Disparities Associated With Patient Adherence to BI-RADS 3 Assessment Follow-up Recommendations for Mammography and Ultrasound.

OBJECTIVE: To assess the relationship between sociodemographic factors and adherence rates in patients with a BI-RADS 3 assessment. METHODS: This retrospective cohort study reviewed data from all patients with a BI-RADS 3 assessment on mammography and ultrasound examinations at a single, multisite academic institution, which serves a diverse urban-suburban population, from January 1, 2015, to December 13, 2017. Appropriate follow-up was defined as returning for the first follow-up examination 3 to 9 months after the index examination. Associations between BI-RADS 3 adherence rates and patient sociodemographic characteristics were evaluated using logistic regression. RESULTS: There were 4,038 patients in our study period; 2,437 patients (60%) had appropriate follow-up, 765 (19%) patients had delayed follow-up, and 836 patients (21%) were lost to follow-up. The overall malignancy rate was 1.4% (46 of 3,202). Older age, retired employment status, and Medicare insurance status were associated with increased adherence to BI-RADS 3 follow-up recommendations. Black race, single relationship status, Medicaid and self-pay insurance status, and living in a top 15% disadvantaged zip code were associated with decreased adherence. On multivariate analysis, older age remained associated with increased adherence and Medicaid insurance status with decreased adherence. Time between index examination and cancer diagnosis was shorter in patients who had timely follow-up (202 days [interquartile range 183-358] versus 392 days [interquartile range 365-563], P ≤ .001), although there was not a significant difference in stage at diagnosis (P = .46). DISCUSSION: Multiple sociodemographic factors are associated with low adherence to BI-RADS 3 follow-up recommendations suggesting that more frequent and targeted interventions are needed to close disparity gaps.

Generation of a genome scale lentiviral vector library for EF1α promoter-driven expression of human ORFs and identification of human genes affecting viral titer.

The bottleneck in elucidating gene function through high-throughput gain-of-function genome screening is the limited availability of comprehensive libraries for gene overexpression. Lentiviral vectors are the most versatile and widely used vehicles for gene expression in mammalian cells. Lentiviral supernatant libraries for genome screening are commonly generated in the HEK293T cell line, yet very little is known about the effect of introduced sequences on the produced viral titer, which we have shown to be gene dependent. We have generated an arrayed lentiviral vector library for the expression of 17,030 human proteins by using the GATEWAY® cloning system to transfer ORFs from the Mammalian Gene Collection into an EF1alpha promoter-dependent lentiviral expression vector. This promoter was chosen instead of the more potent and widely used CMV promoter, because it is less prone to silencing and provides more stable long term expression. The arrayed lentiviral clones were used to generate viral supernatant by packaging in the HEK293T cell line. The efficiency of transfection and virus production was estimated by measuring the fluorescence of IRES driven GFP, co-expressed with the ORFs. More than 90% of cloned ORFs produced sufficient virus for downstream screening applications. We identified genes which consistently produced very high or very low viral titer. Supernatants from select clones that were either high or low virus producers were tested on a range of cell lines. Some of the low virus producers, including two previously uncharacterized proteins were cytotoxic to HEK293T cells. The library we have constructed presents a powerful resource for high-throughput gain-of-function screening of the human genome and drug-target discovery. Identification of human genes that affect lentivirus production may lead to improved technology for gene expression using lentiviral vectors.

A high-throughput platform for lentiviral overexpression screening of the human ORFeome.

In response to the growing need for functional analysis of the human genome, we have developed a platform for high-throughput functional screening of genes overexpressed from lentiviral vectors. Protein-coding human open reading frames (ORFs) from the Mammalian Gene Collection were transferred into lentiviral expression vector using the highly efficient Gateway recombination cloning. Target ORFs were inserted into the vector downstream of a constitutive promoter and upstream of an IRES controlled GFP reporter, so that their transfection, transduction and expression could be monitored by fluorescence. The expression plasmids and viral packaging plasmids were combined and transfected into 293T cells to produce virus, which was then used to transduce the screening cell line. We have optimised the transfection and transduction procedures so that they can be performed using robotic liquid handling systems in arrayed 96-well microplate, one-gene-per-well format, without the need to concentrate the viral supernatant. Since lentiviruses can infect both dividing and non-dividing cells, this system can be used to overexpress human ORFs in a broad spectrum of experimental contexts. We tested the platform in a 1990 gene pilot screen for genes that can increase proliferation of the non-tumorigenic mammary epithelial cell line MCF-10A after removal of growth factors. Transduced cells were labelled with the nucleoside analogue 5-ethynyl-2'-deoxyuridine (EdU) to detect cells progressing through S phase. Hits were identified using high-content imaging and statistical analysis and confirmed with vectors using two different promoters (CMV and EF1α). The screen demonstrates the reliability, versatility and utility of our screening platform, and identifies novel cell cycle/proliferative activities for a number of genes.