A study of over 35,000 women with breast cancer tested with a 25-gene panel of hereditary cancer genes.

BACKGROUND: As panel testing becomes more common in clinical practice, it is important to understand the prevalence and trends associated with the pathogenic variants (PVs) identified. This is especially true for genetically heterogeneous cancers, such as breast cancer (BC), in which PVs in different genes may be associated with various risks and cancer subtypes. The authors evaluated the outcomes of genetic testing among women who had a personal history of BC. METHODS: A total of 35,409 women with a single diagnosis of BC who underwent clinical genetic testing with a 25-gene panel were included in the current analysis. Women with multiple BCs and men with BC were excluded. The frequency and distribution of PVs were assessed for the overall cohort, among women with triple-negative BC (TNBC) (n = 4797), and by age at diagnosis. RESULTS: PVs were identified in 9.3% of women tested; 51.5% of PVs were identified in genes other than breast cancer 1 (BRCA1) and BRCA2, including checkpoint kinase 2 (CHEK2) (11.7%), ataxia telangiectasia mutated (ATM; ATM serine/threonine kinase) (9.7%), and partner and localizer of BRCA2 (PALB2) (9.3%). The prevalence of PVs in BRCA1, PALB2, BRCA1-associated RING domain 1 (BARD1), BRCA1-interacting protein C-terminal helicase 1 (BRIP1), and RAD51 paralog C (RAD51C) was statistically higher among women with TNBC. The PV rate was higher among women aged <40 years, lower among women aged >59 years, and relatively constant (8.5%-9.0%) among women who were diagnosed between ages 40 and 59 years. CONCLUSIONS: These results demonstrate that panel testing increased the number of women identified as carrying a PV in this cohort compared with BRCA testing alone. Furthermore, the proportion of women identified who carried a PV in this cohort did not decrease between ages 40 and 59 years. Cancer 2017;123:1721-1730. © 2017 American Cancer Society.

Assessment of in silico protein sequence analysis in the clinical classification of variants in cancer risk genes.

Missense variants represent a significant proportion of variants identified in clinical genetic testing. In the absence of strong clinical or functional evidence, the American College of Medical Genetics recommends that these findings be classified as variants of uncertain significance (VUS). VUSs may be reclassified to better inform patient care when new evidence is available. It is critical that the methods used for reclassification are robust in order to prevent inappropriate medical management strategies and unnecessary, life-altering surgeries. In an effort to provide evidence for classification, several in silico algorithms have been developed that attempt to predict the functional impact of missense variants through amino acid sequence conservation analysis. We report an analysis comparing internally derived, evidence-based classifications with the results obtained from six commonly used algorithms. We compiled a dataset of 1118 variants in BRCA1, BRCA2, MLH1, and MSH2 previously classified by our laboratory's evidence-based variant classification program. We compared internally derived classifications with those obtained from the following in silico tools: Align-GVGD, CONDEL, Grantham Analysis, MAPP-MMR, PolyPhen-2, and SIFT. Despite being based on similar underlying principles, all algorithms displayed marked divergence in accuracy, specificity, and sensitivity. Overall, accuracy ranged from 58.7 to 90.8% while the Matthews Correlation Coefficient ranged from 0.26-0.65. CONDEL, a weighted average of multiple algorithms, did not perform significantly better than its individual components evaluated here. These results suggest that the in silico algorithms evaluated here do not provide reliable evidence regarding the clinical significance of missense variants in genes associated with hereditary cancer.

An independent validation of a gene expression signature to differentiate malignant melanoma from benign melanocytic nevi.

BACKGROUND: Recently, a 23-gene signature was developed to produce a melanoma diagnostic score capable of differentiating malignant and benign melanocytic lesions. The primary objective of this study was to independently assess the ability of the gene signature to differentiate melanoma from benign nevi in clinically relevant lesions. METHODS: A set of 1400 melanocytic lesions was selected from samples prospectively submitted for gene expression testing at a clinical laboratory. Each sample was tested and subjected to an independent histopathologic evaluation by 3 experienced dermatopathologists. A primary diagnosis (benign or malignant) was assigned to each sample, and diagnostic concordance among the 3 dermatopathologists was required for inclusion in analyses. The sensitivity and specificity of the score in differentiating benign and malignant melanocytic lesions were calculated to assess the association between the score and the pathologic diagnosis. RESULTS: The gene expression signature differentiated benign nevi from malignant melanoma with a sensitivity of 91.5% and a specificity of 92.5%. CONCLUSIONS: These results reflect the performance of the gene signature in a diverse array of samples encountered in routine clinical practice. Cancer 2017;123:617-628. © 2016 American Cancer Society.

Lynch Syndrome Patients with Limited Family History Identified in a Laboratory Setting: A Descriptive Study.

OBJECTIVE: Patients diagnosed with colorectal cancer before the age of 50 years are recommended for Lynch syndrome (LS) testing according to current clinical guidelines. However, many patients are not identified because of the stringent guidelines on existing diagnostic criteria. The aim of this analysis was to evaluate the ability of existing criteria to adequately ascertain patients appropriate for LS genetic testing. METHOD: To determine whether existing clinical diagnostic criteria underascertain individuals who would be appropriate candidates for hereditary cancer risk assessment, we stratified the detection rate of deleterious mismatch repair (MMR) mutations in 9,109 patients with a personal history of colorectal cancer who were diagnosed between the ages of 30 and 74 years with little or no family history suggestive of LS by 5-year age-at-detection intervals. RESULTS: There was little difference in the aggregate positive mutation rate in individuals diagnosed between the ages of 50 and 59 years compared to the positive mutation rate in patients diagnosed before the age of 50 years. CONCLUSION: These results suggest that cancer diagnosis under the age of 50 years is an insufficiently sensitive predictor of hereditary cancer susceptibility.

Phosphatidylglycerol Flip-Flop Suppression due to Headgroup Charge Repulsion.

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.

Analytical validation of a proliferation-based molecular signature used as a prognostic marker in early stage lung adenocarcinoma.

AIMS: The aim of these studies was to validate the analytical performance of a cell cycle progression (CCP) gene signature that provides prognostic information for early stage lung adenocarcinomas. MATERIALS & METHODS: Formalin-fixed paraffin-embedded (FFPE) lung resections were evaluated by quantitative RT-PCR for the expression of 31 target and 15 housekeeper genes comprising the CCP score. RESULTS: The signature had a standard deviation (SD) of 0.06 score units and a dynamic range spanning CCP scores between -13 and 14. The average amplicon efficiencies for target and housekeeper genes were 107% and 105%, respectively. All but one amplicon had a SD <0.5 CT. CONCLUSION: These studies demonstrate that the gene signature is robust and reproducible, making it suitable for use in a clinical setting.

Phase II neoadjuvant clinical trial of carboplatin and eribulin in women with triple negative early-stage breast cancer (NCT01372579).

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.

Lipid flip-flop in binary membranes composed of phosphatidylserine and phosphatidylcholine.

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 effect of cholesterol on the intrinsic rate of lipid flip-flop as measured by sum-frequency vibrational spectroscopy.

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 CH3 vs 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 (deltaH(double dagger)), entropy (deltaS(double dagger)) and free energy (deltaG(double dagger)) 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.

Electrostatic induction of lipid asymmetry.

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.

Phospholipid flip-flop modulated by transmembrane peptides WALP and melittin.

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 1mol.%, 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.

Bis(di-2-pyridylmethane-diol-κN,O,N')copper(II) bis-(tetra-fluorido-borate) dihydrate.

The title complex, [Cu(C(11)H(10)N(2)O(2))(2)](BF(4))(2)·2H(2)O, was isolated as a dihydrate from a 1:2 molar mixture of copper(II) tetra-fluoridoborate hexa-hydrate with di-2-pyridyl ketone in aqueous solution. The centrosymmetric complex cation is structurally similar to that found in previously reported salts and exhibits Cu-O bonds deviating by 25 degrees from an octa-hedral geometry by the so-called 'off-axis angle' distortion. The BF(4) (-) anion exhibits a two site disorder of the fluorine atoms [ratio 0.210 (8):0.790 (8)].