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.

Novel CRTC1::MRTFB(MKL2) Gene Fusion Detected in Myxoid Mesenchymal Neoplasms With Myogenic Differentiation Involving Bone and Soft Tissues.

Appropriate classification of fusion-driven bone and soft tissue neoplasms continues to evolve, often relying on the careful integration of morphologic findings with immunohistochemical, molecular, and clinical data. Herein, we present 3 cases of a morphologically distinct myxoid mesenchymal neoplasm with myogenic differentiation and novel CRTC1::MRTFB (formerly MKL2) gene fusion. Three tumors occurred in 1 male and 2 female patients with a median age of 72 years (range: 28-78). Tumors involved the left iliac bone, the right thigh, and the left perianal region with a median size of 4.0 cm (4.0-7.6 cm). Although 1 tumor presented as an incidental finding, the other 2 tumors were noted, given their persistent growth. At the time of the last follow-up, 1 patient was alive with unresected disease at 6 months, 1 patient was alive without evidence of disease at 12 months after surgery, and 1 patient died of disease 24 months after diagnosis. On histologic sections, the tumors showed multinodular growth and were composed of variably cellular spindle to round-shaped cells with distinct brightly eosinophilic cytoplasm embedded within a myxoid stroma. One tumor showed overt smooth muscle differentiation. Cytologic atypia and mitotic activity ranged from minimal (2 cases) to high (1 case). By immunohistochemistry, the neoplastic cells expressed focal smooth muscle actin, h-caldesmon, and desmin in all tested cases. Skeletal muscle markers were negative. Next-generation sequencing detected nearly identical CRTC1::MRTFB gene fusions in all cases. We suggest that myxoid mesenchymal tumors with myogenic differentiation harboring a CRTC1::MRTFB fusion may represent a previously unrecognized, distinctive entity that involves soft tissue and bone. Continued identification of these novel myxoid neoplasms with myogenic differentiation will be important in determining appropriate classification, understanding biologic potential, and creating treatment paradigms.

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.

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.

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.

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.

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.

Investigation on the characteristics of gut microbiota in critically endangered blue-crowned laughingthrush (Garrulax courtoisi).

Blue-crowned laughingthrush (Garrulax courtoisi), passeriformes, is a critically endangered bird endemic to China. Gut microbiota is well known to play a pivotal role in host health and survival. Thus, the understanding of the microbial communities associated with Garrulax courtoisi could be beneficial to save this species from the brink of extinction. In this study, we used 16 s rDNA amplicon sequencing to investigate the gut community composition and microbial diversity of the Garrulax courtoisi population reared in Nanchang Zoo. The results showed that there were 31 phyla that were dominated by Firmicutes, Proteobacteria, Bacteroidetes, and Cyanobacteria in the intestine of Garrulax courtoisi. Compared with previous studies on birds, the Cyanobacteria exhibited an excessive abundance, which may be largely related to the personal lifestyle of Garrulax courtoisi. At the genus level, a total of 552 genera were identified, among which, 21 key genera constituted the core microbiome, including some culturable bacterial genera such as Lactobacillus, Acinetobacter, and Deinococcus. In the meanwhile, we found that there were remarkable intraspecific differences both in terms of microbial community structures, representative biomarkers and predicted functions between the parental generation and their offspring of the population investigated in this study. Furthermore, we also summarized their different eating behaviors and predicted its association with gut microbiota. This study provided the needed pieces of information about these extremely rare birds, Garrulax courtoisi, whose community composition and microbial diversity are hardly known. Importantly, these findings could contribute to our knowledge of the gut health of Garrulax courtoisi and advance the comprehensive conservation of this endangered bird.

An auto-antibody identified from phenotypic directed screening platform shows host immunity against EV-A71 infection.

BACKGROUND: Enterovirus A71 (EV-A71) is a neurotropic virus which may cause severe neural complications, especially in infants and children. The clinical manifestations include hand-foot-and-mouth disease, herpangina, brainstem encephalitis, pulmonary edema, and other severe neurological diseases. Although there are some vaccines approved, the post-marketing surveillance is still unavailable. In addition, there is no antiviral drugs against EV-A71 available. METHODS: In this study, we identified a novel antibody that could inhibit viral growth through a human single chain variable fragment (scFv) library expressed in mammalian cells and panned by infection with lethal dose of EV-A71. RESULTS: We identified that the host protein α-enolase (ENO1) is the target of this scFv, and anti-ENO1 antibody was found to be more in mild cases than severe EV-A71 cases. Furthermore, we examined the antiviral activity in a mouse model. We found that the treatment of the identified 07-human IgG1 antibody increased the survival rate after virus challenge, and significantly decreased the viral RNA and the level of neural pathology in brain tissue. CONCLUSIONS: Collectively, through a promising intracellular scFv library expression and screening system, we found a potential scFv/antibody which targets host protein ENO1 and can interfere with the infection of EV-A71. The results indicate that the usage and application of this antibody may offer a potential treatment against EV-A71 infection.

Rapid SARS-CoV-2 Spike Protein Detection by Carbon Nanotube-Based Near-Infrared Nanosensors.

To effectively track and eliminate COVID-19, it is critical to develop tools for rapid and accessible diagnosis of actively infected individuals. Here, we introduce a single-walled carbon nanotube (SWCNT)-based optical sensing approach toward this end. We construct a nanosensor based on SWCNTs noncovalently functionalized with ACE2, a host protein with high binding affinity for the SARS-CoV-2 spike protein. The presence of the SARS-CoV-2 spike protein elicits a robust, 2-fold nanosensor fluorescence increase within 90 min of spike protein exposure. We characterize the nanosensor stability and sensing mechanism and passivate the nanosensor to preserve sensing response in saliva and viral transport medium. We further demonstrate that these ACE2-SWCNT nanosensors retain sensing capacity in a surface-immobilized format, exhibiting a 73% fluorescence turn-on response within 5 s of exposure to 35 mg/L SARS-CoV-2 virus-like particles. Our data demonstrate that ACE2-SWCNT nanosensors can be developed into an optical tool for rapid SARS-CoV-2 detection.

Pre-HAART CD4+ T-lymphocytes as biomarkers of post-HAART immune recovery in HIV-infected children with or without TB co-infection.

BACKGROUND: Infection with the Human Immunodeficiency Virus (HIV) dramatically increases the risk of developing active tuberculosis (TB). Several studies have indicated that co-infection with TB increases the risk of HIV progression and death. Sub-Saharan Africa bears the brunt of these dual epidemics, with about 2.4 million HIV-infected people living with TB. The main objective of our study was to assess whether the pre-HAART CD4+ T-lymphocyte counts and percentages could serve as biomarkers for post-HAART treatment immune-recovery in HIV-positive children with and without TB co-infection. METHODS: The data analyzed in this retrospective study were collected from a cohort of 305 HIV-infected children being treated with HAART. A Lehmann family of ROC curves were used to assess the diagnostic performance of pre- HAART treatment CD4+ T-lymphocyte count and percentage as biomarkers for post-HAART immune recovery. The Kaplan-Meier estimator was used to compare differences in post-HAART recovery times between patients with and without TB co-infection. RESULTS: We found that the diagnostic performance of both pre-HARRT treatment CD4+ T-lymphocyte count and percentage was comparable and achieved accuracies as high as 74%. Furthermore, the predictive capability of pre-HAART CD4+ T-lymphocyte count and percentage were slightly better in TB-negative patients. Our analyses also indicate that TB-negative patients have a shorter recovery time compared to the TB-positive patients. CONCLUSIONS: Pre-HAART CD4+ T-lymphocyte count and percentage are stronger predictors of immune recovery in TB-negative pediatric patients, suggesting that TB co-infection complicates the treatment of HIV in this cohort. These findings suggest that the detection and treatment of TB is essential for the effectiveness of HAART in HIV-infected pediatric patients.

Risk factors associated with death in infants <120 days old with severe pertussis: a case-control study.

BACKGROUND AND PURPOSE: Pertussis is a serious infectious disease in young infants, and severe cases frequently cause death. Our study explored risk factors for death from severe pertussis. METHOD: A case-control study of infants with severe pertussis admitted to the paediatric intensive care unit (PICU) in the Children's Hospital of Chongqing Medical University, China, from January 1, 2013, to June 30, 2019, was conducted. Pertussis was confirmed by clinical features and laboratory examinations. Severe pertussis was defined as patients with pertussis resulting in PICU admission or death. To understand the risk factors for death, we compared fatal and nonfatal cases of severe pertussis in infants aged < 120 days by collecting clinical and laboratory data. RESULTS: The participants included 63 infants < 120 days of age with severe pertussis. Fifteen fatal cases were confirmed and compared with 44 nonfatal severe pertussis cases, Four patients with termination of treatment were excluded. In the univariate analysis, the risk factors associated with death included apnoea (P = 0.001), leukocytosis (white blood cell (WBC) count≥30 × 109/L (P = 0.001) or ≥ 50 × 109/L (P = 0)), highest lymphocyte count (P = 0), pulmonary hypertension (P = 0.001), and length of PICU stay (P = 0.003). The multivariate analysis revealed that apnoea (OR 23.722, 95%CI 2.796-201.26, P = 0.004), leukocytosis (OR 63.708, 95%CI 3.574-1135.674, P = 0.005) and pulmonary hypertension (OR 26.109, 95%CI 1.800-378.809, P = 0.017) were significantly associated with death. CONCLUSION: Leukocytosis and pulmonary hypertension exhibited the greatest associations with death in infants with severe pertussis admitted to the PICU. Vaccination is still the most effective protection method against pertussis.

An Accurate and Comprehensive Clinical Sequencing Assay for Cancer Targeted and Immunotherapies.

BACKGROUND: Incorporation of next-generation sequencing (NGS) technology into clinical utility in targeted and immunotherapies requires stringent validation, including the assessment of tumor mutational burden (TMB) and microsatellite instability (MSI) status by NGS as important biomarkers for response to immune checkpoint inhibitors. MATERIALS AND METHODS: We designed an NGS assay, Cancer Sequencing YS panel (CSYS), and applied algorithms to detect five classes of genomic alterations and two genomic features of TMB and MSI. RESULTS: By stringent validation, CSYS exhibited high sensitivity and predictive positive value of 99.7% and 99.9%, respectively, for single nucleotide variation; 100% and 99.9%, respectively, for short insertion and deletion (indel); and 95.5% and 100%, respectively, for copy number alteration (CNA). Moreover, CSYS achieved 100% specificity for both long indel (50-3,000 bp insertion and deletion) and gene rearrangement. Overall, we used 33 cell lines and 208 clinical samples to validate CSYS's NGS performance, and genomic alterations in clinical samples were also confirmed by fluorescence in situ hybridization, immunohistochemistry, and polymerase chain reaction (PCR). Importantly, the landscape of TMB across different cancers of Chinese patients (n = 3,309) was studied. TMB by CSYS exhibited a high correlation (Pearson correlation coefficient r = 0.98) with TMB by whole exome sequencing (WES). MSI measurement showed 98% accuracy and was confirmed by PCR. Application of CSYS in a clinical setting showed an unexpectedly high occurrence of long indel (6.3%) in a cohort of tumors from Chinese patients with cancer (n = 3,309), including TP53, RB1, FLT3, BRCA2, and other cancer driver genes with clinical impact. CONCLUSION: CSYS proves to be clinically applicable and useful in disclosing genomic alterations relevant to cancer target therapies and revealing biomarkers for immune checkpoint inhibitors. IMPLICATIONS FOR PRACTICE: The study describes a specially designed sequencing panel assay to detect genomic alterations and features of 450 cancer genes, including its overall workflow and rigorous clinical and analytical validations. The distribution of pan-cancer tumor mutational burden, microsatellite instability, gene rearrangement, and long insertion and deletion mutations was assessed for the first time by this assay in a broad array of Chinese patients with cancer. The Cancer Sequencing YS panel and its validation study could serve as a blueprint for developing next-generation sequencing-based assays, particularly for the purpose of clinical application.

A Genome-Wide Association Study to Identify Single-Nucleotide Polymorphisms for Acute Kidney Injury.

RATIONALE: Acute kidney injury is a common and severe complication of critical illness and cardiac surgery. Despite significant attempts at developing treatments, therapeutic advances to attenuate acute kidney injury and expedite recovery have largely failed. OBJECTIVES: Identifying genetic loci associated with increased risk of acute kidney injury may reveal novel pathways for therapeutic development. METHODS: We conducted an exploratory genome-wide association study to identify single-nucleotide polymorphisms associated with genetic susceptibility to in-hospital acute kidney injury. MEASUREMENTS AND MAIN RESULTS: We genotyped 609,508 single-nucleotide polymorphisms and performed genotype imputation in 760 acute kidney injury cases and 669 controls. We then evaluated polymorphisms that showed the strongest association with acute kidney injury in a replication patient population containing 206 cases with 1,406 controls. We observed an association between acute kidney injury and four single-nucleotide polymorphisms at two independent loci on metaanalysis of discovery and replication populations. These include rs62341639 (metaanalysis P = 2.48 × 10-7; odds ratio [OR], 0.64; 95% confidence interval [CI], 0.55-0.76) and rs62341657 (P = 3.26 × 10-7; OR, 0.65; 95% CI, 0.55-0.76) on chromosome 4 near APOL1-regulator IRF2, and rs9617814 (metaanalysis P = 3.81 × 10-6; OR, 0.70; 95% CI, 0.60-0.81) and rs10854554 (P = 6.53 × 10-7; OR, 0.67; 95% CI, 0.57-0.79) on chromosome 22 near acute kidney injury-related gene TBX1. CONCLUSIONS: Our findings reveal two genetic loci that are associated with acute kidney injury. Additional studies should be conducted to functionally evaluate these loci and to identify other common genetic variants contributing to acute kidney injury.

Comprehensive genomic profiling of different subtypes of nasopharyngeal carcinoma reveals similarities and differences to guide targeted therapy.

BACKGROUND: To date, no targeted therapy has been approved for nasopharyngeal carcinoma (NPC), and this underscores the need for an in-depth understanding of clinically relevant genomic alterations (CRGAs). METHODS: Comprehensive genomic profiling was performed for 190 NPC patients, including 20 patients with nasopharyngeal adenocarcinoma (NPAC), 62 patients with nasopharyngeal squamous cell carcinoma (NPSCC), and 108 patients with nasopharyngeal undifferentiated carcinoma (NPUC). The associations of genes and pathways with subtypes, Epstein-Barr virus (EBV) infections, and the tumor mutation burden (TMB) were statistically evaluated. RESULTS: Although the overall rates of genomic alterations were similar, the 3 NPC subtypes exhibited different mutational landscapes. Notably, mutations in a proven-treatable target gene, isocitrate dehydrogenase 2 (IDH2), were significantly associated with NPUC but not with NPAC or NPSCC. The top 5 ranked CRGAs included CDKN2A (29%), IDH2 (16%), SMARCB1 (7%), PIK3CA (6%), and NF1 (5%) in NPUC; CDKN2A (27%), PIK3CA (23%), FBXW7 (11%), PTEN (11%), and EGFR (8%) in NPSCC; and CDKN2A (20%), KRAS (15%), CCND1 (10%), MAP3K1 (10%), and NOTCH1 (10%) in NPAC. The incidence of EBV infections significantly correlated with the subtypes and with TP53, CDKN2A, and CDKN2B. The TMB status correlated with the subtypes and with LRP1B, FBXW7, and PIK3CA mutations as well as DNA repair, phosphoinositide 3-kinase/mammalian target of rapamycin, and mitogen-activated protein kinase pathways. CONCLUSIONS: These results indicate that different NPC subtypes harbor different CRGAs. Both EBV infections and the TMB are associated with the NPC subtypes as well as the alterations of individual genes and pathways. The high frequency of IDH2 mutations in NPUC may facilitate potential targeted therapy and will ultimately point to new therapeutic strategies. Cancer 2017;123:3628-37. © 2017 American Cancer Society.

Colorectal carcinomas with submucosal invasion (pT1): analysis of histopathological and molecular factors predicting lymph node metastasis.

Submucosally invasive colorectal carcinoma (pT1) has the potential to be cured by local excision. In US surgical intervention is reserved for tumors with high-grade morphology, lymphvascular invasion, and close/positive margin. In other countries, particularly Japan, surgical therapy is also recommended for mucinous tumors, tumors with >1000 µm of submucosal invasion, and those with high tumor budding. These histological features have not been well evaluated in a western cohort of pT1 carcinomas. In a cohort of 116 surgically resected pT1 colorectal carcinomas, high tumor budding (P<0.001), lymphatic invasion (P=0.003), depth of submucosal invasion >1000 µm (P=0.04), and high-grade morphology (P=0.04) were significantly associated with lymph node metastasis on univariate analysis. Mucinous differentiation, tumor location, tumor growth pattern, and size of invasive component were not significant. On multivariate analysis, only high tumor budding was associated with lymph node metastasis with an odds ratio of 4.3 (P=0.004). A subset of 48 tumors (22 node-positive and 26 node-negative) was analyzed for mutations in 50 oncogenes and tumor suppressors. No statistically significant molecular alterations in these 50 genes were associated with lymph node status. However, lymphatic invasion was associated with BRAF mutations (P=0.01). Furthermore, high tumor budding was associated with mutations in TP53 (P=0.03) and inversely associated with mutations in the mTOR pathway (PIK3CA and AKT, P=0.02). In conclusion, this study demonstrates the importance of identifying high tumor budding in pT1 carcinomas when considering additional surgical resection. Molecular alterations associated with adverse histological features are identified.

Leveraging protein quaternary structure to identify oncogenic driver mutations.

BACKGROUND: Identifying key "driver" mutations which are responsible for tumorigenesis is critical in the development of new oncology drugs. Due to multiple pharmacological successes in treating cancers that are caused by such driver mutations, a large body of methods have been developed to differentiate these mutations from the benign "passenger" mutations which occur in the tumor but do not further progress the disease. Under the hypothesis that driver mutations tend to cluster in key regions of the protein, the development of algorithms that identify these clusters has become a critical area of research. RESULTS: We have developed a novel methodology, QuartPAC (Quaternary Protein Amino acid Clustering), that identifies non-random mutational clustering while utilizing the protein quaternary structure in 3D space. By integrating the spatial information in the Protein Data Bank (PDB) and the mutational data in the Catalogue of Somatic Mutations in Cancer (COSMIC), QuartPAC is able to identify clusters which are otherwise missed in a variety of proteins. The R package is available on Bioconductor at: http://bioconductor.jp/packages/3.1/bioc/html/QuartPAC.html . CONCLUSION: QuartPAC provides a unique tool to identify mutational clustering while accounting for the complete folded protein quaternary structure.

A graph theoretic approach to utilizing protein structure to identify non-random somatic mutations.

BACKGROUND: It is well known that the development of cancer is caused by the accumulation of somatic mutations within the genome. For oncogenes specifically, current research suggests that there is a small set of "driver" mutations that are primarily responsible for tumorigenesis. Further, due to recent pharmacological successes in treating these driver mutations and their resulting tumors, a variety of approaches have been developed to identify potential driver mutations using methods such as machine learning and mutational clustering. We propose a novel methodology that increases our power to identify mutational clusters by taking into account protein tertiary structure via a graph theoretical approach. RESULTS: We have designed and implemented GraphPAC (Graph Protein Amino acid Clustering) to identify mutational clustering while considering protein spatial structure. Using GraphPAC, we are able to detect novel clusters in proteins that are known to exhibit mutation clustering as well as identify clusters in proteins without evidence of prior clustering based on current methods. Specifically, by utilizing the spatial information available in the Protein Data Bank (PDB) along with the mutational data in the Catalogue of Somatic Mutations in Cancer (COSMIC), GraphPAC identifies new mutational clusters in well known oncogenes such as EGFR and KRAS. Further, by utilizing graph theory to account for the tertiary structure, GraphPAC discovers clusters in DPP4, NRP1 and other proteins not identified by existing methods. The R package is available at: http://bioconductor.org/packages/release/bioc/html/GraphPAC.html. CONCLUSION: GraphPAC provides an alternative to iPAC and an extension to current methodology when identifying potential activating driver mutations by utilizing a graph theoretic approach when considering protein tertiary structure.

A spatial simulation approach to account for protein structure when identifying non-random somatic mutations.

BACKGROUND: Current research suggests that a small set of "driver" mutations are responsible for tumorigenesis while a larger body of "passenger" mutations occur in the tumor but do not progress the disease. Due to recent pharmacological successes in treating cancers caused by driver mutations, a variety of methodologies that attempt to identify such mutations have been developed. Based on the hypothesis that driver mutations tend to cluster in key regions of the protein, the development of cluster identification algorithms has become critical. RESULTS: We have developed a novel methodology, SpacePAC (Spatial Protein Amino acid Clustering), that identifies mutational clustering by considering the protein tertiary structure directly in 3D space. By combining the mutational data in the Catalogue of Somatic Mutations in Cancer (COSMIC) and the spatial information in the Protein Data Bank (PDB), SpacePAC is able to identify novel mutation clusters in many proteins such as FGFR3 and CHRM2. In addition, SpacePAC is better able to localize the most significant mutational hotspots as demonstrated in the cases of BRAF and ALK. The R package is available on Bioconductor at: http://www.bioconductor.org/packages/release/bioc/html/SpacePAC.html. CONCLUSION: SpacePAC adds a valuable tool to the identification of mutational clusters while considering protein tertiary structure.

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.