NCI Workshop Report: Clinical and Computational Requirements for Correlating Imaging Phenotypes with Genomics Signatures

The National Cancer Institute (NCI) Cancer Imaging Program organized two related workshops on June 26–27, 2013, entitled “Correlating Imaging Phenotypes with Genomics Signatures Research” and “Scalable Computational Resources as Required for Imaging-Genomics Decision Support Systems.” The first workshop focused on clinical and scientific requirements, exploring our knowledge of phenotypic characteristics of cancer biological properties to determine whether the field is sufficiently advanced to correlate with imaging phenotypes that underpin genomics and clinical outcomes, and exploring new scientific methods to extract phenotypic features from medical images and relate them to genomics analyses. The second workshop focused on computational methods that explore informatics and computational requirements to extract phenotypic features from medical images and relate them to genomics analyses and improve the accessibility and speed of dissemination of existing NIH resources. These workshops linked clinical and scientific requirements of currently known phenotypic and genotypic cancer biology characteristics with imaging phenotypes that underpin genomics and clinical outcomes. The group generated a set of recommendations to NCI leadership and the research community that encourage and support development of the emerging radiogenomics research field to address short-and longer-term goals in cancer research.     

Maternal cardiovascular disease after twin pregnancies complicated by hypertensive disorders of pregnancy: a population-based cohort study

Background:People whose singleton pregnancy is affected by hypertensive disorders of pregnancy (HDP) are at risk of future cardiovascular disease. It is unclear, however, whether this association can be extrapolated to twin pregnancies. We aimed to compare the association between HDP and future cardiovascular disease after twin and singleton pregnancies.Methods:We conducted a population-based retrospective cohort study that included nulliparous people in Ontario, Canada, 1992–2017. We compared the future risk of cardiovascular disease among pregnant people from the following 4 groups: those who delivered a singleton without HDP (referent) and with HDP, and those who delivered twins either with or without HDP.Results:The populations of the 4 groups were as follows: 1 431 651 pregnant people in the singleton birth without HDP group; 98 631 singleton birth with HDP; 21 046 twin birth without HDP; and 4283 twin birth with HDP. The median duration of follow-up was 13 (interquartile range 7–20) years. The incidence rate of cardiovascular disease was lowest among those with a singleton or twin birth without HDP (0.72 and 0.74 per 1000 person-years, respectively). Compared with people with a singleton birth without HDP, the risk of cardiovascular disease was highest among those with a singleton birth and HDP (1.47 per 1000 person-years; adjusted hazard ratio [HR] 1.81 [95% confidence interval (CI) 1.72–1.90]), followed by people with a twin pregnancy and HDP (1.07 per 1000 person-years; adjusted HR 1.36 [95% CI 1.04–1.77]). The risk of the primary outcome after a twin pregnancy with HDP was lower than that after a singleton pregnancy with HDP (adjusted HR 0.74 [95% CI 0.57–0.97]), when compared directly.Interpretation:In a twin pregnancy, HDP are weaker risk factors for postpartum cardiovascular disease than in a singleton pregnancy.

Cardiovascular disease has been shown to be the leading cause of death among women.^1–3 Classic risk factors for cardiovascular disease include obesity, diabetes mellitus, hypertension and family history of cardiovascular disease. ³ More recently, an association has been established between a history of hypertensive disorders of pregnancy (HDP) — gestational hypertension and pre-eclampsia — and future risk of cardiovascular disease.^1,4–11 Consequently, some recommend using a history of HDP for cardiovascular disease risk stratification in women.^3,12The leading hypothesis for the pathogenesis of HDP is that it results from abnormal placentation due to impaired trophoblast invasion,^13–16 resulting in reduced placental perfusion.^17–19 This, in turn, leads to abnormal secretion of the angiogenic factors soluble FMS-like tyrosine kinase 1 (sFlt1) and soluble endoglin (sEng),²⁰ which induce endothelial dysfunction and the clinical manifestations of HDP.^19,21–24 The mechanisms underlying the association between HDP and future cardiovascular disease are under debate.²⁵ One hypothesis is that HDP are merely a marker of underlying subclinical or clinical vascular risk factors that predispose a person to both HDP and future cardiovascular disease.A person who is pregnant with twins is at about 3–4 times higher risk of HDP than a person with a singleton pregnancy,^26–33 with rates of 14% and 5%, respectively.³⁴ The higher risk of HDP in twin pregnancies may be due to higher circulating sFlt1 and sEng owing to greater placental mass in twin pregnancies, ^35–37 and less related to the classic vascular risk factors for HDP in a singleton pregnancy. Therefore, a logical question is whether the established higher risk of future cardiovascular disease after singleton pregnancies with HDP also occurs in twin pregnancies with HDP. Limited data are available to answer this question.³⁸ In the current study, we aimed to test the hypothesis that the association between HDP and future cardiovascular disease is less pronounced in twin versus singleton pregnancies.     

Perturbation of the Intermolecular Contact Regions (Molecular Surface) of Hemoglobin S by Intramolecular, Low-O2-Affinity-Inducing Central Cavity Cross-Bridges

The general assumption among researchers on hemoglobin is that the intramolecular central cavity cross-bridging of Hb does not result in any generalized perturbations at the protein surface. A corollary of this is that central cavity cross-bridges are unlikely to influence the polymerization of deoxy HbS, since polymerization is a protein surface phenomenon involving the participation of multiple protein surface amino acid residues. In an attempt to evaluate this experimentally, we have introduced two low-O₂-affinity-inducing central cavity cross-bridges into HbS, ββ-sebacyl [between the two Lys-82(β) residues] and αα-fumaryl [between the two Lys-99(α) residues], and investigated their influence on the polymerization of the deoxy protein. The O₂ affinities of the cross-bridged HbS exhibited sensitivity toward the buffer ions and pH in a cross-link-specific fashion. The modulation of the O₂ affinity of these cross-bridged HbS in the presence of allosteric effectors, DPG and L-35, is also very distinct, reflecting the differences in the conformational features these two cross-bridges induce within the central cavity at the respective effector-binding domains. In addition, the αα-fumaryl cross bridge inhibited the polymerization, reflecting the perturbation of the microenvironment of one or more intermolecular contact residues, protein surface residues, as a consequence of the central cavity cross-bridge. On the other hand, the ββ-sebacyl cross-bridge exerted a slight potentiating effect on the polymerization of HbS. This reflects the fact that the perturbations at the protein surface are limited and favor polymerization. The results presented demonstrate that the structural changes induced by the central cavity cross-bridges are very specific and not simply restricted to the sites of modification, but are propagated to distant sites/domains, both within and outside the central cavity. It is conceivable that other surface regions that are not involved in the polymerization could also experience similar structural/conformational consequences. These results should be taken into consideration in designing intramolecularly cross-bridged asymmetric hybrid HbS for mapping the contribution of the intermolecular contact residues in the cis and trans dimers of deoxy HbS during polymerization.     

Quantification of Bacterial Groups within Human Fecal Flora by Oligonucleotide Probe Hybridization

To investigate the population structure of the predominant phylogenetic groups within the human adult fecal microbiota, a new oligonucleotide probe designated S-G-Clept-1240-a-A-18 was designed, validated, and used with a set of five 16S rRNA-targeted oligonucleotide probes. Application of the six probes to fecal samples from 27 human adults showed additivity of 70% of the total 16S rRNA detected by the bacterial domain probe. The Bacteroides group-specific probe accounted for 37% ± 16% of the total rRNA, while the enteric group probe accounted for less than 1%. Clostridium leptum subgroup and Clostridium coccoides group-specific probes accounted for 16% ± 7% and 14% ± 6%, respectively, while Bifidobacterium and Lactobacillus groups made up less than 2%.     

Sulfate-Reducing Bacteria Methylate Mercury at Variable Rates in Pure Culture and in Marine Sediments

Differences in methylmercury (CH₃Hg) production normalized to the sulfate reduction rate (SRR) in various species of sulfate-reducing bacteria (SRB) were quantified in pure cultures and in marine sediment slurries in order to determine if SRB strains which differ phylogenetically methylate mercury (Hg) at similar rates. Cultures representing five genera of the SRB (Desulfovibrio desulfuricans, Desulfobulbus propionicus, Desulfococcus multivorans, Desulfobacter sp. strain BG-8, and Desulfobacterium sp. strain BG-33) were grown in a strictly anoxic, minimal medium that received a dose of inorganic Hg 120 h after inoculation. The mercury methylation rates (MMR) normalized per cell were up to 3 orders of magnitude higher in pure cultures of members of SRB groups capable of acetate utilization (e.g., the family Desulfobacteriaceae) than in pure cultures of members of groups that are not able to use acetate (e.g., the family Desulfovibrionaceae). Little or no Hg methylation was observed in cultures of Desulfobacterium or Desulfovibrio strains in the absence of sulfate, indicating that Hg methylation was coupled to respiration in these strains. Mercury methylation, sulfate reduction, and the identities of sulfate-reducing bacteria in marine sediment slurries were also studied. Sulfate-reducing consortia were identified by using group-specific oligonucleotide probes that targeted the 16S rRNA molecule. Acetate-amended slurries, which were dominated by members of the Desulfobacterium and Desulfobacter groups, exhibited a pronounced ability to methylate Hg when the MMR were normalized to the SRR, while lactate-amended and control slurries had normalized MMR that were not statistically different. Collectively, the results of pure-culture and amended-sediment experiments suggest that members of the family Desulfobacteriaceae have a greater potential to methylate Hg than members of the family Desulfovibrionaceae have when the MMR are normalized to the SRR. Hg methylation potential may be related to genetic composition and/or carbon metabolism in the SRB. Furthermore, we found that in marine sediments that are rich in organic matter and dissolved sulfide rapid CH₃Hg accumulation is coupled to rapid sulfate reduction. The observations described above have broad implications for understanding the control of CH₃Hg formation and for developing remediation strategies for Hg-contaminated sediments.     

Terrestrial carbon balance in a drier world: the effects of water availability in southwestern North America

Global modeling efforts indicate semiarid regions dominate the increasing trend and interannual variation of net CO₂ exchange with the atmosphere, mainly driven by water availability. Many semiarid regions are expected to undergo climatic drying, but the impacts on net CO₂ exchange are poorly understood due to limited semiarid flux observations. Here we evaluated 121 site‐years of annual eddy covariance measurements of net and gross CO₂ exchange (photosynthesis and respiration), precipitation, and evapotranspiration (ET) in 21 semiarid North American ecosystems with an observed range of 100 – 1000 mm in annual precipitation and records of 4–9 years each. In addition to evaluating spatial relationships among CO₂ and water fluxes across sites, we separately quantified site‐level temporal relationships, representing sensitivity to interannual variation. Across the climatic and ecological gradient, photosynthesis showed a saturating spatial relationship to precipitation, whereas the photosynthesis–ET relationship was linear, suggesting ET was a better proxy for water available to drive CO₂ exchanges after hydrologic losses. Both photosynthesis and respiration showed similar site‐level sensitivity to interannual changes in ET among the 21 ecosystems. Furthermore, these temporal relationships were not different from the spatial relationships of long‐term mean CO₂ exchanges with climatic ET. Consequently, a hypothetical 100‐mm change in ET, whether short term or long term, was predicted to alter net ecosystem production (NEP) by 64 gCm^?2 yr^?1. Most of the unexplained NEP variability was related to persistent, site‐specific function, suggesting prioritization of research on slow‐changing controls. Common temporal and spatial sensitivity to water availability increases our confidence that site‐level responses to interannual weather can be extrapolated for prediction of CO₂ exchanges over decadal and longer timescales relevant to societal response to climate change.     

Suppressed patterns alter vision during binocular rivalry

Binocular rivalry occurs when incongruent patterns are presented to corresponding regions of the retinas, leading to fluctuations of awareness between the patterns . One attribute of a stimulus may rival whereas another may combine between the eyes , but it is typically assumed that the dominant features are perceived veridically. Here, we show this is not necessarily the case and that a suppressed visual feature can alter dominant perception. The cortical representations of oriented gratings can interact even when one of them is perceptually suppressed, such that the perceived orientation of the dominant grating is systematically biased depending on the orientation of the suppressed grating. A suppressed inducing pattern has the same qualitative effect as a visible one, but suppression reduces effective contrast by a factor of around six. A simple neural model quantifies and helps explain these illusions. These results demonstrate that binocular rivalry suppression operates in a graded fashion across multiple sites in the visual hierarchy rather than truncating processing at a single site and that suppressed visual information can alter dominant vision in real-time.     

Allele-Specific PCR with Competitive Probe Blocking for Sensitive and Specific Detection of BRAF V600E in Thyroid Fine-Needle Aspiration Specimens

Objective: To detect BRAF V600E mutation in thyroid fine-needle aspiration (FNA) slides and needle rinses (NR). Study Design: Tumor-enriched DNA was extracted from FNA smears, formalin-fixed paraffin-embedded (FFPE) sections, or NR specimens from 37 patients with confirmed papillary thyroid carcinoma or benign findings. An allele-specific primer selectively amplified the 1799 T>A BRAF mutation while simultaneously blocking amplification of wild-type (WT) BRAF with an unlabeled probe during PCR. Mutation detection was accomplished by melting analysis of the probe. Results: Allele-specific/blocking probe PCR confirmed the BRAF mutation status for 20 of 24 paired FNA/FFPE samples previously tested by fluorescent probe real-time PCR. For the other 4 cases, the sensitive PCR method detected the BRAF mutation in all paired FNA/FFPE samples. Previously, the mutation had been detected in only the FFPE samples. The BRAF mutation was also detected in some NR specimens. Conclusion: Treatment of patients with thyroid nodules is guided by FNA biopsy, which can be scantly cellular, necessitating a sensitive test that can detect low levels of BRAF V600E mutation in a WT background. We report increased detection of BRAF V600E in FNA specimens using allele-specific/blocking probe PCR, which has an analytical sensitivity of 0.01%.     

Localization of protein kinase C epsilon to macrophage vacuoles perforated by Listeria monocytogenes cytolysin

Three proteins secreted by Listeria monocytogenes facilitate escape from macrophage vacuoles: the cholesterol-dependent cytolysin listeriolysin O (LLO), a phosphoinositide-specific phospholipase C (PI-PLC) and a broad-range phospholipase C (PC-PLC). LLO and PI-PLC can activate several members of the protein kinase C (PKC) family during infection. PKCepsilon is a novel PKC that contributes to macrophage activation, defence against bacterial infection, and phagocytosis; however, a role for PKCepsilon in Lm infections has not been described. To study PKCepsilon dynamics, PKCepsilon-YFP chimeras were visualized in macrophages during Lm infection. PKCepsilon-YFP was recruited to forming vacuoles during macrophage phagocytosis of Lm and again later to fully formed Lm vacuoles. The PKCepsilon-YFP localization to the fully formed Lm vacuole was LLO-dependent but independent of PI-PLC or PC-PLC. PKCepsilon-YFP recruitment often followed LLO perforation of the membrane, as indicated by localization of PKCepsilon-YFP to Lm vacuoles after they released small fluorescent dyes into the cytoplasm. PKCepsilon-YFP recruitment to vesicles also followed phagocytosis of LLO-containing liposomes or osmotic lysis of endocytic vesicles, indicating that vacuole perforation by LLO was the chief cause of the PKCepsilon response. These studies implicate PKCepsilon in a cellular mechanism for recognizing damaged membranous organelles, including the disrupted vacuoles created when Lm escapes into cytoplasm.