We have mapped GRB2, a signal transduction gene whose protein product is an essential component of the pathway between tyrosine kinases (such as the epidermal growth factor receptor) and downstream ...proteins (such as Ras and Sos). We assigned GRB2 to human chromosome 17 by hybridization to a somatic cell hybrid mapping panel. To position the locus at a much finer resolution, we have isolated the human GRB2 gene in three different cosmids, which we have mapped by fluorescence in situ hybridization to the long arm of human chromosome 17 (17q24-q25). We have hybridized a human GRB2 open reading frame probe to mouse DNAs from the European Interspecific Backcross. The segregation patterns reveal that the mouse Grb2 locus maps distally on chromosome 11, and an additional Grb2-related locus is present on chromosome 4 of one of the parental strains, Mus spretus/CRC.
Decisions to stop randomized trials are often based on traditional P value thresholds and are often unconvincing to clinicians. To familiarize clinical investigators with the application and ...advantages of Bayesian monitoring methods, we illustrate the steps of Bayesian interim analysis using a recent major trial that was stopped based on frequentist analysis of safety and futility.
We conducted Bayesian reanalysis of a factorial trial in newborn infants with hypoxic-ischemic encephalopathy that was designed to investigate whether outcomes would be improved by deeper (32 °C) or longer cooling (120 h), as compared with those achieved by standard whole body cooling (33.5 °C for 72 h). Using prior trial data, we developed neutral and enthusiastic prior probabilities for the effect on predischarge mortality, defined stopping guidelines for a clinically meaningful effect, and derived posterior probabilities for predischarge mortality.
Bayesian relative risk estimates for predischarge mortality were closer to 1.0 than were frequentist estimates. Posterior probabilities suggested increased predischarge mortality (relative risk > 1.0) for the three intervention groups; two crossed the Bayesian futility threshold.
Bayesian analysis incorporating previous trial results and different pre-existing opinions can help interpret accruing data and facilitate informed stopping decisions that are likely to be meaningful and convincing to clinicians, meta-analysts, and guideline developers.
ClinicalTrials.gov NCT01192776 . Registered on 31 August 2010.
Functional imaging studies have demonstrated a relationship between fatigue and altered cerebral activation patterns in multiple sclerosis patients, but no relationship between fatigue and brain ...atrophy has been demonstrated. We hypothesized that the subjective complaint of fatigue would predict the severity of destructive brain pathology. We assessed the relationship between fatigue and brain atrophy longitudinally in a cohort of 134 patients previously enrolled in a 2-year clinical trial of interferon β-1a and re-evaluated 8 years after randomization into the trial. Brain atrophy was measured using the brain parenchymal fraction (BPF), and disability was measured using the Multiple Sclerosis Functional Composite at baseline, year 2 and year 8. Fatigue was measured using the Sickness Impact Profile's Sleep and Rest Scale (SIPSR) at baseline, year 2 and year 8. Linear regression analyses were used to assess the relationship between changes in fatigue and atrophy progression
. Worsening fatigue on the SIPSR during the initial 2 years was significantly associated with progressive brain atrophy over the subsequent 6 years. The relationships between fatigue and brain atrophy were independent of changes in disability, mood, or other MRI characteristics. This suggests that the subjective complaint of fatigue is linked to destructive pathologic processes in RRMS patients.
Every year individuals experience symptoms that remain undiagnosed by healthcare providers. In the United States, these rare diseases are defined as a condition that affects fewer than 200,000 ...individuals. However, there are an estimated 7000 rare diseases, and there are an estimated 25-30 million Americans in total (7.6-9.2% of the population as of 2018) affected by such disorders. The NIH Common Fund Undiagnosed Diseases Network (UDN) seeks to provide diagnoses for individuals with undiagnosed disease. Mass spectrometry-based metabolomics and lipidomics analyses could advance the collective understanding of individual symptoms and advance diagnoses for individuals with heretofore undiagnosed disease. Here, we report the mass spectrometry-based metabolomics and lipidomics analyses of blood plasma, urine, and cerebrospinal fluid from 148 patients within the UDN and their families, as well as from a reference population of over 100 individuals with no known metabolic diseases. The raw and processed data are available to the research community so that they might be useful in the diagnoses of current or future patients suffering from undiagnosed disorders.
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Therapy for pediatric acute myeloid leukemia (AML) requires long term central vascular access, which puts patients at risk for central line-associated bloodstream infections and their associated ...morbidity and mortality. Options for central venous catheter (CVC) access include tunneled external catheters, implanted ports, or peripherally inserted central catheters (PICC). Multiple studies provide conflicting reports on which CVC type is least likely to be associated with bloodstream infections in pediatric hematology-oncology patients. This multicenter cohort of pediatric AML patients is the first to investigate the course-specific incidence of positive CVC blood cultures by line type.
The study included patients receiving frontline chemotherapy for AML between 1/1/2012 and 6/30/2017 at 12 US institutions. Trained personnel completed manual chart abstraction from 12/1/2015 to 07/15/2017 to obtain information on demographics, diagnosis, treatment, antibacterial prophylaxis, CVC type at the start of each course, neutropenia duration, and blood culture results obtained from CVC. Abstractors followed a standardized protocol and were required to achieve >95% concordance with trainer before starting formal chart review.
Course-specific follow-up began the first day absolute neutrophil count (ANC) fell below 200 cells/uL and continued to the earliest of first positive CVC culture (overall and separately for gram-positive and gram-negative species), ANC recovery, start of conditioning for stem cell transplant, relapse, death or start of next course. Per National Healthcare Safety Network guidance, common commensals were not considered true events with the exception of viridans group streptococci (VGS). Nonbacterial pathogens were also excluded. Covariate distributions were compared by CVC type using chi-square tests. Variability in CVC type by site was examined graphically. Incidence rates for CVC positive blood cultures were reported by line type as per 1000 neutropenic days. Multivariate Poisson regression with log of neutropenia duration as offset was used to compute adjusted rate ratios (aIRR) comparing rates of positive CVC blood cultures by line type, controlling for covariates itemized in Table 1. General estimating equation methods were used to account for non-independence of observations from patients from the same institution.
The study population included 357 patients who received 1,164 chemotherapy courses. Median course duration was 38 days (IQR: 34-44) with a median neutropenia duration of 19 days (IQR: 14-25). CVC were present in all patients at the start of each course, with the exception of 11 (3.3%) patients lacking a CVC at the start of Induction I. While tunneled CVC were most prevalent representing 59.1% of courses, PICC and implanted ports were utilized in 28.9% and 11.6% of courses, respectively. There was considerable variation in the distribution of CVC type by treating institution (Figure 1). Age, race, and ethnicity distributions varied by line type (Table 1). Antibacterial prophylaxis was utilized in 47% of courses.
In total, there were 300 (25.8%) courses in 199 (55.7%) patients complicated by a positive CVC blood culture during the neutropenic period. The overall incidence rate of positive CVC blood cultures was 6.5 per 1000 neutropenic days. Rates increased over the course of frontline treatment from 3.1 per 1000 neutropenic days in Induction I to 9.6 per 1000 neutropenic days in Intensification II. The rate of gram-positive events (4.2 per 1000 neutropenic days) was higher than gram-negative events (2.0 per 1000 neutropenic days). The most common isolated organisms were VGS at 57.3% and less than 1% of events were polymicrobial infections. Overall rates of positive CVC blood cultures were higher for PICC relative to tunneled CVC (aIRR=1.19, 95% CI: 1.02, 1.38), while rates for implanted ports and tunneled CVC were similar (aIRR=1.06, 95% CI: 0.75, 1.51).
While bacteremia is an expected complication during post-chemotherapy neutropenia and not always due to the presence of a CVC, the risk for a positive CVC blood culture is significantly increased for children with PICC compared with those with tunneled CVC or implanted port. Also, the risk was higher after Induction I and increased with age. Given the notable practice variation in line choice, these data highlight an opportunity for standardization with a goal to reduce infectious complications.
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Fisher:Pfizer: Research Funding; Merck: Research Funding. Gramatges:Bristol Meyer Squibb: Research Funding.
Abstract The SENTINEL study showed that the addition of natalizumab improved outcomes for patients with relapsing multiple sclerosis (MS) who had experienced disease activity while receiving ...interferon beta-1a (IFNβ-1a) alone. Previously unreported secondary and tertiary magnetic resonance imaging (MRI) measures are presented here. Patients received natalizumab 300 mg ( n = 589) or placebo ( n = 582) intravenously every 4 weeks plus IFNβ-1a 30 µg intramuscularly once weekly. Annual MRI scans allowed comparison of a range of MRI end points versus baseline. Over 2 years, 67% of patients receiving natalizumab plus IFNβ-1a remained free of new or enlarging T2-lesions compared with 30% of patients receiving IFNβ-1a alone. The mean change from baseline in T2 lesion volume over 2 years decreased in patients receiving natalizumab plus IFNβ-1a and increased in those receiving IFNβ-1a alone (–277.5 mm3 versus 525.6 mm3 ; p < 0.001). Compared with IFNβ-1a alone, add-on natalizumab therapy resulted in a smaller increase in mean T1-hypointense lesion volume after 2 years (1821.3 mm3 versus 2210.5 mm3 ; p < 0.001), a smaller mean number of new T1-hypointense lesions over 2 years (2.3 versus 4.1; p < 0.001), and a slower rate of brain atrophy during the second year of therapy (–0.31% versus –0.40%; p = 0.020). Natalizumab add-on therapy reduced gadolinium-enhancing, T1-hypointense, and T2 MRI lesion activity and slowed brain atrophy progression in patients with relapsing MS who experienced disease activity despite treatment with IFNβ-1a alone.
Heterotrimeric guanine nucleotide binding proteins of the G12/13 subfamily, which includes the α-subunits Gα12 and Gα13, stimulate the monomeric G protein RhoA through interaction with a distinct ...subset of Rho-specific guanine nucleotide exchange factors (RhoGEFs). The structural features that mediate interaction between Gα13 and RhoGEFs have been examined in crystallographic studies of the purified complex, whereas a Gα12:RhoGEF complex has not been reported. Several signaling responses and effector interactions appear unique to Gα12 or Gα13, despite their similarity in amino acid sequence.
To comprehensively examine Gα12 for regions involved in RhoGEF interaction, we screened a panel of Gα12 cassette substitution mutants for binding to leukemia-associated RhoGEF (LARG) and for activation of serum response element mediated transcription.
We identified several cassette substitutions that disrupt Gα12 binding to LARG and the related p115RhoGEF. These Gα12 mutants also were impaired in activating serum response element mediated signaling, a Rho-dependent response. Most of these mutants matched corresponding regions of Gα13 reported to contact p115RhoGEF, but unexpectedly, several RhoGEF-uncoupling mutations were found within the N- and C-terminal regions of Gα12. Trypsin protection assays revealed several mutants in these regions as retaining conformational activation. In addition, charge substitutions near the Gα12 N-terminus selectively disrupted binding to LARG but not p115RhoGEF.
Several structural aspects of the Gα12:RhoGEF interface differ from the reported Gα13:RhoGEF complex, particularly determinants within the C-terminal α5 helix and structurally uncharacterized N-terminus of Gα12. Furthermore, key residues at the Gα12 N-terminus may confer selectivity for LARG as a downstream effector.
Long COVID is a major problem affecting patient health, the health service, and the workforce. To optimise the design of future interventions against COVID-19, and to better plan and allocate health ...resources, it is critical to quantify the health and economic burden of this novel condition. We aimed to evaluate and estimate the differences in health impacts of long COVID across sociodemographic categories and quantify this in Quality-Adjusted Life-Years (QALYs), widely used measures across health systems.
With the approval of NHS England, we utilised OpenPROMPT, a UK cohort study measuring the impact of long COVID on health-related quality-of-life (HRQoL). OpenPROMPT invited responses to Patient Reported Outcome Measures (PROMs) using a smartphone application and recruited between November 2022 and October 2023. We used the validated EuroQol EQ-5D questionnaire with the UK Value Set to develop disutility scores (1-utility) for respondents with and without Long COVID using linear mixed models, and we calculated subsequent Quality-Adjusted Life-Months (QALMs) for long COVID.
The total OpenPROMPT cohort consisted of 7575 individuals who consented to data collection, with which we used data from 6070 participants who completed a baseline research questionnaire where 24.6% self-reported long COVID. In multivariable regressions, long COVID had a consistent impact on HRQoL, showing a higher likelihood or odds of reporting loss in quality-of-life (Odds Ratio (OR): 4.7, 95% CI: 3.72–5.93) compared with people who did not report long COVID. Reporting a disability was the largest predictor of losses of HRQoL (OR: 17.7, 95% CI: 10.37–30.33) across survey responses. Self-reported long COVID was associated with an 0.37 QALM loss.
We found substantial impacts on quality-of-life due to long COVID, representing a major burden on patients and the health service. We highlight the need for continued support and research for long COVID, as HRQoL scores compared unfavourably to patients with conditions such as multiple sclerosis, heart failure, and renal disease.
This research was supported by the National Institute for Health and Care Research (NIHR) (OpenPROMPT: COV-LT2-0073).
ABSTRACT
Background
Currently available structural variant (SV) detection methods do not span the complete spectrum of disease‐causing SVs. Optical genome mapping (OGM), an emerging technology with ...the potential to resolve diagnostic dilemmas, was performed to investigate clinically‐relevant SVs in a 4‐year‐old male with an epileptic encephalopathy of undiagnosed molecular origin.
Methods
OGM was utilized to image long, megabase‐size DNA molecules, fluorescently labeled at specific sequence motifs throughout the genome with high sensitivity for detection of SVs greater than 500 bp in size. OGM results were confirmed in a CLIA‐certified laboratory via mate‐pair sequencing.
Results
OGM identified a mosaic, de novo 90 kb deletion and inversion on the X chromosome disrupting the CDKL5 gene. Detection of the mosaic deletion, which had been previously undetected by chromosomal microarray, an infantile epilepsy panel including exon‐level microarray for CDKL5, exome sequencing as well as genome sequencing, resulted in a diagnosis of X‐linked dominant early infantile epileptic encephalopathy‐2.
Conclusion
OGM affords an effective technology for the detection of SVs, especially those that are mosaic, since these remain difficult to detect with current NGS technologies and with conventional chromosomal microarrays. Further research in undiagnosed populations with OGM is warranted.
In a participant with an epileptic encephalopathy of undiagnosed molecular origin, optical genome mapping (OGM), identified a mosaic, de novo 90 kb deletion on the X chromosome disrupting the CDKL5 gene. Detection of the mosaic deletion, which had been previously undetected by chromosomal microarray, an infantile epilepsy panel including exon‐level microarray for CDKL5, exome sequencing as well as genome sequencing, resulted in a diagnosis of X‐linked dominant early infantile epileptic encephalopathy‐2. OGM affords an effective technology for detection of SVs, especially those that are mosaic, since these remain difficult to detect with current NGS technologies and with conventional chromosomal microarrays.
Lymphomas represent nearly 70 distinct diseases with unique clinical presentations, therapeutic responses and underlying biology. There is a pressing shortage of publically available cell line and in ...vivo models of nearly all of these diseases, which has severely hampered efforts to understand and target their biology. To address this issue, we have established a repository of patient-derived xenografts (PDX) of lymphomas by engrafting human tumors into immunodeficient NOD/SCID/IL2rgnull (NSG) mice. These lymphomas, along with a spectrum of other PDXs of hematologic malignancies, are available to collaborators through the online portal PRoXe (Public Repository of Xenografts) at http://PRoXe.org. Blood and bone marrow specimens involved with tumor are injected by tail vein (IV) injection. Lymph node and extranodal biopsy specimens are implanted under the renal capsule as a 1x1x2mm tumor seed (renal), which maintains the in situ microarchitecture. A full description of xenografted lymphomas is included in the Table. Table 1DiseaseType of implant# in 1st passage# in 2nd passage or higherT-cell prolymphocytic leukemiaIV1Angioimmunoblastic T-cell lymphomaIV11Mantle cell lymphomaIV12Double-hit DLBCLIV2Sézary SyndromeIV1Adult T-cell Leukemia/LymphomaIV1Diffuse large B cell lymphomaIV2Diffuse large B cell lymphomarenal2Marginal zone lymphomarenal11NK/T-cell lymphomarenal1Peripheral T-cell lymphoma-NOSrenal1Breast implant-associated anaplastic large cell lymphomarenal1
Engrafted PDXs have been extensively characterized by immunohistochemistry, flow cytometry, transcriptome sequencing and targeted DNA sequencing. Flow cytometric analysis of patient tumors and their respective xenografts consistently revealed highly concordant immunophenotypes compared to the original tumors. Similarly, immunohistochemistry of involved tissues confirmed retention of tumor immunophenotypes, architecture, and even tissue tropism in the PDXs. Examples include a Sézary syndrome PDX that was injected by tail vein and trafficked to spleen, bone marrow, blood and skin, a diffuse large B-cell lymphoma (DLBCL) PDX that infiltrated the CNS, and a second DLBCL PDX that was implanted into the renal capsule of the left kidney and progressed within 8 weeks to bilateral renal involvement. Other notable models include a breast implant-associated, ALK-negative anaplastic large cell lymphoma implanted under the renal capsule that metastasized to the liver and spleen while uniformly retaining CD30 positivity. Two double-hit lymphoma (DHL) PDXs maintained their CD20-negative phenotype through serial passage to P1. A peripheral T-cell lymphoma-NOS (PTCL) specimen implanted under the renal capsule engrafted in the spleen, with a notable admixture of nonmalignant T cells and scattered EBV-positive B cells. T-cell receptor gene rearrangement PCR performed on this PTCL demonstrated an identical rearrangement pattern in the primary tumor and the PDX. Luciferized mantle cell lymphoma and DHL PDXs clearly home to bone marrow, lymph nodes, spleen, and liver as early as two weeks after injection. These findings support the utility of these PDX lines as in vivo models that more accurately recapitulate the human disease than commonly used subcutaneous cell line models. In addition to generating PDXs that remain faithful to their source tumors, we have witnessed interesting examples of in vivo histologic transformation, opening the door to studies of disease progression. One primary follicular lymphoma specimen injected into a cohort of mice transformed to DLBCL in one mouse and a lymphoblastic lymphoma-like disease in another mouse, as confirmed by IHC and flow cytometry. Further xenografting of primary tumors is underway with the goal of establishing a large repository of lymphoma PDXs useful for biologic interrogation and preclinical trials.
Davids:Genentech: Other: ad board; Pharmacyclics: Consultancy; Janssen: Consultancy. Shipp:Gilead: Consultancy; Sanofi: Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees.
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