Abstract: The Muller F element (4.2 Mb, ~80 protein-coding genes) is an unusual autosome of Drosophila melanogaster; it is mostly heterochromatic with a low recombination rate. To investigate how these properties impact the evolution of repeats and genes, we manually improved the sequence and annotated the genes on the D. erecta, D. mojavensis, and D. grimshawi F elements and euchromatic domains from the Muller D element. We find that F elements have greater transposon density (25–50%) than euchromatic reference regions (3–11%). Among the F elements, D. grimshawi has the lowest transposon density (particularly DINE-1: 2% vs. 11–27%). F element genes have larger coding spans, more coding exons, larger introns, and lower codon bias. Comparison of the Effective Number of Codons with the Codon Adaptation Index shows that, in contrast to the other species, codon bias in D. grimshawi F element genes can be attributed primarily to selection instead of mutational biases, suggesting that density and types of transposons affect the degree of local heterochromatin formation. F element genes have lower estimated DNA melting temperatures than D element genes, potentially facilitating transcription through heterochromatin. Most F element genes (~90%) have remained on that element, but the F element has smaller syntenic blocks than genome averages (3.4–3.6 vs. 8.4–8.8 genes per block), indicating greater rates of inversion despite lower rates of recombination. Overall, the F element has maintained characteristics that are distinct from other autosomes in the Drosophila lineage, illuminating the constraints imposed by a heterochromatic milieu.

Abstract: Anti–vascular endothelial growth factor (VEGF) injections in eyes with nonproliferative diabetic retinopathy (NPDR) without center-involved diabetic macular edema (CI-DME) reduce development of vision-threatening complications from diabetes over at least 2 years, but whether this treatment has a longer-term benefit on visual acuity is unknown. Objective To compare the primary 4-year outcomes of visual acuity and rates of vision-threatening complications in eyes with moderate to severe NPDR treated with intravitreal aflibercept compared with sham. The primary 2-year analysis of this study has been reported. Design, Setting, and Participants Randomized clinical trial conducted at 64 clinical sites in the US and Canada from January 2016 to March 2018, enrolling 328 adults (399 eyes) with moderate to severe NPDR (Early Treatment Diabetic Retinopathy Study [ETDRS] severity level 43-53) without CI-DME. Interventions Eyes were randomly assigned to 2.0 mg aflibercept (n = 200) or sham (n = 199). Eight injections were administered at defined intervals through 2 years, continuing quarterly through 4 years unless the eye improved to mild NPDR or better. Aflibercept was given in both groups to treat development of high-risk proliferative diabetic retinopathy (PDR) or CI-DME with vision loss. Main Outcomes and Measures Development of PDR or CI-DME with vision loss (≥10 letters at 1 visit or ≥5 letters at 2 consecutive visits) and change in visual acuity (best corrected ETDRS letter score) from baseline to 4 years. Results Among participants (mean age 56 years; 42.4% female; 5% Asian, 15% Black, 32% Hispanic, 45% White), the 4-year cumulative probability of developing PDR or CI-DME with vision loss was 33.9% with aflibercept vs 56.9% with sham (adjusted hazard ratio, 0.40 [97.5% CI, 0.28 to 0.57] ; P   & amp;lt; .001). The mean (SD) change in visual acuity from baseline to 4 years was −2.7 (6.5) letters with aflibercept and −2.4 (5.8) letters with sham (adjusted mean difference, −0.5 letters [97.5% CI, −2.3 to 1.3]; P  = .52). Antiplatelet Trialists’ Collaboration cardiovascular/cerebrovascular event rates were 9.9% (7 of 71) in bilateral participants, 10.9% (14 of 129) in unilateral aflibercept participants, and 7.8% (10 of 128) in unilateral sham participants. Conclusions and Relevance Among patients with NPDR but without CI-DME, at 4 years treatment with aflibercept vs sham, initiating aflibercept treatment only if vision-threatening complications developed, resulted in statistically significant anatomic improvement but no improvement in visual acuity. Aflibercept as a preventive strategy, as used in this trial, may not be generally warranted for patients with NPDR without CI-DME. Trial Registration ClinicalTrials.gov Identifier: NCT02634333

Abstract: The DRCR Retina Network Protocol AC showed no significant difference in visual acuity outcomes over 2 years between treatment with aflibercept monotherapy and bevacizumab first with switching to aflibercept for suboptimal response in treating diabetic macular edema (DME). Understanding the estimated cost and cost-effectiveness of these approaches is important. Objective To evaluate the cost and cost-effectiveness of aflibercept monotherapy vs bevacizumab-first strategies for DME treatment. Design, Setting, and Participants This economic evaluation was a preplanned secondary analysis of a US randomized clinical trial of participants aged 18 years or older with center-involved DME and best-corrected visual acuity of 20/50 to 20/320 enrolled from December 15, 2017, through November 25, 2019. Interventions Aflibercept monotherapy or bevacizumab first, switching to aflibercept in eyes with protocol-defined suboptimal response. Main Outcomes and Measures Between February and July 2022, the incremental cost-effectiveness ratio (ICER) in cost per quality-adjusted life-year (QALY) over 2 years was assessed. Efficacy and resource utilization data from the randomized clinical trial were used with health utility mapping from the literature and Medicare unit costs. Results This study included 228 participants (median age, 62 [range, 34-91 years; 116 [51%] female and 112 [49%] male; 44 [19%] Black or African American, 60 [26%] Hispanic or Latino, and 117 [51%] White) with 1 study eye. The aflibercept monotherapy group included 116 participants, and the bevacizumab-first group included 112, of whom 62.5% were eventually switched to aflibercept. Over 2 years, the cost of aflibercept monotherapy was $26 504 (95% CI, $24 796-$28 212) vs $13 929 (95% CI, $11 984-$15 874) for the bevacizumab-first group, a difference of $12 575 (95% CI, $9987-$15 163). The aflibercept monotherapy group gained 0.015 (95% CI, −0.011 to 0.041) QALYs using the better-seeing eye and had an ICER of $837 077 per QALY gained compared with the bevacizumab-first group. Aflibercept could be cost-effective with an ICER of $100 000 per QALY if the price per dose were $305 or less or the price of bevacizumab was $1307 per dose or more. Conclusions and Relevance Variability in individual needs will influence clinician and patient decisions about how to treat specific eyes with DME. While the bevacizumab-first group costs still averaged approximately $14 000 over 2 years, this approach, as used in this study, may confer substantial cost savings on a societal level without sacrificing visual acuity gains over 2 years compared with aflibercept monotherapy.

Abstract: Elevated mammographic breast density is a strong breast cancer risk factor with poorly understood etiology. Increased deposition of collagen, one of the main fibrous proteins present in breast stroma, has been associated with increased mammographic density. Collagen fiber architecture has been linked to poor outcomes in breast cancer. However, relationships of quantitative collagen fiber features assessed in diagnostic biopsies with mammographic density and lesion severity are not well-established. Methods Clinically indicated breast biopsies from 65 in situ or invasive breast cancer cases and 73 frequency matched-controls with a benign biopsy result were used to measure collagen fiber features (length, straightness, width, alignment, orientation and density (fibers/µm 2 )) using second harmonic generation microscopy in up to three regions of interest (ROIs) per biopsy: normal, benign breast disease, and cancer. Local and global mammographic density volumes were quantified in the ipsilateral breast in pre-biopsy full-field digital mammograms. Associations of fibrillar collagen features with mammographic density and severity of biopsy diagnosis were evaluated using generalized estimating equation models with an independent correlation structure to account for multiple ROIs within each biopsy section. Results Collagen fiber density was positively associated with the proportion of stroma on the biopsy slide ( p   〈  0.001) and with local percent mammographic density volume at both the biopsy target ( p  = 0.035) and within a 2 mm perilesional ring ( p  = 0.02), but not with global mammographic density measures. As severity of the breast biopsy diagnosis increased at the ROI level, collagen fibers tended to be less dense, shorter, straighter, thinner, and more aligned with one another ( p   〈  0.05). Conclusions Collagen fiber density was positively associated with local, but not global, mammographic density, suggesting that collagen microarchitecture may not translate into macroscopic mammographic features. However, collagen fiber features may be markers of cancer risk and/or progression among women referred for biopsy based on abnormal breast imaging.

Abstract: Background: High percent mammographic density (MD), which reflects the relative fibroglandular tissue content of the breast, is one of the strongest breast cancer risk factors; however, the pathologic mediators of this risk are unknown. We hypothesize that analysis of breast tissue sections using deep learning approaches may characterize histologic features that underpin risk associated with high MD. Methods: Non-targeted H & E stained breast tissue sections of diagnostic image-guided breast biopsies were evaluated among 588 women enrolled following an abnormal mammogram in the Breast Radiology Evaluation and Study of Tissues (BREAST) Stamp Project (2007-2010). Overall volumetric percent MD for the biopsied breast and localized volumetric percent MD surrounding the biopsy site were determined for each participant. A deep convolutional neural network (CNN) model was trained to identify and quantitatively assess breast epithelial, stroma and fat tissue and their organizational and spatial arrangements. Least absolute shrinkage and selection operator (Lasso) regression was used to determine relationships between MD measures and pathological features. To ensure reliability of the model, a cross-validation strategy was employed to build and assess the performance of the fitted model. Finally, Spearman correlation coefficients were estimated to test the association between the predicted density values by each model (predicting overall or localized MD) and the actual MD measurements. We report the average and standard deviation (SD) of the correlation coefficients. Results: In an independent validation set, the CNN model was 95.5% accurate in classifying epithelial, stromal and fat tissue. The mean (SD) correlations between the predicted model and the actual measurements for overall and localized MD were 0.70 (0.06) and 0.65 (0.06) respectively. The amount of stroma identified (normalized to tissue area) had the highest selection probability (P-value) by the Lasso model and thus the strongest positive relationship with MD (P-value & gt;0.9 for each MD measurement). In contrast, the amount of normalized epithelial tissue was not related to MD (P-value=0.01 for each MD measurement). No association was observed for the total normalized fat area with MD (P-value & lt;0.31 for each MD measurement). In addition, the number of distributed epithelial regions was positively associated, whereas the distance between epithelial regions was inversely associated with overall MD (P-value & lt;0.87 and 0.62, respectively). Conclusions: These results show that greater stromal tissue amount and spatial distribution patterns of epithelial regions, rather than total epithelial amounts, had the strongest relationships with elevated MD. Future work will determine the relationship of these MD features with biopsy diagnosis. Citation Format: Maeve Mullooly, Babak Ehteshami Bejnordi, Maya Palakal, Pamela M. Vacek, Donald L. Weaver, John A. Shepherd, Bo Fan, Amir Pasha Mahmoudzadeh, Jeff Wang, Jason M. Johnson, Sally D. Herschorn, Brian L. Sprague, Ruth M. Pfeiffer, Louise A. Brinton, Mark E. Sherman, Andrew Beck, Gretchen L. Gierach. Application of convolutional neural networks to breast biopsies to uncover tissue correlates of mammographic breast density [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4235. doi:10.1158/1538-7445.AM2017-4235

Abstract: Introduction: The etiology of breast cancer remains an area of ongoing investigation. Improving our understanding of factors associated with breast cancer development will strengthen the utility of risk prediction strategies and improve risk stratification. Mammographic breast density remains one of the most influential breast cancer risk factors, with 4 to 6 fold elevated risk observed among women with the highest compared to the lowest levels. These associations have consistently been observed irrespective of the method (visual or automated), used to quantify breast density, showing the robustness of the associations. An understudied breast cancer risk factor is the bilateral asymmetry of mammographic features, with prior studies suggesting that women with higher levels of breast asymmetry may be at elevated breast cancer risk. Increasingly, studies are recognizing the potential of bilateral breast asymmetry defined by mammographic breast density in determining breast cancer risk. Recently, Eriksson and colleagues highlighted a strong influence of breast asymmetry within a model they developed to predict short-term breast cancer risk among women attending breast screening. They observed that the contribution to the model of asymmetry in mammographic breast density, microcalcifications, and masses between the breasts was as substantial as the total number of microcalcifications and masses found within a mammogram, indicating that differences between the breasts may be an important risk marker. To help further understand associations between breast cancer risk factors and asymmetry of breast density, particularly among women at elevated risk for breast cancer, we evaluated risk factor relationships with the bilateral asymmetry of volumetric measures of global and local breast density across the spectrum of premalignant and invasive breast cancer diagnoses. Methods: This study included 882 women enrolled as part of the National Cancer Institute's Breast Radiology Evaluation and Study of Tissues (BREAST)-Stamp Project (2007-2010). The BREAST-Stamp Project is a cross-sectional molecular epidemiologic study that aims to understand how novel breast density measures are related to breast cancer etiology. Women were enrolled if they were referred for diagnostic image-guided breast biopsy following an abnormal mammogram at the University of Vermont Medical Center, and had not previously been diagnosed or treated for cancer, undergone breast surgery within one year or received chemoprevention. Risk factor data were collected at study enrolment via interview and self-administered questionnaires. Breast density measures were estimated using Single X-ray Absorptiometry (SXA), a technique in which an SXA breast density phantom was affixed to the compression paddle of the mammography machine during routine mammography so that it was included in the X-ray field. It served as a reference standard to estimate volumetric breast density. Breast density was assessed using pre-biopsy craniocaudal full-field digital mammograms of both the ipsilateral (affected) and contralateral (unaffected) breast. Firstly, global density from each laterality was determined as percent fibroglandular volume (%FGV). Secondly, localized volumetric density measures were estimated following identification of the biopsy site on the ipsilateral pre-biopsy mammogram by the study radiologist and identifying the corresponding site on the contralateral mammogram. The SXA estimated %FGV in a perilesional volume twice the size of, but excluding, the biopsy target, centered at the biopsy site. Previous estimates of reproducibility for the SXA test phantoms demonstrated a repeatability SD of 2%, with a ±2% accuracy for the entire thickness and density ranges. Breast density asymmetry was defined as an absolute bilateral difference in measures when subtracting the breast density measures of the contralateral breast from the ipsilateral measures. Spearman's correlations (rho) examined associations between breast density measures of the left and right breasts. To determine relationships between breast cancer risk factors (defined as categorical variables) and measures of bilateral breast density asymmetry, analysis of covariance (ANCOVA) models (PROC GLM) were used. Analyses were conducted at the per woman level using SAS. Probability values of & lt;0.05 were considered statistically significant, and all tests were two-tailed. Results:We initially investigated asymmetry within each woman by examining correlations between breast density measures in the left and right breasts. Strong, positive correlations between the ipsilateral and contralateral breast were observed for each breast density measure (rho for global %FGV=0.89, p-value & lt;0.0001; rho for local %FGV=0.79, p-value & lt;0.0001). Breast asymmetry was observed in the majority of the study population. For global %FGV, 76% of women had a bilateral difference ≥2%, with 43% of women having higher %FGV in their ipsilateral affected breast and 33% having higher in their contralateral unaffected breast. For localized %FGV, the majority of women had differences between their two breasts (89%), with 61% of women having higher localized %FGV in their ipsilateral affected breast compared to the remaining 28% who had higher localized breast density in their unaffected breast. We next examined relationships between breast cancer risk factors and breast asymmetry. Overall, no associations were observed between any of the risk factors examined, including age, race, body mass index, education, menopausal status, menopausal hormone therapy use with absolute bilateral differences in global or localized %FGV. Among the study population, most women had a benign breast disease diagnosis, with 33% and 43% being diagnosed with benign non-proliferative and benign proliferative lesions, respectively. Of the study population, 15% were diagnosed with invasive breast cancer. Overall, no differences were observed in bilateral differences in global %FGV according to diagnosis; however, higher mean bilateral differences in localized %FGV were observed for women with invasive compared to other diagnoses including benign and in-situ lesions (p=0.056). Discussion and conclusions: Our findings showed that breast asymmetry, defined by bilateral differences in global and localized mammographic breast density, was mostly unrelated to breast cancer risk factors among women undergoing an image guided breast biopsy. We observed that localized measures, defined according to within-woman bilateral differences in localized %FGV surrounding a suspicious lesion as compared with localized %FVG in a comparable location in the contralateral breast, may be an indication of cancer. This investigation is currently ongoing and efforts are underway to replicate findings using evolving image analysis methods such as Volpara Density Maps, an FDA-approved breast imaging tool which provides volumetric estimates of local glandular tissue distribution. Further, the ascertainment of 10-year follow-up data within this study population is in progress, which will facilitate prospective investigations of the relationship between breast asymmetry and breast cancer risk. Ongoing work will also extend our understanding of whether breast asymmetry is related to individualized breast cancer risk, defined using tailored breast cancer risk assessment tools. In conclusion, further understanding of breast asymmetry is needed to better exploit how bilateral differences in mammographic features may be used to inform breast cancer etiology as well as future breast cancer risk. Citation Format: Maeve Mullooly, Shaoqi Fan, Ruth M. Pfeiffer, Brian Sprague, Pamela M. Vacek, Donald L. Weaver, John A. Shepherd, Amir Pasha Mahmoudzadeh, Jeff Wang, Serghei Malkov, Jason M. Johnson, Sally D. Herschorn, Gretchen L. Gierach. Investigation of relationships between breast cancer risk factors and bilateral mammographic breast density asymmetry among women undergoing diagnostic image-guided breast biopsies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr NG15.

Abstract: Elevated mammographic density (MD) is an established breast cancer risk factor. Reduced involution of terminal duct lobular units (TDLU), the histologic source of most breast cancers, has been associated with higher MD and breast cancer risk. We investigated relationships of TDLU involution with area and volumetric MD, measured throughout the breast and surrounding biopsy targets (perilesional). Three measures inversely related to TDLU involution (TDLU count/mm2, median TDLU span, median acini count/TDLU) assessed in benign diagnostic biopsies from 348 women, ages 40–65, were related to MD area (quantified with thresholding software) and volume (assessed with a density phantom) by analysis of covariance, stratified by menopausal status and adjusted for confounders. Among premenopausal women, TDLU count was directly associated with percent perilesional MD (P trend = 0.03), but not with absolute dense area/volume. Greater TDLU span was associated with elevated percent dense area/volume (P trend & lt;0.05) and absolute perilesional MD (P = 0.003). Acini count was directly associated with absolute perilesional MD (P = 0.02). Greater TDLU involution (all metrics) was associated with increased nondense area/volume (P trend ≤ 0.04). Among postmenopausal women, TDLU measures were not significantly associated with MD. Among premenopausal women, reduced TDLU involution was associated with higher area and volumetric MD, particularly in perilesional parenchyma. Data indicating that TDLU involution and MD are correlated markers of breast cancer risk suggest that associations of MD with breast cancer may partly reflect amounts of at-risk epithelium. If confirmed, these results could suggest a prevention paradigm based on enhancing TDLU involution and monitoring efficacy by assessing MD reduction. Cancer Prev Res; 9(2); 149–58. ©2015 AACR.