IMPORTANCE: Variant reclassification is an important component of hereditary cancer genetic testing; however, there are few published data quantifying the prevalence of reclassification. OBJECTIVE: ...Retrospective cohort study of individuals who had genetic testing from 2006 through 2016 at a single commercial laboratory. DESIGN, SETTING, AND PARTICIPANTS: A retrospective cohort of individuals who had genetic testing between 2006 and 2016 at a single commercial laboratory was assessed. Variants were classified as benign, likely benign, variant of uncertain significance, likely pathogenic, or pathogenic. Retrospective chart reviews were conducted for patients from the University of Texas Southwestern (UTSW) Medical Center. EXPOSURES: Hereditary cancer genetic testing. MAIN OUTCOMES AND MEASURES: Frequency of and time to amended reports; frequency and types of variant reclassification. RESULTS: From 2006 through 2018, 1.45 million individuals (median interquartile range age at testing, 49 years 40.69-58.31 years, 95.6% women) had genetic testing, and 56.6% (n = 821 724) had a personal history of cancer. A total of 1.67 million initial tests were reported and 59 955 amended reports were issued due to variant reclassification. Overall, 6.4% (2868 of 44 777) of unique variants were reclassified. Reclassification to a different clinical category was rare among unique variants initially classified as pathogenic or likely pathogenic (0.7%, 61 of 9112) or benign or likely benign (0.2%, 15 of 8995). However, 7.7% (2048 of 26 670) of unique variants of uncertain significance were reclassified: 91.2% (1867 of 2048) were downgraded to benign or likely benign (median time to amended report, 1.17 years), 8.7% (178 of 2048) were upgraded to pathogenic or likely pathogenic variants (median time to amended report, 1.86 years). Because most variants were observed in more than 1 individual, 24.9% (46 890 of 184 327) of all reported variants of uncertain significance were reclassified. CONCLUSIONS AND RELEVANCE: Following hereditary cancer genetic testing at a single commercial laboratory, 24.9% of variants of uncertain significance were reclassified, which included both downgrades and upgrades. Further research is needed to assess generalizability of the findings for other laboratories, as well as the clinical consequences of the reclassification as a component of a genetic testing program.
Current prescribing practices for major depressive disorder (MDD) produce limited treatment success. Although pharmacogenomics may improve outcomes by identifying genetically inappropriate ...medications, studies to date were limited in scope. Outpatients (N = 1167) diagnosed with MDD and with a patient- or clinician-reported inadequate response to at least one antidepressant were enrolled in the Genomics Used to Improve DEpression Decisions (GUIDED) trial – a rater- and patient-blind randomized controlled trial. Patients were randomized to treatment as usual (TAU) or a pharmacogenomics-guided intervention arm in which clinicians had access to a pharmacogenomic test report to inform medication selections (guided-care). Medications were considered congruent (‘use as directed’ or ‘use with caution’ test categories) or incongruent (‘use with increased caution and with more frequent monitoring’ test category) with test results. Unblinding occurred after week 8. Primary outcome was symptom improvement change in 17-item Hamilton Depression Rating Scale (HAM-D17) at week 8; secondary outcomes were response (≥50% decrease in HAM-D17) and remission (HAM-D17 ≤ 7) at week 8. At week 8, symptom improvement for guided-care was not significantly different than TAU (27.2% versus 24.4%, p = 0.107); however, improvements in response (26.0% versus 19.9%, p = 0.013) and remission (15.3% versus 10.1%, p = 0.007) were statistically significant. Patients taking incongruent medications prior to baseline who switched to congruent medications by week 8 experienced greater symptom improvement (33.5% versus 21.1%, p = 0.002), response (28.5% versus 16.7%, p = 0.036), and remission (21.5% versus 8.5%, p = 0.007) compared to those remaining incongruent. Pharmacogenomic testing did not significantly improve mean symptoms but did significantly improve response and remission rates for difficult-to-treat depression patients over standard of care (ClinicalTrials.gov NCT02109939).
The kinetics and thermodynamics of lipid flip-flop in bilayers composed of 1,2-dipalmitoyl-sn-glycero-3-phospho-l-serine (DPPS) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) were studied ...using sum-frequency vibrational spectroscopy. The kinetics of DSPC and DPPS flip-flop were examined as a function of temperature and bilayer composition. The rate of DSPC flip-flop did not exhibit any significant dependence on bilayer composition while the rate of DPPS flip-flop was inversely dependent on the mole fraction of DPPS. The transition-state thermodynamics for DSPC and DPPS lipids in these mixed bilayers were determined in order to identify the energetic impact of the phosphatidylserine headgroup on lipid flip-flop. The thermodynamics for the DSPC component remained statistically identical to bilayers composed entirely of DSPC. The activation energy for the DPPS component showed a linear correlation with the mole fraction of DPPS for all bilayer compositions. The enthalpy and entropy for DPPS flip-flop did not increase linearly with the fraction of DPPS but did directly correlate with the molecular area. The DPPS component also exhibited enthalpy–entropy compensation which suggests that lipid hydration may play a significant role in membrane dynamics.
The kinetics and thermodynamics of 1,2-distearoyl-sn-glycero-3-phospho(1′-rac-glycerol) (DSPG) flip-flop in 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) membranes were examined by sum-frequency ...vibrational spectroscopy (SFVS). The effect of DSPG concentration in the membrane and the influence of electrolyte concentration were examined in an attempt to decipher the role the anionic PG headgroup plays in dictating the dynamics of PG flip-flop for this biologically important lipid species. DSPG flip-flop dynamics and the activation barrier to exchange were found to be directly dependent on the amount of DSPG present in the bilayer. Analysis of the activation free energy for DSPG flip-flop in mixed DSPG + DSPC bilayers reveals that charge repulsion between neighboring PG headgroups modulates the free energy barrier and subsequently, the rate of translocation. Specifically, when DSPG comprises a small portion of the bilayer, the electrostatic potential of neighboring PG lipids are effectively shielded from each other under high ionic strength conditions and little to no charge repulsion occurs. When DSPG lipids are close enough to experience charge repulsion from neighboring PG lipids, as in bilayers containing a large fraction of DSPG, or for bilayers in low ionic strength solutions, the influence of charge repulsion on the energetics of lipid flip-flop are measurable. For biological membranes, where the concentration of PG is relatively low, the neighboring PG lipids are spaced far enough apart that their anionic charges are effectively shielded, such that under physiological conditions the charged nature of the headgroup does little to modulate its lipid flip-flop energetics and corresponding rate of translocation.
The asymmetric arrangement of phospholipids between the two leaflets of the plasma membrane of eukaryotic cells is an integral part of cellular function. ATP-dependent translocases capable of ...selective lipid transport across the membrane are believed to play a role in this lipid asymmetry, but our understanding of this process is incomplete. Here we show the first direct and quantitative experiments demonstrating the induction of phosphatidylserine asymmetry in a membrane by electrostatic association of poly-l-lysine in an attempt to elucidate the complex factors which govern the establishment and maintenance of lipid compositional asymmetry in the plasma membrane on a fundamental level. The attractive electrostatic interactions between the charged surface-associated polylysine and phosphatidylserine are sufficient to both induce and maintain an asymmetric arrangement of phosphatidylserine in a planar supported membrane, as measured by sum-frequency vibrational spectroscopy. These studies provide a glimpse of the physical and chemical underpinnings of lipid asymmetry in the eukaryotic plasma membrane.
Select transmembrane proteins found in biogenic membranes are known to facilitate rapid bidirectional flip-flop of lipids between the membrane leaflets, while others have no little or no effect. The ...particular characteristics which determine the extent to which a protein will facilitate flip-flop are still unknown. To determine if the relative polarity of the transmembrane protein segment influences its capacity for facilitation of flip-flop, we have studied lipid flip-flop dynamics for bilayers containing the peptides WALP
23 and melittin. WALP
23 is used as a model hydrophobic peptide, while melittin consists of both hydrophobic and hydrophilic residues. Sum-frequency vibrational spectroscopy (SFVS) was used to characterize the bilayers and determine the kinetics of flip-flop for the lipid component, 1,2-distearoyl-
sn-glycero-3-phosphocholine (DSPC), within the mixed bilayers. The kinetic data were utilized to determine the activation thermodynamics for DSPC flip-flop in the presence of the peptides. Melittin was found to significantly reduce the free energy barrier to DSPC flip-flop when incorporated into the bilayer at 1
mol.%, while incorporation of WALP
23 at the same concentration led to a more modest reduction of the free energy barrier. The possible mechanisms by which these peptides facilitate flip-flop are analyzed and discussed in terms of the observed activation thermodynamics.
Cholesterol is a major constituent of biological membranes in mammalian cells. Experiments have shown that cholesterol influences the physical properties of the plasma membrane, such as lateral ...diffusion and phase equilibrium. In addition to controlling the 2-dimensional phase behaviour and mobility of lipids in membranes, cholesterol has also been implicated in the transbilayer diffusion of lipids across the bilayer. Sum-frequency vibrational spectroscopy (SFVS) is used to measure the intrinsic rate of lipid flip-flop for 1,2-distearoyl-
sn
-glycero-3-phosphocholine (DSPC) in the presence of cholesterol using planar supported lipid bilayer (PSLB) model membranes. Asymmetric PSLBs were prepared using the Langmuir-Blodgett (LB) method by placing a perdeuterated lipid analogue in one leaflet of the PSLB. SFVS was used to directly measure the asymmetric distribution of DSPC within the membrane by measuring the decay in the CH
3
v
s
intensity at 2875 cm
−1
with time and as a function of temperature. A complete kinetic analysis of DSPC flip-flop and the effect of cholesterol on the DSPC dynamics are presented. An analysis of the kinetic data in the framework of Eyring theory provides important insight into the transition state enthalpy (Δ
H
‡
), entropy (Δ
S
‡
) and free energy (Δ
G
‡
) for this important biological process. In addition, the transmembrane migration of cholesterol molecules was also explored by SFVS. These combined studies are aimed at providing new insight in to the transbilayer migration of phospholipids and cholesterol in biological membranes and the effects cholesterol plays in membrane dynamics.
The purpose of this study is to evaluate the efficacy and safety of neoadjuvant treatment with carboplatin and eribulin in patients with early-stage triple negative breast cancer (TNBC), and to ...explore biomarkers based on DNA and protein expression profiles as predictors of response. Patients with histologically confirmed early-stage TNBC received carboplatin AUC 6 iv every 21 days, and eribulin 1.4 mg/m
2
day 1 and day 8 every 21 days for four cycles. The primary endpoint of the study was pathologic complete response (pCR), with secondary endpoints including clinical response and safety of the combination. Exploratory studies assessed DNA-based biomarkers homologous recombination deficiency (HRD) score, and HR deficiency status (HRD score +
BRCA1/BRCA2
mutation status), protein-based biomarkers (Ki67, TP53, androgen receptor, Cyclin E, CDK2, Cyclin D, CDK4, Pin1 and Smad3), and clinical pretreatment factors as predictors of pCR. 13/30 (43.3 %) patients enrolled in the study achieved pCR. 24 (80.0 %) had a clinical complete or partial response. The combination was safe with mostly grade 1 and 2 toxicities. HRD score (
P
= 0.0024) and HR deficiency status (
P
= 0.0012) significantly predicted pCR. Pretreatment cytoplasmic CDK2 was also associated with pCR (
P
= 0.021). Significant differences in pre- versus post-treatment expression levels of nuclear Cyclin D (
P
= 0.020), nuclear CDK4 (
P
= 0.0030), and nuclear Smad3 (
P
= 0.015) were detected. The combination of carboplatin and eribulin is safe and efficacious in the treatment of early-stage TNBC. HRD score, HR deficiency status, and cytoplasmic CDK2 predicted pCR in this patient population.
PDF
