Addressing Sex Disparities in Lung Cancer Screening Eligibility: USPSTF vs PLCOm2012 Criteria.

BACKGROUND: Lung cancer is the leading cause of cancer death in women in the United States. Prospective randomized lung screening trials suggest a greater lung cancer mortality benefit from screening women compared with men. RESEARCH QUESTION: Do the United States Preventative Services Task Force (USPSTF) lung screening guidelines that are based solely on age and smoking history contribute to sex disparities in eligibility, and if so, does the use of the PLCOm2012 risk prediction model that is based on 11 predictors of lung cancer reduce sex disparities? STUDY DESIGN AND METHODS: This retrospective analysis of 883 lung cancer cases in the Chicago Race Eligibility for Screening Cohort (CREST) determined the sensitivity of USPSTF vs PLCOm2012 eligibility criteria, stratified according to sex. For comparisons vs the USPSTF 2013 and the recently published USPSTF 2021 (released March 9, 2021) eligibility criteria, the PLCOm2012 model was used with risk thresholds of ≥ 1.7%/6 years (6y) and ≥ 1.0%/6y, respectively. RESULTS: The sensitivities for screening by the USPSTF 2013 were 46.7% for women and 64.6% for men (P = .003) and by the USPSTF 2021 were 56.8% and 71.8%, respectively (P = .02). In contrast, the PLCOm2012 ≥ 1.7%/6y sensitivities were 64.6% and 70.4%, and the PLCOm2012 ≥ 1.0%/6y sensitivities were 77.4% and 82.4%. The PLCOm2012 differences in sensitivity using ≥ 1.7%/6y and ≥ 1.0%/6y thresholds between women and men were nonsignificant (both, P = .07). Compared with men, women were more likely to be ineligible according to the USPSTF 2021 criteria because their smoking exposures were < 20 pack-years (22.8% vs 14.8%; ORWomen vs Men, 1.70; 95% CI, 1.19-2.44; P = .002), and 27% of these ineligible women were eligible according to the PLCOm2012 ≥ 1.0%/6y criteria. INTERPRETATION: Although the USPSTF 2021 eligibility criteria are more sensitive than the USPSTF 2013 guidelines, sex disparities in eligibility remain. Adding the PLCOm2012 risk prediction model to the USPSTF guidelines would improve sensitivity and attenuate sex disparities.

Brief Report: Risk Prediction Model Versus United States Preventive Services Task Force 2020 Draft Lung Cancer Screening Eligibility Criteria-Reducing Race Disparities.

INTRODUCTION: Eligibility criteria for lung cancer screening based solely on age and smoking history are less sensitive than validated risk prediction models. The U.S. Preventive Services Task Force (USPSTF) has proposed new guidelines to improve the sensitivity for selecting high-risk individuals and to decrease race disparity. In this retrospective study, termed the Chicago Race Eligibility for Screening Cohort, we compare the sensitivity of the proposed USPSTF2020 criteria versus the PLCOm2012 risk prediction model for selecting a racially diverse lung cancer population with a smoking history for lung cancer screening. METHODS: This Chicago Race Eligibility for Screening Cohort study applies the PLCOm2012 model with a risk threshold of 1.0%/6 years and the USPSTF2020 criteria (age 50-80 y, pack-years ≥ 20 y, quit-years ≤ 15 y) to 883 individuals with a smoking history diagnosed with having lung cancer. RESULTS: The PLCOm2012 was more sensitive than the USPSTF2020 overall (79.1% versus 68.6%, p < 0.0001) in White (81.5% versus 75.4%, p = 0.029) and in African American (82.8% versus 70.6% p < 0.0001) individuals. Of the total cohort, 254 (28.8%) would not have qualified owing to less than 20 pack-years, quit-time of more than 15 years, and age less than 50 years. Of these 254 cases, 40% would have qualified by the PLCOm2012 model. For the 20 pack-year criterion, of the 497 African American individuals, 19.3% did not meet this criterion, and of these, an additional 31.3% would have qualified by the PLCOm2012 model (p = 0.002). CONCLUSIONS: Although more sensitive than USPSTF2013, the proposed USPSTF2020 draft guidelines still have a race disparity in eligibility for screening. This study provides "real world" evidence that use of the PLCOm2012 risk prediction model eliminates this race disparity.

Risk Prediction Model Versus United States Preventive Services Task Force Lung Cancer Screening Eligibility Criteria: Reducing Race Disparities.

INTRODUCTION: Disparities exist in lung cancer outcomes between African American and white people. The current United States Preventive Services Task Force (USPSTF) lung cancer screening eligibility criteria, which is based solely on age and smoking history, may exacerbate racial disparities. We evaluated whether the PLCOm2012 risk prediction model more effectively selects African American ever-smokers for screening. METHODS: Lung cancer cases diagnosed between 2010 and 2019 at an urban medical center serving a racially and ethnically diverse population were retrospectively reviewed for lung cancer screening eligibility based on the USPSTF criteria versus the PLCOm2012 model. RESULTS: This cohort of 883 ever-smokers comprised the following racial and ethnic makeup: 258 white (29.2%), 497 African American (56.3%), 69 Hispanic (7.8%), 24 Asian (2.7%), and 35 other (4.0%). Compared with the USPSTF criteria, the PLCOm2012 model increased the sensitivity for the African American cohort at lung cancer risk thresholds of 1.51%, 1.70%, and 2.00% per 6 years (p < 0.0001). For example, at the 1.70% risk threshold, the PLCOm2012 model identified 71.3% African American cases, whereas the USPSTF criteria only identified 50.3% (p < 0.0001). In contrast, in case of whites there was no difference (66.0% versus 62.4%, respectively [p = 0.203]). Of the African American ever-smokers who were PLCO1.7%-positive and USPSTF-negative, the criteria missed from the USPSTF were those with pack-years less than 30 (67.7%), quit time of greater than 15 years (22.5%), and age less than 55 years (13.0%). CONCLUSIONS: The PLCOm2012 model was found to be preferable over the USPSTF criteria at identifying African American ever-smokers for lung cancer screening. The broader use of this model in racially diverse populations may help overcome disparities in lung cancer screening and outcomes.

Neuroprotective and neurotrophic effects of Lanthionine Ketimine Ester.

Lanthionine ketimine ethyl ester (LKE) is a synthetic derivative of the naturally occurring amino acid lanthionine ketimine. We previously showed that LKE reduced clinical signs in a mouse model of multiple sclerosis (MS) associated with reductions in axonal damage; however, whether LKE has direct beneficial actions on mammalian neuronal cells was not examined. In the current study, we tested the effects of LKE in SH-SY5Y human neuronal cells and in primary mouse cerebellar granule neurons. In both cell types, LKE dose-dependently reduced the cell death that occurred spontaneously followed a change in media. LKE also reduced cell death due to glutamate excitoxicity, accompanied by a reduction in production of reactive oxygen species. LKE induced neuritogenesis in both undifferentiated SH-SY5Y cells and in primary neuron, increasing process numbers and lengths. These results demonstrate that direct neuroprotective and neurotrophic effects of LKE likely contribute to its beneficial actions in vivo.

Expression of cross-tolerance to a wide range of conditions in a human lung cancer cell line after adaptation to nitric oxide.

Previously, we have shown that A549, a human lung adenocarcinoma, can be adapted to nitric oxide (NO●). NO● is a nitrogen-based free radical that is synthesized by a family of enzymes known as nitric oxide synthases. NO● has been shown to be overexpressed in patient populations of different cancers. In addition, it has been observed that patients who express high levels of nitric oxide synthases tend to have poorer clinical outcomes than those with low levels of expression. The original cell line A549 (parent) and the adapted A549-HNO (high nitric oxide) cell line serve as a useful model system to investigate the role of NO● in tumor progression and prognosis. We have previously shown that the A549-HNO-adapted cells grow aggressively when compared to A549-parent cells. Furthermore, we have shown that the A549-HNO-adapted cells exhibit a higher percentage of cell viability when exposed to ultraviolet and X-ray radiation than the A549-parent cells. Cancer patients who develop resistance to one treatment often become resistant to other previously unencountered forms of treatment. This phenomenon is known as cross-tolerance. To determine whether NO● is a potential cross-tolerance causing agent, we have expanded our research by conducting parallel studies to a variety of other agents and conditions beyond radiation and ultraviolet exposure. We exposed both cell lines to varying levels of chemotherapeutic drugs (taxol and doxorubicin), temperature, pH, calcium chloride, cadmium chloride, copper chloride, sodium chloride, ferrous chloride, and sodium-R-lipoic acid. Our results show that the A549-HNO cells exhibit greater viability than the A549-parent cells when exposed to each of the various conditions. Therefore, NO● is one potential driving force that can make tumor cells exhibit cross-tolerance.

Heparanase: Potential roles in multiple sclerosis.

Heparanase is a heparan sulfate degrading enzyme that cleaves heparan sulfate (HS) chains present on HS proteoglycans (HSPGs), and has been well characterized for its roles in tumor metastasis and inflammation. However, heparanase is emerging as a contributing factor in the genesis and severity of a variety of neurodegenerative diseases and conditions. This is in part due to the wide variety of HSPGs on which the presence or absence of HS moieties dictates protein function. This includes growth factors, chemokines, cytokines, as well as components of the extracellular matrix (ECM) which in turn regulate leukocyte infiltration into the CNS. Roles for heparanase in stroke, Alzheimer's disease, and glioma growth have been described; roles for heparanase in other disease such as multiple sclerosis (MS) are less well established. However, given its known roles in inflammation and leukocyte infiltration, it is likely that heparanase also contributes to MS pathology. In this review, we will briefly summarize what is known about heparanase roles in the CNS, and speculate as to its potential role in regulating disease progression in MS and its animal model EAE (experimental autoimmune encephalitis), which may justify testing of heparanase inhibitors for MS treatment.

Asxl2-/- Mice Exhibit De Novo Cardiomyocyte Production during Adulthood.

Heart attacks affect more than seven million people worldwide each year. A heart attack, or myocardial infarction, may result in the death of a billion cardiomyocytes within hours. The adult mammalian heart does not have an effective mechanism to replace lost cardiomyocytes. Instead, lost muscle is replaced with scar tissue, which decreases blood pumping ability and leads to heart failure over time. Here, we report that the loss of the chromatin factor ASXL2 results in spontaneous proliferation and cardiogenic differentiation of a subset of interstitial non-cardiomyocytes. The adult Asxl2-/- heart displays spontaneous overgrowth without cardiomyocyte hypertrophy. Thymidine analog labeling and Ki67 staining of 12-week-old hearts revealed 3- and 5-fold increases of proliferation rate for vimentin⁺ non-cardiomyocytes in Asxl2-/- over age- and sex-matched wildtype controls, respectively. Approximately 10% of proliferating non-cardiomyocytes in the Asxl2-/- heart express the cardiogenic marker NKX2-5, a frequency that is ~7-fold higher than that observed in the wildtype. EdU lineage tracing experiments showed that ~6% of pulsed-labeled non-cardiomyocytes in Asxl2-/- hearts differentiate into mature cardiomyocytes after a four-week chase, a phenomenon not observed for similarly pulse-chased wildtype controls. Taken together, these data indicate de novo cardiomyocyte production in the Asxl2-/- heart due to activation of a population of proliferative cardiogenic non-cardiomyocytes. Our study suggests the existence of an epigenetic barrier to cardiogenicity in the adult heart and raises the intriguing possibility of unlocking regenerative potential via transient modulation of epigenetic activity.

Additional sex combs-like family genes are required for normal cardiovascular development.

Congenital heart disease (CHD) is the most common birth defect. However, the majority of CHD cases have unknown etiology. Here we report the identification of ASXL2 and ASXL1, two homologous chromatin factors, as novel regulators of heart development. Asxl2(-/-) fetuses have reduced body weight and display congenital heart malformations including thickened compact myocardium in the left ventricle, membranous ventricular septal defect, and atrioventricular valval stenosis. Although most Asxl2(-/-) animals survive to term, the neonates have patent ductus arteriosus and consequent lung hemorrhage and die soon after birth. Asxl1(-/-) fetuses have reduced body weight and display cleft palate, anophthalmia as well as ventricular septal defects and a failure in lung maturation. From these results, we conclude that normal heart development requires both ASXL proteins. In particular, ASXL2 plays an important role in heart morphogenesis and the transition from fetal to postnatal circulation.

Part I-mechanism of adaptation: high nitric oxide adapted A549 cells show enhanced DNA damage response and activation of antiapoptotic pathways.

Our previous studies demonstrate that A549, a human lung adenocarcinoma line, could be adapted to the free radical nitric oxide (NO([Symbol: see text])). NO([Symbol: see text]) has been shown to be overexpressed in human tumors. The original cell line, A549 (parent), and the newly adapted A549-HNO (which has a more aggressive phenotype) serves as a useful model system to study the role of NO([Symbol: see text]) in tumor biology. It is well known that DNA damage response (DDR) is altered in cancer cells and NO([Symbol: see text]) is known to cause DNA damage. Modulations in molecular mechanisms involved in DNA damage response in A549-HNO cells can provide better insights into the enhanced growth behavior of these cells. Thus, here, we carried out a series of time course experiments by treating A549 and A549-HNO cells with NO([Symbol: see text]) donor and examining levels of proteins involved in the DDR pathway. We observed induced expression of key components of DDR pathway in A549-HNO cells. The HNO cells showed sustained expression of key proteins involved in both nonhomologous end joining (NHEJ) and homologous recombination pathways, whereas parent cells only expressed low levels of NHEJ pathway proteins. Further with prolonged NO([Symbol: see text]) exposure, ATR, Chk1, and p53 were activated and upregulated in HNO cells. Activation of p53 results in inhibition of apoptosis through induced Mcl1 expression. It also leads to cell cycle modulation. Interestingly, several reports show that cancer stem cells have enhanced expression of proteins involved in DNA damage response and also activated an antiapoptotic response. Our results here suggest that our HNO adapted A549 cells have increased activation of DNA damage response pathway proteins which can lead to better DNA repair function. Enhanced DDR leads to activation of antiapoptosis response and modulation in the cell cycle which may lead to better survival of these cells under harsh conditions. Thus, our present investigation further supports the hypothesis that HNO exposure leads to survival of these cells.

Part II-mechanism of adaptation: A549 cells adapt to high concentration of nitric oxide through bypass of cell cycle checkpoints.

Previous work has shown enhanced survival capacity in high nitric oxide (HNO)-adapted tumor cells. In Part I of this series of manuscripts, we have shown that A549-HNO cells demonstrate an improved growth profile under UV and X-ray radiation treatment. These cells exhibit increased expression of proteins involved in DNA damage recognition and repair pathway, both the non-homologous end joining pathway and homologous recombination. These include Ku80, DNA-PK, XLF ligase and MRN complex proteins. Further, the A549-HNO cells show high levels of ATM, ATR, Chk1 and Chk2, and phospho-p53. Activation of these molecules may lead to cell cycle arrest and apoptosis due to DNA damage. This is observed in parent A549 cells in response to NO donor treatment; however, the A549-HNO cells proliferate and inhibit apoptosis. Cell cycle analysis showed slowed progression through S phase which will allow time for DNA repair. Thus, to better understand the increased growth rate in A549-HNO when compared to the parent cell line A549, we studied molecular mechanisms involved in cell cycle regulation in A549-HNO cells. During the initial time period of NO donor treatment, we observe high levels of cyclin/Cdk complexes involved in regulating various stages of the cell cycle. This would lead to bypass of G1-S and G2-M checkpoints. The HNO cells also show much higher expression of Cdc25A. Cdc25A activates Cdk molecules involved in different phases of the cell cycle. In addition, there is enhanced phosphorylation of the Rb protein in HNO cells. This leads to inactivation of Rb/E2F checkpoint regulating G1-S transition. This may lead to faster progression in S phase. Thus, all of these perturbations in HNO cells lead to accelerated cell cycle progression and a higher growth rate. We also assessed expression of cell cycle inhibitors in HNO cells. Interestingly, the HNO cells show a significant decline in p21CIP1 at initial time points, but with prolonged exposure, the levels were much higher than those of the parent cells. This suggests an initial bypass of cell cycle checkpoints as p21CIP1 can inhibit the activity of all cyclin/Cdk complexes. p21CIP1 is also known to inhibit p53-induced apoptosis. This could be important during later phases of the cell cycle to allow time for repair of damaged DNA and thus better survival of HNO cells.

Application of immunohistochemical staining to detect antigen destruction as a measure of tissue damage.

Electrocautery and directed energy devices (DEDs) such as lasers, which are used in surgery, result in tissue damage that cannot be readily detected by traditional histological methods, such as hematoxylin and eosin staining. Alternative staining methods, including 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to stain live tissue, have been reported. Despite providing superior detection of damaged tissue relative to the hematoxylin and eosin (H&E) method, the MTT method possesses a number of drawbacks, most notably that it must be carried out on live tissue samples. Herein, we report the development of a novel staining method, "antigen destruction immunohistochemistry" (ADI), which can be carried out on paraffin-embedded tissue. The ADI method takes advantage of epitope loss to define the area of tissue damage and provides many of the benefits of live tissue MTT staining without the drawbacks inherent to that method. In addition, the authors provide data to support the use of antibodies directed at a number of gene products for use in animal tissue for which there are no species-specific antibodies commercially available, as well as an example of a species-specific direct antibody. Data are provided that support the use of this method in many tissue models, as well as evidence that ADI is comparable to the live tissue MTT method.