A directional switch of integrin signalling and a new anti-thrombotic strategy.

Integrins have a critical role in thrombosis and haemostasis. Antagonists of the platelet integrin αIIbß3 are potent anti-thrombotic drugs, but also have the life-threatening adverse effect of causing bleeding. It is therefore desirable to develop new antagonists that do not cause bleeding. Integrins transmit signals bidirectionally. Inside-out signalling activates integrins through a talin-dependent mechanism. Integrin ligation mediates thrombus formation and outside-in signalling, which requires Gα13 and greatly expands thrombi. Here we show that Gα13 and talin bind to mutually exclusive but distinct sites within the integrin ß3 cytoplasmic domain in opposing waves. The first talin-binding wave mediates inside-out signalling and also ligand-induced integrin activation, but is not required for outside-in signalling. Integrin ligation induces transient talin dissociation and Gα13 binding to an EXE motif (in which X denotes any residue), which selectively mediates outside-in signalling and platelet spreading. The second talin-binding wave is associated with clot retraction. An EXE-motif-based inhibitor of Gα13-integrin interaction selectively abolishes outside-in signalling without affecting integrin ligation, and suppresses occlusive arterial thrombosis without affecting bleeding time. Thus, we have discovered a new mechanism for the directional switch of integrin signalling and, on the basis of this mechanism, designed a potent new anti-thrombotic drug that does not cause bleeding.

Improved specificity of TALE-based genome editing using an expanded RVD repertoire.

Transcription activator-like effector (TALE) proteins have gained broad appeal as a platform for targeted DNA recognition, largely owing to their simple rules for design. These rules relate the base specified by a single TALE repeat to the identity of two key residues (the repeat variable diresidue, or RVD) and enable design for new sequence targets via modular shuffling of these units. A key limitation of these rules is that their simplicity precludes options for improving designs that are insufficiently active or specific. Here we address this limitation by developing an expanded set of RVDs and applying them to improve the performance of previously described TALEs. As an extreme example, total conversion of a TALE nuclease to new RVDs substantially reduced off-target cleavage in cellular studies. By providing new RVDs and design strategies, these studies establish options for developing improved TALEs for broader application across medicine and biotechnology.

Small-airway obstruction and emphysema in chronic obstructive pulmonary disease.

BACKGROUND: The major sites of obstruction in chronic obstructive pulmonary disease (COPD) are small airways (<2 mm in diameter). We wanted to determine whether there was a relationship between small-airway obstruction and emphysematous destruction in COPD. METHODS: We used multidetector computed tomography (CT) to compare the number of airways measuring 2.0 to 2.5 mm in 78 patients who had various stages of COPD, as judged by scoring on the Global Initiative for Chronic Obstructive Lung Disease (GOLD) scale, in isolated lungs removed from patients with COPD who underwent lung transplantation, and in donor (control) lungs. MicroCT was used to measure the extent of emphysema (mean linear intercept), the number of terminal bronchioles per milliliter of lung volume, and the minimum diameters and cross-sectional areas of terminal bronchioles. RESULTS: On multidetector CT, in samples from patients with COPD, as compared with control samples, the number of airways measuring 2.0 to 2.5 mm in diameter was reduced in patients with GOLD stage 1 disease (P=0.001), GOLD stage 2 disease (P=0.02), and GOLD stage 3 or 4 disease (P<0.001). MicroCT of isolated samples of lungs removed from patients with GOLD stage 4 disease showed a reduction of 81 to 99.7% in the total cross-sectional area of terminal bronchioles and a reduction of 72 to 89% in the number of terminal bronchioles (P<0.001). A comparison of the number of terminal bronchioles and dimensions at different levels of emphysematous destruction (i.e., an increasing value for the mean linear intercept) showed that the narrowing and loss of terminal bronchioles preceded emphysematous destruction in COPD (P<0.001). CONCLUSIONS: These results show that narrowing and disappearance of small conducting airways before the onset of emphysematous destruction can explain the increased peripheral airway resistance reported in COPD. (Funded by the National Heart, Lung, and Blood Institute and others.).

CIB1 is an endogenous inhibitor of agonist-induced integrin alphaIIbbeta3 activation.

In response to agonist stimulation, the alphaIIbbeta3 integrin on platelets is converted to an active conformation that binds fibrinogen and mediates platelet aggregation. This process contributes to both normal hemostasis and thrombosis. Activation of alphaIIbbeta3 is believed to occur in part via engagement of the beta3 cytoplasmic tail with talin; however, the role of the alphaIIb tail and its potential binding partners in regulating alphaIIbbeta3 activation is less clear. We report that calcium and integrin binding protein 1 (CIB1), which interacts directly with the alphaIIb tail, is an endogenous inhibitor of alphaIIbbeta3 activation; overexpression of CIB1 in megakaryocytes blocks agonist-induced alphaIIbbeta3 activation, whereas reduction of endogenous CIB1 via RNA interference enhances activation. CIB1 appears to inhibit integrin activation by competing with talin for binding to alphaIIbbeta3, thus providing a model for tightly controlled regulation of alphaIIbbeta3 activation.

Two-Year Follow-Up of a Randomized Controlled Study of Integrated Smoking Cessation in a Lung Cancer Screening Program.

INTRODUCTION: Smoking cessation activities incorporated into lung cancer screening programs have been broadly recommended, but studies to date have not exhibited increased quit rates associated with cessation programs in this setting. We aimed to determine the long-term effectiveness of smoking cessation counseling in smokers presenting for lung cancer screening. METHODS: This was a randomized control trial of an intensive, telephone-based smoking cessation counseling intervention incorporating lung cancer screening results versus usual care (information pamphlet). This analysis reports on the long-term impact (24-mo) of the intervention on abstinence from smoking. RESULTS: A total of 337 active smokers who participated in the screening study were randomized to active smoking cessation counseling (n = 171) or control arm (n = 174) and completed a 24-month assessment. The 30-day smoking abstinence rates at 24 months postrandomization was 18.3% and 21.4% in the control and intervention arms, respectively-a 3.1% difference (95% confidence interval: -5.4 to 11.6, p = 0.48). No statistically significant differences in the 7-day abstinence, the use of pharmacologic cessation aids, nicotine replacement therapies, nor intent to quit in the following 30 days were noted (p > 0.05). The abstinence rates at 24-months were higher overall than at 12-months (19.9% versus 13.3%, p < 0.001), and smoking intensity was lower than at baseline for ongoing smokers. CONCLUSIONS: A telephone-based smoking cessation counseling intervention incorporating lung cancer screening results did not result in increased long-term cessation rates versus written information alone in unselected smokers undergoing lung cancer screening. Overall, quit rates were high and continued to improve throughout participation in the screening program. (ClinicalTrials.govNCT02431962).

Deep Learning for Lung Cancer Detection on Screening CT Scans: Results of a Large-Scale Public Competition and an Observer Study with 11 Radiologists.

PURPOSE: To determine whether deep learning algorithms developed in a public competition could identify lung cancer on low-dose CT scans with a performance similar to that of radiologists. MATERIALS AND METHODS: In this retrospective study, a dataset consisting of 300 patient scans was used for model assessment; 150 patient scans were from the competition set and 150 were from an independent dataset. Both test datasets contained 50 cancer-positive scans and 100 cancer-negative scans. The reference standard was set by histopathologic examination for cancer-positive scans and imaging follow-up for at least 2 years for cancer-negative scans. The test datasets were applied to the three top-performing algorithms from the Kaggle Data Science Bowl 2017 public competition: grt123, Julian de Wit and Daniel Hammack (JWDH), and Aidence. Model outputs were compared with an observer study of 11 radiologists that assessed the same test datasets. Each scan was scored on a continuous scale by both the deep learning algorithms and the radiologists. Performance was measured using multireader, multicase receiver operating characteristic analysis. RESULTS: The area under the receiver operating characteristic curve (AUC) was 0.877 (95% CI: 0.842, 0.910) for grt123, 0.902 (95% CI: 0.871, 0.932) for JWDH, and 0.900 (95% CI: 0.870, 0.928) for Aidence. The average AUC of the radiologists was 0.917 (95% CI: 0.889, 0.945), which was significantly higher than grt123 (P = .02); however, no significant difference was found between the radiologists and JWDH (P = .29) or Aidence (P = .26). CONCLUSION: Deep learning algorithms developed in a public competition for lung cancer detection in low-dose CT scans reached performance close to that of radiologists.Keywords: Lung, CT, Thorax, Screening, Oncology Supplemental material is available for this article. © RSNA, 2021.

Adhesion-induced unclasping of cytoplasmic tails of integrin alpha(IIb)beta3.

Integrin alpha(IIb)beta(3) plays a pivotal role in hemostasis and thrombosis by mediating adhesive interactions of platelets. Binding of alpha(IIb)beta(3) to its physiological ligands, immobilized fibrinogen and fibrin, induces outside-in signaling in platelets, leading to their adhesion and spreading even without prior stimulation by agonists. Implicit in these phenomena is a requirement for the linkage between integrins' cytoplasmic tails and intracellular proteins. However, the nature of the initiating signal has not been established. In this study, we examined whether binding of alpha(IIb)beta(3) to immobilized fibrin(ogen), per se, triggers interaction of the integrin with cytoplasmic proteins. Using the integrin-binding skelemin fragment as a marker of exposure of residues involved in the clasp between alpha(IIb) and beta(3) cytoplasmic tails, we showed that its binding site in the membrane-proximal beta(3) 715-730 segment is cryptic and becomes exposed as a result of binding of isolated alpha(IIb)beta(3) to immobilized ligands. Furthermore, the skelemin-like protein present in platelets and CHO cells does not associate with alpha(IIb)beta(3) in resting platelets or suspended alpha(IIb)beta(3)-expressing CHO cells but is recruited to integrin during cell adhesion. In addition, not only beta(3) but also the membrane-proximal 989-1000 segment of the alpha(IIb) cytoplasmic tail binds the skelemin fragment. Finally, the same residues, alpha(IIb) Val(990), alpha(IIb) Arg(995), and beta(3) His(722), involved in the formation of the clasp between the tails are also required for skelemin binding. These studies suggest that ligation of alpha(IIb)beta(3) by immobilized ligands during platelet adhesion induces a transmembrane conformation change in the integrin, resulting in unclasping of the complex between the membrane-proximal parts of cytoplasmic tails, thereby unmasking residues involved in binding the skelemin-like protein. Thus, the junction between alpha(IIb) and beta(3) cytoplasmic tails may contain the critical structural information for the initiation of outside-in signaling.

Lung nodules: CT-guided placement of microcoils to direct video-assisted thoracoscopic surgical resection.

PURPOSE: To prospectively assess the safety and effectiveness of computed tomography (CT)-guided placement of fiber-coated microcoils used to guide video-assisted thoracoscopic surgical (VATS) excision of small peripheral lung nodules, with successful excision as the primary outcome and successful CT-guided microcoil placement and procedural complications as secondary outcomes. MATERIALS AND METHODS: The institutional review board approved the study protocol. Informed consent was obtained from all 69 enrolled patients (30 men, 39 women; mean age, 60.7 years +/- 10.1 [standard deviation]) with 75 nodules. At CT, one end of an 80-mm long, 0.018-inch-diameter fiber-coated microcoil was placed deep to the small peripheral lung nodule, and the other end was coiled in the pleural space. VATS excision of lung tissue, nodules, and the microcoil was performed with fluoroscopic guidance. RESULTS: Seventy-three (97%) 4-24-mm nodules were successfully removed at fluoroscopically guided VATS excision; two nodules could not be removed. CT-guided microcoil placement was successful in all cases; however, two (3%) of 75 coils were displaced at VATS excision. Pneumothorax requiring chest tube placement occurred in two (3%) patients, and asymptomatic hemothorax occurred in one (1%) patient. The microcoil did not impede intraoperative frozen-section histopathologic analysis, which facilitated accurate clinical management in all patients. For 19 (28%) patients, the preoperative treatment plan based on bronchoscopy, needle biopsy, and positron emission tomography findings changed after VATS excision. CONCLUSION: Microcoil localization of small peripheral lung nodules enabled fluoroscopically guided VATS resection of 97% of the nodules, with a low rate of intervention (3%) for procedural complications.

Critical roles for the COOH-terminal NITY and RGT sequences of the integrin beta3 cytoplasmic domain in inside-out and outside-in signaling.

Bidirectional signaling of integrin alphaIIbbeta3 requires the beta3 cytoplasmic domain. To determine the sequence in the beta3 cytoplasmic domain that is critical to integrin signaling, cell lines were established that coexpress the platelet receptor for von Willebrand factor (vWF), glycoprotein Ib-IX, integrin alphaIIb, and mutants of beta3 with truncations at sites COOH terminal to T741, Y747, F754, and Y759. Truncation at Y759 did not affect integrin activation, as indicated by vWF-induced fibrinogen binding, but affected cell spreading and stable adhesion. Thus, the COOH-terminal RGT sequence of beta3 is important for outside-in signaling but not inside-out signaling. In contrast, truncation at F754, Y747, or T741 completely abolished integrin activation. A point mutation replacing Y759 with alanine also abolished integrin activation. Thus, the T755NITY759 sequence of beta3, containing an NXXY motif, is critical to inside-out signaling, whereas the intact COOH terminus is important for outside-in signaling. In addition, we found that the calcium-dependent protease calpain preferentially cleaves at Y759 in a population of beta3 during platelet aggregation and adhesion, suggesting that calpain may selectively regulate integrin outside-in signaling.

The role of integrin alpha D beta2 (CD11d/CD18) in monocyte/macrophage migration.

Integrin alpha(D)beta(2) (CD11d/CD18) is a multiligand macrophage receptor with recognition specificity identical to that of the major myeloid cell-specific integrin alpha(M)beta(2) (CD11b/CD18, Mac-1). Despite its prominent upregulation on inflammatory macrophages, the role of alpha(D)beta(2) in monocyte and macrophage migration is unknown. In this study, we have generated model and natural cell lines expressing different densities of alpha(D)beta(2) and examined their migration to various extracellular matrix proteins. When expressed at a low density, alpha(D)beta(2) on the surface of recombinant HEK293 cells and murine IC-21 macrophages cooperates with beta(1)/beta(3) integrins to support cell migration. However, its increased expression on the alpha(D)beta(2)-expressing HEK293 cells and its upregulation by PMA on the IC-21 macrophages result in increased cell adhesiveness and inhibition of cell migration. Furthermore, ligation of alpha(D)beta(2) with anti-alpha(D) blocking antibodies restores beta(1)/beta(3)-driven cell migration by removing the excess alpha(D)beta(2)-mediated adhesive bonds. Consistent with in vitro data, increased numbers of inflammatory macrophages were recovered from the inflamed peritoneum of mice after the administration of anti-alpha(D) antibody. These results demonstrate that the density of alpha(D)beta(2) is critically involved in modulating macrophage adhesiveness and their migration, and suggest that low levels of alpha(D)beta(2) contribute to monocyte migration while alpha(D)beta(2) upregulation on differentiated macrophages may facilitate their retention at sites of inflammation.

A Randomized Controlled Study of Integrated Smoking Cessation in a Lung Cancer Screening Program.

INTRODUCTION: Smoking cessation activities incorporated into lung cancer screening programs have been broadly recommended, but studies to date have not shown increased quit rates associated with cessation programs in this setting. We aimed to determine the effectiveness of smoking cessation counseling in smokers presenting for lung cancer screening. METHODS: This study is a randomized control trial of an intensive telephone-based smoking cessation counseling intervention incorporating lung cancer screening results versus usual care (information pamphlet). All active smokers enrolled in the Alberta Lung Cancer Screening Study cohort were randomized on a 1:1 ratio with a primary endpoint of self-reported 30-day abstinence at 12 months. RESULTS: A total of 345 active smokers participating in the screening study were randomized to active smoking cessation counseling (n = 171) or control arm (n = 174). Thirty-day smoking abstinence at 12 months post-randomization was noted in 22 of 174 (12.6%) and 24 of 171 (14.0%) of participants in the control and intervention arms, respectively, a 1.4% difference (95% confidence interval: -5.9 to 8.7, p = 0.7). No statistically significant differences in 7-day or point abstinence were noted, nor were differences at 6 months or 24 months. CONCLUSIONS: A telephone-based smoking cessation counseling intervention incorporating lung cancer screening results did not result in increased 12-month cessation rates versus written information alone in unselected smokers undergoing lung cancer screening. Routine referral of all current smokers to counseling-based cessation programs may not improve long-term cessation in this patient cohort. Future studies should specifically focus on this subgroup of older long-term smokers to determine the optimal method of integrating smoking cessation with lung cancer screening (clinicaltrials.govNCT02431962).

Enhancing gene editing specificity by attenuating DNA cleavage kinetics.

Engineered nucleases have gained broad appeal for their ability to mediate highly efficient genome editing. However the specificity of these reagents remains a concern, especially for therapeutic applications, given the potential mutagenic consequences of off-target cleavage. Here we have developed an approach for improving the specificity of zinc finger nucleases (ZFNs) that engineers the FokI catalytic domain with the aim of slowing cleavage, which should selectively reduce activity at low-affinity off-target sites. For three ZFN pairs, we engineered single-residue substitutions in the FokI domain that preserved full on-target activity but showed a reduction in off-target indels of up to 3,000-fold. By combining this approach with substitutions that reduced the affinity of zinc fingers, we developed ZFNs specific for the TRAC locus that mediated 98% knockout in T cells with no detectable off-target activity at an assay background of ~0.01%. We anticipate that this approach, and the FokI variants we report, will enable routine generation of nucleases for gene editing with no detectable off-target activity.

Diversifying the structure of zinc finger nucleases for high-precision genome editing.

Genome editing for therapeutic applications often requires cleavage within a narrow sequence window. Here, to enable such high-precision targeting with zinc-finger nucleases (ZFNs), we have developed an expanded set of architectures that collectively increase the configurational options available for design by a factor of 64. These new architectures feature the functional attachment of the FokI cleavage domain to the amino terminus of one or both zinc-finger proteins (ZFPs) in the ZFN dimer, as well as the option to skip bases between the target triplets of otherwise adjacent fingers in each zinc-finger array. Using our new architectures, we demonstrate targeting of an arbitrarily chosen 28 bp genomic locus at a density that approaches 1.0 (i.e., efficient ZFNs available for targeting almost every base step). We show that these new architectures may be used for targeting three loci of therapeutic significance with a high degree of precision, efficiency, and specificity.

Application of Lung-Screening Reporting and Data System Versus Pan-Canadian Early Detection of Lung Cancer Nodule Risk Calculation in the Alberta Lung Cancer Screening Study.

BACKGROUND: False-positive scans and resultant needless early recalls can increase harms and reduce cost-effectiveness of low-dose CT (LDCT) lung cancer screening. How LDCT scans are interpreted and classified may impact these metrics. METHODS: The Pan-Canadian Early Detection of Lung Cancer risk calculator was used to determine nodule risk of malignancy on baseline screening LDCTs in the Alberta Lung Cancer Screening Study, which were then classified according to Nodule Risk Classification (NRC) categories and ACR Lung Screening Reporting and Data System (Lung-RADS). Test performance characteristics and early recall rates were compared for each approach. RESULTS: In all, 775 baseline screens were analyzed. After a mean of 763 days (±203) of follow-up, lung cancer was detected in 22 participants (2.8%). No statistically significant differences in sensitivity, specificity, or area under the receiver operator characteristic curve occurred between the NRC and Lung-RADS nodule management approaches. Early recall rates were 9.2% and 9.3% for NRC and Lung-RADS, with the NRC unnecessarily recalling some ground glass nodules, and the Lung-RADS recalling many smaller solid nodules with low risk of malignancy. CONCLUSION: Performances of both the NRC and Lung-RADS in this cohort were very good with a trend to higher sensitivity for the NRC. Early recall rates were less than 10% with each approach, significantly lower than rates using the National Lung Screening Trial cutoffs. Further reductions in early recall rates without compromising sensitivity could be achieved by increasing the NRC threshold to 20% for ground glass nodules or by applying the nodule risk calculator with a 5% threshold to 6- to 10-mm solid nodules under Lung-RADS.

Health-related quality of life and anxiety in the PAN-CAN lung cancer screening cohort.

OBJECTIVES: The impact of lung cancer screening with low-dose chest CT (LDCT) on participants' anxiety levels and health-related quality of life (HRQoL) is an important consideration in the implementation of such programmes. We aimed to describe changes in anxiety and HRQoL in a high-risk Canadian cohort undergoing LDCT lung cancer screening. METHODS: 2537 subjects who had 2% or greater lung cancer risk over 6 years using a risk prediction tool were recruited from eight centres across Canada in the Pan-Canadian Early Detection of Lung Cancer Study (2008-2010). We compared HRQoL and anxiety levels before and after screening of 1237 participants with LDCT (excluding a subset of 1300 participants who also underwent autofluorescence bronchoscopy screening), as well as after investigations performed because of a positive screening examination. The 12-item short-form Physical and Mental Component Scales (SF-12), EQ-5D-3L scores and State Trait Anxiety Inventory-State anxiety were used at each assessment. RESULTS: Overall, there were no clinically significant differences in HRQoL outcomes between baseline and each of the survey time points following initial screening. No mean change in anxiety in the overall cohort was noted following baseline LDCT, but more participants had clinically significant increase in anxiety versus decrease after baseline screening (increase >minimal clinically important difference (MCID) (n=180) vs decrease >MCID (n=50), p<0.001). This finding persisted but to a lesser degree at the 12 month time point (increase >MCID (n=146) vs decrease >MCID (n=87), p<0.001). CONCLUSIONS: CT screening for lung cancer has no major overall impact on HRQoL among participants, although a minority of participants (number-needed-to-harm=7 after baseline screening and 18 at 1 year) demonstrated clinically significant increased anxiety levels. TRIALREGISTRATION NUMBER: NCT00751660; Results.

Tobacco use and motivation to stop smoking among long-term smokers who are ineligible for lung cancer screening.

BACKGROUND: The importance of smoking cessation interventions in lung cancer screening participants has been highlighted. This study aimed to describe the smoking habits of individuals who were ineligible for lung cancer screening and to investigate whether this encounter may represent an opportunity to reduce tobacco use. METHODS: Ever smokers between the ages of 55 and 80 and ≥1.5% lung cancer risk over 6 years or having smoked ≥30 pack-years and with no more than 15 years of smoking abstinence were eligible to participate in the Alberta Lung Cancer Screening Program (ALCSP). A baseline questionnaire exploring tobacco use was administered to all interested individuals as part of the eligibility determination for the program. RESULTS: Among 504 individuals, 254 (50.4%) met the criteria for the ALCSP and 250 (49.6%) were non-eligible for screening. Non-eligible individuals were slightly younger (mean=60.2 vs. 63.1 years, p-value <0.001), and less likely to be current smokers (26.0% vs. 48.8%, p-value <0.001). Non-eligible smokers had a lower degree of addiction compared to eligible group, as measured by the Fagerström Test of Nicotine Dependence (Median=4.0 vs 6.0, p-value=0.001), but still in the "moderately dependent" range for this test. There were no significant differences in motivation to quit (98.5% vs. 97.6%, p-value=0.689), or motivation to receive help with their quit attempt (89.2% vs. 90.3%, p-value=0.813) between these two groups. Only 7.7% of non-eligible and 2.4% of eligible current smokers were currently in a smoking cessation program. CONCLUSION: A significant proportion of individuals applying to, but not qualifying for a lung cancer screening program are active smokers with significant nicotine dependence. Very few are currently participating in active smoking cessation programs but almost all are interested in quitting and in receiving help with quit attempts. Future studies need to investigate the most effective approaches for smoking cessation in this substantial group of older, long-term smokers, capitalizing on their motivation to receive cessation assistance.

TA205, an anti-talin monoclonal antibody, inhibits integrin-talin interaction.

Talin mediates integrin signaling by binding to integrin cytoplasmic tails through its FERM domain which consists of F1, F2 and F3 subdomains. TA205, an anti-talin monoclonal antibody, disrupts actin stress fibers and focal adhesion when microinjected into fibroblasts. Here, we showed that TA205 caused an allosteric inhibition of integrin alphaIIb beta3 binding to the talin FERM domain and mapped the TA205 epitope to residues 131-150 in talin F1. Furthermore, binding of a talin rod fragment to talin head was partially inhibited by TA205. These findings suggest that talin F1 may be important in regulation of integrin binding and talin head-rod interaction.

Low Prevalence of High-Grade Lesions Detected With Autofluorescence Bronchoscopy in the Setting of Lung Cancer Screening in the Pan-Canadian Lung Cancer Screening Study.

BACKGROUND: Lung cancer screening with low-dose CT (LDCT) scan has been demonstrated to reduce lung cancer mortality. Preliminary reports suggested that up to 20% of lung cancers may be CT scan occult but detectable by autofluorescence bronchoscopy (AFB). We evaluated the prevalence of CT scan occult, invasive, and high-grade preinvasive lesions in high-risk participants undergoing screening for lung cancer. METHODS: The first 1,300 participants from seven centers in the Pan-Canadian Early Detection of Lung Cancer Study who had ≥ 2% lung cancer risk over 5 years were invited to have an AFB in addition to a LDCT scan. We determined the prevalence of CT scan and AFB abnormalities and analyzed the association between selected predictor variables and preinvasive lesions plus invasive cancer. RESULTS: A total of 776 endobronchial biopsies were performed in 333 of 1,300 (25.6%) participants. Dysplastic or higher-grade lesions were detected in 5.3% of the participants (n = 68; mild dysplasia: n = 36, moderate dysplasia: n = 25, severe dysplasia: n = 3, carcinoma in situ [CIS]: n = 1, and carcinoma: n = 4). Only one typical carcinoid tumor and one CIS lesion were detected by AFB alone, for a rate of CT scan occult cancer of 0.15% (95% CI, 0.0%-0.6%). Fifty-six prevalence lung cancers were detected by LDCT scan (4.3%). The only independent risk factors for finding of dysplasia or CIS on AFB were smoking duration (OR, 1.05; 95% CI, 1.02-1.07) and FEV1 percent predicted (OR, 0.99; 95% CI, 0.98-0.99). CONCLUSIONS: The addition of AFB to LDCT scan in a high lung cancer risk cohort detected too few CT occult cancers (0.15%) to justify its incorporation into a lung cancer screening program. TRIAL REGISTRY: ClinicalTrials.gov; No.: NCT00751660; URL: www.clinicaltrials.gov.

The angiogenic factor cysteine-rich 61 (CYR61, CCN1) supports vascular smooth muscle cell adhesion and stimulates chemotaxis through integrin alpha(6)beta(1) and cell surface heparan sulfate proteoglycans.

Cysteine-rich 61 (CYR61, CCN1) is a heparin-binding, extracellular, matrix-associated protein of the cysteine-rich 61/nephroblastoma family, which also includes connective tissue growth factor, nephroblastoma overexpressed, Wnt-induced secreted protein-1 (WISP-1), WISP-2, and WISP-3. CYR61 induces angiogenesis in vivo and supports cell adhesion, promotes cell migration, and enhances growth factor-stimulated mitogenesis in fibroblasts and endothelial cells. Although the expression of CYR61 has been observed in arterial walls, its function in vascular smooth muscle cells (VSMCs) has not been examined to date. Here we show that purified CYR61 supports VSMC adhesion in a dose-dependent, saturable manner through integrin alpha(6)beta(1) with an absolute requirement of cell surface heparan sulfate proteoglycans. In addition, CYR61 induces VSMC chemotaxis, but not chemokinesis, through integrin alpha(6)beta(1) and heparan sulfate proteoglycans. Heparin-binding defective CYR61 mutants are unable to support VSMC adhesion but can still induce chemotaxis at a reduced level. Following balloon angioplasty in rat carotid artery, CYR61 protein level is elevated in the media and neointima of the injured vessel by d 4 post angioplasty, peaks from d 7 to 14, and remains high for at least 28 d. These data demonstrate the activities of CYR61 in VSMCs, identify the receptors that mediate its functions, and show that CYR61 is synthesized in arterial smooth muscle walls during proliferative restenosis. Together, these results implicate CYR61 as a novel factor that modulates the responses of VSMCs to vascular injury.

Incremental value of pulmonary function and sputum DNA image cytometry in lung cancer risk prediction.

Lung cancer is the leading cause of cancer death worldwide. Accurate prediction of lung cancer risk is of value for individuals, clinicians, and researchers. The aims of this study were to characterize the associations between pulmonary function and sputum DNA image cytometry (SDIC) and lung cancer, and their contributions to risk prediction. During 1990 to 2007, 2,596 high-risk individuals were enrolled and followed prospectively for development of lung cancer (n = 139; median follow-up 7.7 years) in trials at the British Columbia Cancer Agency. At baseline, an epidemiologic questionnaire was administered, sputum was collected for aneuploidy measurement and spirometry was obtained. Multivariable logistic models were prepared including known lung cancer predictors (model 1), that additionally included percent-expected-forced expiratory volume in 1 second [forced expiratory volume in 1 second (FEV(1)%), model 2], and that additionally included SDIC (model 3). Prediction was assessed by evaluating discrimination (receiver operator characteristic area under the curve (ROC AUC)) and calibration. Net reclassification indices (NRI) were calculated with cutoff points for 8-year risks identifying low, intermediate, and high risk at 1.5% and 3%. Lung cancer risk increased with decline in FEV(1)%, but did so more for men than for women (interaction P < 0.001). SDIC demonstrated a dose-response with lung cancer (P = 0.022). The ROC AUCs for models 1, 2, and 3 were 0.718 (95% CI: 0.671-0.765), 0.767 (95% CI: 0.725-0.809), and 0.773 (95% CI: 0.732-0.815), respectively. Model 2 versus 1 had a NRI of 12.6% (P < 0.0001) and model 3 versus 2 had a NRI of 3.1% (P = 0.059). Spirometry and SDIC data substantially and minimally improved lung cancer prediction, respectively.