A Challenging Correlation between Tumor Cellularity and Somatic Variant Allele Fraction in Lung and Colorectal Cancers-Specimens of Low Tumor Percentage Should Be Analyzed with Caution.

Background and aims: The percentage of tumor cells (tumor cellularity) in a cancerous tissue has been assumed to correlate with the variant allele fraction (VAF) of an identified pathogenic variant. Many laboratories use the tumor cellularity as part of a quality criteria for specimen processing and clinical reporting. However, a systematic study of such correlation has yet to be shown. We performed a relatively large-scale study to determine whether pathologist-estimated tumor cellularity is correlated with next-generation sequencing (NGS)-derived VAF. Materials and Methods: A total of 1511 non-small cell lung cancer (NSCLC) and colorectal cancer (CRC) specimens, including formalin-fixed paraffin-embedded (FFPE) and fine needle aspirated (FNA) tissues, were analyzed by cancer hotspot NGS. For a given specimen, pathogenic variants of BRAF, EGFR, KRAS, and NRAS were identified and the determined VAFs were correlated with the corresponding tissue tumor cellularity. Results: The coefficient of determination R-squared (R2) values were calculated for each correlation. All R2 values were lower than 0.25, indicating poor correlations. Pathogenic variants were found, not uncommonly, in tumor specimens that carried 10% or lower tumor cellularity. There were no apparent differences of R2 values between the FFPE and FNA specimens. Conclusion: In both NSCLC and CRC, the lack of linear relationship between tumor cellularity and VAF was found across a wide range of tumor cell percentages. Caution should be used when using tumor cellularity to triage specimens for NGS testing. The tumor cellularity should be considered in relation to the limit of detection of the specific assay for the proper interpretation of a negative test result.

Addressing sequential and concurrent treatment regimens in a small n sequential, multiple assignment, randomized trial (snSMART) in the MISTIC study.

Multisystem Inflammatory Syndrome in children (MIS-C) is a rare and novel pediatric complication linked to COVID-19 exposure, which was first identified in April 2020. A small n, Sequential, Multiple Assignment, Randomized Trial (snSMART) was applied to the Multisystem Inflammatory Syndrome Therapies in Children Comparative Effectiveness Study (MISTIC) to efficiently evaluate multiple competing treatments. In the MISTIC snSMART study, participants are randomized to one of three interventions (steroids, infliximab or anakinra), and potentially re-randomized to the remaining two treatments depending on their response to the first randomized treatment. However, given the novelty and urgency of the MIS-C disease, in addition to patient welfare concerns, treatments were not always administered sequentially, but allowed to be administered concurrently if deemed medically necessary. We propose a pragmatic modification to the original snSMART design to address the analysis of concurrent versus sequential treatments in the MISTIC study. A modified Bayesian joint stage model is developed that can distinguish a concurrent treatment effect from a sequential treatment effect. A simulation study is conducted to demonstrate the improved accuracy and efficiency of the primary aim to estimate the first stage treatments' response rates and the secondary aim to estimate the combined first and second stage treatments' responses in the proposed model compared to the standard snSMART Bayesian joint stage model. We observed that the modified model has improved efficiency in terms of bias and rMSE under large sample size settings.

Mitohormesis.

Perturbation of mitochondrial function can trigger a host of cellular responses that seek to restore cellular metabolism, cytosolic proteostasis, and redox homeostasis. In some cases, these responses persist even after the stress is relieved, leaving the cell or tissue in a less vulnerable state. This process-termed mitohormesis-is increasingly viewed as an important aspect of normal physiology and a critical modulator of various disease processes. Here, we review aspects of mitochondrial stress signaling that, among other things, can rewire the cell's metabolism, activate the integrated stress response, and alter cytosolic quality-control pathways. We also discuss how these pathways are implicated in various disease states from pathogen challenge to chemotherapeutic resistance and how their therapeutic manipulation can lead to new strategies for a host of chronic conditions including aging itself.

Design of Superlubricity System Using Si3N4/Polyimide as the Friction Pair and Nematic Liquid Crystals as the Lubricant.

Polyimide (PI) is a high-performance engineering plastic used as a bearing material. A superlubricity system using Si3N4/PI as the friction pair and nematic liquid crystals (LCs) as the lubricant was designed. The superlubricity performance was studied by simulating the start-stop condition of the machine, and it was found that the superlubricity system had good reproducibility and stability. In the superlubricity system, friction aligned with the PI molecules, and this alignment was less relevant compared to which substance was rubbing on the PI. Oriented PI molecules induced LC molecule alignment when the pretilt angle was very small, and the LC molecules were almost parallel to the PI molecules due to the one-dimensional ordered arrangement of LC molecules and low viscosity, which is conducive to the occurrence of the superlubricity phenomenon.

Identification and interpretation of TET2 noncanonical splicing site intronic variants in myeloid neoplasm patients.

Background: DNA hypermethylation and instability due to inactivation mutations in Ten-eleven translocation 2 (TET2) is a key biomarker of hematological malignancies. This study aims at characterizing two intronic noncanonical splice-site variants, c.3954+5_3954+8delGTTT and c.3954+5G>A. Methods: We used in silico prediction tools, reverse transcription (RT)-PCR, and Sanger sequencing on blood/bone marrow-derived RNA specimens to determine the aberrant splicing. Results: In silico prediction of both variants exhibited reduced splicing strength at the TET2 intron 7 splicing donor site. RT-PCR and Sanger sequencing identified a 62-bp deletion at the exon 7, producing a frameshift mutation, p.Cys1298*. Conclusion: This study provides functional evidence for two intronic TET2 variants that cause alternative splicing and frameshift mutation.

ZC3H7B-BCOR Fusion High-grade Endometrial Stromal Sarcoma With Morphologic Features of Low-grade Endometrial Stromal Sarcoma. A Case Report and Review of Literature.

High-grade endometrial stromal sarcomas with ZC3H7B-BCOR fusion are rare. They are predominantly located in the endomyometrium, with morphologic features characterized as haphazardly arranged fascicles of spindle cells with mild to moderate atypia, abundant myxoid matrix, high mitotic index, and tongue-like/pushing patterns of myometrial invasion. Furthermore, conventional or variant low-grade endometrial stromal sarcomas are often not present. Clinically, they present at a higher stage and are associated with worse prognosis compared with low-grade endometrial stromal sarcoma. Given the limited number of reported cases, we describe the case of a ZC3H7B-BCOR fusion high-grade endometrial stromal sarcoma initially diagnosed on the hysterectomy specimen as low-grade endometrial stromal sarcoma based on an endometrial stromal tumor showing tongue-like myometrial and lymphovascular invasion, minimal cytologic atypia, low-mitotic activity (0-1/10 high-power field), round/spindle cell component and immunohistochemical stain results (positive for CD10, estrogen receptor, progesterone receptor, and focally positive for cyclin D1). At the time of pathologic diagnosis, she was Stage Ia and managed conservatively. Subsequent molecular analysis revealed a ZC3H7B (exon 10)- BCOR (BCL-6 corepressor) (exon 7) gene fusion. On follow-up, she showed no evidence of disease at 37 months from the time of diagnosis. This case report expands the morphologic spectrum of ZC3H7B-BCOR fusion high-grade ESS, which includes an intramural location, morphologic and immunophenotypic features similar to LG-ESS, as well as the presence of round and spindle cell components. This case also underscores the value of molecular analysis in the proper classification of ESS.

Reproducible SERS substrates manipulated by interparticle spacing and particle diameter of gold nano-island array using in-situ thermal evaporation.

Gold (Au) nano-island arrays were deposited on the glass substrate to fabricate surface-enhanced Raman scattering (SERS) substrates by in-situ thermal evaporation (deposited and annealed samples at the same time). The optimal SERS intensity deposited by various thicknesses and in-situ annealing temperatures of Au nano-island arrays would be investigated. The biomolecules (adenine) were dropped on the well-designed SERS substrate for precise and quantitative SERS detection. The characterization of Au nano-island arrays SERS substrate would be evaluated by scanning electron microscope (SEM) and Raman spectroscopy. The results showed that the optimal deposition thickness and annealing temperature of Au nano-island arrays SERS substrate is about 14 nm and 200 °C respectively, which can construct the smallest interparticle spacing (W)/ particle diameter (D) ratio and the lowest reflection (%) and transmittance (%) to form the strongest SERS intensity. Moreover, finite-difference time-domain (FDTD) simulation of the electromagnetic field distributions on Au nano-island arrays displays the similar trend with the experimental results. The 14 nm deposition with 200 °C in-situ annealing temperature would display the highest density of hot-spots by FDTD simulation. The reproducible Au nano-island arrays SERS substrates with tunable surface roughness, W/D ratio, and lower reflection and transmittance show promising potential for SERS detection of biomolecules, bacteria, and viruses.

Keratin-Positive Giant Cell-Rich Tumor of Bone Harboring an HMGA2::NCOR2 Fusion: Two Cases, Including a Patient With Metastatic Disease, and Review of the Literature.

Giant cell-rich lesions of bone represent a heterogeneous group of entities which classically include giant cell tumor of bone, aneurysmal bone cyst, nonossifying fibroma, and Brown tumor of hyperparathyroidism. A recently described subset of giant cell-rich tumors involving bone and soft tissue has been characterized by recurrent HMGA2::NCOR2 fusions and keratin expression. The overlapping clinical, radiographic, and morphological features of these giant cell-rich lesions provide a unique diagnostic challenge, particularly on biopsy. We present 2 additional cases of keratin-positive giant cell-rich tumor of bone with HMGA2::NCOR2 fusions, including 1 patient who developed metastatic disease.

Proteome reveals the mechanism of selenium-sulfur interaction in regulating isothiocyanate biosynthesis and the physiological metabolism of broccoli sprouts.

Broccoli sprouts have a strong ability to accumulate isothiocyanate and selenium. In this study, the isothiocyanate content increased significantly as a result of ZnSO4 stress. Particularly, based on the isothiocyanate content is not affected, the combined ZnSO4 and Na2SeO3 treatment alleviated the inhibition of ZnSO4 and induced selenium content. Gene transcription and protein expression analyses revealed the changes in isothiocyanate and selenium metabolite levels in broccoli sprouts. ZnSO4 combined with Na2SeO3 was proven to activate a series of isothiocyanate metabolite genes (UGT74B1, OX1, and ST5b) and selenium metabolite genes (BoSultr1;1, BoCOQ5-2, and BoHMT1). The relative abundance of the total 317 and 203 proteins, respectively, in 4-day-old broccoli sprouts varied, and the metabolic and biosynthetic pathways for secondary metabolites were significantly enriched in ZnSO4/control and ZnSO4 combined Na2SeO3/ZnSO4 comparisons. The findings demonstrated how ZnSO4 combined with Na2SeO3 treatment reduced stress inhibition and the accumulation of encouraged selenium and isothiocyanates during the growth of broccoli sprouts.

Intra-host mutation rate of acute SARS-CoV-2 infection during the initial pandemic wave.

SARS-CoV-2 mutation is minimized through a proofreading function encoded by NSP-14. Most estimates of the SARS-CoV-2 mutation rate are derived from population based sequence data. Our understanding of SARS-CoV-2 evolution might be enhanced through analysis of intra-host viral mutation rates in specific populations. Viral genome analysis was performed between paired samples and mutations quantified at allele frequencies (AF) ≥ 0.25, ≥ 0.5 and ≥ 0.75. Mutation rate was determined employing F81 and JC69 evolution models and compared between isolates with (ΔNSP-14) and without (wtNSP-14) non-synonymous mutations in NSP-14 and by patient comorbidity. Forty paired samples with median interval of 13 days [IQR 8.5-20] were analyzed. The estimated mutation rate by F81 modeling was 93.6 (95%CI 90.8-96.4], 40.7 (95%CI 38.9-42.6) and 34.7 (95%CI 33.0-36.4) substitutions/genome/year at AF ≥ 0.25, ≥ 0.5, ≥ 0.75 respectively. Mutation rate in ΔNSP-14 were significantly elevated at AF ≥ 0.25 vs wtNSP-14. Patients with immune comorbidities had higher mutation rate at all allele frequencies. Intra-host SARS-CoV-2 mutation rates are substantially higher than those reported through population analysis. Virus strains with altered NSP-14 have accelerated mutation rate at low AF. Immunosuppressed patients have elevated mutation rate at all AF. Understanding intra-host virus evolution will aid in current and future pandemic modeling.

iTRAQ-Based Proteomic Analyses of Regulation of Isothiocyanate and Endogenous Selenium Metabolism in Broccoli Sprouts by Exogenous Sodium Selenite.

Broccoli sprouts have high isothiocyanate and selenium accumulation capacity. This study used a combination of methods, including physiological and biochemical, gene transcription and proteomic, to investigate the isothiocyanate and endogenous selenium accumulation mechanisms in broccoli sprouts under exogenous sodium selenite treatment during germination. Compared with the control, the sprouts length of broccoli sprouts under exogenous selenium treatment was significantly lower, and the contents of total phenol and malondialdehyde in 6-day-old broccoli sprouts were substantially higher. The contents of isothiocyanate and sulforaphane in 4-day-old were increased by up-regulating the relative expression of genes of UGT74B1, OX-1, and ST5b. The relative expression of BoSultr1;1, BoSMT, BoHMT1, and BoCOQ5-2 genes regulating selenium metabolism was significantly up-regulated. In addition, 354 proteins in 4-day-old broccoli sprouts showed different relative abundance compared to the control under selenium treatment. These proteins were classified into 14 functional categories. It was discovered that metabolic pathways and biosynthetic pathways of secondary metabolites were significantly enriched. The above results showed that exogenous selenium was beneficial in inducing the accumulation of isothiocyanate and selenium during the growth of broccoli sprouts.

Application of the network scale-up method to estimate the sizes of key populations for HIV in Singapore using online surveys.

INTRODUCTION: Singapore lacks robust data on the sizes of the key populations that are most at risk for HIV. Using the network scale-up method for hidden or hard-to-reach populations, we estimate the sizes of five key populations-male clients of female sex workers (MCFSW), men who have sex with men (MSM), female sex workers (FSW), people who inject drugs (PWID) and transgender people-and profile the ages and ethnicities of respondents with the high-risk contacts they report knowing. METHODS: We conducted a cross-sectional online survey between March and May 2019 (n = 2802) using a network scale-up instrument previously developed for Singapore. Participants were recruited using an existing panel and online advertising, and the sample reweighted by age, sex, ethnicity and education attained to represent the general adult population. We built a Bayesian hierarchical model to estimate the sizes of the five key populations for HIV in Singapore. RESULTS: After adjustment, the sizes of the at-risk populations are estimated to be: 76,800 (95% credible interval [CI]: 64,200-91,800) MCFSW; 139,000 (95% CI: 120,000-160,000) MSM; 8030 (95% CI: 3980-16,200) FSW; 3470 (95% CI: 1540-7830) PWID and 18,000 (95% CI: 14,000-23,200) transgender people. Generally, men reported knowing more people in all the high-risk groups; older people reported knowing more MCFSW, FSW and transgender people; and younger people reported knowing more MSM. There was a bimodal effect of age on those who reported knowing more PWIDs: people in their 20s and 60s reported more contacts. CONCLUSIONS: This study demonstrates that a size estimation study of hidden populations is quickly and efficiently scalable through using online surveys in a socially conservative society, like Singapore, where key populations are stigmatized or criminalized. The approach may be suitable in other countries where stigma is prevalent and where barriers to surveillance and data collection are numerous.

The role of medication adherence in the association between depressive symptoms and quality of life in older adults with type 2 diabetes mellitus.

BACKGROUND: At present, the role of medication adherence in the association between depressive symptoms and quality of life (QOL) in older adults with type 2 diabetes mellitus (T2DM) was unclear. The purpose of this study was to explore the associations among depressive symptoms, medication adherence and QOL in older adults with T2DM. METHODS: In this cross-sectional study, 300 older adults with T2DM from the First Affiliated Hospital of Anhui Medical University were enrolled. Among them, 115 patients had depressive symptoms and 185 had no depressive symptoms. Univariate linear regression analysis was conducted to identify possible covariates. Univariate and multivariable linear regression analyses were performed to explore the associations between depressive symptoms and medication adherence or QOL in older adults with T2DM. Multiplicative interaction analysis was evaluated whether there was interaction effect between medication adherence and depressive symptoms on QOL of patients. Mediating effect analysis was used to analyze the medication effect of medication adherence on depressive symptoms and QOL in older adults with T2DM. RESULTS: Decreased medication adherence was observed in patients with depressive symptoms (ß = -0.67, 95%CI: -1.10, -0.24) after adjusting for covariates. Depressive symptoms were associated with decreased QOL in older adults with T2DM (ß = -5.99, 95%CI: -7.56, -4.42). The mediating analysis revealed that depressive symptoms were associated with decreased medication adherence (ß = -0.67, 95%CI: -1.09, -0.25). Medication adherence was linked with increased QOL of older adults with T2DM (ß = 0.65, 95%CI: 0.24, 1.06). Depressive symptoms were correlated with decreased QOL of older adults with T2DM (ß = -5.56, 95%CI: -7.10, -4.01). The percentage mediated by medication adherence on depressive symptoms and QOL in older adults with T2DM was 10.61%. CONCLUSION: Medication adherence might mediate depressive symptoms and QOL of older adults with T2DM, which might provide a reference for the improvement of QOL of these patients.

Biallelic TET2 mutations and canonical ASXL1 mutations are frequent and cooccur in Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN): An institutional experience and review of literature.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is recurrently mutated in epigenetic pathway genes. We studied the myeloid-related genetic mutations in a cohort of five patients with BPDCN and one paired relapse case at our institution and identified a high frequency of biallelic TET2 and canonical ASXL1 (c.1934dupG) mutations. The number of cases is small, but the variant allele fraction (VAF) sums of the TET2 mutations, as well as the persistence of TET2 mutations in a case of relapsed BPDCN, suggest an ancestral/founder nature of TET2 clones in the cases. Further literature review shows a high frequency of biallelic TET2 mutations in reported cases of BPDCN.

Highly Sensitive Blocker Displacement Amplification for Detection of Low-Level JAK2V617F Variant.

BACKGROUND: Key criteria in the diagnostic workup and risk stratification for myeloproliferative neoplasms (MPN) include molecular testing for JAK2V617F and other mutant alleles. Multiple methods for quantitatively detecting nucleotide sequence changes exist, but the lower limit of detection can limit identification of the low-level allele fraction of a variant. We evaluated a recently developed blocker displacement amplification (BDA)-based quantitative PCR platform for detection and quantitation of JAK2V617F variant allele fraction (VAF). METHODS: Clinical samples were tested using BDA, next-generation sequencing (NGS), and droplet digital PCR (ddPCR) in a head-to-head comparison of sensitivity and specificity in detecting the JAK2V617F variant. In total, 112 human genomic DNA specimens previously tested for JAK2V617F gene mutation status with NGS were analyzed, including 12 samples with low-level variants with VAF ≤2%, 6 samples with VAF >2%, and 94 samples with no variant previously identified by NGS. RESULTS: BDA and ddPCR results correlated well across a range of VAFs, with both methods identifying the JAK2V617F variant down to at least 0.05% VAF. NGS of routine sequencing depth was less sensitive, identifying JAK2V617F only at 0.6% VAF. CONCLUSIONS: BDA can provide a cost-effective alternative means to identify low-level variants using instrumentation commonly found in laboratories.

Renal Genetics Clinic: 3-Year Experience in the Cleveland Clinic.

Rationale & Objective: There has been an increasing demand for the expertise provided by a renal genetics clinic. Such programs are limited in the United States and typically operate in a genomics research setting. Here we report a 3-year, real-world, single-center renal genetics clinic experience. Study Design: Retrospective cohort. Setting & Participants: Outpatient cases referred to the renal genetics clinic of the Cleveland Clinic between January 2019 and March 2022 were reviewed. Analytical Approach: Clinical and laboratory characteristics were analyzed. All genetic testing was performed in clinical labs. Results: 309 new patients referred from 15 specialties were evaluated, including 118 males and 191 females aged 35.1 ± 20.3 years. Glomerular diseases were the leading presentation followed by cystic kidney diseases, electrolyte disorders, congenital anomalies of kidneys and urinary tract, nephrolithiasis, and tubulointerstitial kidney diseases. Dysmorphic features were noted in 27 (8.7%) patients. Genetic testing was recommended in 292 (94.5%) patients including chromosomal microarray (8.9%), single-gene tests (19.5%), multigene panels (77.3%), and exome sequencing (17.5%). 80.5% of patients received insurance coverage for genetic testing. 45% (115/256) of patients had positive results, 25% (64/256) had variants of unknown significance, and 22.3% (57/256) had negative results. 43 distinct monogenic disorders were diagnosed. Family history of kidney disease was present in 52.8% of patients and associated with positive genetic findings (OR, 2.28; 95% CI, 1.40-3.74). 69% of patients with positive results received a new diagnosis and/or a change in the diagnosis. Among these, 39.7% (31/78) of patients received a significant change in disease management. Limitations: Retrospective and single-center study. Conclusions: The renal genetics clinic plays important roles in the diagnosis and management of patients with genetic kidney diseases. Multigene panels are the most frequently used testing modality with a high diagnostic yield. Family history of kidney disease is a strong indication for renal genetics clinic referral.

Biphenotypic Sinonasal Sarcoma with a Novel PAX3::FOXO6 Fusion: A Case Report and Review of the Literature.

BACKGROUND: Biphenotypic sinonasal sarcoma (BSS) is a low-grade, locally aggressive sarcoma unique to the sinonasal region. BSS is most common in middle aged patients and affects women more frequently than men. It is characterized by a bland spindled cell proliferation with neural and myogenic differentiation. BSS are usually associated with rearrangement t(2;4)(q35;q31.1) resulting in a PAX3::MAML3 fusion. Less commonly, other genes are found in combination with PAX3 and some cases reported in the literature have an unknown fusion partner. METHODS: A 54-year-old man presented with nasal mass. Endoscopic resection showed a low-grade spindle cell neoplasm with morphologic features of BSS and immunohistochemical and next generation sequencing were performed to confirm the diagnosis. RESULTS: The tumor was positive for S100 and smooth muscle actin but negative for SOX10. Next generation sequencing demonstrated a novel PAX3::FOXO6 gene fusion. CONCLUSIONS: Although a PAX3::FOXO6 gene fusion has never been reported, this finding combined with the morphologic and immunophenotypic features supports the diagnosis of supports the diagnosis of BSS.

Alpha to Omicron: Disease Severity and Clinical Outcomes of Major SARS-CoV-2 Variants.

BACKGROUND: Four severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants predominated in the United States since 2021. Understanding disease severity related to different SARS-CoV-2 variants remains limited. METHOD: Viral genome analysis was performed on SARS-CoV-2 clinical isolates circulating March 2021 through March 2022 in Cleveland, Ohio. Major variants were correlated with disease severity and patient outcomes. RESULTS: In total 2779 patients identified with either Alpha (n 1153), Gamma (n 122), Delta (n 808), or Omicron variants (n 696) were selected for analysis. No difference in frequency of hospitalization, intensive care unit (ICU) admission, and death were found among Alpha, Gamma, and Delta variants. However, patients with Omicron infection were significantly less likely to be admitted to the hospital, require oxygen, or admission to the ICU (2 12.8, P .001; 2 21.6, P .002; 2 9.6, P .01, respectively). In patients whose vaccination status was known, a substantial number had breakthrough infections with Delta or Omicron variants (218/808 [26.9] and 513/696 [73.7], respectively). In breakthrough infections, hospitalization rate was similar regardless of variant by multivariate analysis. No difference in disease severity was identified between Omicron subvariants BA.1 and BA.2. CONCLUSIONS: Disease severity associated with Alpha, Gamma, and Delta variants is comparable while Omicron infections are significantly less severe. Breakthrough disease is significantly more common in patients with Omicron infection.

ERK and c-Myc signaling in host-derived tumor endothelial cells is essential for solid tumor growth.

The limited efficacy of the current antitumor microenvironment strategies is due in part to the poor understanding of the roles and relative contributions of the various tumor stromal cells to tumor development. Here, we describe a versatile in vivo anthrax toxin protein delivery system allowing for the unambiguous genetic evaluation of individual tumor stromal elements in cancer. Our reengineered tumor-selective anthrax toxin exhibits potent antiproliferative activity by disrupting ERK signaling in sensitive cells. Since this activity requires the surface expression of the capillary morphogenesis protein-2 (CMG2) toxin receptor, genetic manipulation of CMG2 expression using our cell-type-specific CMG2 transgenic mice allows us to specifically define the role of individual tumor stromal cell types in tumor development. Here, we established mice with CMG2 only expressed in tumor endothelial cells (ECs) and determined the specific contribution of tumor stromal ECs to the toxin's antitumor activity. Our results demonstrate that disruption of ERK signaling only within tumor ECs is sufficient to halt tumor growth. We discovered that c-Myc is a downstream effector of ERK signaling and that the MEK-ERK-c-Myc central metabolic axis in tumor ECs is essential for tumor progression. As such, disruption of ERK-c-Myc signaling in host-derived tumor ECs by our tumor-selective anthrax toxins explains their high efficacy in solid tumor therapy.

Identification of Novel MET Exon 14 Skipping Variants in Non-Small Cell Lung Cancer Patients: A Prototype Workflow Involving in Silico Prediction and RT-PCR.

Background and aims: The MET exon 14 skipping (METex14) is an oncogenic driver mutation that provides a therapeutic opportunity in non-small cell lung cancer (NSCLCs) patients. This event often results from sequence changes at the MET canonical splicing sites. We characterize two novel non-canonical splicing site variants of MET that produce METex14. Materials and Methods: Two variants were identified in three advanced-stage NSCLC patients in a next-generation sequencing panel. The potential impact on splicing was predicted using in silico tools. METex14 mutation was confirmed using reverse transcription (RT)-PCR and a Sanger sequencing analysis on RNA extracted from stained cytology smears. Results: The interrogated MET (RefSeq ID NM_000245.3) variants include a single nucleotide substitution, c.3028+3A>T, in intron 14 and a deletion mutation, c.3012_3028del, in exon 14. The in silico prediction analysis exhibited reduced splicing strength in both variants compared with the MET normal transcript. The RT-PCR and subsequent Sanger sequencing analyses confirmed METex14 skipping in all three patients carrying these variants. Conclusion: This study reveals two non-canonical MET splice variants that cause exon 14 skipping, concurrently also proposes a clinical workflow for the classification of such non-canonical splicing site variants detected by routine DNA-based NGS test. It shows the usefulness of in silico prediction to identify potential METex14 driver mutation and exemplifies the opportunity of routine cytology slides for RNA-based testing.