Aligning tumor mutational burden (TMB) quantification across diagnostic platforms: phase II of the Friends of Cancer Research TMB Harmonization Project.

BACKGROUND: Tumor mutational burden (TMB) measurements aid in identifying patients who are likely to benefit from immunotherapy; however, there is empirical variability across panel assays and factors contributing to this variability have not been comprehensively investigated. Identifying sources of variability can help facilitate comparability across different panel assays, which may aid in broader adoption of panel assays and development of clinical applications. MATERIALS AND METHODS: Twenty-nine tumor samples and 10 human-derived cell lines were processed and distributed to 16 laboratories; each used their own bioinformatics pipelines to calculate TMB and compare to whole exome results. Additionally, theoretical positive percent agreement (PPA) and negative percent agreement (NPA) of TMB were estimated. The impact of filtering pathogenic and germline variants on TMB estimates was assessed. Calibration curves specific to each panel assay were developed to facilitate translation of panel TMB values to whole exome sequencing (WES) TMB values. RESULTS: Panel sizes >667 Kb are necessary to maintain adequate PPA and NPA for calling TMB high versus TMB low across the range of cut-offs used in practice. Failure to filter out pathogenic variants when estimating panel TMB resulted in overestimating TMB relative to WES for all assays. Filtering out potential germline variants at >0% population minor allele frequency resulted in the strongest correlation to WES TMB. Application of a calibration approach derived from The Cancer Genome Atlas data, tailored to each panel assay, reduced the spread of panel TMB values around the WES TMB as reflected in lower root mean squared error (RMSE) for 26/29 (90%) of the clinical samples. CONCLUSIONS: Estimation of TMB varies across different panels, with panel size, gene content, and bioinformatics pipelines contributing to empirical variability. Statistical calibration can achieve more consistent results across panels and allows for comparison of TMB values across various panel assays. To promote reproducibility and comparability across assays, a software tool was developed and made publicly available.

Comparative reliability and diagnostic performance of conventional 3T magnetic resonance imaging and 1.5T magnetic resonance arthrography for the evaluation of internal derangement of the hip.

OBJECTIVE: To compare the diagnostic accuracy of conventional 3T MRI against 1.5T MR arthrography (MRA) in patients with clinical femoroacetabular impingement (FAI). METHODS: Sixty-eight consecutive patients with clinical FAI underwent both 1.5T MRA and 3T MRI. Imaging was prospectively analysed by two musculoskeletal radiologists, blinded to patient outcomes and scored for internal derangement including labral and cartilage abnormality. Interobserver variation was assessed by kappa analysis. Thirty-nine patients subsequently underwent hip arthroscopy and surgical results and radiology findings were analysed. RESULTS: Both readers had higher sensitivities for detecting labral tears with 3T MRI compared to 1.5T MRA (not statistically significant p=0.07). For acetabular cartilage defect both readers had higher statistically significant sensitivities using 3T MRI compared to 1.5T MRA (p=0.02). Both readers had a slightly higher sensitivity for detecting delamination with 1.5T MRA compared to 3T MRI, but these differences were not statistically significant (p=0.66). Interobserver agreement was substantial to perfect agreement for all parameters except the identification of delamination (3T MRI showed moderate agreement and 1.5T MRA substantial agreement). CONCLUSION: Conventional 3T MRI may be at least equivalent to 1.5T MRA in detecting acetabular labrum and possibly superior to 1.5T MRA in detecting cartilage defects in patients with suspected FAI. KEY POINTS: • Conventional 3T MRI is equivalent to 1.5T MRA for diagnosing labral tears. • Conventional 3T MRI is superior to 1.5T MRA for diagnosing acetabular cartilage defect. • Conventional 3T MRI is equivalent to 1.5T MRA for diagnosing cartilage delamination. • Symptom severity score was significantly higher (p<0.05) in group proceeding to surgery.

NAPB - a novel SNARE-associated protein for early-onset epileptic encephalopathy.

Next-generation sequencing has accelerated the identification of disease genes in many rare genetic disorders including early-onset epileptic encephalopathies (EOEEs). While many of these disorders are caused by neuronal channelopathies, the role of synaptic and related neuronal proteins are increasingly being described. Here, we report a 6-year-old girl with unexplained EOEE characterized by multifocal seizures and profound global developmental delay. Recessive inheritance was considered due to parental consanguinity and Irish Traveller descent. Exome sequencing was performed. Variant prioritization identified a homozygous nonsense variant in the N-ethylmaleimide-sensitive factor attachment protein, beta (NAPB) gene resulting in a premature stop codon and 46% loss of the protein. NAPB plays a role in soluble N-ethylmaleimide-sensitive fusion attachment protein receptor (SNARE)-complex dissociation and recycling (synaptic vesicle docking). Knockout mouse models of the murine ortholog Napb have been previously reported. These mice develop recurrent post-natal epileptic seizures in the absence of structural brain changes. The identification of a disease-causing variant in NAPB further recognizes the importance of the SNARE complex in the development of epilepsy and suggests that this gene should be considered in patients with unexplained EOEE.

Viewing the immune checkpoint VISTA: landscape and outcomes across cancers.

BACKGROUND: Optimizing immune checkpoint inhibitor (ICI) therapy may require identification of co-targetable checkpoint pathways via immune profiling. Herein, we analyzed the transcriptomic expression and clinical correlates of V-domain immunoglobulin suppressor of T-cell activation (VISTA), a promising targetable checkpoint. PATIENTS AND METHODS: RNA sequencing was carried out on 514 tissues reflecting diverse advanced/metastatic cancers. Expression of eight immune checkpoint markers [lymphocyte-activation gene 3 (LAG-3), tumor necrosis factor receptor superfamily 14 (TNFRSF14), programmed cell death protein 1 (PD-1), programmed death-ligand 1 (PD-L1), programmed death-ligand 2 (PD-L2), B- and T-lymphocyte attenuator (BTLA), T-cell immunoglobulin and mucin domain-containing protein 3 (TIM-3), cytotoxic T-lymphocyte antigen 4 (CTLA-4)], in addition to VISTA, was analyzed, along with clinical outcomes. RESULTS: High VISTA RNA expression was observed in 32% of tumors (66/514) and was the most common highly expressed checkpoint among the nine assessed. High VISTA expression was independently correlated with high BTLA, TIM-3, and TNFRSF14, and with a diagnosis of pancreatic, small intestine, and stomach cancer. VISTA transcript levels did not correlate with overall survival (OS) from metastatic/advanced disease in the pan-cancer cohort or with immunotherapy outcome (progression-free survival and OS from the start of ICI) in 217 ICI-treated patients. However, in ICI-treated pancreatic cancer patients (n = 16), median OS was significantly shorter (from immunotherapy initiation) for the high- versus not-high-VISTA groups (0.28 versus 1.21 years) (P = 0.047); in contrast, VISTA levels were not correlated with OS in 36 pancreatic cancer patients who did not receive ICI. CONCLUSION: High VISTA expression correlates with high BTLA, TIM-3, and TNFRSF14 checkpoint-related molecules and with poorer post-immunotherapy survival in pancreatic cancer, consistent with prior literature indicating that VISTA is prominently expressed on CD68+ macrophages in pancreatic cancers and requiring validation in larger prospective studies. Immunomic analysis may be important for individualized precision immunotherapy.

Genome-wide association study of osteochondrosis in the tarsocrural joint of Dutch Warmblood horses identifies susceptibility loci on chromosomes 3 and 10.

Equine osteochondrosis is a developmental joint disease that is a significant source of morbidity affecting multiple breeds of horse. The genetic variants underlying osteochondrosis susceptibility have not been established. Here, we describe the results of a genome-wide association study of osteochondrosis using 90 cases and 111 controls from a population of Dutch Warmblood horses. We report putative associations between osteochondrosis and loci on chromosome 3 (BIEC2-808543; P = 5.03 × 10(-7) ) and chromosome 10 (BIEC2-121323; P = 2.62 × 10(-7) ).

Assembly of microarrays for genome-wide measurement of DNA copy number.

We have assembled arrays of approximately 2,400 BAC clones for measurement of DNA copy number across the human genome. The arrays provide precise measurement (s.d. of log2 ratios=0.05-0.10) in cell lines and clinical material, so that we can reliably detect and quantify high-level amplifications and single-copy alterations in diploid, polyploid and heterogeneous backgrounds.

Kinda baboons (Papio kindae) and grayfoot chacma baboons (P. ursinus griseipes) hybridize in the Kafue river valley, Zambia.

The ranges of small kinda (Papio kindae) and much larger grayfooted chacma (P. ursinus griseipes) baboons adjoin in the Kafue National Park, Zambia. In a visual survey of baboons at 48 sites in the Kafue River drainage we found that, contrary to previous reports, groups at the species interface near the town of Ngoma are phenotypically diverse and presumably formed by multigenerational hybridization. Mitochondrial and/or Y-chromosome genetic markers from fecal samples (N=164) collected at 29 sites support this conclusion. Groups with phenotypic signs of a history of hybridization also had taxon-specific mitochondria and Y-haplotypes from both parental species. Although the distribution of mitochondrial haplotypes largely mirrored that of external phenotypes, a significant proportion of male specimens from grayfoot as well as hybrid groups carried kinda Y-chromosomes, and kinda Y-chromosomes were involved in all observed cases of mitochondrial/Y-chromosome discordance. These observations are consistent with, though they do not prove, a population history in which the range of chacmas and the hybrid zone have advanced at the expense of the kinda range. They also suggest that, unexpectedly, kinda male×chacma female matings are much more common than the reciprocal cross in the ancestry of hybrids. We suggest that distinctive male kinda behavior and the "juvenile" appearance of kinda baboons of both sexes, perhaps combined with obstetric difficulties of a small kinda female carrying the large offspring of a chacma male, may account for this bias.

Clonal expansion of abnormal B cells in old NZB mice.

The spleens of old NZB mice have an abnormal population of B cells with extra chromosomes. These hyperdiploid B cells manifest increased proliferative capacity; they grow in (NZB X DBA/2)F1 spleens after intravenous injections. Molecular analysis of individual old NZB and F1 passaged spleens demonstrate that hyperdiploid cells represent a clonal or oligoclonal expansion of B cells. All spleens with at least 10% hyperdiploid cells demonstrated both heavy and kappa light chain immunoglobulin gene rearrangements by Southern blot hybridization. None of the hyperdiploid spleens from old NZB mice had lambda rearrangements and only one of five showed evidence of clonal rearrangement of the TCR-beta gene. One also had a VK10 clonal rearrangement. Elevated p53 oncogene protein was observed in NZB hyperdiploid spleen cells; however, no p53 or other oncogene rearrangements or amplifications were seen. Hyperdiploid cells were IgM-bright, IgD-dull, Ia+, dull B220, Thy-1-, and Ly-1-dull. Spleens with hyperdiploid B cells had increased percentages of Ly-1 B cells. The data suggest that hyperdiploid cells in old NZB mice represent clonal expansion of B cells and that they may represent an intermediate stage between autoimmunity and malignancy.

Genome-wide SNP association-based localization of a dwarfism gene in Friesian dwarf horses.

The recent completion of the horse genome and commercial availability of an equine SNP genotyping array has facilitated the mapping of disease genes. We report putative localization of the gene responsible for dwarfism, a trait in Friesian horses that is thought to have a recessive mode of inheritance, to a 2-MB region of chromosome 14 using just 10 affected animals and 10 controls. We successfully genotyped 34,429 SNPs that were tested for association with dwarfism using chi-square tests. The most significant SNP in our study, BIEC2-239376 (P(2df)=4.54 × 10(-5), P(rec)=7.74 × 10(-6)), is located close to a gene implicated in human dwarfism. Fine-mapping and resequencing analyses did not aid in further localization of the causative variant, and replication of our findings in independent sample sets will be necessary to confirm these results.

Case report: Rhodococcus erythropolis osteomyelitis in the toe.

The Rhodococcus species rarely cause musculoskeletal infections, with only two cases reported in the literature. We report the case of a 53-year-old woman who had an infection develop after first metatarsophalangeal joint fusion. A year after surgery, she continued to have pain and swelling with nonunion. She underwent revision of the arthrodesis and tissue samples from surgery revealed Rhodococcus erythropolis. The patient's symptoms improved with oral antibiotics. One year after the revision surgery, the fusion had united. We believe this is the first report of a case of a musculoskeletal infection caused by Rhodococcus erythropolis.

Ischemia-induced alterations in myocardial (Na+ + K+)-ATPase and cardiac glycoside binding.

The effects of ischemia on the canine myocardial (Na+ + K+)-ATPase complex were examined in terms of alterations in cardiac glycoside binding and enzymatic activity. Ability of the myocardial cell to bind tritiated ouabain in vivo was assessed after 1, 2, and 6 h of coronary occlusion followed by 45 min of reperfusion, and correlated with measurements of in vitro (Na+ + K+)-ATPase activity and in vitro [3H]ouabain binding after similar periods of ischemia. Regional blood flow alterations during occlusion and reperfusion were simultaneously determined utilizing 15 mum radioactive microspheres to determine the degree to which altered binding of ouabain might be flow related. Anterior wall infarction was produced in 34 dogs by snaring of confluent branches of the left coronary system. Epicardial electrograms delineated ischemic and border zone areas. Coronary reperfusion after 2 and 6 h of occlusion was associated with impaired reflow of blood and markedly impaired uptake of [3H]ouabain in ischemic myocardium. In both groups, in vivo [3H]ouabain binding by ischemic tissue was reduced out of proportion to the reduction in flow. Despite near-complete restoration of flow in seven dogs occluded for 1 h and reperfused, [3H]ouabain remained significantly reduced to 58 +/- 9% of nonischemic uptake in subendocardial layers of the central zone of ischemia. Thus, when coronary flow was restored to areas of myocardium rendered acutely ischemia for 1 or more hours, ischemic zones demonstrated progressively diminished ability to bind ouabain. To determine whether ischemia-induced alteration in myocardial (Na+ + K+)-ATPase might underlie these changes, (Na+ + K+)-ATPase activity and [3H]ouabain binding were measured in microsomal fractions from ischemic myocardium after 1, 2, and 6 h of coronary occlusion. In animals occluded for 6 h, (Na+ + K+)-ATPase activity was significantly reduced by 40% in epicardial and by 35% in endocardial layers compared with nonischemic myocardium. Comparable reductions in in vitro [3H]ouabain binding were also demonstrated. Reperfusion for 45 min after occlusion for 6 h resulted in no significant restoration of enzyme activity when compared to the nonreperfused animals. In six animals occluded for 2 h, a time at which myocardial creatine phosphokinase activity remains unchanged, (Na+ + K+)-ATPase activity was reduced by 25% compared with nonischemic enzyme activity. In five dogs occluded for 1 h, (Na+ + K+)-ATPase activity in ischemic myocardium was unchanged from control levels. We conclude that reduced regional myocardial blood flow, local alterations in cellular milieu, and altered glycoside-binding properties of (Na+ + K+)-ATPase all participate in the reduction of cardiac glycoside binding observed after reperfusion of ischemic myocardium. In addition, after 2 or more hours of severe ischemia, myocardial (Na+ + K+)-ATPase catalytic activity is significantly reduced despite incubation in the presence of optimal substrate concentrations.

Temperature effects on wood anatomy, wood density, photosynthesis and biomass partitioning of Eucalyptus grandis seedlings.

Wood density, a gross measure of wood mass relative to wood volume, is important in our understanding of stem volume growth, carbon sequestration and leaf water supply. Disproportionate changes in the ratio of wood mass to volume may occur at the level of the whole stem or the individual cell. In general, there is a positive relationship between temperature and wood density of eucalypts, although this relationship has broken down in recent years with wood density decreasing as global temperatures have risen. To determine the anatomical causes of the effects of temperature on wood density, Eucalyptus grandis W. Hill ex Maiden seedlings were grown in controlled-environment cabinets at constant temperatures from 10 to 35 degrees C. The 20% increase in wood density of E. grandis seedlings grown at the higher temperatures was variously related to a 40% reduction in lumen area of xylem vessels, a 10% reduction in the lumen area of fiber cells and a 10% increase in fiber cell wall thickness. The changes in cell wall characteristics could be considered analogous to changes in carbon supply. Lumen area of fiber cells declined because of reduced fiber cell expansion and increased fiber cell wall thickening. Fiber cell wall thickness was positively related to canopy CO2 assimilation rate (Ac), which increased 26-fold because of a 24-fold increase in leaf area and a doubling in leaf CO2 assimilation rate from minima at 10 and 35 degrees C to maxima at 25 and 30 degrees C. Increased Ac increased seedling volume, biomass and wood density; but increased wood density was also related to a shift in partitioning of seedling biomass from roots to stems as temperature increased.

Why does phosphorus limitation increase wood density in Eucalyptus grandis seedlings?

Wood density influences both the physiological function and economic value of tree stems. We examined the relationship between phosphorus (P) supply and stem wood density of Eucalyptus grandis Hill ex Maiden seedlings grown with varying soil P additions and determined how changes in wood anatomy and biomass partitioning affect the relationship. Plant height, stem diameter and total biomass increased by 400-500% with increasing P supply. Stem wood density decreased sharply from 520 to 380 kg m(-3) as P supply increased to 70 mg P kg(soil) (-1). Further increases in P supply to 1000 mg P kg(soil) (-1) had no effect on wood density. The increase in wood density at low soil P supply arose principally from enhanced secondary wall thickening of stem fiber cells. Cell wall thickness increased from 3.6 to 4.5 microm as soil P supply decreased. Because fiber cell diameter was independent of soil P (12 microm +/- 0.3), the proportion of the stem occupied by cell wall material increased as P supply declined. The enhanced secondary wall thickening of stem fiber cells at low P supply was not associated with changes in whole-plant biomass partitioning. Instead, low P supply appeared to alter biomass partitioning within the stem in favor of secondary wall thickening. Thus, increased wood density in E. grandis seedlings grown at low P soil supply was associated with inhibited stem cambial activity, resulting in an increased proportion of photoassimilates available for secondary wall thickening of fiber cells.

Intrachromosomal genomic instability in human sporadic colorectal cancer measured by genome-wide allelotyping and inter-(simple sequence repeat) PCR.

We have used genome-wide allelotyping with 348 polymorphic autosomal markers spaced, on average, 10 cM apart to quantitate the extent of intrachromosomal instability in 59 human sporadic colorectal carcinomas. We have compared instability measured by this method with that measured by inter-(simple sequence repeat) PCR and microsatellite instability assays. Instability quantitated by fractional allelic loss rates was found to be independent of that detected by microsatellite instability analyses but was weakly associated with that measured by inter-(simple sequence repeat) PCR. A set of seven loci were identified that were most strongly associated with elevated rates of fractional allelic loss and/or inter-(simple sequence repeat) PCR instability; these seven loci were on chromosomes 3, 8, 11, 13, 14, 18, and 20. A lesser association was seen with two loci flanking p53 on chromosome 17. Coordinate loss patterns for these loci suggest that at least two separate sets of cooperating loci exist for intrachromosomal genomic instability in human colorectal cancer.

Canine pulmonary angiotensin-converting enzyme. Physicochemical, catalytic and immunological properties.

Antiontensin-converting enzyme (peptidyldipeptide hydrolase, EC 3.4.15.1) has been solubilized from canine pulmonary particles and purified to apparent homogeneity. A value of approx. 140000 was estimated for the molecular weight of the native and the reduced, denatured forms of the enzyme. No free NH2-terminal residue was detected by the dansylation procedure. Carbohydrate accounted for 17% of the weight of the enzyme, and the major residues were galactose, mannose and N-acetylglucosamine with smaller amounts of sialic acid and fucose. Removal of sialic acid residues with neuraminidase did not alter enzymatic activity. The enzyme contained one molar equivalent of zinc. Addition of this metal reversed stimulation and inhibition of activity observed in the presence of Co2+ and Mn2+, respectively. Immunologic homology of pure dog and rabbit enzymes was demonstrable with goat antisera. Fab fragments and intact IgG antibodies displayed similar inhibition dose vs. response curves with homologous enzyme, whereas the fragments were poor inhibitors of heterologous activity compared to the holoantibodies. The canine glycoprotein was much less active than the rabbit preparation in catalyzing hydrolysis of Hip-His-Leu. In contrast, the two enzymes exhibited comparable kinetic parameters with angiotensin I as substrate.

Diurnal Regulation of Leaf Blade Elongation in Rice by CO2 (Is it Related to Sucrose-Phosphate Synthase Activity?).

The relationship between leaf blade elongation rates (LER) and sucrose-phosphate synthase (SPS) activity was investigated at different times during ontogeny of rice (Oryza sativa L. cv Jarrah) grown in flooded soil at either 350 or 700 [mu]L CO2 L-1. High CO2 concentrations increased LER of expanding blades and in vivo activity (Vlimiting) SPS activity of expanded blades during the early vegetative stage (21 d after planting [DAP]), when tiller number was small and growing blades were strong carbohydrate sinks. Despite a constant light environment, there was a distinct diurnal pattern in LER, Vlimiting SPS activity, and concentration of soluble sugars, with an increase in the early part of the light period and a decrease later in the light period. The strong correlation (r = 0.65) between LER and Vlimiting SPS activity over the diurnal cycle indicated that SPS activity played an important role in controlling blade growth. The higher Vlimiting SPS activity at elevated CO2 at 21 DAP was caused by an increase in the activation state of the enzyme rather than an increase in Vmax. Fructose and glucose accumulated to a greater extent than sucrose at high CO2 and may have been utilized for synthesis of cell-wall components, contributing to higher specific leaf weight. By the mid-tillering stage (42 DAP), CO2 enrichment enhanced Vlimiting and Vmax activities of source blades. Nevertheless, LER was depressed by high CO2, probably because tillers were stronger carbohydrate sinks than growing blades.

Accelerated Early Growth of Rice at Elevated CO2 (Is It Related to Developmental Changes in the Shoot Apex?).

The influence of elevated CO2 on the development of the shoot apex and on subsequent vegetative growth and grain yield was investigated using rice (Oryza sativa L. cv Jarrah) grown in flooded soil at either 350 or 700 [mu]L CO2 L-1. At 8 d after planting (DAP), elevated CO2 increased the height and diameter of the apical dome and lengths of leaf primordia and tiller buds but had no effect on their numbers. By 16 DAP, there were five tiller buds in the apex at 700 [mu]L CO2 L-1 compared with only three tiller buds at 350 [mu]L CO2 L-1. These changes in development of the shoot apex at high CO2 were forerunners to faster development of the vegetative shoot at elevated CO2 between 11 and 26 DAP as evidenced by increases in the relative growth rates of the shoot and tillers. Accelerated development at high CO2 was responsible for the 42% increase in tiller number at the maximum tillering stage and the 57% enhancement of grain yield at the final harvest. The link between high CO2 effects on development during the first 15 DAP and final tiller number and grain yield was demonstrated by delaying exposure of plants to high CO2 for 15 d. The delay totally inhibited the tillering response to high CO2, and the increase in grain yield of 20% arose from a greater number of grains per panicle. Consequently, it can be concluded that accelerated development in the shoot apex early in development is crucial for obtaining maximum increases in grain yield at elevated atmospheric CO2 concentrations.

Inhibition of horseradish peroxidase activity by specific antibody: determinant specificity of anticatalytic antibodies.

Rabbit antisera specific for horseradish peroxidase inhibit the catalytic activity of the enzyme. All antibodies prepared against the holoenzyme react with the peroxidase apoenzyme. However, only a minority (30-45%) of the total antiperoxidase pool cross react with reduced and alkylated apoenzymes. The antibodies inhibiting peroxidase activity do not bind to S-carboxymethyl of S-carboxamidomethylated apoenzyme derivatives as measured by absorption and competition of inhibition experiments. Glycopeptides derived from horseradish peroxidase also failed to bind anticatalytic antibodies. Antibodies that inhibit enzyme activity have specificity for noncarbohydrate conformation dependent antigenic determinants of horseradish peroxidase. Additional experiments probed the mechanism by which inhibitory antibody decreases the catalytic activity of horseradish peroxidase. Absorption spectra of horseradish peroxidase that has bound Fab fragments sufficient to cause 90% inhibition of the enzyme activity determined that the enzyme retained the ability to bind hydrogen peroxide. Thus, anticatalytic antibodies do not prevent the formation of the first enzyme-substrate intermediate but mediate their inhibitory effects by disrupting a later step in the reaction mechanism.