Proximity Scores Research Articles

Radiographic determination of the tracheal indices at caudal cervical, thoracic-inlet, and intra-thoracic trachea in non-bulldog brachycephalic breeds without evidence of cardiorespiratory disease

BackgroundCongenital tracheal hypoplasia is a component of airway syndrome affecting a wide variety of brachycephalic dog breeds. Several radiographic procedures were utilized to assess vertical tracheal diameter (VTD) in dogs. The objective of this study was to calculate the tracheal indices at the caudal cervical, thoracic-inlet, and intra-thoracic tracheal regions on the right lateral thoracic radiograph to further establish a screening protocol for diagnosis of tracheal hypoplasia in non-bulldog brachycephalic breeds. Dogs without clinical or radiographic evidence of tracheal, respiratory, or cardiovascular abnormalities were investigated. The absolute and average VTDs were normalized by manubrium length (ML), thoracic-inlet distance (Ti-D), and proximal 3rd rib width (PR3-W). Manubrium-tracheal index (M-TI), thoracic inlet-tracheal index (Ti-TI), and proximal R3-tracheal score (PR3-TS) were calculated. Correlations between averaged VTD and each of the normalizing parameters (ML, Ti-D, and PR3-W), and between M-TI and each of the previously established procedures (Ti-TI and PR3-TS) were determined.ResultsEighty healthy subjects met the inclusion criteria for the study. There were significant differences (P ≤ 0.0001) among the means of absolute and normalized VTDs at the 3 tracheal levels. The smallest VTD was identified at the thoracic inlet. The average tracheal diameter showed a better correlation with ML (rs=0.81, P < 0.0001) compared to Ti-D and PR3-W. There was a strong correlation (rs=0.83, P < 0.0001) between the averaged M-TI and Ti-TI.ConclusionRadiographic M-TI could be an alternative to traditional procedures to assess the tracheal lumen in non-bulldog brachycephalic dogs. M-TI < 0.39, < 0.30, or < 0.34 at caudal cervical, thoracic inlet, or intrathoracic trachea, respectively, may indicate tracheal hypoplasia in non-bulldog brachycephalic breeds. Screening of tracheal diameter using M-TI should be recommended. However, further investigation of non-bulldog brachycephalic breeds with cardiac and/or respiratory disease is indicated.

Distinguishing Distinct Neural Systems for Proximal vs Distal Upper Extremity Motor Control After Acute Stroke.

The classic and singular pattern of distal greater than proximal upper extremity motor deficits after acute stroke does not account for the distinct structural and functional organization of circuits for proximal and distal motor control in the healthy CNS. We hypothesized that separate proximal and distal upper extremity clinical syndromes after acute stroke could be distinguished and that patterns of neuroanatomical injury leading to these 2 syndromes would reflect their distinct organization in the intact CNS. Proximal and distal components of motor impairment (upper extremity Fugl-Meyer score) and strength (Shoulder Abduction Finger Extension score) were assessed in consecutively recruited patients within 7 days of acute stroke. Partial correlation analysis was used to assess the relationship between proximal and distal motor scores. Functional outcomes including the Box and Blocks Test (BBT), Barthel Index (BI), and modified Rankin scale (mRS) were examined in relation to proximal vs distal motor patterns of deficit. Voxel-based lesion-symptom mapping was used to identify regions of injury associated with proximal vs distal upper extremity motor deficits. A total of 141 consecutive patients (49% female) were assessed 4.0 ± 1.6 (mean ± SD) days after stroke onset. Separate proximal and distal upper extremity motor components were distinguishable after acute stroke (p = 0.002). A pattern of proximal more than distal injury (i.e., relatively preserved distal motor control) was not rare, observed in 23% of acute stroke patients. Patients with relatively preserved distal motor control, even after controlling for total extent of deficit, had better outcomes in the first week and at 90 days poststroke (BBT, ρ = 0.51, p < 0.001; BI, ρ = 0.41, p < 0.001; mRS, ρ = 0.38, p < 0.001). Deficits in proximal motor control were associated with widespread injury to subcortical white and gray matter, while deficits in distal motor control were associated with injury restricted to the posterior aspect of the precentral gyrus, consistent with the organization of proximal vs distal neural circuits in the healthy CNS. These results highlight that proximal and distal upper extremity motor systems can be selectively injured by acute stroke, with dissociable deficits and functional consequences. Our findings emphasize how disruption of distinct motor systems can contribute to separable components of poststroke upper extremity hemiparesis.

Functional status and spatial architecture of tumor-infiltrating CD8+ T cells are associated with lymph node metastases in non-small cell lung cancer

BackgroundAnti-PD-(L)1 immunotherapy has been recommended for non-small cell lung cancer (NSCLC) patients with lymph node metastases (LNM). However, the exact functional feature and spatial architecture of tumor-infiltrating CD8 + T cells remain unclear in these patients.MethodsTissue microarrays (TMAs) from 279 IA-IIIB NSCLC samples were stained by multiplex immunofluorescence (mIF) for 11 markers (CD8, CD103, PD-1, Tim3, GZMB, CD4, Foxp3, CD31, αSMA, Hif-1α, pan-CK). We evaluated the density of CD8 + T-cell functional subsets, the mean nearest neighbor distance (mNND) between CD8 + T cells and neighboring cells, and the cancer-cell proximity score (CCPS) in invasive margin (IM) as well as tumor center (TC) to investigate their relationships with LNM and prognosis.ResultsThe densities of CD8 + T-cell functional subsets, including predysfunctional CD8 + T cells (Tpredys) and dysfunctional CD8 + T cells (Tdys), in IM predominated over those in TC (P < 0.001). Multivariate analysis identified that the densities of CD8 + Tpredys cells in TC and CD8 + Tdys cells in IM were significantly associated with LNM [OR = 0.51, 95%CI (0.29–0.88), P = 0.015; OR = 5.80, 95%CI (3.19–10.54), P < 0.001; respectively] and recurrence-free survival (RFS) [HR = 0.55, 95%CI (0.34–0.89), P = 0.014; HR = 2.49, 95%CI (1.60–4.13), P = 0.012; respectively], independent of clinicopathological factors. Additionally, shorter mNND between CD8 + T cells and their neighboring immunoregulatory cells indicated a stronger interplay network in the microenvironment of NSCLC patients with LNM and was associated with worse prognosis. Furthermore, analysis of CCPS suggested that cancer microvessels (CMVs) and cancer-associated fibroblasts (CAFs) selectively hindered CD8 + T cells from contacting with cancer cells, and were associated with the dysfunction of CD8 + T cells.ConclusionTumor-infiltrating CD8 + T cells were in a more dysfunctional status and in a more immunosuppressive microenvironment in patients with LNM compared with those without LNM.

The influence of arable crops on ALS risk and age at onset: a population-based study (S19.006)

<h3>Objective:</h3> We investigated the effect of living near to specific agricultural crops on the ALS risk, age at onset and other phenotypical variables. <h3>Background:</h3> Several environmental risk factors, such as agricultural workers, have been related to ALS risk, but with inconsistent results. Regarding phenotypic heterogeneity, a recent study failed to identify possible environmental factors underlying ALS clinical presentation. <h3>Design/Methods:</h3> All ALS patients (diagnosis 2007–2014) belong to the Piemonte and Valle d’Aosta Register (PARALS). Using Regional Environmental Protection Agency (ARPA) data, we established the geographical distribution of agricultural areas in the same period. We calculated the area covered by each culture and patients smoothed incidence and we compared them using linear regression. A proximity score for each environmental factor was calculated using the area of the environmental component enclosed by a circle centered on the residence address, (variable radii 100–2000 meters). <h3>Results:</h3> The regression model for arable crops confirmed a linear increase in ALS incidence in the municipalities with a larger area covered by arable crops. Median incidence increased from 0.75 (IQR 0.00–1.26) cases/100.000/year in municipalities with no area covered by arable crops to 1.81 (IQR 0.75–4.11) cases/100.000/year where arable crops covered more than 60% of total municipality area. The proximity score analysis confirmed that arable crops proximity (considering a 500, 1000, 1500 meters radii) significantly reduced the age at onset by about 2 years. Linear regression for proximity score and age at onset, significantly confirm this trend (R= −0.51675, p=0.0245). We confirmed the same trend by subgrouping patients according to sex and site of onset. <h3>Conclusions:</h3> We confirmed an higher ALS patients incidence for the municipality with high percentage of arable land. Arable land proximity scores resulted to be the related to a significantly reduced median onset age, confirming the presence of possible shared environmental factors that could anticipate disease onset in the exposed population <b>Disclosure:</b> Dr. Calvo has nothing to disclose. Mr. Callegaro has nothing to disclose. Dr. Vasta has nothing to disclose. Mr. Grassano has nothing to disclose. Antonio Canosa has nothing to disclose. Sara Cabras has nothing to disclose. Dr. Di Pede has nothing to disclose. Dr. Matteoni has nothing to disclose. Fabiola De Marchi has nothing to disclose. Letizia Mazzini has nothing to disclose. Cristina Moglia has nothing to disclose. Dr. Chio has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Cytokinetics. Dr. Chio has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Mitsubishi. Dr. Chio has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Biogen. Dr. Manera has nothing to disclose.

Abstract P2-09-03: Quantification of HER2 expression and spatial biology using computational pathology: A cross-assay validation study in breast cancer

Abstract Background Conventional pathologic scoring of HER2 by IHC is proven to distinguish potential responders to trastuzumab but has not been effective for next generation antibody drug conjugates (ADCs) such as trastuzumab deruxtecan (T-DXd), which is capable of bystander killing. Several alternative approaches have been deployed to measure HER2, including immunofluorescence and mRNA sequencing. We have developed a novel and fully automated computational pathology technique, Quantitative Continuous Scoring (QCS), to quantify the level and distribution of HER2 from digitized HER2 IHC slides in an objective, quantifiable, and reproducible manner on a per-cell basis [Gustavson et al., SABCS 2020]. To further validate this approach, we performed a systematic multi-omic comparison of QCS to orthogonal methods of HER2 quantitation on a cohort of primary and metastatic breast cancer cases (N=30). Methods HER2 was evaluated using three independent methods on serial tissue sections obtained from 30 archival FFPE breast cancer samples distributed over the full range of HER2 expression, from 0 to 3+. HER2-IHC staining (clone 4B5, Roche Tissue Diagnostics) was performed using standard methods and cases were scored by two pathologists using CAP/ASCO guidelines and H-scores were assigned. We performed FISH (HER2 IQFISH pharmDx [Dako]; PathVysion HER-2 DNA Probe Kit [Vysis]), mRNA quantification of ERBB2 transcript levels (Nano String), and immunofluorescence (IF; HER2 clone 29D8, CST). Imaged with Vectra (Akoya) and analyzed with Halo (Indica). QCS readouts were generated from the above-mentioned digital images of IHC slides by using a fully automated image analysis pipeline; readouts included per-cell staining intensity measurements of membranes and cytoplasmic sub-compartments in terms of optical density (OD) [Van der Laak, JQCS 2000], which were aggregated to a single slide-level score. Additionally, using the OD measurements and the cell locations, a Spatial Proximity Score (SPS) was computed, summing the percentage of cells with OD≥10 (corresponding to the limit of visual detection of IHC staining) as well as the percentage of cells with OD&amp;lt; 10 within a prespecified radius (25µm) of a neighboring cell with OD≥10. Results Our analysis demonstrated that QCS-based scoring correlates with orthogonal measurements used in this study. Comparing protein-based assays, the observed Pearson correlation was R=0.88 between QCS median membrane OD and IHC H-scores, R=0.86 with IF-based HER2 mean cell expression intensity, and R=0.85 with IF-based H-scores. Correlation with transcriptomic profiling was R=0.81 for OD vs. mRNA, however ERBB2 transcript levels did not distinguish between HER2 0, 1+, and 2+ FISH negative cases, while QCS was able to do so. Correlation between protein-based and nucleic-acid based assays were numerically worse, with R=0.64 for OD vs. FISH. All samples (including those with HER2 IHC scores of 0 and H-Scores &amp;lt; 10) had at least ~20% of cells with quantifiable HER2 expression by OD, the presence of which was confirmed using IF. For cases in the lowest quartile of HER2 expression by OD, SPS identified 20-50% additional HER2-null cells that were in close proximity to HER2-expressing cells that may be vulnerable to bystander killing. Conclusion QCS-based scoring is consistent with orthogonal protein-based measurements across the range of HER2 expression. Most importantly, QCS derived-spatial analysis features identify additional patients in the lower end of HER2 expression that might be highly relevant for ADC response prediction, particularly if a drug exerts bystander activity. Further clinical verification and validation on large cohorts is needed. Footnote: This study was approved by the IRB at MSKCC. Citation Format: Joshua Drago, Zonera Hassan, Jan Zaucha, Ansh Kapil, Fatemeh Derakhshan, Fresia Pareja, Shimulov Anatoliy, Fanni Ratzon, Travis J Hollman, Claire Myers, Jessica Chan, Andrea Spitzmuller, Mark Gustavson, Danielle Carroll, Dara Ross, Jorge Reis-Filho, Carl barrett, Sihem Khalifa, Schmidt Guenter, Hadassah Sade, Sarat Chandarlapaty. Quantification of HER2 expression and spatial biology using computational pathology: A cross-assay validation study in breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-09-03.

Abstract P6-04-03: Computational pathology based HER2 expression quantification in HER2-low breast cancer

Abstract Background HER2 directed therapies for breast cancer (BC) rely on accurate estimation of HER2 expression by pathologist scoring of immunohistochemically (IHC) stained tissue according to ASCO/CAP guidelines Emerging HER2-targeted antibody drug conjugates (ADCs) like trastuzumab deruxtecan (T-DXd), have demonstrated efficacy in the HER2-low (IHC 1+ or IHC 2+/ISH-) population (Modi, NEJM 2022). A deeper understanding of the spectrum of HER2 expression and its spatial distribution could provide insights about the mode of action of ADCs, including potential bystander activity. Computational pathology-based methods like Quantitative Continuous Scoring (QCS) can help here by objectively quantifying HER2 expression levels on a per cell basis from digitized HER2 IHC slides (Gustavson, SABCS 2020). We applied QCS to a cohort of HER2-negative (HER2-neg) patients (pts) from a retrospective study (NCT04807595) to quantify the prevalence of HER2 expression in this population and investigate the relationship with manual scoring. Methods To analyze the prevalence of HER2 expression in the HER2-neg population, we used available digital images (N=207 pts) from retrospectively rescored HER2 slides from tumors categorized as HER2-low or IHC 0 (IHC 0 or &amp;gt;0&amp;lt; 1+). QCS algorithm was applied to perform an instance segmentation of each tumor cell into the membrane, cytoplasmic and nuclear sub-compartments. HER2 expression levels on the membrane were estimated from a Hue-Saturation-Density model (Van der Laak, JQCS 2000) in terms of optical density (OD). Descriptive statistics and spatial modelling were used to aggregate cell-level information to a slide level score using the membrane OD values and tumor cell locations. A novel Spatial Proximity Score (SPS) was used to mathematically model the proportion of tumor cells that could potentially be targeted either directly or via bystander activity of ADCs. The analysis is ongoing, complete results with additional patient data to be presented. Analytical validation of the QCS algorithm demonstrated high correlation between OD values as measured on the automatically detected membranes from QCS and those measured on consolidated manual membrane annotations (N=2157 cells) from three pathologists (R = 0.993). This is very similar to the correlation observed between individual pathologists (R = 0.995). Results In the analyzed cohort (N=207), median OD of HER2-low tumors was significantly higher compared to IHC 0 tumors (one-sided Wilcoxon p-value &amp;lt; 0.001). A significant increase of OD values was observed for increasing IHC categories from 0 through &amp;gt;0 &amp;lt; 1+ and 1+ to 2+/ISH- (one-sided Jonckheere-Terpstra p-value &amp;lt; 0.001). OD values within each IHC category showed considerable variability, particularly in IHC 1+ and IHC 2+. In 49% of pts (N=101), greater than 88% of tumor cells expressed HER2 at any intensity (OD≥10). Among the remaining 106 pts, the number of potentially ADC-susceptible cells (within 25μm radius of HER2 expressing cells) as estimated by SPS was at least double the amount estimated by single cell-based scores alone in 45 cases (42%) and increased by at least 50% in another 12 cases (11%). Conclusions Computational approaches such as QCS can help us to objectively characterize the spectrum and spatial distribution of HER2 expression. These mathematical models contribute to our understanding of potential mechanisms of action of ADCs. While this study confirmed a general association of QCS-based scores with manual IHC categories, we also saw considerable variation, as some IHC 1+ or 2+ samples had low OD. Building on these and other promising initial results (Gustavson et al, SABCS 2020), we will further explore clinical relevance of QCS-based scoring. Eventually, digital scoring may be able to define data-driven signatures to select HER2-low pts that might benefit from HER2 targeted therapies. Citation Format: Andreas Spitzmüller, Ansh Kapil, Anatoliy Shumilov, Jessica Chan, Lemonia Konstantinidou, Zonera Hassan, Mark Gustavson, Danielle Carroll, Della Varghese, Gareth D. James, Akira Moh, Andrew Livingston, Victoria de Giorgio-Miller. Computational pathology based HER2 expression quantification in HER2-low breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-04-03.

Contraintuitive fallacy in a big-risk-no-gain stroke scenario? Please verify!

Contraintuitive fallacy in a big-risk-no-gain stroke scenario? Please verify!

Computational pathology–based HER2 quantification to identify novel biomarkers in gastric cancer (GC).

449 Background: Trastuzumab deruxtecan (T-DXd) is an antibody-drug conjugate (ADC) approved for treatment of HER2-positive GC/gastroesophageal junction adenocarcinoma (GEJA) after chemotherapy (Japan) and for advanced/metastatic GC/GEJA after a trastuzumab-containing regimen (US/Israel/Singapore). Conventional HER2 immunohistochemistry (IHC) scoring identifies potential responders to trastuzumab but may not be suitable for next-generation ADCs capable of bystander killing, such as T-DXd [Modi NEJM 2022]. Use of computational pathology–based tools enables precise and accurate quantification of HER2 expression and spatial analysis, which enables design of better scores to reflect the T-DXd mechanism of action. Methods: Quantitative continuous scoring (QCS) was performed to quantify HER2 expression and distribution per cell in digitized images of HER2-stained (IHC, Ventana 4B5) GC. A QCS-derived signature was identified, called the continuous spatial proximity score (cSPS), which considers spatial location of tumor cells in addition to HER2 expression level and can be used as a surrogate for potential bystander activity. We retrospectively stratified patients in DESTINY-Gastric01 (DG-01; NCT03329690; T-DXd arm: n=123a; standard of care [SOC] arm: n=59a) using cSPS to give a binary QCS biomarker positive (BM+) or negative (BM−) status. QCS cSPS signature was independently validated on the DESTINY-Gastric02 cohort (DG-02; NCT04014075; n=71a). Results: In the DG-01 cohort, enrolled per HER2-positive status by IHC using ASCO/CAP GC scoring guidelines, median progression-free survival (mPFS) was 5.5a mo with T-DXd and 2.8a mo with SOC. QCS-derived cSPS scores showed significant survival benefit for BM+ vs BM− in the DG-01 T-DXd arm ( P&lt;0.0001; HR=0.37 [0.24-0.58]) and longer mPFS for BM+ (8.3 mo) vs BM− (3.9 mo). Within the SOC arm, this signature showed the inverse prognosis ( P=0.034; HR=2.17 [1.06-4.44]; mPFS, 2.8 mo for BM+ vs 4.9 mo for BM−), strongly suggesting the signature is predictive of T-DXd response. This signature was validated in the DG-02 cohort (mPFS, 5.5 mo on enrollment by IHC status, 10.1 mo for BM+ vs 3.7 mo for BM−; HR=0.24 [0.13-0.45]; P&lt;0.0001). Conclusions: We established QCS, a novel scoring method for tissue biomarkers. Our validated data and results support possible future use of this quantitative approach against manual scoring for identification of patients with GC who may benefit from T-DXd therapy. Clinical trial information: NCT03329690 , NCT04014075 . [Table: see text]

Abstract PR001: Spatial proximity between CD8+ T cells and tumor cells correlates with invasive recurrence in DCIS

Abstract Purpose: The immune microenvironment in ductal carcinoma in situ (DCIS) and its significance are not well established. In this study, we characterized the immune microenvironment of DCIS to determine if immune cell densities, or their spatial relationships with tumor cells, are predictive of recurrence with invasive breast cancer. Methods: One hundred thirty-two cases of DCIS were included in this study. Seventy (53%) cases were high grade, 72 (54%) were HER2+, and 91 (69%) were HR+. Twenty cases recurred with invasive breast cancer. Immune infiltrates were characterized by multiplex immunofluorescence staining of FFPE sections using two 6-plex panels. Panel IP1.2 included markers for CD3, CD20, Foxp3, pan-cytokeratins, Ki67, and HLADR. Panel IP2.2 included markers for CD3, CD8, CD68, pan-cytokeratins, PD-1, and PD-L1. Staining was performed on a Leica Bond autostainer and images were acquired on a Vectra Polaris imaging system. Image analysis was performed utilizing inForm and QuPath software packages, as well as R scripts. Counts for each cell population were determined and spatial point pattern analyses were performed to quantitate the spatial relationships between tumor cells and various immune cell types. Results: High infiltrates of T cells, CD8+ T cells (Tc), Foxp3+ T cells, and B cells were associated with high grade, hormone receptor (HR) negativity, HER2 positivity, and the presence of comedonecrosis. PD-1+ T cells and PD-1+ Tc were associated with high grade, HR negativity, and HER2 positivity. PD-L1+ immune cells, but not PD-L1+ tumor cells, were also associated with high grade, HR negativity, and HER2 positivity. None of the cell populations were associated with invasive recurrence. However, the spatial proximity of tumor cells with T cells, in particular Tc, was significantly associated with a subsequent invasive event. Patients whose DCIS had a high tumor-to-Tc proximity score (indicating close proximity of tumor and Tc) were less likely to have an invasive recurrence. Conclusion: These findings suggest that the tumor immune microenvironment is an important factor in identifying DCIS cases at risk for invasive recurrence and that manipulating the immune microenvironment may be an efficacious strategy to alter or prevent disease progression. Citation Format: Michael J. Campbell, Nicole Schindler, Alexa Glencer, Jennifer Bolen, Hidetoshi Mori, Tristan Loveday, Harriet Rothschild, Gillian Hirst, Scott Vandenberg, Alexander Borowsky, Laura Esserman. Spatial proximity between CD8+ T cells and tumor cells correlates with invasive recurrence in DCIS [abstract]. In: Proceedings of the AACR Special Conference on Rethinking DCIS: An Opportunity for Prevention?; 2022 Sep 8-11; Philadelphia, PA. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_1): Abstract nr PR001.

Occupational characteristics associated with SARS-CoV-2 infection in the UK Biobank during August–November 2020: a cohort study

BackgroundOccupational exposures may play a key role in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection risk. We used a job-exposure matrix linked to the UK Biobank to measure occupational characteristics and estimate associations with a positive SARS-CoV-2 test.MethodsPeople reporting job titles at their baseline interview in England who were < 65 years of age in 2020 were included. Healthcare workers were excluded because of differential access to testing. Jobs were linked to the US Occupational Information Network (O*NET) job exposure matrix. O*NET-based scores were examined for occupational physical proximity, exposure to diseases/infection, working outdoors exposed to weather, and working outdoors under cover (score range = 1–5). Jobs were classified as remote work using two algorithms. SARS-CoV-2 test results were evaluated between August 5th-November 10th, 2020, when the UK was released from lockdown. Cox regression was used to calculate adjusted hazard ratios (aHRs), accounting for age, sex, race, education, neighborhood deprivation, assessment center, household size, and income.ResultsWe included 115,451 people with job titles, of whom 1746 tested positive for SARS-CoV-2. A one-point increase in physical proximity score was associated with 1.14 times higher risk of SARS-CoV-2 (95%CI = 1.05–1.24). A one-point increase in the exposure to diseases/infections score was associated with 1.09 times higher risk of SARS-CoV-2 (95%CI = 1.02–1.16). People reporting jobs that could not be done remotely had higher risk of SARS-CoV-2 regardless of the classification algorithm used (aHRs = 1.17 and 1.20). Outdoors work showed an association with SARS-CoV-2 (exposed to weather aHR = 1.06, 95%CI = 1.01–1.11; under cover aHR = 1.08, 95%CI = 1.00–1.17), but these associations were not significant after accounting for whether work could be done remotely.ConclusionPeople in occupations that were not amenable to remote work, required closer physical proximity, and required more general exposure to diseases/infection had higher risk of a positive SARS-CoV-2 test. These findings provide additional evidence that coronavirus disease 2019 (COVID-19) is an occupational disease, even outside of the healthcare setting, and indicate that strategies for mitigating transmission in in-person work settings will remain important.

Retrospective study on clinical manifestation, thigh MRI and electrophysiology characteristics of immune-mediated necrotizing myopathy

Objective: To summarize the clinical, thigh magnetic resonance (tMRI) and electromyographic (EMG) characteristics in patients with immune-mediated necrotizing myopathy (IMNM). Methods: A total of 32 IMNM patients who were admitted to the Department of Neurology from April 2019 to April 2021 were enrolled at the First Medical Centre of Chinese PLA General Hospital. According to the type of antibody, the patients were divided into anti-SRP antibody positive (SRP+) group, anti-HMGCR antibody positive (HMGCR+) group and seronegative (SN) group. The gender, age, course of disease, myositis antibodies, extramuscular manifestations, EMG were collected and analyzed among three groups. The characteristics of skeletal muscle were assessed by tMRI inflammatory edema and fat infiltration scores. Analysis of variance, Kruskal-Wallis test and Chi-square test were used to compare the differences in different clinical characteristics and tMRI scores among the three groups. When there was a statistical difference among the three groups, the comparison between the two groups was corrected by the Bonferroni method. Result: (1) Of the 32 patients, 20 were females (62.5%).The median age of onset was 47±14 years, 25 (78.1%) patients had an acute or subacute course.There were 17 (53.1%) with SRP+, 8 (25.0%) with HMGCR+, and 7 (21.9%) with MSAs (myositis specific antibodies) negative. Anti-Ro52 antibody was the most common combined antibody (12/32, 37.5%), among which 10 were in SRP+group.(2) The CK of all patients were elevated, median was 5 948 (4 229, 7 664) U/L. There was no statistical difference of MMT scores among three groups. The proximal limb score was lower than distal limb (P<0.01). The axial muscle score was lower than the distal limb score (P<0.05).(3) Extramuscular manifestations of HMGCR+ group were lower than those of the other two groups (12.5% vs. 71.4% and 76.5%, P<0.017). Rash (60.0% vs.14.3%, P<0.05) and interstitial pulmonary diseases (70.0% vs. 14.3%, P<0.05) were more common in patients with anti-SRP coexistence with anti-Ro52 than those with isolated anti-SRP. Connective tissue disease was more common in SN group (57.1% vs. 11.8% and 0, P<0.017).(4) tMRI showed fascial edema of SN group was more obvious than that of the other two groups (P<0.017). There was no statistical difference in the degree of fat infiltration and inflammatory edema among three groups, but SRP+ group had more cases of early fat infiltration.(5) Myotonic potentials (25.0% vs. 0 and 0, P<0.017) and compound repetitive discharges (CRDs) (50.0% vs. 5.9% and 0, P<0.017) were common in HMGCR+ group. Proteomic analysis found significantly different expressed proteins in skeletal muscle of patients with myotonic potentials or CRDs were associated with cytoskeleton, cell junction and extracellular matrix. Conclusion: IMNM with pure anti-SRP antibody positive and anti-HMGCR positive were mainly affected by skeletal muscles. Those who were co-positive for anti-SRP antibody and anti-Ro52 antibody had more extramuscular manifestations, which might be a special subtype of SRP+ group. This study proposed for the first time that myofascial inflammatory edema is an early sign of SN-IMNM injury. EMG of HMGCR+group were more prone to myotonia potential and CRDs.

The Proximal Humerus Outcome Score at One Year (POSY) Predicts Which Patients Have Poor Functional Outcomes Following Operative Fixation of Proximal Humerus Fractures.

Background: The ability to predict long-term outcomes following surgical fixation of proximal humerus fractures would help identify patients at risk of poor functional outcomes. The purpose of this study was to develop a simple score based on preoperative data that can accurately predict functional outcomes for patients following operative management of proximal humerus fractures.Methods: Over a 12-year period, all proximal humerus fractures surgically treated with a locked proximal humerus plate at a single institution were enrolled in a prospective database. Inclusion criteria in this analysis were any patient with a minimum of a one-year functional outcome score. Patients were assigned to the poor outcome cohort if their Disabilities of the Arm, Shoulder, and Hand (DASH) score at that time point was greater than 10 points above the mean DASH score. Logistic regression was used to build a predictive formula for cohort membership using p < 0.15 and an area under the receiver operator characteristic curve (AUROC) value was calculated to define the overall predictive capacity.Results: A total of 165 patients with an average age of 60.91±13.5 years met the inclusion criteria, with 47 (28.5%) patients assigned to the poor outcome group and 118 (71.5%) patients assigned to the good outcome group. Older age (p = 0.088), BMI (p = 0.019), age-adjusted CCI (p = 0.001), non-Caucasian race (p = 0.017), no college degree (p < 0.0005), unemployed (p < 0.0005), and worker’s compensation case (p = 0.002) were found to be significant predictors of poorer outcome and were used to create a final formula through logistic regression which predicted the probability of a poor outcome (Nagelkerke R Square = 0.403; Hosmer and Lemeshow = 0.902; AUROC = 0.839 [CI: 0.762-0.917]). Once each patient was assigned a score, cutoff values were defined that divided the cohort into three groups. High-risk patients had a score above 50%, and 19 (73.1%) of these patients had a poor outcome.Conclusions: The POSY score is a tool that can predict the functional outcome at one year or greater following surgical intervention for a proximal humerus fracture. Patients who score above 50% are considered at high risk for a poor functional outcome. In the era of value-based care, the POSY score may be used to direct resource utilization while improving outcomes.

Identifying Candidate Flavonoids for Non-Alcoholic Fatty Liver Disease by Network-Based Strategy.

Nonalcoholic fatty liver disease (NAFLD) is the most common type of chronic liver disease and lacks guaranteed pharmacological therapeutic options. In this study, we applied a network-based framework for comprehensively identifying candidate flavonoids for the prevention and/or treatment of NAFLD. Flavonoid-target interaction information was obtained from combining experimentally validated data and results obtained using a recently developed machine-learning model, AI-DTI. Flavonoids were then prioritized by calculating the network proximity between flavonoid targets and NAFLD-associated proteins. The preventive effects of the candidate flavonoids were evaluated using FFA-induced hepatic steatosis in HepG2 and AML12 cells. We reconstructed the flavonoid-target network and found that the number of re-covered compound-target interactions was significantly higher than the chance level. Proximity scores have successfully rediscovered flavonoids and their potential mechanisms that are reported to have therapeutic effects on NAFLD. Finally, we revealed that discovered candidates, particularly glycitin, significantly attenuated lipid accumulation and moderately inhibited intracellular reactive oxygen species production. We further confirmed the affinity of glycitin with the predicted target using molecular docking and found that glycitin targets are closely related to several proteins involved in lipid metabolism, inflammatory responses, and oxidative stress. The predicted network-level effects were validated at the levels of mRNA. In summary, our study offers and validates network-based methods for the identification of candidate flavonoids for NAFLD.

Prognostic significance of spatial and density analysis of T lymphocytes in colorectal cancer

BackgroundAlthough high T cell density is a strong favourable prognostic factor in colorectal cancer, the significance of the spatial distribution of T cells is incompletely understood. We aimed to evaluate the prognostic significance of tumour cell-T cell co-localisation and T cell densities.MethodsWe analysed CD3 and CD8 immunohistochemistry in a study cohort of 983 colorectal cancer patients and a validation cohort (N = 246). Individual immune and tumour cells were identified to calculate T cell densities (to derive T cell density score) and G-cross function values, estimating the likelihood of tumour cells being co-located with T cells within 20 µm radius (to derive T cell proximity score).ResultsHigh T cell proximity score associated with longer cancer-specific survival in both the study cohort [adjusted HR for high (vs. low) 0.33, 95% CI 0.20–0.52, Ptrend < 0.0001] and the validation cohort [adjusted HR for high (vs. low) 0.15, 95% CI 0.05–0.45, Ptrend < 0.0001] and its prognostic value was independent of T cell density score.ConclusionsThe spatial point pattern analysis of tumour cell-T cell co-localisation could provide detailed information on colorectal cancer prognosis, supporting the value of spatial measurement of T cell infiltrates as a novel, robust tumour-immune biomarker.

Abstract P5-13-12: Immune signatures and MammaPrint (ultra) high risk class (MP2) as predictors of response to pembrolizumab combined with the TLR9 agonist SD101 in the neoadjuvant I-SPY 2 TRIAL

Abstract Background: Pembrolizumab, an anti-PD-1 immune checkpoint inhibitor, is approved for treatment in multiple cancers and has been shown to increase pathologic complete response (pCR) and survival in the neoadjuvant setting in breast cancer. Pembrolizumab combined with paclitaxel followed by doxorubicin/cyclophosphamide (P+T-&amp;gt;AC) was evaluated in HER2- patients in the neoadjuvant I-SPY 2 TRIAL and graduated in the HER2-, HR+HER2- and triple negative (TN) signatures. Our biomarker analysis revealed that immune cell abundance and MP2 class predicts response in HR+HER2- patients whereas tumor-immune proximity scores (multiplex-IF) and signaling signatures (mRNA) predict response in TN patients. In an effort to further improve response, the TLR9 agonist SD101 was added to Pembro (P+S+T -&amp;gt; AC) for testing in I-SPY 2. While P+S increased estimated pCR rates relative to control (T-&amp;gt;AC), it did not graduate for efficacy. To better understand the biology underlying response to P+S, we evaluated 31 expression based biomarkers relating to immune, ER and proliferation as predictors of response to P+S overall and within subtypes. Methods: Data from 72 patients (HR+HER2-: 45; TN: 27) treated with P+S were available. Pre-treatment FFPE biopsies were assayed using Agilent gene expression arrays. We evaluated genes/signatures representing 6 immune checkpoint/targets (CD274, PDCD1, TLR9, TIGIT, LAG3, and TIM3), 14 immune cell types (e.g., TILs, T cells, CD8 T cells, Tregs, cytotoxic cells, dendritic cells, mast cells, B cells, macrophages, and neutrophils), 3 T/B-cell prognostic (e.g., ICS5), 5 Tumor-immune signaling (e.g., STAT1, Chemokine12, TIS, and Geparsixto), and 1 TGFB signaling signatures as predictors of response to P+S. We also assess ESR1/PGR and proliferation, and the prognostic marker MP2 class. Signature scores were calculated as previously published. We used logistic modeling to assess biomarker performance (likelihood ratio test, p&amp;lt;0.05). This analysis was also performed in a model adjusting for HR status, and within receptor subsets. For the dichotomous MP1/2, we used Bayesian modeling to estimate the pCR rates of patients in each class. Multiple hypothesis testing adjustment was performed using the Benjamini-Hochberg method. Our statistics are descriptive rather than inferential and do not adjust for multiplicities of other biomarkers outside this study. Results: Higher levels of most (24/29) immune biomarkers associate with pCR in the population as a whole (BH LR p&amp;lt;0.05). Among target genes, CD274 and PDCD1 strongly associate with pCR; however, TLR9 did not associate with response. As seen in previous biomarker analyses of IO agents including Pembro, there are major differences in predictive biology between receptor subsets. Immune cell subpopulation abundance signatures (13/14) and T/B-cell prognostic signatures (3/3) associate with pCR in HR+HER2- but not TN subsets. Whereas tumor-immune signaling signatures (4/5) dominated by chemokines and cytokines associate with pCR in both HR+HER2- and TN subsets. In addition, high ESR1/PGR and low proliferation signature levels associate with response in HR+HER2-, as does MP1/2 class, with an estimated 45% pCR in MP2 versus 17% pCR in MP1. Analysis of multiplex-IF immune markers is pending. Conclusion: Though TN patients are more responsive to Pembro+SD101 and other immunotherapies than HR+HER2- patients, many more immune biomarkers associate with pCR in the latter group. Only tumor-immune signaling signatures associate with pCR in both HR+HER2- and TN subsets. Response in the HR+HER2- subset is higher in MP2 class, high-proliferation, lower-ER tumors. Lack of predictive signal for TLR9 may help explain why the addition of SD101 to Pembro failed to further boost response. Citation Format: Denise M Wolf, Christina Yau, Michael Campbell, Hatem Soliman, Mark Magbanua, Ruixiao Lu, Nicholas O'Grady, Lamorna Brown-Swigart, Gillian Hirst, Laura Sit, Yvonne M Florence, I-SPY 2 TRIAL Investigators, Smita Asare, Doug Yee, Angie DeMichele, Don Berry, Laura Esserman, Jo Chien, Laura van 't Veer. Immune signatures and MammaPrint (ultra) high risk class (MP2) as predictors of response to pembrolizumab combined with the TLR9 agonist SD101 in the neoadjuvant I-SPY 2 TRIAL [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-13-12.

Abstract P4-01-03: Multiomics data reveal novel biomarkers for CDK4/6 resistance

Abstract Background: Cyclin-dependent 4/6 kinase inhibitors (CDK4/6i) and endocrine therapy (ET) have improved progression-free survival (PFS) and overall survival in hormone-receptor-positive (HR+) metastatic breast cancer (MBC), but endocrine resistance is a major challenge. PROMISE [NCT0281902; n=63] is a multicenter study that enrolled women with HR+ HER2- MBC commencing palbociclib (Pb) with letrozole (1st line [1L]) or fulvestrant (2nd line [2L]), and was designed to perform a comprehensive “omic” assessment of prospectively collected biospecimens (pre-treatment (M1), at 2 months (M2), and at disease progression). The goal is to identify novel genomic variants and pathways associated with resistance to CDK4/6i and ET and PFS outcomes. Here we report the association between the proteomic, metabolomics, and lipidomics data generated from pre-Pb and 2-month serum samples and PFS. Methods: Untargeted mass spectrometry data was generated from Metabolon, assaying 1308 metabolites and 831 lipids. Additionally, 1436 proteins were assayed on the Olink platform. Cox proportional hazard models were used to evaluate the univariate hazard ratio (HR) for all features with respect to PFS. The analyses were performed on samples from 45 patients (N=33 1Lwith 9 progression events and 12 2L with 8 progression events), obtained from M1 and M2 timepoints on Pb + ET. Enrichment analysis p-values are calculated using Fisher’s exact test. Results: Proteomics: In the M1 timepoint, 93 and 43 proteins were associated with PFS in the 1L and 2L settings, respectively; inflammation genes were enriched among the 1L setting (p= 0.034); 33 proteins presented HRs ranging between 0.026 and 0.56. The FABP9 protein (HR of 1.98, 95% CI 1.02-3.83) was associated with worse PFS. Conversely, inflammation genes were not observed to be enriched in 2L. In the M2 timepoint, we observed 60 and 21 proteins significantly associated with PFS, but no biological function was enriched in 1L and 2L. Metabolites: In the M1 timepoint, metabolism of the sulfur-containing amino acids (methionine, cysteine, SAM and taurine) were enriched in the 1L setting (p= 0.035, HR range 0.15-0.33); and the branched-chain amino acids (leucine, isoleucine, and valine) were significantly associated with PFS in the 2L setting (p= 0.028, HR range 0.013-0.33). At the M2 timepoint, the amino acids were no longer enriched, but fatty acid metabolism was significantly enriched for both 1L and 2L (p= 0.048 and 0.067, respectively). Pathways involving lipids, amino acids, and xenobiotics were enriched in metabolites related to PFS (p &amp;lt;0.05) for both treatment lines at M1 and M2. Lipidomics: In the M1 timepoint, 10 and 19 lipids were associated with PFS for 1L and 2L, respectively. The most notable lipid associated with worse PFS in the 1L was an 18 carbon phosphatidylinositol, PI(18:1/18:2), (HR 7.34 (CI 1.27-42.50); 8 triglycerides were associated with improved PFS (HR range 0.39 and 0.55). In 2L, the 19 lipids associated with PFS included 12 phosphatidylcholines (enrichment p = 5.6X10-8). In the M2 timepoint, 15 and 8 lipids were significantly associated with PFS for 1L and 2L. An enrichment of phosphatidylinositols was observed in 1L (p= 1.2X10-5); none were observed in the 2L.Future Directions: Networks are being constructed using the proximity scores of the proteins, lipids, and metabolites associated with PFS in M1 and M2 for 1L and 2L. Network similarities and analyses will be conducted.Conclusion: Distinct multi-omic changes identified in serum samples obtained from PROMISE participants M1 and M2 on Pb correlate with disease progression in both 1L and 2L settings. Additionally, validation studies will determine the significance of these findings. Citation Format: Krishna R. Kalari, Kevin J. Thompson, Jason Sinnwell, Xiaojia Tang, Vera J. Suman, Jun He, Seul Kee Byeon, Akhilesh Pandey, Abe Eyman Casey, Peter T. Vedell, Ann M. Moyer, Alvaro Moreno-Aspitia, Donald W. Northfelt, Minetta C. Liu, Tufia C. Haddad, Saranya Chumsri, Prema Peethambaram, Kathryn J. Ruddy, Karthik V. Giridhar, Roberto A. Leon-Ferre, Richard M. Weinshilboum, Liewei Wang, Ciara C. O’ Sullivan, Matthew P. Goetz. Multiomics data reveal novel biomarkers for CDK4/6 resistance [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-01-03.

Abstract WMP40: Premovement Sensorimotor Oscillations Are Associated With Motor Status In Chronic Stroke

Introduction: Oscillations in the beta frequency range within sensorimotor cortex play a key role in normal motor control. Beta band activity decreases after stroke in association with motor impairment. Changes in sensorimotor beta band electrical activity in the time period preceding movement onset have received limited study in patients with stroke, although these events are critical to genesis of movement. Here we test the hypothesis that premovement beta band sensorimotor activity is associated with level of motor impairment. Methods: 27 subjects with stroke (age 60±14 yr, 46±57 mo post-stroke) and 23 healthy subjects (age 59±12 yr) performed a cued shoulder rotation movement during EEG (256 channels). EEG was analyzed in the window -1500ms to +500ms relative to the movement onset. Event-related desynchronization (ERD) in the beta (15 - 30 Hz) frequency range was computed in a cluster surrounding lead C1. Results: Subjects with stroke showed significantly decreased beta ERD amplitude in the -350 ms to -130 ms premovement time window compared to controls (gray box in Fig A, p&lt;0.01). Fig B shows the topographic distribution of beta ERD during this time window, which is smaller in area among subjects with stroke. Beta ERD amplitude correlated with the proximal Fugl-Meyer motor score during the time window -300 ms to +100 ms relative to movement onset (Fig C), maximally correlating at 200ms before movement (r=-0.56, p &lt; 0.01). Conclusion: Premovement beta ERD is reduced in amplitude and spatial distribution after stroke. This decrease in premovement beta oscillatory activity is associated with greater motor impairment. Measures of premovement electrical activity provide useful insights into motor system dysfunction after stroke and are attractive biomarker candidates given their ease of collection at the bedside.

Radiographic vertical tracheal diameter assessment at different levels along the trachea as an alternative method for the evaluation of the tracheal diameter in non-brachycephalic small breed dogs

BackgroundTracheal narrowing due to congenital tracheal hypoplasia, acquired tracheal stenosis and tracheal collapse can lead to life-threatening respiratory distress. Tracheal hypoplasia has been identified in brachycephalic dog breeds, predominantly English Bulldogs, by measuring the tracheal diameter compared to the diameter of the thoracic inlet and creating a ratio. However, reference ranges for tracheal diameter have not been established for non-brachycephalic small breed dogs. It would be advantageous to have established tracheal diameters for non-brachycephalic small breed dogs, as these are the dogs most at risk of tracheal collapse. The main objective, of this study was to radiographically evaluate vertical tracheal diameter (VTD) at three standardized locations along the trachea of non-brachycephalic small breed dogs, in an attempt to further establish a screening diagnostic protocol for canine tracheal hypoplasia. Medical records and thoracic radiographs of non-brachycephalic small breed dogs without respiratory disease were reviewed. Right lateral radiographs were reviewed. The absolute and average VTDs at three locations (location A: caudal cervical VTD; location B thoracic inlet VTD; location C: intrathoracic VTD) were standardized by manubrium length (ML), as well as by the previously utilized thoracic inlet distance (Ti-D) and proximal 3rd rib width (PR3-W) to calculate manubrium-tracheal index (M-TI), thoracic inlet-tracheal index (Ti-TI), and proximal R3-tracheal score (PR3-TS), respectively. Correlations between averaged tracheal diameter and each of the ML, Ti-D, and PR3-W, and between M-TI and each of Ti-TI and PR3-TS were calculated.ResultsEighty-one healthy dogs met the criteria for inclusion. Significant differences (P < 0.0001) were identified among the mean values of the absolute and standardized VTDs at levels A, B, and C. The smallest tracheal diameter was identified at the level of the thoracic inlet (Level B).The average VTD correlated better with ML (rs = 0.82, P < 0.0001) compared to Ti-D and PR3-W. A relatively strong correlation (rs = 0.77, P < 0.0001) was identified between the averaged manubrium tracheal index (M-TI) and thoracic inlet tracheal index (Ti-TI).ConclusionM-TI is an appropriate alternative to Ti-TI and PR3-TS to radiographically evaluate VTD in dogs. M-TI < 0.43, < 0.34, or < 0.38 at level A, B, or C, respectively, may indicate tracheal hypoplasia in non-brachycephalic small breed dogs. Screening of canine VTD could be achieved using M-TI.

Agreement and Reliability Analysis of Machine Learning Scaling and Wireless Monitoring in the Assessment of Acute Proximal Weakness by Experts and Non-Experts: A Feasibility Study.

Assessing the symptoms of proximal weakness caused by neurological deficits requires the knowledge and experience of neurologists. Recent advances in machine learning and the Internet of Things have resulted in the development of automated systems that emulate physicians’ assessments. The application of those systems requires not only accuracy in the classification but also reliability regardless of users’ proficiency in the real environment for the clinical point-of-care and the personalized health management. This study provides an agreement and reliability analysis of using a machine learning-based scaling of Medical Research Council (MRC) proximal scores to evaluate proximal weakness by experts and non-experts. The system trains an ensemble learning model using the signals from sensors attached to the limbs of patients in a neurological intensive care unit. For the agreement analysis, we investigated the percent agreement of MRC proximal scores and Bland-Altman plots of kinematic features between the expert- and non-expert scaling. We also analyzed the intra-class correlation coefficients (ICCs) of kinematic features and Krippendorff’s alpha of the observers’ scaling for the reliability analysis. The mean percent agreement between the expert- and the non-expert scaling was 0.542 for manual scaling and 0.708 for autonomous scaling. The ICCs of kinematic features measured using sensors ranged from 0.742 to 0.850, whereas the Krippendorff’s alpha of manual scaling for the three observers was 0.275. The autonomous assessment system can be utilized by the caregivers, paramedics, or other observers during an emergency to evaluate acute stroke patients.

A proposal for a technology-assisted approach to wildlife management in Singapore

The long-tailed macaque (Macaca fascicularis fascicularis) is a non-human primate species that is native to Singapore. From mid-2020, the Nanyang Technological University campus witnessed a sharp increase in the number of human–macaque encounters. In this article, we will first identify a set of technological tools that we have developed to guide decision- and policy-making on the wildlife management front and mitigate human–macaque conflict. Thereafter, we will describe how we applied these tools to the activity log cataloguing the macaque activity on campus and offer an analysis of the trends in macaque behaviour that may be shared with members of the public and affected residents. Among the results that will be discussed are the following: a significant decline in the number of macaque sightings at Student Residences over the weekend, a majority of the reported macaque sightings occurring in the early evening, no significant difference in the damage and proximity scores over time, and a trend in observed macaque activity at critical sleep-relevant time intervals and areas on campus. We hope that our approach will promote, through an improved and data-driven understanding of macaque behavioural trends, technology-assisted decision-making and a more harmonious co-existence between humans and macaques on campus and in Singapore. Last but not least, we anticipate further extensions of our approach with respect to other forms of wildlife.