Search

Purpose: Anaplastic lymphoma kinase (ALK) aberrations have been identified in pediatric-type infant gliomas, but their occurrence across age groups, functional effects, and treatment response has not been broadly established. Experimental Design: We performed a comprehensive analysis of ALK expression and genomic aberrations in both newly generated and retrospective data from 371 glioblastomas (156 adult, 205 infant/pediatric, and 10 congenital) with in vitro and in vivo validation of aberrations. Results: ALK aberrations at the protein or genomic level were detected in 12% of gliomas (45/371) in a wide age range (0–80 years). Recurrent as well as novel ALK fusions (LRRFIP1–ALK, DCTN1–ALK, PRKD3–ALK) were present in 50% (5/10) of congenital/infant, 1.4% (3/205) of pediatric, and 1.9% (3/156) of adult GBMs. ALK fusions were present as the only candidate driver in congenital/infant GBMs and were sometimes focally amplified. In contrast, adult ALK fusions co-occurred with other oncogenic drivers. No activating ALK mutations were identified in any age group. Novel and recurrent ALK rearrangements promoted STAT3 and ERK1/2 pathways and transformation in vitro and in vivo. ALK-fused GBM cellular and mouse models were responsive to ALK inhibitors, including in patient cells derived from a congenital GBM. Relevant to the treatment of infant gliomas, we showed that ALK protein appears minimally expressed in the forebrain at perinatal stages, and no gross effects on perinatal brain development were seen in pregnant mice treated with the ALK inhibitor ceritinib. Conclusions: These findings support use of brain-penetrant ALK inhibitors in clinical trials across infant, pediatric, and adult GBMs.

Low-capital-layout sequencing options from Oxford Nanopore Technologies (ONT) could assist in expanding HIV drug resistance testing to resource-limited settings. HIV drug resistance mutations often occur as mixtures, but current ONT pipelines provide a consensus sequence only. Moreover, there is no integrated pipeline that provides a drug resistance report from an ONT sequence file without intervention from skilled bioinformaticists. We therefore investigated Nano-RECall, which provides seamless drug resistance interpretation while requiring low-read coverage ONT sequence data from affordable Flongle or MinION flow cells and which provides mutation mixtures similar to Sanger Sequencing.We compared Sanger sequencing to ONT sequencing of the same HIV-1 subtype C polymerase chain reaction (PCR) amplicons, respectively using RECall and the novel Nano-RECall bioinformatics pipelines. Amplicons were from separate assays: (a) Applied Biosystems HIV-1 Genotyping Kit (ThermoFisher) spanning protease (PR) to reverse transcriptase (RT) (PR-RT) (n=46) and (b) homebrew integrase (IN) (n=21). The agreement between Sanger sequences and ONT sequences was assessed at nucleotide level, and at codon level for Stanford HIV drug resistance database mutations at an optimal ONT read depth of 400 reads only.The average sequence similarity between ONT and Sanger sequences was 99.3% (95% CI: 99.1-99.4%) for PR-RT and 99.6% (95% CI: 99.4-99.7%) for INT. Drug resistance mutations did not differ for 21 IN specimens; 8 mutations were detected by both ONT- and Sanger sequencing. For the 46 PR and RT specimens, 245 mutations were detected by either ONT or Sanger, of these 238 (97.1%) were detected by both.The Nano-RECall pipeline, freely available as a downloadable application on a Windows computer, provides Sanger-equivalent HIV drug resistance interpretation. This novel pipeline combined with a simple workflow and multiplexing samples on ONT flow-cells would contribute to making HIV drug resistance sequencing feasible for resource limited settings.

Long-term morbidity including hypertension often persists in coarctation patients despite current guidelines. Coarctation severity can be invasively assessed via peak-to-peak catheter pressure gradient (PPCG), which is estimated noninvasively via simplified Bernoulli equation and conventionally reported as peak instantaneous Doppler gradient (PIDG). However, underlying simplifications of the equation limit diagnostic accuracy. We studied the diagnostic performance of a new Doppler-based diastolic index called the continuous flow pressure gradient (CFPG) versus conventional indices in assessing coarctation severity.In a rabbit model mimicking human aortic coarctation, temporal blood pressure waveforms revealed the diastolic instantaneous pressure gradients and spectral Doppler features impacted by coarctation severity. We therefore hypothesized that CFPG provides superior correlation with coarctation gradients measured invasively. PIDG and CFPG were quantified using color flow echocardiography in humans and rabbits with discrete coarctations. Results were compared with PPCG in rabbits (n = 34) and arm-leg systolic gradients (n = 25) in humans via 1-way analysis of variance, Pearson's correlation, linear regression, and Bland-Altman analysis.A threshold of CFPG ≥ 4.6 mm Hg was identified via the Youden index as representative of PPCG ≥ 20 mm Hg (the current guideline value for coarctation intervention) in rabbits, while a CFPG ≥1.0 mm Hg represented an arm-leg systolic gradient ≥20 mm Hg in humans. Accuracy measures revealed superior correlation of CFPG (R

Insufficient invasion of conceptus-derived trophoblast cells in the maternal decidua is a key event in the development of early-onset preeclampsia (PE), a subtype of PE associated with high maternal and fetal morbidity and mortality. Kisspeptins, a family of peptides previously shown to inhibit trophoblast cell invasion, have been implicated in the pathogenesis of early-onset PE. However, a role of kisspeptin signaling during the genesis of this syndrome has not been elucidated. Herein, we used the preeclamptic-like BPH/5 mouse model to investigate kisspeptin expression and potential upstream regulatory mechanisms in a PE-like syndrome. Expression of the kisspeptin encoding gene, Kiss1, and the 10-amino-acid kisspeptide (Kp-10), are upregulated in the non-pregnant uterus of BPH/5 females during diestrus and in the maternal-fetal interface during embryonic implantation and decidualization. Correspondingly, the dysregulation of molecular pathways downstream to kisspeptins also occurs in this mouse model. BPH/5 females have abnormal sex steroid hormone profiles during early gestation. In this study, the normalization of circulating concentrations of 17β-estradiol (E2) and progesterone (P4) in pregnant BPH/5 females not only mitigated Kiss1 upregulation, but also rescued the expression of multiple molecules downstream to kisspeptin and ameliorated adverse fetoplacental outcomes. Those findings suggest that uterine Kiss1 upregulation occurs pre-pregnancy and persists during early gestation in a PE-like mouse model. Moreover, this study highlights the role of sex steroid hormones in uteroplacental Kiss1 dysregulation and the improvement of placentation by normalization of E2, P4 and Kiss1.

Supplementary Figure 3: PPP1CB-ALK fusion is oncogenic and sensitive to targeted therapy. A. Immunoblot analysis of ALK downstream signaling proteins in cortical mNSC (CTX#6) and brainstem mNSC (BS#3) expressing PPP1CB-ALK. Cells were treated for 4 hours with ceritinib and lorlatinib at the indicated concentrations.B. Left panel: Soft agar colony-forming assay using stable mNSC PPP1CB-ALK demonstrated colony formation with dose-dependent inhibition by ceritinib. mNSC KRAS did not respond to ceritinib validating the drug specificity. Right panel: Quantification of colony formation under targeted drug inhibition using CellProfiler. Values represent colony counts relative to the untreated group ± s.d. The mean of three independent replicates is shown. Significance between treatments determined by the Mann-Whitney test. *P < 0.05, **P < 0.01.C. Cell viability of ceritinib-treated PPP1CB-ALK NIH-3T3 relative to eGFP-positive cells by CellTiterGlo.D. Relative viability of the ALK wild-type GBM line (BT164) and BT1857 lorlatinib-treated cells versus DMSO control.E. Relative viability of BT164 and BT1857 ceritinib-treated cells versus DMSO control.F. Relative viability of BT164 and BT1857 cells treated with an EGFR tyrosine kinase inhibitor neratinib.G. Pharmacodynamic analysis of NIH 3T3-PPP1CB-ALK s.c. tumors treated with vehicle or ceritinib at 30mg/kg for 5 days. Representative H&E stain, ALK, pSTAT3, pAKT S473 and Ki-67 IHC.H. Quantification of Ki-67-positive cells in vehicle and ceritinib-treated tumors using CellProfiler software. Error bars show standard error of the mean. *P < 0.05.I. Quantification of pSTAT3-positive cells in vehicle and ceritinib-treated tumors using CellProfiler software. Error bars show standard error of the mean. *P < 0.05.J. Tumor growth following subcutaneous implantation of PPP1CB-ALK and eGFP-expressing fibroblasts.K. Kaplan-Meier survival curves of SCID mice injected subcutaneously with mNSC CTX-PPP1CB-ALK (red, n = 5) or mNSC CTX-eGFP (black, n = 4) and treated with ceritinib at 30mg/kg/d for 15 days. Mice were euthanized at endpoint (tumor volume of 2000mm3). The grey area represents the treatment period.L. Vehicle or ceritinib-treated mouse weights at start and end of treatment.

Supplementary Figure 5: Single-cell RNA Sequencing and IHC examination of ALK in human neural development and immature neural cells. A. 2D representation of human brain cell subtypes (48 samples) during cortex development using a single cell RNA-seq atlas (cortex-dev.cells.ucsc.edu) shows that ALK transcript appears to be enriched in a certain cell type. Color-coded by ALK gene expression (beige to red) and age in weeks (rainbow).B. ALK-stained sagittal section of normal human cerebellum at 15 postconceptional weeks (pcw).C. ALK-stained sagittal section of human fetal neural stem cell pellets at 13 postconceptional weeks (pcw).D. ALK-stained sagittal section of human fetal neural stem cell pellets at 15 postconceptional weeks (pcw).

Purpose:Anaplastic lymphoma kinase (ALK) aberrations have been identified in pediatric-type infant gliomas, but their occurrence across age groups, functional effects, and treatment response has not been broadly established.Experimental Design:We performed a comprehensive analysis of ALK expression and genomic aberrations in both newly generated and retrospective data from 371 glioblastomas (156 adult, 205 infant/pediatric, and 10 congenital) with in vitro and in vivo validation of aberrations.Results:ALK aberrations at the protein or genomic level were detected in 12% of gliomas (45/371) in a wide age range (0–80 years). Recurrent as well as novel ALK fusions (LRRFIP1–ALK, DCTN1–ALK, PRKD3–ALK) were present in 50% (5/10) of congenital/infant, 1.4% (3/205) of pediatric, and 1.9% (3/156) of adult GBMs. ALK fusions were present as the only candidate driver in congenital/infant GBMs and were sometimes focally amplified. In contrast, adult ALK fusions co-occurred with other oncogenic drivers. No activating ALK mutations were identified in any age group. Novel and recurrent ALK rearrangements promoted STAT3 and ERK1/2 pathways and transformation in vitro and in vivo. ALK-fused GBM cellular and mouse models were responsive to ALK inhibitors, including in patient cells derived from a congenital GBM. Relevant to the treatment of infant gliomas, we showed that ALK protein appears minimally expressed in the forebrain at perinatal stages, and no gross effects on perinatal brain development were seen in pregnant mice treated with the ALK inhibitor ceritinib.Conclusions:These findings support use of brain-penetrant ALK inhibitors in clinical trials across infant, pediatric, and adult GBMs.

Supplementary Figure 6: Single cell RNA sequencing comparison of ALK expression and other infant/pediatric glioma kinase drivers in developing human and mouse brain. A. 2D representation of human brain cell subtypes (48 samples) during cortex development using a single cell RNA-seq atlas (http://cells.ucsc.edu/?ds=cortex-dev) shows that ALK positive cells make up less than 1% of all cells in the dataset and aren't localized within a specific cluster.B. Cluster-level summary of the human scRNA dataset shows that ALK expression (TPM) is decreased compared to EGFR, NTRK2, and NTRK3, and ALK positive cells make up a much lower percentage of each cluster compared to those 3 genes.C. 2D representation of developmental mouse forebrain (E12.5 – P6) in a single cell RNA-seq atlas (http://cc-shiny-01.functionalgenomics.ca/braindex/clusters) shows similar results as seen in the human scRNA Nowakowski dataset.D. Cluster-level summary of the mouse scRNA dataset corroborates results seen in the Nowkowski scRNA dataset; that Alk expression (log-normalized) is decreased, and less prevalent than EGFR, NTRK2, and NTRK3.E. 2D representation of RNA sequencing data for NTRK1-3 genes in the Nowakowski developing human cortex dataset. Compared to ALK expression, the NTRK genes have increased expression levels, increased prevalence in the dataset, and show increased localization to specific cluster.F. Similar to (E), the 2D representation of Ntrk genes in the developing mouse scRNA dataset shows increased expression and prevalence compared to Alk.

Supplementary Figure 2: T1 post-contrast axial and sagittal images of ALK-fused and non ALK-altered GBMs and ALK staining in ALK-fused congenital GBMs. A. Example images from patients with ALK aberrations (76, 89, 116, 197)B. In contrast to those without ALK aberrations (right columns). Contrast is gadolinium for those where applicable and show enhancement within both the tumor categories.C. Representative H&E and ALK IHCs of congenital GBM harboring an ALK fusion.

Supplementary Figure 4: Characterization of the lung cancer recurrent DCTN1-ALK fusion identified in cGBM (case ALK.232). A. Integrative Genomic Viewer (IGV) screenshot of the DCTN1-ALK variant.B. Predicted sequence of DCTN1-ALK fusion (DCTN1 exon 1-26, ALK exon 20-29 conserved).C. PDX DCTN1-ALK sub-renal capsule engraftment confimed by IHC.C. Relative viability of the ALK wild-type GBM line (BT164) and BT1857 ceritinib-treated cells versus DMSO control.D. Relative viability of BT164 and BT1857 lorlatinib-treated cells versus DMSO control.E. Relative viability of BT164 and BT1857 cells treated with an EGFR tyrosine kinase inhibitor neratinib.F. PDX DCTN1-ALK sub-renal capsule engraftment confirmed by IHC.

Supplementary Figure 1: Characterization of the novel LRRFIP1-ALK fusion (case ALK.223) A. Visualization of the read coverage at the fusion sites in ALK and LRRFIP1 genes using Integrative Genomic Viewer.B. Pile-up plot generated by SvABA using whole-genome sequencing data shows the breakpoint coordinates.C. Copy number ratio profile by WGS showing no somatic copy number alterations anywhere in the genome. The x-axis represents the chromosomes.D. Copy number ratio profile on chromosome 2 showing no copy number change of ALK and LRRFIP1 genes.E. Two RT-PCR products of 186bp (variant 1) and 189bp (variant 3) were detected by agarose gel electrophoresis with total RNA from tumor specimen ALK.223.F. Primer sequences of ALK-LRRFIP1 (variants 1 and 2) and LRRFIP1-ALK (variant 3) and the predicted DNA product size.G. Western Blot analysis of ALK signaling pathway in subcutaneous tumors treated with vehicle or lorlatinib. Normal brain was used as control.H. Representative 40x images and CellProfiler quantification of Cleaved Caspase 3- and Ki-67- stained subcutaneous tumors.

Additional information tables 1 to 8.

BackgroundPediatric low-grade gliomas (pLGGs) are the most common central nervous system tumor in children, characterized by RAS/MAPK pathway driver alterations. Genomic advances have facilitated the use of molecular targeted therapies, however, their long-term impact on tumor behavior remains critically unanswered.MethodsWe performed an IRB-approved, retrospective chart and imaging review of pLGGs treated with off-label targeted therapy at Dana-Farber/Boston Children’s from 2010 to 2020. Response analysis was performed for BRAFV600E and BRAF fusion/duplication-driven pLGG subsets.ResultsFifty-five patients were identified (dabrafenib n = 15, everolimus n = 26, trametinib n = 11, and vemurafenib n = 3). Median duration of targeted therapy was 9.48 months (0.12–58.44). The 1-year, 3-year, and 5-year EFS from targeted therapy initiation were 62.1%, 38.2%, and 31.8%, respectively. Mean volumetric change for BRAFV600E mutated pLGG on BRAF inhibitors was −54.11%; median time to best volumetric response was 8.28 months with 9 of 12 (75%) objective RAPNO responses. Median time to largest volume post-treatment was 2.86 months (+13.49%); mean volume by the last follow-up was −14.02%. Mean volumetric change for BRAF fusion/duplication pLGG on trametinib was +7.34%; median time to best volumetric response was 6.71 months with 3 of 7 (43%) objective RAPNO responses. Median time to largest volume post-treatment was 2.38 months (+71.86%); mean volume by the last follow-up was +39.41%.ConclusionsOur integrated analysis suggests variability in response by pLGG molecular subgroup and targeted therapy, as well as the transience of some tumor growth following targeted therapy cessation.

In hypoplastic left heart syndrome, the size and function of the left ventricle vary and are dependent on the patency of the aortic valve. A patent native aortic valve, permitting left ventricular ejection, can augment cardiac output. We performed a retrospective chart review of patients with hypoplastic left heart syndrome and a stenotic aortic valve who underwent native aortic valvuloplasty at the time of Norwood and found that none of the eight patients identified had clinically significant aortic insufficiency. This case series suggests that surgical aortic valvuloplasty at Norwood is associated with aortic valve patency/augmented systemic cardiac output without the development of clinically significant aortic regurgitation at intermediate follow-up in a limited cohort.

Multiple system atrophy (MSA) is a fatal neurodegenerative disease of unknown etiology characterized by widespread aggregation of the protein alpha-synuclein in neurons and glia. Its orphan status, biological relationship to Parkinson’s disease (PD), and rapid progression have sparked interest in drug development. One significant obstacle to therapeutics is disease heterogeneity. Here, we share our process of developing a clinical trial-ready cohort of MSA patients (69 patients in 2 years) within an outpatient clinical setting, and recruiting 20 of these patients into a longitudinal “n-of-few” clinical trial paradigm. First, we deeply phenotype our patients with clinical scales (UMSARS, BARS, MoCA, NMSS, and UPSIT) and tests designed to establish early differential diagnosis (including volumetric MRI, FDG-PET, MIBG scan, polysomnography, genetic testing, autonomic function tests, skin biopsy) or disease activity (PBR06-TSPO). Second, we longitudinally collect biospecimens (blood, CSF, stool) and clinical, biometric, and imaging data to generate antecedent disease-progression scores. Third, in our Mass General Brigham SCiN study (stemcellsinneurodegeneration), we generate induced pluripotent stem cell (iPSC) models from our patients, matched to biospecimens, including postmortem brain. We present 38 iPSC lines derived from MSA patients and relevant disease controls (spinocerebellar ataxia and PD, including alpha-synuclein triplication cases), 22 matched to whole-genome sequenced postmortem brain. iPSC models may facilitate matching patients to appropriate therapies, particularly in heterogeneous diseases for which patient-specific biology may elude animal models. We anticipate that deeply phenotyped and genotyped patient cohorts matched to cellular models will increase the likelihood of success in clinical trials for MSA.

Background Cardiac troponin is commonly raised in patients with malignancy and may aid clinicians in risk prediction. The prognostic significance of raised troponin in these patients with known malignancies remains unclear. Purpose We sought to investigate the relation between troponin and mortality in a large, well characterised cohort of patients undergoing cardiac troponin testing with a concomitant malignancy. Methods A retrospective cohort study was carried out using the National Institute for Health Research Health Informatics Collaborative Cardiovascular dataset of all consecutive patients who had a troponin measured at five hospitals between 2010 and 2017. Patients with a primary inpatient diagnosis of malignancy who had at least one cTn measurement during their hospital stay were identified. Patients were classified into solid tumour or haematological malignancy subgroups. Survival analyses were performed using multivariate Cox regression analyses and Kaplan-Meier plots. Cox regression analyses were adjusted for age, gender, C-reactive protein, haemoglobin, platelet count, white cell count, acute coronary syndrome, diabetes mellitus, heart failure, chronic kidney disease, chronic obstructive pulmonary disease, atrial fibrillation and angiography. The peak cTn level (highest level measured), standardised to the upper limit of normal (ULN), was used for all analyses. Results 5571 patients undergoing troponin testing had a primary diagnosis of malignancy and comprised of twenty-one different cancer types. 4649 patients were diagnosed with solid tumours and 922 patients were diagnosed with haematological malignancies. Patients with raised troponin had a higher burden of cardiovascular comorbidities compared to patients with a troponin level below the ULN. The median follow-up in the cohort was 14 months (interquartile range 2–39 months). At 1-year follow-up, 2495 (42%) of patients died. Figure 1 shows Kaplan-Meier plots for patients stratified by troponin level. Patients with a troponin level ≥1xULN had a higher risk of death compared to patients with a troponin level Conclusion A raised troponin was associated with an increased mortality risk in patients with malignancy regardless of cancer subtype. Stratification of mortality risk using troponin may help guide clinicians in making management decisions for patients with malignancy. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): NIHR Imperial Biomedical Research Centre, as part of the NIHR Health Informatics Collaborative, and 2) British Heart Foundation

Background A positive cardiac troponin (cTn) is an independent predictor of short-term mortality in individuals presenting with acute pulmonary embolism (PE). However, there is limited evidence regarding the impact age has on the association between cTn levels and mortality in patients with PE. Purpose The aim of our study was to investigate the relationship between cTn level, age, and all-cause mortality, in hospitalised patients diagnosed with an acute PE. Methods A retrospective cohort study using the National Institute for Health Research Health Informatics Collaborative Cardiovascular dataset of all consecutive patients who had a troponin measured at five hospitals between 2010 and 2017. Patients admitted to hospital with a primary diagnosis of PE with at least one cTn measurement were included. We modelled the relation between peak troponin level and all-cause mortality using multivariable adjusted restricted cubic spline Cox regression analyses. Effect estimates were adjusted for age, gender, high-sensitivity troponin assay, C-reactive protein, haemoglobin, platelet count, white cell count, creatinine, sodium, potassium, diabetes, hypertension, hypercholesterolaemia, acute coronary syndrome, atrial fibrillation, heart failure, acute kidney injury, chronic kidney disease, obstructive lung disease, inflammatory disorders, pneumonia and malignancy. The peak cTn level (highest level measured), standardised to the upper limit of normal (ULN), was used for all analyses. Results 1,477 patients with at least one cTn measurement and a diagnosis of acute PE were included. During a median follow-up of 34.8 months, there were 290 (19.6%) deaths. Elevated cTn (>1xULN) was associated with a hazard ratio (HR) of 3.29 (95% confidence interval [CI] 1.95–5.53) for 30-day mortality and 2.12 (95% CI 1.63–2.75) for 3-year mortality. Higher cTn levels were progressively associated with a higher mortality risk, reaching a maximum HR of 2.59 (95% CI 1.64–4.09) at 141xULN (Figure 1). Younger patients ( Conclusion Elevated cTn, at all ages, is associated with an increased mortality risk in patients presenting with PE, with increasing cTn levels conferring a progressively worse long-term prognosis. Elevated cTn, no matter how small, needs to be taken seriously, particularly in young patients with an acute PE. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): 1) NIHR Imperial Biomedical Research Centre, as part of the NIHR Health Informatics Collaborative, and 2) British Heart Foundation

Introduction Acute stroke accounts for significant morbidity and mortality globally. The role of troponin for risk stratification in stroke is unclear. Purpose The aims of this study were to assess the relationship between peak troponin and mortality in patients with ischaemic stroke, haemorrhagic stroke, or subarachnoid haemorrhage and to compare this with the predictive value of first troponin or dynamic troponin change. Methods A retrospective cohort study was carried out using the National Institute for Health Research Health Informatics Collaborative Cardiovascular dataset of all consecutive patients who had a troponin measured at five hospitals between 2010 and 2017. Patients with at least one troponin measurement and a primary diagnosis of ischaemic stroke, haemorrhagic stroke or subarachnoid haemorrhage during a hospital admission were included. The main exposure variables were first and peak troponin, and dynamic troponin change, and the main outcome was all-cause mortality. Results were analysed using multivariable adjusted restricted cubic spline Cox regression. Survival analyses were adjusted for troponin assay, assay sensitivity (standard or highly sensitive), number of troponin measurements, age, sex, C-reactive protein level, white blood cell count, platelet count, haemoglobin, estimated glomerular filtration rate, angiography during admission, acute coronary syndrome during admission, and cardiovascular history (history of diabetes mellitus, myocardial infarction, heart failure, hypertension, stroke or atrial fibrillation). Receiver Operator Characteristic (ROC) curves were used to assess the predictive value of each exposure variable. Results 4,712 patients were included in the analysis (ischaemic stroke: 3,346; haemorrhagic stroke: 718; subarachnoid haemorrhage: 648). Peak troponin was above the upper limit of normal in 47.4% of ischaemic stroke patients, 52.8% of haemorrhagic stroke patients, and 57.1% of subarachnoid haemorrhage patients. Patients with elevated peak troponin were older and had more cardiovascular risk factors. A direct positive relationship was seen between peak troponin level and mortality hazard ratio in all three stroke subtypes (Figure 1). This relationship was consistent when considering dynamic troponin fold change for ischaemic or haemorrhagic stroke. For all three stroke subtypes, there was no added predictive value of peak troponin or dynamic troponin change over first troponin in predicting mortality (Figure 2). Conclusions A positive peak troponin was associated with increased mortality in patients presenting with ischaemic stroke, haemorrhagic stroke, or subarachnoid haemorrhage. Overall, serial troponin measurements may not improve mortality prediction beyond a single measurement. These findings may have implications for risk stratification of patients with acute stroke syndromes. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): NIHR Imperial Biomedical Research Centre, as part of the NIHR Health Informatics Collaborative, and 2) British Heart Foundation

Kisspeptins (KP), encoded by Kiss1 , are reported to play a role in early-onset preeclampsia (ePE), a life-threatening hypertensive disorder of pregnancy. KP is higher in term ePE placentae than in normal pregnancies. However, the role of KP in PE has not been confirmed. The Blood Pressure High Subline 5 (BPH/5) mouse model spontaneously develops the main features of ePE, including impaired placental expansion and maternal hypertension. We have shown that Kiss1 is higher at the BPH/5 maternal-fetal interface on embryonic days (e) 4.5 and 7.5. BPH/5 females have abnormal circulating sex steroid hormones (SSH) during early gestation (e2.5), with lower estradiol-17ß (E2) and higher progesterone (P4). Since E2 and P4 may modulate uterine Kiss1 , we aimed to investigate the effects of artificial synchronization of SSH (AS-SSH) in BPH/5 uteroplacental KP signaling and placental development. We hypothesized that AS-SSH would prevent BPH/5 uteroplacental Kiss1 upregulation and normalize KP downstream expression of tissue inhibitor of metalloproteinase (TIMP) 2 and placental defects. AS-SSH was performed after ovariectomy of pregnant BPH/5 and C57 females (n = 8/strain) at e2.5 with one dose of E2 (25 ng/mouse, subcutaneously), and daily subcutaneous injections of P4 (1ng/mouse) until embryonic implantation sites (eIS) were collected 3 days post-surgery (n = 8/group). Contrary to natural (Nat) BPH/5 pregnancies, eIS Kiss1 and TIMP2 expression was not different between AS-SSH BPH/5 and C57 (p > 0.05). Nat and AS-SSH pregnant mice were anesthetized at e12.5 to assess umbilical cord blood flow via ultrasound and uteroplacental units were harvested for placental morphometry (n = 4-6/group). Placental expansion into the maternal decidua was measured and expressed as the ratio of the placenta depth in relation to the placenta + decidua. Nat BPH/5 had lower placental expansion and umbilical blood flow than Nat C57, whereas AS-SSH BPH/5 had higher placental expansion and umbilical blood flow than Nat BPH/5 (p < 0.05). In conclusion, synchronization of the E2 and P4 profile in the BPH/5 mouse early gestation mitigated Kiss1 and TIMP2 upregulation at the maternal-fetal interface and ameliorated the placental defects previously reported in this model.

Empathy is an innate ability to understand the emotional states of others along with the motivation to improve it. It has evolved over time into highly complex behaviors, the basis of which can be described using the Perception Action Model (PAM), where shared affect promotes an action that eliminates the distress of both the passive “Target” and, by extension, the active “Observer.” There are myriad biological variables that may modulate empathic behavior, including sex, sensory modalities, and neural activity. In the following studies, using our lab’s model of social contact-independent targeted helping, we first tested whether sex differences exist in helping behavior. Next, we explored sex differences in sensory and affective signaling, including the impact of direct visualization of a distressed conspecific and the type of ultrasonic vocalizations (USV) made between animal pairs during the task. Finally, we examined the neural activity of multiple cortical and subcortical regions of interest across time during targeted helping between males and females. We show both sexes exhibit similar helping behavior, but sensory and affective signaling differs between sexes. Further, changes in neural activity exhibited distinct sex-specific patterns across time. Our results indicate sex differences are not a ubiquitous presence in targeted helping. Instead, it is likely sex differences may be a convergent phenomenon in which the behavior is similar, but the underlying biological mechanisms are distinct. These results lay the groundwork for future studies to explore the similarities and differences that drive empathic behavior in both males and females.

IntroductionOxford Nanopore Technologies (ONT) offer sequencing with low-capital-layout sequencing options, which could assist in expanding HIV drug resistance testing to resource limited settings. However, sequence analysis remains time time-consuming and reliant on skilled personnel. Moreover, current ONT bioinformatic pipelines provide a single consensus sequence that is not equivalent to Sanger sequencing, as drug resistance is often detected in mixed populations. We have therefore investigated an integrated bioinformatic pipeline, Nano-RECall, for seamless drug resistance of low read coverage ONT sequence data from affordable Flongle or MinION flow cells.MethodsWe compared Sanger sequencing to ONT sequencing of the same HIV-1 subtype C polymerase chain reaction (PCR) amplicons, respectively using RECall and the novel Nano-RECall bioinformatics pipelines. Amplicons were from separate assays a) Applied Biosystems HIV-1 Genotyping Kit (ThermoFisher) spanning protease (PR) to reverse transcriptase (RT) (PR-RT) (n=46) and b) homebrew integrase (IN) (n=21). We investigated optimal read-depth by assessing the coefficient of variation (CV) of nucleotide proportions for various read-depths; and between replicates of 400 reads. The agreement between Sanger sequences and ONT sequences were assessed at nucleotide level, and at codon level for Stanford HIV drug resistance database mutations.ResultsThe coefficient of variation of ONT minority variants plateaued after a read depth of 400-fold implying limited benefit of additional depth and replicates of 400 reads showed a CV of ∼6 % for a representative position. The average sequence similarity between ONT and Sanger sequences was 99.3% (95% CI: 99.1-99.4%) for PR-RT and 99.6% (95% CI: 99.4-99.7%) for INT. Drug resistance mutations did not differ for 21 IN sequences; 16 mutations were detected by both ONT- and Sanger sequencing. For the 46 PR and RT sequences, 245 mutations were detected by either ONT or Sanger, of these 238 (97.1%) were detected by both.ConclusionsThe Nano-RECall pipeline, freely available as a downloadable application on a Windows computer, provides Sanger-equivalent HIV drug resistance interpretation. This novel pipeline combined with a simple workflow and multiplexing samples on ONT flow-cells would contribute to making HIV drug resistance sequencing feasible for resource limited settings.

Background Mortality after stage 1 palliation of hypoplastic left heart syndrome remains significant. Both cardiac output (CO) and systemic vascular resistance (SVR) contribute to hemodynamic vulnerability. Simultaneous measures of mean arterial pressure and somatic regional near infrared spectroscopy saturation can classify complex hemodynamics into 4 distinct states, with a low-CO state of higher risk. We sought to identify interventions associated with low-CO state occupancy and transition. Methods Perioperative data were prospectively collected in an institutional review board–approved database. Hemodynamic state was classified as high CO, high SVR, low SVR, and low CO using bivariate analysis. Associations of static and dynamic support levels and state classifications over 48 postoperative hours were tested between states and across transitions using mixed regression methods in a quasi-experimental design. Results Data from 10,272 hours in 214 patients were analyzed. A low-CO state was observed in 142 patients for 1107 hours. Both low CO and extracorporeal membrane oxygenation had increased mortality risk. The low-CO state was characterized by lower milrinone but higher catecholamine dose. Successful transition out of low CO was associated with increased milrinone dose and hemoglobin concentration. Increasing milrinone and hemoglobin levels predicted reduced risk of low CO in future states. Conclusions Bivariate classification objectively defines hemodynamic states and transitions with distinct support profiles. Maintaining or increasing inodilator and hemoglobin levels were associated with improved hemodynamic conditions and were predictive of successful future transitions from the low-CO state.

Background: Shone syndrome is characterized by coincident mitral valve stenosis and left ventricular outflow tract obstruction. Although first described in 1963, little research has expounded surgical outcomes. We sought to evaluate our experience with this cohort, emphasizing outcomes including mortality, morbidity, and cardiac function. Methods: A retrospective chart review of 46 patients who underwent operation for Shone syndrome between 1990 and May 2018 was conducted. Index operations included 32 repairs of the left ventricular outflow tract, four mitral valve repair/replacements, nine combined repairs, and one non-Shone’s repair. Median age at index procedure was 22 days (2 days-10 years). Mean follow-up was 9.1 years (2 months-21 years), and 70 additional operations (51 reoperations) were required. Three patients were lost to follow-up. Results: Overall survival was 95.7% with two late deaths. Freedom from death or transplant was 93.5%. Thirteen (28.3%) patients remained free from reoperation. Thirty-three patients required 51 reoperations of the left ventricle outflow tract (n = 12), mitral valve (n = 16), combined repairs (n = 21), and transplant (n = 1). At most recent follow-up, patients exhibited mitral stenosis (n = 21), aortic stenosis (n = 7), and diminished LV function (n = 2). Conclusion: Surgical correction of Shone’s offers excellent survival benefit, but reoperation burden is high, with >70% of patients requiring reintervention in the follow-up period. A total of 65% of patients developed recurrent obstruction of left ventricular inflow or outflow, however, ventricular function is preserved in the majority of patients. All but one patient had no functional deficits, classified as New York Heart Association I with > 60% requiring no medication.

Background and purpose The goal of this prospective study is to validate the use of periodic imaging during treatment with a fiducial marker detection algorithm using radiofrequency transponders for prostate cancer patients undergoing treatment for radiation therapy. Materials and methods Ten male patients were enrolled in this study and treated for prostate cancer with implanted electromagnetic monitoring beacons. We evaluated the accuracy and limitations of Intrafraction Motion Review (IMR) by comparing the known locations of the beacons using the electromagnetic monitoring system to the position data reported from IMR images. Results A total of 4054 images were taken during treatment. The difference in vector magnitude of the two methods is centered around zero (mean: 0.03 cm, SD: 0.16 cm) and Lin’s Concordance Correlation Coefficient (CCC) is 0.99 (95% CI: 0.98, 1) overall. The Euclidean distance between the two methods was close to zero (median: 0.09 cm, IQR: 0.06, 0.14 cm). The difference in distance between any two markers was centered around zero (mean: 0.01 cm, SD: 0.12 cm) and Lin’s CCC is 0.97 (95% CI: 0.96, 0.98) overall. Conclusion The accuracy of the algorithm for detected markers within the 2D images is comparable to electromagnetic monitoring for fiducial identification when detected. IMR could provide an alternate solution for patients with contraindications of use of an electromagnetic monitoring system and a cost effective alternative to the acquisition of an additional system for patient monitoring, but does not provide data for pre-treatment set-up verification and real-time 3D positioning during treatment.

Purpose: Anaplastic Lymphoma Kinase (ALK) aberrations have been identified in pediatric type infant gliomas, but their occurrence across age groups, functional effects, and treatment response have not been broadly established. Experimental Design: We performed a comprehensive analysis of ALK expression and genomic aberrations in both newly-generated and retrospective data from 371 glioblastomas (156 adult, 205 infant/pediatric and 10 congenital) with in vitro and in vivo validation of aberrations. Results: ALK aberrations at the protein or genomic level were detected in 12% of gliomas (45/371) in a wide age range (0-80 years). Recurrent as well as novel ALK fusions (LRRFIP1-ALK, DCTN1-ALK, PRKD3-ALK) were present in 50% (5/10) of congenital/infant, 1.4% (3/205) of pediatric, and 1.9% (3/156) of adult GBMs. ALK fusions were present as the only candidate driver in congenital/infant GBMs, and were sometimes focally amplified. In contrast, adult ALK fusions co-occurred with other oncogenic drivers. No activating ALK mutations were identified in any age group. Novel and recurrent ALK rearrangements promoted STAT3 and ERK1/2 pathways and transformation in vitro and in vivo. ALK-fused GBM cellular and mouse models were responsive to ALK inhibitors, including in patient cells derived from a congenital GBM. Relevant to treatment of infant gliomas, we showed that ALK protein appears minimally expressed in the forebrain at perinatal stages and no gross effects on perinatal brain development was seen in pregnant mice treated with the ALK inhibitor ceritinib. Conclusions: These findings support expanded evaluation of brain-penetrant ALK inhibitors in clinical trials across infant, pediatric, and adult GBMs. Citation Format: Anne-Florence Blandin, Ross Giglio, Maya Srikanth Graham, Guadalupe Garcia, Seth Malinowski, Jared K. Woods, Shakti Ramkissoon, Lori Ramkissoon, Frank Dubois, Kate Schoolcraft, Jessica W. Tsai, Dayle K. Wang, Robert Jones, Jayne Vogelzang, Kristine Pelton, Sarah Becker, Fiona Watkinson, Claire Sinai, Elizabeth Cohen, Matthew Booker, Michael Tolstorukov, Veerle Haemels, Liliana Goumnerova, Karen Wright, Mark Kieran, Katie Fehnel, David Reardon, Arnault Tauziede-Espariat, Rishi Lulla, Benjamin Carcamo, Stanley Chaleff, Alain Charest, Frederik De Smet, Azra H. Ligon, Adrian Dubuc, Melanie Pagès, Pascale Varlet, Patrick Wen, Brian Alexander, Susan Chi, Sanda Alexandrescu, Ralf Kittler, Robert Bachoo, Rameen Beroukhim, Pratiti Bandopadhayay, Keith L. Ligon. ALK amplification and rearrangements are recurrent targetable events in congenital and adult glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1201.

Aortopulmonary collaterals (APCs) develop universally, but to varying degrees, in patients with single ventricle congenital heart disease (CHD). Despite their ubiquitous presence, APCs remain poorly understood. We sought to evaluate the association between APC burden and common non-invasive clinical variables. We conducted a single center, retrospective study of patients with single ventricle CHD and previous Glenn palliation who underwent pre-Fontan cardiac magnetic resonance (CMR) imaging from 3/2018 to 3/2021. CMR was used to quantify APC flow, which was normalized to aortic (APC/Q

We sought to characterize the clinical course and outcomes of intervention for Tetralogy of Fallot (TOF) with associated conal septal hypoplasia (CSH) compared to those with identifiable conal septum on initial newborn echocardiogram. We performed a retrospective, 1:2 case-control study of children with TOF anatomy, 33 with CSH and 66 with typical TOF, who underwent surgical repair from 1991-2019 at Children's Wisconsin. Data on echocardiographic anatomic features, systemic oxygen saturations, medical therapies, admissions, palliative interventions, operative strategies, and long-term follow-up were compared. The CSH group had fewer hypercyanotic spells (6% vs 42%, p 0.001), beta-blockers prescribed (12% vs 41%, p = 0.005), and hospital admissions for cyanosis (12% vs 44%; p = 0.001) prior to any intervention. Of 14 who required palliative intervention, 8 had balloon pulmonary valvuloplasty (BPV) (7 from the CSH group and 1 from the control group), and 6 had systemic-to-pulmonary artery shunts (all from the control group). Definitive repair was performed at a significantly older age in the CSH group (10.2 ± 10 vs 5.6 ± 5.9 months, p = 0.011), with less subpulmonary muscle resection (57.6% in vs 92.4%, p 0.001) and higher use of a transannular patch (84.8% vs 65.2%, p = 0.040). The average time to surgical reintervention was similar in both groups (9.7 ± 5.9 vs 8.6 ± 6.4 years in controls). We conclude that infants with TOF and CSH have a milder preoperative clinical course with fewer hypercyanotic spells or need for medical therapy. They also respond well to palliative BPV and can safely undergo later definitive repair compared to typical TOF with a well-developed conal septum.

Prevention of infection and propagation of SARS-CoV-2 is of high priority in the COVID-19 pandemic. Here, we describe S-nitrosylation of multiple proteins involved in SARS-CoV-2 infection, including angiotensin converting enzyme 2 (ACE2), the receptor for viral entry. This reaction prevents binding of ACE2 to the SARS-CoV-2 Spike protein, thereby inhibiting viral entry, infectivity, and cytotoxicity. Aminoadamantane compounds also inhibit coronavirus ion channels formed by envelope (E) protein. Accordingly, we developed dual-mechanism aminoadamantane nitrate compounds that inhibit viral entry and thus spread of infection by S-nitrosylating ACE2 via targeted delivery of the drug after E-protein channel blockade. These non-toxic compounds are active in vitro and in vivo in the Syrian hamster COVID-19 model, and thus provide a novel avenue for therapy.

Interest in integrating magnetic resonance imaging (MRI) in radiation therapy (RT) practice has increased dramatically in recent years owing to its unique advantages such as excellent soft tissue contrast and capability of measuring biological properties. Continuous real-time imaging for intrafractional motion tracking without ionizing radiation serves as a particularly attractive feature for applications in RT. Despite its many advantages, the integration of MRI in RT workflows is not straightforward, with many unmet needs. MR safety remains one of the key challenges and concerns in the clinical implementation of MR simulators and MR-guided radiation therapy systems in radiation oncology. Most RT staff are not accustomed to working in an environment with a strong magnetic field. There are specific requirements in RT that are different from diagnostic applications. A large variety of implants and devices used in routine RT practice do not have clear MR safety labels. RT-specific imaging pulse sequences focusing on fast acquisition, high spatial integrity, and continuous, real-time acquisition require additional MR safety testing and evaluation. This article provides an overview of MR safety tailored toward RT staff, followed by discussions on specific requirements and challenges associated with MR safety in the RT environment. Strategies and techniques for developing an MR safety program specific to RT are presented and discussed.

The purpose of the current study was to investigate the unique effects of a commonly used skill incorporated into treatment packages for borderline personality disorder (BPD), countering emotion-driven behavioral urges. Individuals with BPD (N = 8) participated in a single-case experimental design, specifically a multiple baseline, in which they were randomly assigned to complete a baseline assessment-only phase of 2 or 4 weeks. Participants then received four sessions of the countering emotional behaviors module from the unified protocol, followed by a 4-week follow-up phase. Throughout the duration of the study, daily data capture was used to assess real-time changes in the frequency of emotionally avoidant behaviors in response to emotional experiences. Symptoms of BPD, depression, and anxiety were also assessed. By follow-up, the majority of patients demonstrated a meaningful reduction (per single-case experimental design guidelines for evaluating improvements) in their use of avoidant behaviors. There was also preliminary evidence that encouraging participants to act counter to avoidant urges is associated with decreases in BPD, depression, and anxiety symptoms, as well as negative affectivity. The countering emotional behaviors skill from the unified protocol indeed engages its putative target of emotionally avoidant behavioral coping, indicating it is an active ingredient in multicomponent treatment packages for BPD, with implications for downstream clinical endpoints such as BPD and depressive and anxiety symptoms. (PsycInfo Database Record (c) 2020 APA, all rights reserved).

Oxyntomodulin (OXM) is an intestinal peptide hormone that activates both glucagon-like peptide-1 (GLP-1) and glucagon (GCG) receptors. The natural peptide reduces body weight in obese subjects and exhibits direct acute glucoregulatory effects in patients with type II diabetes. However, the clinical utility of OXM is limited due to its lower in vitro potency and short in vivo half-life. To overcome these issues, we developed stapled, long-acting, and highly potent OXM analogs with balanced activities at both GLP-1 and GCG receptors. The lead molecule O14 exhibits potent and long-lasting effects on glucose control, body weight loss, and reduction of hepatic fat reduction in DIO mice. Importantly, O14 significantly reversed hepatic steatosis; reduced liver weight, total cholesterol, and hepatic triglycerides; and improved markers of liver function in a nonalcoholic steatohepatitis (NASH) mouse model. A symmetrical version of the peptide was also shown to be more efficacious and long-lasting in controlling glucose than semaglutide and the clinical candidate cotadutide in wild-type mice, highlighting the utility of our designs of the dual agonist as a potential new therapy for diabetes and liver diseases.

Background Mortality after stage 1 palliation of hypoplastic left heart syndrome remains significant. Hemodynamic changes result from interaction of cardiac output (CO) and systemic vascular resistance (SVR). We sought to identify time-dependent changes in postoperative hemodynamic states and their associations with mortality. Methods Perioperative data were prospectively collected in an institutional review board–approved database. Hemodynamic state was classified as high CO, high SVR, low SVR, and low CO using bivariate analysis of mean arterial pressure and somatic regional near-infrared spectroscopic oximetry saturation. State classifications over 48 postoperative hours were modelled using multinomial logistic regressions for association with mortality. Results Data from 9614 of 10,272 hours in 214 patients were analyzed. Operative survival was 91%. The predominant state was high CO (46% time). Low CO state without extracorporeal membrane oxygenation (ECMO) was found in 52% of patients for 9.7% time. ECMO was employed in 24 (11.2%) patients for 10.4% time. State stability was 33%, with high SVR the least stable (17%) and high CO the most stable (53%). Transition from high CO increased in hours 1 to 12, mainly to low SVR. Transition to low CO was 18.4%, increasing in hours 1 to 12, mainly from high SVR. The transition risk to ECMO was 0.32%, and 0.74% during hours 1 to 12, only from low CO. Both low CO and ECMO had increased mortality risk. Conclusions Bivariate classification defines hemodynamic states with distinct physiologic, transition, and mortality risk profiles. High SVR state was unstable. Transition to ECMO occurred only from low CO, while the low SVR and high CO states were safest.

BACKGROUND: Difficulty weaning from cardiopulmonary bypass (CPB) or the need to return to CPB (collectively D-CPB) may occur after the Norwood procedure. We sought to evaluate the relationship between D-CBP and survival. METHODS: This was a retrospective chart review of all patients undergoing a Norwood procedure at our institution during the interval 2005-2017. Primary outcome was survival for the Norwood procedure. Secondary outcomes included various measures of morbidity. Successful wean from CBP (S-CPB) was defined as no need to return to full flow CPB during the initial definitive wean or after separation from CPB; otherwise the classification was difficulty with wean (D-CBP). Successful rescue in the D-CPB group was defined as not requiring extracorporeal life support (ECLS) either in the operating room or within the first three postoperative days. RESULTS: Of the 196 patients in the cohort, 49 were D-CPB. Survival for S-CPB was 92.5% (136/147) vs 71.4%% (35/49) for D-CPB (p = 0.001). Major morbidity occurred in 29.9% (44/147) in S-CPB vs 69.4 % (34/49) in D-CPB (p < 0.001). With multivariable analysis, D-CPB was significantly associated with mortality (OR=8.09; CI 2.72 – 24.05; p