteria. The tumor and nodal stages ranged from T1 to T4 and N1 to N2 by AJCC 8th edition staging. All patients were p16-positive by immunohistochemistry, and eight with available in situ hybridization were confirmed to be HPV-positive. The 1-year overall survival from the time of diagnosis was 57%, and the 2-year overall survival was 17%. TP53 mutations were present in three of five (60%) persistent tumors compared to 2% (one of 47) of treatment-responsive HPV-positive tumors (p = 0.008). Other genes with recurrent mutations in persistent HPV-positive OPSCC tumors were NF1, KMT2D, PIK3C2B, and TFGBR2. Compared to treatment-responsive HPV-positive tumors, persistent tumors demonstrated activation of DNA Repair and p53, EMT, MYC, SRC, and TGF-beta signaling pathways, with post-treatment samples demonstrating significant activation of the PI3K-EMT-Stem pathways compared to pretreatment samples. Conclusion: Chemoradiation-resistant HPV-positive OPSCC occurs infrequently but portends a poor prognosis. These tumors demonstrate higher rates of p53 mutation and activation of MYC, SRC, and TGF-beta pathways. A comparison of tumors before and after treatment demonstrates PI3K-EMT-Stem pathways post-treatment in HPV-positive tumors with persistent disease after platinum-based chemoradiation.

Deciphering the Functional Roles of Individual Cancer Alleles Across Comprehensive Cancer Genomic Studies.

Cancer genome data has been growing in both size and complexity, primarily driven by advances in next-generation sequencing technologies, such as Pan-cancer data from TCGA, ICGC, and single-cell sequencing. Yet, discerning the functional role of individual genomic lesions remains a substantial challenge due to the complexity and scale of the data. Previously, we introduced REVEALER, which identifies groups of genomic alterations that significantly associate with target functional profiles or phenotypes, such as pathway activation, gene dependency, or drug response. In this paper, we present a new mathematical formulation of the algorithm. This version (REVEALER 2.0) is considerably more powerful than the original, allowing for rapid processing and analysis of much larger datasets and facilitating higher-resolution discoveries at the level of individual alleles. REVEALER 2.0 employs the Conditional Information Coefficient (CIC) to pinpoint features that are either complementary or mutually exclusive but still correlate with the target functional profile. The aggregation of these features provides a better explanation for the target functional profile than any single alteration on its own. This is indicative of scenarios where several activating genomic lesions can initiate or stimulate a key pathway or process. We replaced the initial three-dimensional kernel estimation with multiple precomputed one-dimensional kernel estimations, resulting in an approximate 150x increase in speed and efficiency. This improvement, combined with its efficient execution, makes REVEALER 2.0 suitable for much larger datasets and a more extensive range of genomic challenges.

Activation of KrasG12D in Subset of Alveolar Type II Cells Enhances Cellular Plasticity in Lung Adenocarcinoma.

We have previously identified alveolar type II cell as the cell-of-origin of KrasG12D-induced lung adenocarcinoma using cell lineage-specific inducible Cre mouse models. Using gain-of-function and loss-of-function genetic models, we discovered that active Notch signaling and low Sox2 levels dictate the ability of type II cells to proliferate and progress into lung adenocarcinoma upon KrasG12D activation. Here, we examine the phenotype of type II cells after Kras activation and find evidence for proliferation of cells that coexpress type I and type II markers. Three-dimensional organoid culture and transplantation studies determine that these dual-positive cells are highly plastic and tumor initiating in vivo. RNA sequencing analysis reveals that these dual-positive cells are enriched in Ras/MAPK, EGFR, and Notch pathways. Furthermore, the proliferation of these cells requires active Notch signaling and is inhibited by genetic/chemical Sox2 upregulation. Our findings could provide new therapeutic strategies to target KRAS-activated lung adenocarcinomas. SIGNIFICANCE: Identification of progenitor like tumor-initiating cells in KRAS-mutant lung adenocarcinoma may allow development of novel targeted therapeutics.

KITlow Cells Mediate Imatinib Resistance in Gastrointestinal Stromal Tumor.

Gastrointestinal stromal tumor (GIST) is commonly driven by oncogenic KIT mutations that are effectively targeted by imatinib (IM), a tyrosine kinase inhibitor (TKI). However, IM does not cure GIST, and adjuvant therapy only delays recurrence in high-risk tumors. We hypothesized that GIST contains cells with primary IM resistance that may represent a reservoir for disease persistence. Here, we report a subpopulation of CD34+KITlow human GIST cells that have intrinsic IM resistance. These cells possess cancer stem cell-like expression profiles and behavior, including self-renewal and differentiation into CD34+KIThigh progeny that are sensitive to IM treatment. We also found that TKI treatment of GIST cell lines led to induction of stem cell-associated transcription factors (OCT4 and NANOG) and concomitant enrichment of the CD34+KITlow cell population. Using a data-driven approach, we constructed a transcriptomic-oncogenic map (Onco-GPS) based on the gene expression of 134 GIST samples to define pathway activation during GIST tumorigenesis. Tumors with low KIT expression had overexpression of cancer stem cell gene signatures consistent with our in vitro findings. Additionally, these tumors had activation of the Gas6/AXL pathway and NF-κB signaling gene signatures. We evaluated these targets in vitro and found that primary IM-resistant GIST cells were effectively targeted with either single-agent bemcentinib (AXL inhibitor) or bardoxolone (NF-κB inhibitor), as well as with either agent in combination with IM. Collectively, these findings suggest that CD34+KITlow cells represent a distinct, but targetable, subpopulation in human GIST that may represent a novel mechanism of primary TKI resistance, as well as a target for overcoming disease persistence following TKI therapy.

Can modifying shielding, field of view, and exposure settings make the effective dose of a cone-beam computed tomography comparable to traditional radiographs used for orthodontic diagnosis?

OBJECTIVES: To analyze the effect of changes in exposure settings, field of view (FOV), and shielding on radiation to an adult and child phantom from cone-beam computed tomography (CBCT) imaging compared to panoramic and lateral cephalometric radiographs. MATERIALS AND METHODS: The effective dose to an adult and child anthropomorphic phantom by the CS 9300 using various scan protocols was recorded. Absorbed radiation was measured with optically stimulated luminescence dosimeters and effective dose calculated using 2007 International Commission on Radiological Protection tissue weighting factors. Scan protocols included different FOVs, voxel sizes, and standard versus low-dose protocols. Radiation shielding was used when it did not interfere with FOV. Panoramic and lateral cephalometric radiographs were taken with the Orthophos SL. RESULTS: Even with shielding, smaller FOVs, and increased voxel sizes, the effective dose of standard CBCT scans was higher than panoramic and lateral cephalometric radiographs. A shielded limited FOV standard scan combined with a lateral cephalometric radiograph resulted in a lower dose (P < .001) than a full FOV standard scan. Low-dose shielded scans resulted in significant dose reductions to the adult (P < .05) and child (P < .001) phantoms compared to the respective panoramic and lateral cephalometric radiographs combined. Image quality analysis was not possible with radiation equivalent phantoms. CONCLUSIONS: Unlike standard CBCTs, shielded low-dose CBCT protocols in the CS 9300 have lower effective doses than conventional radiographs for adult and child phantoms. If high resolution and cranial base visualization are necessary, combining a shielded LFOV standard exposure with a cephalometric radiograph is recommended.

Dose reduction in head and neck organs through shielding and application of different scanning parameters in cone beam computed tomography: an effective dose study using an adult male anthropomorphic phantom.

OBJECTIVES: The aim of this study was to determine the effect of shielding and scanning parameters on radiation dose reduction to the organs in the head and neck region in cone beam computed tomography (CBCT). STUDY DESIGN: An anthropomorphic phantom and optically stimulated luminescent dosimeters were used to calculate the changes in effective or equivalent doses to 9 anatomic structures through the addition of a thyroid collar, radiation safety glasses, and a radiation safety cap and by using different scanning protocols on a CS 9300 CBCT unit. RESULTS: The thyroid collar alone yielded dose reductions of 46% to the thyroid gland and at least 38% to the esophagus, but no more than 12% to the salivary glands. The radiation safety cap significantly reduced doses to the brain and the pituitary gland. Full shielding resulted in dose reductions of at least 50% to the thyroid gland, at least 47% to the esophagus, and approximately 35% to the brain and the pituitary gland. Significant dose reductions were recorded for all tissues with the "low dose" setting compared with the standard setting. CONCLUSIONS: Increased protection of the organs in the head and neck regions can be achieved by using various forms of shielding in CBCT imaging, with selection of the most appropriate scanning parameters based on the purpose of the examination.

Decomposing Oncogenic Transcriptional Signatures to Generate Maps of Divergent Cellular States.

The systematic sequencing of the cancer genome has led to the identification of numerous genetic alterations in cancer. However, a deeper understanding of the functional consequences of these alterations is necessary to guide appropriate therapeutic strategies. Here, we describe Onco-GPS (OncoGenic Positioning System), a data-driven analysis framework to organize individual tumor samples with shared oncogenic alterations onto a reference map defined by their underlying cellular states. We applied the methodology to the RAS pathway and identified nine distinct components that reflect transcriptional activities downstream of RAS and defined several functional states associated with patterns of transcriptional component activation that associates with genomic hallmarks and response to genetic and pharmacological perturbations. These results show that the Onco-GPS is an effective approach to explore the complex landscape of oncogenic cellular states across cancers, and an analytic framework to summarize knowledge, establish relationships, and generate more effective disease models for research or as part of individualized precision medicine paradigms.

Modulation of the Effect of Transforming Growth Factor-ß3 by Low-Intensity Pulsed Ultrasound on Scaffold-Free Dedifferentiated Articular Bovine Chondrocyte Tissues.

The aim of this study was to evaluate how low-intensity pulsed ultrasound (LIPUS) modulates the effect of transforming growth factor-ß3 (TGF-ß3) on the differentiation of scaffold-free dedifferentiated bovine articular chondrocyte tissues toward a cartilage-like phenotype. Specifically, the effect of these stimuli on the expression of hypertrophic markers collagen type I, collagen type X, and cartilage-degrading collagenase gene expression for a scaffold-free model was analyzed. A bioreactor that applied LIPUS directly from the transducer through a silicone gel to a six-well plate containing the tissues allowed simple, sterile, and large-scale experiments. Tissues were subjected to LIPUS of 55 mW/cm(2) in a 200 µs burst sine wave of 1 MHz over a 10-day period with or without TGF-ß3 (10 ng/mL). Tissues exposed to TGF-ß3 had significantly increased glycosaminoglycan and total collagen protein production along with upregulated cartilage-specific gene expression, resulting in tissues with a higher Young's Modulus. However, these tissues had also upregulated gene expression for hypertrophic markers collagen type I, collagen type X, MMP-1, MMP-13, MMP-2, and also an increase in the phosphorylation of p38. The expression of these matrix-degrading enzymes was remediated by hypertrophic development and differentiate dedifferentiated bovine articular chondrocytes towards a chondrogenic lineage allowing it to be a valuable tool in cartilage tissue engineering.

Influence of a tungsten metal conditioner on the adhesion and residual stress of porcelain bonded to cobalt-chromium alloy.

STATEMENT OF PROBLEM: Cobalt-chromium (CoCr) metal ceramic restorations are known to be more susceptible to cracking and interfacial failures. This is partially related to their high potential for oxidation compared with restorations made with high noble alloys. One approach that may improve their compatibility is the use of bonding agents. PURPOSE: The purpose of this study was to investigate the influence of a tungsten metal conditioner on the adhesion and residual stress of porcelain bonded to a cobalt-chromium alloy. MATERIAL AND METHODS: Eighty-one metal-porcelain bilayered specimens were manufactured and tested with a 4-point bend for adhesion and with Vickers indentation for residual stress determination. The strain energy release rate for adhesion energy and indentation residual stress was evaluated for specimens layered with and without tungsten (W) metal conditioner. Subsequent scanning electron microscopy and energy dispersive spectrometry were performed to identify fracture behavior and chemical and phase compositions. RESULTS: The average strain energy release rate of the specimen group tested without the W metal conditioner was significantly higher (P<.05) (44.70 J/m(2)) than that of the group with the W metal conditioner (28.65 J/m(2)). The average residual stress of the specimen group with (0.1 MPa) and without (1.61 MPa) the W metal conditioner did not differ significantly. Scanning electron microscopy and energy dispersive spectrometry analysis enabled the modes of failure to be determined and indicated the mechanisms by which the W metal conditioner influenced the bond. CONCLUSIONS: The W metal conditioner used in this study significantly lowered the strain energy release rate of the porcelain-cobalt-chromium interface and did not have a significant influence on the residual stress state of the porcelain.

Effect of autoclave induced low-temperature degradation on the adhesion energy between yttria-stabilized zirconia veneered with porcelain.

OBJECTIVE: To investigate the effect of autoclave induced low-temperature degradation on the adhesion energy between yttria-stabilized zirconia veneered with porcelain. METHODS: The strain energy release rate using a four-point bending stable fracture test was evaluated for two different porcelains [leucite containing (VM9) and glass (Zirox) porcelain] veneered to zirconia. Prior to veneering the zirconia had been subjected to 0 (control), 1, 5, 10 and 20 autoclave cycles. The specimens were manufactured to a total bi-layer dimension of 30 mm × 8 mm × 3 mm. Subsequent scanning electron microscopy/energy dispersive spectrometry, electron backscatter diffraction and X-ray diffraction analysis were performed to identify the phase transformation and fracture behavior. RESULTS: The strain energy release rate for debonding of the VM9 specimens were significantly higher (p<0.05) compared to the Zirox specimens across all test groups. Increasing autoclave cycles lowered the strain energy release rate significantly (p<0.05) from 18.67 J/m(2) (control) to the lowest of 12.79 J/m(2) (cycle 10) for only the VM9 specimens. SEM analyses showed predominant cohesive fracture within the porcelain for all cycle groups. XRD analysis of the substrate prior to veneering confirmed a tetragonal to monoclinic phase transformation with increasing the number of autoclave cycles between 5 and 20. The monoclinic phase reverted back to tetragonal phase after undergoing conventional porcelain firing cycles. EBSD data showed significant changes of the grain size distribution between the control and autoclaved specimen (cycle 20). SIGNIFICANCE: Increasing autoclave cycles only significantly decreased the adhesion of the VM9 layered specimens. In addition, a conventional porcelain firing schedule completely reverted the monoclinic phase back to tetragonal.