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Top genotyped SNP associations with metastasis at diagnosis in the discovery and all replication studies.

Top 30 loci associated with metastasis at diagnosis in the discovery set of 541 European osteosarcoma cases.

Relationship of variables to metastasis at diagnosis in the discovery set.

Figures S1-S7

SNPs in the NFIB locus associated with metastasis at diagnosis with P{less than or equal to}1x10-5 in the discovery stage, and functionally annotated with information from the ENCODE and 1000 Genomes Project data.

Associations of the top GWAS and imputed SNP with metastasis at diagnosis in the discovery stage (European ancestry) by inheritance model.

Statistical Data and SNPS

Supplementary Figure 1. Manhattan plot of the Discovery stage. SNPs in the NFIB risk locus are highlighted in green. Supplementary Figure 2. LD structure of the NFIB risk locus. Plot was made using the CEU population of the 1000 Genomes Project data with LDlink (ref 54). Supplementary Figure 3. Significant relationship between the genotype of the metastasis associated SNP rs7034162, and expression of NFIB and IGFBP5 in osteosarcoma cell lines and tumors. Significance is based on linear regression comparing the distribution of expression of NFIBbetween the TT homozygous non-risk genotype (combined N=34) of rs7034162 genotypes to the heterozygous risk AT (combined N=10) and the homozygous risk AA genotypes (combined N=2). The green triangle represents the expression levels in the OSA cell line, the blue circle is expression levels in the HOS cell line, and the red square the U2OS cell line expression. These analyses were based on the publically available genotype and expression data from 17 osteosarcoma cell lines and 29 osteosarcoma tumors (ref 20). Supplementary Figure 4. No significant relationship between the genotype of the metastasis associated SNP, rs7034162, and expression of protein-encoding genes FREM1, ZDHHC1, and MPDZ in the neighborhood of NFIB in osteosarcoma cell lines and tumors. Significance is based on linear regression comparing the distribution of expression of FREM1, ZDHHC1, and MPDZ between the TT homozygous non-risk genotype (combined N=34) of rs7034162 genotypes to the heterozygous risk AT (combined N=10) and the homozygous risk AA genotypes (combined N=2). The green triangle represents the expression levels in the OSA cell line, the blue circle is expression levels in the HOS cell line, and the red square the U2OS cell line expression. These analyses were based on the publically available genotype and expression data from 17 osteosarcoma cell lines and 29 osteosarcoma tumors (ref 20). Supplementary Figure 5. The human osteosarcoma cell lines, U2OS and HOS, had higher NFIB protein levels then OSA cells. NFIB levels were determined using immunoblot analysis in the OSA, HOS and U2OS cells both basal (A) and after treatment with siRNA against NFIB (B). Supplementary Figure 6. NFIB expression significantly correlates with IGFBP5 expression in osteosarcoma cell lines and tumors. Significance is based on linear regression comparing the expression levels of NFIB with the corresponding IGFBP5 expression levels. Supplementary Figure 7. Expression levels of IGFBP5 decreases after NFIB suppression of human osteosarcoma cell lines. IGFBP5 expression of OSA, HOS and U2OS cells was determined 48 hours after transfection with control siRNA (si-NEG) or siRNA targeting NFIB (si-NFIB). Graph showing relative expression compared to control treated U2OS cells. Supplementary Figure 8. A model of how the NFIB risk locus leads to changes in NFIB expression and potentially may affect osteosarcoma metastatic potential. The risk allele (right panel) leads to lowered expression of NFIB and NFIB-mediated lower expression of IGFPB5, which then leads to less IGFBP5-mediated inhibition of IGF-1 (right panel, grey arrows), leading to an increase in proliferation, survival and metastasis of osteosarcoma cells (right panel, black arrows). The reference allele (left panel) leads to both higher expression of NFIB and higher expression of NFIB-mediated IGFPB5. In turn, higher IGFBP5 levels leads to increased inhibition of IGF-1 (left panel, black arrows), leading to a decrease in proliferation, survival and metastasis of osteosarcoma cells (left panel, grey arrows). Supplementary Figure 9. Plot of the PCA Eigenvectors of the Discovery stage. Supplementary Figure 10. QQ-plot of the Discovery stage.

Cancer treatments often require combinations of molecularly targeted agents to be effective. mTORi (rapamycin) and HDACi (MS-275/entinostat) inhibitors have been shown to be effective in limiting tumor growth, and here we define part of the cooperative action of this drug combination. More than 60 human cancer cell lines responded synergistically (CIin vivo. Mol Cancer Ther; 16(9); 2008–21. ©2017 AACR.

supplementary statistical methods and primer sequences

Description of participating case studies.

OBJECTIVE To establish a pathoepidemiological model to evaluate the role of SARS-CoV-2 infection in the first 10 companion animals that died while infected with SARS-CoV-2 in the US. ANIMALS 10 cats and dogs that tested positive for SARS-CoV-2 and died or were euthanized in the US between March 2020 and January 2021. PROCEDURES A standardized algorithm was developed to direct case investigations, determine the necessity of certain diagnostic procedures, and evaluate the role, if any, that SARS-CoV-2 infection played in the animals’ course of disease and death. Using clinical and diagnostic information collected by state animal health officials, state public health veterinarians, and other state and local partners, this algorithm was applied to each animal case. RESULTS SARS-CoV-2 was an incidental finding in 8 animals, was suspected to have contributed to the severity of clinical signs leading to euthanasia in 1 dog, and was the primary reason for death for 1 cat. CONCLUSIONS AND CLINICAL RELEVANCE This report provides the global community with a standardized process for directing case investigations, determining the necessity of certain diagnostic procedures, and determining the clinical significance of SARS-CoV-2 infections in animals with fatal outcomes and provides evidence that SARS-CoV-2 can, in rare circumstances, cause or contribute to death in pets.

Background Diagnosis of invasive fungal infections from formalin-fixed paraffin-embedded (FFPE) tissues by PCR amplification is a developing technology. One of the difficulties of establishing a validated protocol for this testing is that the gold standard, culture, is much less sensitive than the test being validated. Objectives To validate FFPE PCR as a refence laboratory identification methodology in the absence of abundant gold standard specimens. Methods In this validation, PCR from FFPE tissue was compared to other diagnostic methods for genus/species identification. Four different groups of correlative data from FFPE tissues were used to validate this procedure. Thirteen specimens had culture or serology results and FFPE PCR results, 49 specimens had both immunohistochemistry (IHC) identification and FFPE PCR results, 118 specimens had histological evidence of fungal elements, 64 of which also had FFPE PCR results, and 36 fungal mock tissues or fungal negative tissues were used. Results The sensitivity determined from the tissues with positive fungal histopathology was 54%. The specificity of the cases for which there were both culture and FFPE PCR results was 100%. For the correlation with IHC, the specificity was 98%. For the mock tissues and fungal-negative tissues the calculated analytical sensitivity was 94%, specificity was 95% and accuracy was 94%. Conclusions By uniquely combining various data sources this study provides a comprehensive framework for how validation can be achieved in the absence of a gold standard and outlines the excellent performance of PCR from FFPE tissue, despite relatively the low sensitivity when compared to histopathology.

On April 20, 2018, the Kween District Health Office in Kween District, Uganda reported 7 suspected cases of human anthrax. A team from the Uganda Ministry of Health and partners investigated and identified 49 cases, 3 confirmed and 46 suspected; no deaths were reported. Multiple exposures from handling the carcass of a cow that had died suddenly were significantly associated with cutaneous anthrax, whereas eating meat from that cow was associated with gastrointestinal anthrax. Eating undercooked meat was significantly associated with gastrointestinal anthrax, but boiling the meat for >60 minutes was protective. We recommended providing postexposure antimicrobial prophylaxis for all exposed persons, vaccinating healthy livestock in the area, educating farmers to safely dispose of animal carcasses, and avoiding handling or eating meat from livestock that died of unknown causes.

Summary Background Since August, 2019, US public health officials have been investigating a national outbreak of e-cigarette, or vaping, product use-associated lung injury (EVALI). A spectrum of histological patterns consistent with acute to subacute lung injury has been seen in biopsies; however, autopsy findings have not been systematically characterised. We describe the pathological findings in autopsy and biopsy tissues submitted to the US Centers for Disease Control and Prevention (CDC) for the evaluation of suspected EVALI. Methods Between Aug 1, 2019, and Nov 30, 2019, we examined lung biopsy (n=10 individuals) and autopsy (n=13 individuals) tissue samples received by the CDC, submitted by 16 US states, from individuals with: a history of e-cigarette, or vaping, product use; respiratory, gastrointestinal, or constitutional symptoms; and either pulmonary infiltrates or opacities on chest imaging, or sudden death from an undetermined cause. We also reviewed medical records, evaluated histopathology, and performed infectious disease testing when indicated by histopathology and clinical history. Findings 21 cases met surveillance case definitions for EVALI, with a further two cases of clinically suspected EVALI evaluated. All ten lung biopsies showed histological evidence of acute to subacute lung injury, including diffuse alveolar damage or organising pneumonia. These patterns were also seen in nine of 13 (69%) autopsy cases, most frequently diffuse alveolar damage (eight autopsies), but also acute and organising fibrinous pneumonia (one autopsy). Additional pulmonary pathology not necessarily consistent with EVALI was seen in the remaining autopsies, including bronchopneumonia, bronchoaspiration, and chronic interstitial lung disease. Three of the five autopsy cases with no evidence of, or a plausible alternative cause for acute lung injury, had been classified as confirmed or probable EVALI according to surveillance case definitions. Interpretation Acute to subacute lung injury patterns were seen in all ten biopsies and most autopsy lung tissues from individuals with suspected EVALI. Acute to subacute lung injury can have numerous causes; however, if it is identified in an individual with a history of e-cigarette, or vaping, product use, and no alternative cause is apparent, a diagnosis of EVALI should be strongly considered. A review of autopsy tissue pathology in suspected EVALI deaths can also identify alternative diagnoses, which can enhance the specificity of public health surveillance efforts. Funding US Centers for Disease Control and Prevention.

An ongoing pandemic of coronavirus disease (COVID-19) is caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Characterization of the histopathology and cellular localization of SARS-CoV-2 in the tissues of patients with fatal COVID-19 is critical to further understand its pathogenesis and transmission and for public health prevention measures. We report clinicopathologic, immunohistochemical, and electron microscopic findings in tissues from 8 fatal laboratory-confirmed cases of SARS-CoV-2 infection in the United States. All cases except 1 were in residents of long-term care facilities. In these patients, SARS-CoV-2 infected epithelium of the upper and lower airways with diffuse alveolar damage as the predominant pulmonary pathology. SARS-CoV-2 was detectable by immunohistochemistry and electron microscopy in conducting airways, pneumocytes, alveolar macrophages, and a hilar lymph node but was not identified in other extrapulmonary tissues. Respiratory viral co-infections were identified in 3 cases; 3 cases had evidence of bacterial co-infection.

Lassa virus (LASV), a hemorrhagic fever virus endemic to West Africa, causes conjunctivitis in patients with acute disease. To examine ocular manifestations of LASV, we histologically examined eyes from infected guinea pigs. In fatal disease, LASV immunostaining was most prominent in the anterior uvea, especially in the filtration angle, ciliary body, and iris and in and around vessels in the bulbar conjunctiva and peripheral cornea, where it co-localized with an endothelial marker (platelet endothelial cell adhesion molecule). Antigen was primarily associated with infiltration of T-lymphocytes around vessels in the anterior uvea and with new vessel formation at the peripheral cornea. In animals that exhibited clinical signs but survived infection, eyes had little to no inflammation and no LASV immunostaining 6 weeks after infection. Overall, in this model, LASV antigen was restricted to the anterior uvea and was associated with mild chronic inflammation in animals with severe disease but was not detected in survivors.

Transmission of SARS-CoV-2, the virus that causes COVID-19, from people to companion animals has been reported globally. Between March 2020 and January 2021, the United States reported 94 companion animals with SARS-CoV-2. While most animals with SARS-CoV-2 have mild illness, 10 animals (5 dogs, 5 cats) died around the time of SARS-CoV-2 diagnosis. In one dog, histopathologic changes suggest SARS-CoV-2 exacerbated a severe chronic respiratory disease and contributed to death. In one cat, SARS-CoV-2 was associated with histopathologic changes suggesting the virus caused clinical signs that resulted in euthanasia. In the remaining eight animals, SARS-CoV-2 infection was an incidental finding (4 dogs, 4 cats). This report provides evidence that in rare circumstances, SARS-CoV-2 can contribute to or cause death in companion animals with underlying conditions.

Background The coronavirus disease 2019 (COVID-19) pandemic continues to produce substantial morbidity and mortality. To understand the reasons for the wide-spectrum complications and severe outcomes of COVID-19, we aimed to identify cellular targets of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) tropism and replication in various tissues. Methods We evaluated RNA extracted from formalin-fixed, paraffin-embedded autopsy tissues from 64 case patients (age range, 1 month to 84 years; 21 COVID-19 confirmed, 43 suspected COVID-19) by SARS-CoV-2 reverse-transcription polymerase chain reaction (RT-PCR). For cellular localization of SARS-CoV-2 RNA and viral characterization, we performed in situ hybridization (ISH), subgenomic RNA RT-PCR, and whole-genome sequencing. Results SARS-CoV-2 was identified by RT-PCR in 32 case patients (21 COVID-19 confirmed, 11 suspected). ISH was positive in 20 and subgenomic RNA RT-PCR was positive in 17 of 32 RT-PCR–positive case patients. SARS-CoV-2 RNA was localized by ISH in hyaline membranes, pneumocytes, and macrophages of lungs; epithelial cells of airways; and endothelial cells and vessel walls of brain stem, leptomeninges, lung, heart, liver, kidney, and pancreas. The D614G variant was detected in 9 RT-PCR–positive case patients. Conclusions We identified cellular targets of SARS-CoV-2 tropism and replication in the lungs and airways and demonstrated its direct infection in vascular endothelium. This work provides important insights into COVID-19 pathogenesis and mechanisms of severe outcomes., By analyzing tissues from patients with fatal COVID-19, using in situ hybridization and RT-PCR, we identified cellular targets of SARS-CoV-2 tropism and replication in the lungs, airways, and vascular endothelium. These findings provide important insights into COVID-19 pathogenesis and severe outcomes.

PI3K/AKT/mTOR pathway hyperactivation is frequent in T-cell acute lymphoblastic leukemia/lymphoma (T-ALL/LBL). To model inhibition of mTOR, pre–T-cell lymphoblastic leukemia/lymphoma (pre-T LBL) tumor development was monitored in mice with T lymphocyte–specific, constitutively active AKT (Lck-MyrAkt2) that were either crossed to mTOR knockdown (KD) mice or treated with the mTOR inhibitor everolimus. Lck-MyrAkt2;mTOR KD mice lived significantly longer than Lck-MyrAkt2;mTOR wild-type (WT) mice, although both groups ultimately developed thymic pre-T LBL. An increase in survival was also observed when Lck-MyrAkt2;mTOR WT mice were treated for 8 weeks with everolimus. The transcriptional profiles of WT and KD thymic lymphomas were compared, and Ingenuity Pathway Upstream Regulator Analysis of differentially expressed genes in tumors from mTOR WT versus KD mice identified let-7 and miR-21 as potential regulatory genes. mTOR KD mice had higher levels of let-7a and miR-21 than mTOR WT mice, and rapamycin induced their expression in mTOR WT cells. CDK6 was one of the most downregulated targets of both let-7 and miR21 in mTOR KD tumors. CDK6 overexpression and decreased expression of let-7 in mTOR KD cells rescued a G1 arrest phenotype. Combined mTOR (rapamycin) and CDK4/6 (palbociclib) inhibition decreased tumor size and proliferation in tumor flank transplants, increased survival in an intravenous transplant model of disseminated leukemia compared with single agent treatment, and cooperatively decreased cell viability in human T-ALL/LBL cell lines. Thus, mTOR KD mice provide a model to explore drug combinations synergizing with mTOR inhibitors and can be used to identify downstream targets of inhibition.

Sudden-onset sensorineuronal hearing loss (SNHL) is reported in approximately one-third of survivors of Lassa fever (LF) and remains the most prominent cause of Lassa virus (LASV)-associated morbidity in convalescence. Using a guinea pig model of LF, and incorporating animals from LASV vaccine trials, we investigated viral antigen distribution and histopathology in the ear of infected animals to elucidate the pathogenesis of hearing loss associated with LASV infection. Antigen was detected only in animals that succumbed to disease and was found within structures of the inner ear that are intimately associated with neural detection and/or translation of auditory stimuli and in adjacent vasculature. No inflammation or viral cytopathic changes were observed in the inner ear or surrounding structures in these animals. In contrast, no viral antigen was detected in the ear of surviving animals. However, all survivors that exhibited clinical signs of disease during the course of infection developed perivascular mononuclear inflammation within and adjacent to the ear, indicating an ongoing inflammatory response in these animals that may contribute to hearing loss. These data contribute to the knowledge of LASV pathogenesis in the auditory system, support an immune-mediated process resulting in LASV-associated hearing loss, and demonstrate that vaccination protecting animals from clinical disease can also prevent infection-associated auditory pathology.

Granulomatous amoebic encephalitis (GAE) caused by Balamuthia mandrillaris is a rare subacute infection with exceptionally high mortality. Diagnosis is typically made by brain biopsy or at autopsy. Detection of Balamuthia mandrillaris cell-free DNA by next-generation sequencing of plasma enabled rapid, noninvasive diagnosis in a case of amoebic encephalitis.

Summary Bone marrow-derived mesenchymal stem cells (BMSCs) are proposed as the cells of origin of several subtypes of osteosarcoma (OS). However, signals that direct BMSCs to form different subtypes of OS are unclear. Here we show that the default tumor type from spontaneously transformed p53 knockout (p53_KO) BMSCs is osteoblastic OS. The development of this default tumor type caused by p53 loss can be overridden by various oncogenic signals: RAS reprograms p53_KO BMSCs into undifferentiated sarcoma, AKT enhances osteoblastic OS, while cFOS promotes chondroblastic OS formation. We focus on studying the mechanism of cFOS-induced chondroblastic OS formation. Integrated genome-wide studies reveal a regulatory mechanism whereby cFOS binds to the promoter of a key chondroblastic transcription factor, Sox9, and induces its transcription in BMSCs. Importantly, SOX9 mediates cFOS-induced cartilage formation in chondroblastic OS. In summary, oncogenes determine tumor types derived from BMSCs, and the cFOS-SOX9 axis is critical for chondroblastic OS formation., Graphical Abstract, Highlights • The default tumor type from p53_KO BMSCs is osteoblastic OS • Oncogenes reprogram p53_KO BMSCs into different sarcomas • cFOS promotes chondroblastic OS from p53_KO BMSCs • SOX9 is a mediator of cFOS in promoting chondroblastic OS, In this article, Huang and colleagues show that the default tumor type arising from p53 knockout mouse bone marrow-derived mesenchymal stem cells is osteoblastic osteosarcoma (OS), and oncogenic signals can override this default tumor type choice. They focus on studying the mechanisms underlying cFOS-driven chondroblastic OS and demonstrate that SOX9 is one of the mediators of cFOS.

Zika virus is causally linked with congenital microcephaly and may be associated with pregnancy loss. However, the mechanisms of Zika virus intrauterine transmission and replication and its tropism and persistence in tissues are poorly understood. We tested tissues from 52 case-patients: 8 infants with microcephaly who died and 44 women suspected of being infected with Zika virus during pregnancy. By reverse transcription PCR, tissues from 32 (62%) case-patients (brains from 8 infants with microcephaly and placental/fetal tissues from 24 women) were positive for Zika virus. In situ hybridization localized replicative Zika virus RNA in brains of 7 infants and in placentas of 9 women who had pregnancy losses during the first or second trimester. These findings demonstrate that Zika virus replicates and persists in fetal brains and placentas, providing direct evidence of its association with microcephaly. Tissue-based reverse transcription PCR extends the time frame of Zika virus detection in congenital and pregnancy-associated infections.

Background: Infection with Zika virus (ZIKV) during pregnancy is known to cause birth defects and could also be linked to pregnancy loss. Case: A pregnant woman in Puerto Rico contracted ZIKV at 16 weeks gestation. ZIKV RNA persisted in serum from her initial test at 16 weeks through 24 weeks gestation, when fetal demise occurred, and was detected in placental tissue. Conclusion: Prolonged detection of ZIKV RNA in maternal serum was associated with ZIKV RNA detection in the placenta of a patient who experienced fetal demise. While detection of placenta ZIKV RNA does not establish that ZIKV conclusively caused the demise, these findings support emerging evidence that the placenta may serve as a reservoir for ZIKV, which may be associated with prolonged detection of ZIKV RNA in serum.

As influenza viruses continue to jump species barriers to cause human infection, assessments of disease severity and viral replication kinetics in vivo provide crucial information for public health professionals. The ferret model is a valuable resource for evaluating influenza virus pathogenicity; thus, understanding the most effective techniques for sample collection and usage, as well as the full spectrum of attainable data after experimental inoculation in this species, is paramount. This is especially true for scheduled necropsy of virus-infected ferrets, a standard component in evaluation of influenza virus pathogenicity, as necropsy findings can provide important information regarding disease severity and pathogenicity that is not otherwise available from the live animal. In this review, we describe the range of influenza viruses assessed in ferrets, the measures of experimental disease severity in this model, and optimal sample collection during necropsy of virus-infected ferrets. Collectively, this information is critical for assessing systemic involvement after influenza virus infection in mammals.

Nipah virus is an emerging pathogen in the Paramyxoviridae family. Upon transmission of Nipah virus from its natural reservoir, Pteropus spp. fruit bats, to humans, it causes respiratory and neurological disease with a case-fatality rate about 70%. Human-to-human transmission has been observed during Nipah virus outbreaks in Bangladesh and India. A therapeutic treatment for Nipah virus disease is urgently needed. Here, we tested the efficacy of remdesivir (GS-5734), a broad-acting antiviral nucleotide prodrug, against Nipah virus Bangladesh genotype in African green monkeys. Animals were inoculated with a lethal dose of Nipah virus, and a once-daily intravenous remdesivir treatment was initiated 24 hours later and continued for 12 days. Mild respiratory signs were observed in two of four treated animals, whereas all control animals developed severe respiratory disease signs. In contrast to control animals, which all succumbed to the infection, all remsdesivir-treated animals survived the lethal challenge, indicating that remdesivir represents a promising antiviral treatment for Nipah virus infection.

This manuscript describes the Child Health and Mortality Prevention Surveillance (CHAMPS) network approach to pathologic evaluation of minimally invasive tissue sampling (MITS) specimens, including guidelines for histopathologic examination and further diagnostics with special stains, immunohistochemistry, and molecular testing, as performed at the CHAMPS Central Pathology Laboratory (CPL) at the Centers for Disease Control and Prevention, as well as techniques for virtual discussion of these cases (telepathology) with CHAMPS surveillance locations. Based on review of MITS from the early phase of CHAMPS, the CPL has developed standardized histopathology-based algorithms for achieving diagnoses from MITS and telepathology procedures in conjunction with the CHAMPS sites, with the use of whole slide scanners and digital image archives, for maximizing concurrence and knowledge sharing between site and CPL pathologists. These algorithms and procedures, along with lessons learned from initial implementation of these approaches, guide pathologists at the CPL and CHAMPS sites through standardized diagnostics of MITS cases, and allow for productive, real-time case discussions and consultations.

Summary Background Zika virus is an arthropod-borne virus that is a member of the family Flaviviridae transmitted mainly by mosquitoes of the genus Aedes . Although usually asymptomatic, infection can result in a mild and self-limiting illness characterised by fever, rash, arthralgia, and conjunctivitis. An increase in the number of children born with microcephaly was noted in 2015 in regions of Brazil with high transmission of Zika virus. More recently, evidence has been accumulating supporting a link between Zika virus and microcephaly. Here, we describe findings from three fatal cases and two spontaneous abortions associated with Zika virus infection. Methods In this case series, formalin-fixed paraffin-embedded tissue samples from five cases, including two newborn babies with microcephaly and severe arthrogryposis who died shortly after birth, one 2-month-old baby, and two placentas from spontaneous abortions, from Brazil were submitted to the Infectious Diseases Pathology Branch at the US Centers for Disease Control and Prevention (Atlanta, GA, USA) between December, 2015, and March, 2016. Specimens were assessed by histopathological examination, immunohistochemical assays using a mouse anti-Zika virus antibody, and RT-PCR assays targeting the NS5 and envelope genes. Amplicons of RT-PCR positive cases were sequenced for characterisation of strains. Findings Viral antigens were localised to glial cells and neurons and associated with microcalcifications in all three fatal cases with microcephaly. Antigens were also seen in chorionic villi of one of the first trimester placentas. Tissues from all five cases were positive for Zika virus RNA by RT-PCR, and sequence analyses showed highest identities with Zika virus strains isolated from Brazil during 2015. Interpretation These findings provide strong evidence of a link between Zika virus infection and different congenital central nervous system malformations, including microcephaly as well as arthrogryposis and spontaneous abortions. Funding None.

As one of the earliest examples of “chemical biology,” the Mechanistic Target of Rapamycin (mTOR) protein and its chemical inhibitors have been extensively studied across a spectrum of physiologic and pathologic processes at the molecular, organismal, and patient population levels. There are several FDA-approved mTOR inhibitors (sirolimus, everolimus, and temsirolimus) with indications for cancer treatment and for prevention of solid organ rejection. Dozens of mTOR inhibitors are currently being evaluated in hundreds of ongoing clinical trials across a spectrum of diseases, including numerous cancer indications, autoimmune diseases, and a number of congenital disorders. As many of the approved and investigational indications for mTOR inhibitors require long-term treatment, the magnitude and incidence of particular side effects differ from those observed in shorter-term treatments. Here, we focus on the increased risk of infections in patients being treated with mTOR inhibitors. While increased infection rates might be expected from a class of drugs approved as posttransplant immunosuppressants, we review reports from clinical, mechanistic, and genetically engineered mouse model studies detailing a much more nuanced view of mTOR inhibitor drug action and target biology. Clin Cancer Res; 22(2); 277–83. ©2015 AACR.

Metastasis is the leading cause of death in patients with osteosarcoma, the most common pediatric bone malignancy. We conducted a multistage genome-wide association study of osteosarcoma metastasis at diagnosis in 935 osteosarcoma patients to determine whether germline genetic variation contributes to risk of metastasis. We identified an SNP, rs7034162, in NFIB significantly associated with metastasis in European osteosarcoma cases, as well as in cases of African and Brazilian ancestry (meta-analysis of all cases: P = 1.2 × 10−9; OR, 2.43; 95% confidence interval, 1.83–3.24). The risk allele was significantly associated with lowered NFIB expression, which led to increased osteosarcoma cell migration, proliferation, and colony formation. In addition, a transposon screen in mice identified a significant proportion of osteosarcomas harboring inactivating insertions in Nfib and with lowered NFIB expression. These data suggest that germline genetic variation at rs7034162 is important in osteosarcoma metastasis and that NFIB is an osteosarcoma metastasis susceptibility gene. Significance: Metastasis at diagnosis in osteosarcoma is the leading cause of death in these patients. Here we show data that are supportive for the NFIB locus as associated with metastatic potential in osteosarcoma. Cancer Discov; 5(9); 920–31. ©2015 AACR. This article is highlighted in the In This Issue feature, p. 893

Zika virus infection during pregnancy can cause congenital microcephaly and brain abnormalities (1), and detection of Zika virus RNA in clinical and tissue specimens can provide definitive laboratory evidence of recent Zika virus infection. Whereas duration of viremia is typically short, prolonged detection of Zika virus RNA in placental, fetal, and neonatal brain tissue has been reported and can provide key diagnostic information by confirming recent Zika virus infection (2). In accordance with recent guidance (3,4), CDC provides Zika virus testing of placental and fetal tissues in clinical situations where this information could add diagnostic value. This report describes the evaluation of formalin-fixed paraffin-embedded (FFPE) tissue specimens tested for Zika virus infection in 2016 and the contribution of this testing to the public health response. Among 546 live births with possible maternal Zika virus exposure, for which placental tissues were submitted by the 50 states and District of Columbia (DC), 60 (11%) were positive by Zika virus reverse transcription-polymerase chain reaction (RT-PCR). Among 81 pregnancy losses for which placental and/or fetal tissues were submitted, 18 (22%) were positive by Zika virus RT-PCR. Zika virus RT-PCR was positive on placental tissues from 38/363 (10%) live births with maternal serologic evidence of recent unspecified flavivirus infection and from 9/86 (10%) with negative maternal Zika virus immunoglobulin M (IgM) where possible maternal exposure occurred >12 weeks before serum collection. These results demonstrate that Zika virus RT-PCR testing of tissue specimens can provide a confirmed diagnosis of recent maternal Zika virus infection.

Cancer treatments often require combinations of molecularly targeted agents to be effective. mTORi (rapamycin) and HDACi (MS-275/entinostat) inhibitors have been shown to be effective in limiting tumor growth, and here we define part of the cooperative action of this drug combination. More than 60 human cancer cell lines responded synergistically (CI

Summary Four, 1-to 4-week-old ferret kits were submitted to the Diagnostic Center for Population and Animal Health at Michigan State University for post-mortem examination. Grossly, multiple bowel loops in all ferret kits were distended by mucoid faecal material. Microscopically, there was no evidence of inflammation or notable alteration to the normal mucosal morphology. Gram-positive coccoid bacteria colonized variable segments of the small intestine. These bacteria were identified as Staphylococcus delphini by phenotypic and molecular analyses. Enzyme-linked immunosorbent assay for detection of Staphylococcus enterotoxins was positive and polymerase chain reaction detected the gene for Staphylococcus enterotoxin E in the isolates. The hypersecretory diarrhoea in these ferret kits may have been associated with colonization of the small intestine by S. delphini, cultures of which were shown in vitro to be potentially capable of producing enterotoxin E. The condition described in these ferrets is similar to ‘sticky’ kit syndrome in mink.

Melanoma represents a significant malignancy in humans and dogs. Different from genetically engineered models, sporadic canine melanocytic neoplasms share several characteristics with human disease that could make dogs a more relevant preclinical model. Canine melanomas rarely arise in sun-exposed sites. Most occur in the oral cavity, with a subset having intra-epithelial malignant melanocytes mimicking the in situ component of human mucosal melanoma. The spectrum of canine melanocytic neoplasia includes benign lesions with some analogy to nevi, as well as invasive primary melanoma, and widespread metastasis. Growing evidence of distinct subtypes in humans, differing in somatic and predisposing germ-line genetic alterations, cell of origin, epidemiology, relationship to ultraviolet radiation and progression from benign to malignant tumors, may also exist in dogs. Canine and human mucosal melanomas appear to harbor BRAF, NRAS, and c-kit mutations uncommonly, compared with human cutaneous melanomas, although both species share AKT and MAPK signaling activation. We conclude that there is significant overlap in the clinical and histopathological features of canine and human mucosal melanomas. This represents opportunity to explore canine oral cavity melanoma as a preclinical model.

A 17-year-old, male castrated cougar (Puma concolor) was presented minimally responsive and severely depressed, with bilateral mydriasis and absent pupillary light response. On gross examination of the brain, there was a tan-to-gray, invasive mass with a central cavitation on the ventral aspect in the left cerebral hemisphere, rostral to the caudate nucleus. On histopathologic examination, the mass was composed of sheets of medium-sized, round-to-polygonal cells that were multifocally separated by islands of neuropil. Approximately 80% of the neoplastic cells showed strong cytoplasmic labeling for glial fibrillary acidic protein. These findings were consistent with a medium-grade astrocytoma. To the authors' knowledge, neoplastic disease of the central nervous system has not been previously reported in cougars.

Summary A one-year-old, Thoroughbred colt presented for evaluation due to a one month history of fever of unknown origin and progressive weight loss. On initial presentation, the horse was febrile and showed signs localised to the respiratory tract. These included bilaterally increased bronchovesicular sounds and a moderate, diffuse interstitial pattern on thoracic radiographs. A transtracheal wash yielded mucopurulent debris, culture of which grew small numbers of Staphylococcus epidermidis and Aspergillus spp. The horse was discharged with a diagnosis of bronchointerstitial pneumonia and placed on antibiotic therapy. Ten days after initial presentation, he developed abdominal pain that was unresponsive to on-farm treatment. The horse was febrile, displayed increased respiratory rate and effort, and showed moderate signs of abdominal pain. On rectal examination, a firm, 8–10 cm mass was palpated on midline. The colt was admitted to the hospital and scheduled for exploratory laparotomy, but died a short time later before surgery could be performed. This report describes the clinical, diagnostic and histopathological findings of a case of alimentary lymphoma in a yearling colt.

Background Prompt and accurate identification and differentiation of Mycobacterium tuberculosis-complex (MTBC) from non-tuberculous mycobacteria (NTM) is crucial for the selection of antimicrobial treatment and appropriate public health response. Diagnosis and characterization of mycobacteria is challenging due to diverse clinical presentations, lack of sensitivity of smear microscopy, and fastidious culture identification. Moreover, because of clinical suspicion of noninfectious conditions, specimens are often not processed for culture and formalin-fixed, paraffin-embedded (FFPE) tissues are the only specimens available. For rapid and accurate identification of Mycobacterium spp. from patient tissues, sensitive and specific molecular assays combined with other tissue-based methods are vital. Methods We extracted DNA from FFPE tissues from 931 patients with clinical and histopathological suspicion of mycobacterial infection (received during 2013–2016) and evaluated by multistage, multigene targeted Mycobacterium-genus, complexes-and species-specific PCR assays (targets including 16S rRNA, rpoB, groEL, IS6110, RLEP) and sequencing. Tissues were also examined by acid-fast bacilli (AFB) stains and mycobacteria immunohistochemistry (IHC). Assays to detect mutations associated with drug resistance were performed on MTBC cases. Results A Mycobacterium species was detected in 465 (50%) cases by PCR and sequencing. Of these, 380 (82%) were positive by Mycobacterium PCR targeting 16S rRNA. 85 cases (18%), including 9 MTBC, 12 M. avium complex and 3 M. leprae, were positive by other PCRs. Co-infection of MTBC and NTM spp. was detected in 5 cases. Of 465 PCR positive cases, 327 (70%) showed immunostaining and 223 (48%) were AFB-positive. Molecular markers for drug resistance were detected in 9 out of 88 (10%) tested MTBC cases. Conclusion FFPE tissue analysis by multigene targeted PCR assays expands the opportunities for rapid identification of Mycobacterium species, allows differentiation of MTBC from NTM, and helps to detect co-infections. Using multigene targeted PCRs in combination with histopathology and IHC improve the accuracy of diagnosis, particularly in the presence of commensal and environmental pathogens. Disclosures All authors: No reported disclosures.

Zika virus is a mosquito-borne flavivirus that is related to dengue virus and transmitted primarily by Aedes aegypti mosquitoes, with humans acting as the principal amplifying host during outbreaks. Zika virus was first reported in Brazil in May 2015 (1). By February 9, 2016, local transmission of infection had been reported in 26 countries or territories in the Americas.* Infection is usually asymptomatic, and, when symptoms are present, typically results in mild and self-limited illness with symptoms including fever, rash, arthralgia, and conjunctivitis. However, a surge in the number of children born with microcephaly was noted in regions of Brazil with a high prevalence of suspected Zika virus disease cases. More than 4,700 suspected cases of microcephaly were reported from mid-2015 through January 2016, although additional investigations might eventually result in a revised lower number (2). In response, the Brazil Ministry of Health established a task force to further investigate possible connections between the virus and brain anomalies in infants (3).

The mammalian target of rapamycin (mTOR), a protein in the phosphoinositide 3-kinase (PI3K) pathway important to cell cycle regulation, is frequently activated in cancer. This signaling pathway regulates cell growth and metabolism, and when dysregulated in cancer, it contributes to tumor angiogenesis and growth. mTOR contains two distinct complexes, MTORC1, and MTORC2. MTORC1 plays a role in biosynthesis, while MTORC2 helps with cellular metabolism. It has been shown that mTOR knockout models are embryonically lethal to mice. However, lower levels of functional mTOR have been shown to be beneficial and slow cancer progression. Allelic variation occurs within inbred strains of mice, including BALB/c mice. One allelic variant in mTOR, R628C, which contributes to tumor susceptibility has been identified in BALB/c mice. This single amino acid change occurs in the HEAT repeats of mTOR. HEAT repeats have been shown to facilitate binding partner interactions, dimerization, and localization. Studies on mutations found within the HEAT repeats in several proteins have altered protein function and emphasize the structural importance of HEAT repeats. The goal of this study is to further elucidate the functional significance of HEAT repeats in TOR function and how allelic variation in the HEAT repeats may contribute to tumor susceptibility. Characterization of this allelic variant has shown stress inputs, such as irradiation, lead to a more severe phenotype and reduced mTORC1/2 signaling. Subsequent in vitro studies using a tetracycline inducible expression system (T-RExTM) have shown differential binding to mTORC1/2 complex partners in cells expressing the Balb/c allele. Additional studies are being performed to assess other complex binding partners, dimerization state and localization of the allelic variant. Our findings will model mTOR signaling activation mechanisms in humans to aid in development of new cancer treatments. Note: This abstract was not presented at the meeting. Citation Format: Patricia A. Wiley, Joy Gary, Zaw Phyo, Shuling Zhang, Amanda Sciorillo, Dorian Fraizer. Phenotypic characterization of allelic variation within the HEAT repeats of the mechanistic target of rapamycin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3410. doi:10.1158/1538-7445.AM2017-3410

Generation of high-affinity Abs in response to Ags/infectious agents is essential for developing long-lasting immune responses. B cell maturation and Ab responses to Ag stimulation require Ig somatic hypermutation (SHM) and class-switch recombination (CSR) for high-affinity responses. Upon immunization with either the model Ag 4-hydroxy-3-nitrophenylacetyl hapten (NP) conjugated to chicken γ globulin lysine (NP-CGG) or heat-killed Streptococcus pneumoniae capsular type 14 protein (Pn14), knock-in (KI) mice hypomorphic for mTOR function had a decreased ability to form germinal centers, develop high-affinity anti-NP–specific or anti-Pn14–specific Abs, and perform SHM/CSR. Hypomorphic mTOR mice also had a high mortality (40%) compared with wild-type (WT) (0%) littermates and had lower pneumococcal surface protein A–specific Ab titers when immunized and challenged with live S. pneumoniae infection. Mice with mTOR deleted in their B cell lineage (knockout [KO]) also produced fewer splenic germinal centers and decreased high-affinity Ab responses to NP-CGG than did their WT littermates. CSR rates were lower in mTOR KI and KO mice, and pharmacologic inhibition of mTOR in WT B cells resulted in decreased rates of ex vivo CSR. RNA and protein levels of activation-induced cytidine deaminase (AID), a protein essential for SHM and CSR, were lower in B cells from both KI and B cell–specific KO mice, concomitant with increases in phosphorylated AKT and FOXO1. Rescue experiments increasing AID expression in KI B cells restored CSR levels to those in WT B cells. Thus, mTOR plays an important immunoregulatory role in the germinal center, at least partially through AID signaling, in generating high-affinity Abs.