Meta-Analysis of Rates and Risk Factors for Local Recurrence in Surgically Resected Patients With NSCLC and Differences Between Asian and Non-Asian Populations.

Introduction: Postoperative radiotherapy (PORT) reduces local failure in patients with NSCLC, without a clear overall survival benefit. It is unknown whether the subsets of patients benefit. Two recent large randomized controlled trials, PORT-C (People's Republic of China) and Lung ART (Europe), reported widely different locoregional recurrence (LR) rates in the control arms, at 18.3% and 28.1% (46% of which were mediastinal recurrences), respectively. We performed a meta-analysis of patients with pathologic (p) N0 to N2 disease to evaluate the risk factors for LR and to explore possible differences in recurrence risk between Asian population (AP) and non-Asian population (NAP). Methods: We identified all original studies of curative NSCLC surgical resection which reported risk of LR between January 1, 2000, and January 10, 2021, excluding studies with less than 10 LR, patients with metastatic disease, or any neoadjuvant therapy. A total of 87 studies were identified with pN0 to N2 disease; of these, 56 were of high quality (HQ) on the basis of the Newcastle-Ottawa Scale. For each risk factor, we derived pooled relative risk (RR) and 5-year rate estimates using random-effects models. Results: Overall, the three significant highest pooled RRs (95% confidence intervals) for LR were pN2 versus pN0 (3.01, 1.39-6.55), lymphovascular invasion (1.92, 1.58-2.33), and advanced pT3-4 stage versus pT1 (1.86, 1.53-2.25). For HQ studies, the highest RRs for LR were lymphovascular invasion (1.94, 1.57-2.40), sublobar versus lobar resection (1.86, 1.46-2.36), and pN1 versus pN0 (1.84, 1.37-2.47), but pN2 versus pN0 was no longer significant (3.0, 0.57-15.61), on the basis of only two eligible studies. The RRs for LR were consistent for most factors in AP and NAP, although the RR for male versus female sex was higher in AP (1.44, 1.21-1.72) than in NAP (1.09, 0.99-1.19). Where reported, the pooled rate of LR at 5 years was lower in AP (12.0%) than in NAP (22.7%), despite similar overall 5-year recurrence rates (both LR and distal) in both populations: 38.0% in AP and 37.3% in NAP. Nevertheless, a lower 5-year mortality rate was noted in AP (24.3%) than in NAP (45.9%). Conclusions: There is little high-quality evidence to support the hypothesis that pN2 disease is a risk factor for LR, but LR seems to be lower in Asians. Prospective evaluation of LR factors and rates may be necessary before further prospective evaluation of PORT, because it may not depend on nodal status alone. Recurrence rates may differ in Asians. The impact of mutational status and modern treatment including targeted therapies and immune checkpoint inhibitors is inadequately studied.

The role of well-defined patterned substrata on the regeneration of DRG neuron pathfinding and integrin expression dynamics using chondroitin sulfate proteoglycans.

Injured neurons intrinsically adapt to and partially overcome inhibitory proteoglycan expression in the central nervous system by upregulating integrin expression. It remains unclear however, to what extent varying proteoglycan concentrations influence the strength of this response, how rapidly neurons adapt to proteoglycans, and how pathfinding dynamics are altered over time as integrin expression is modulated in response to proteoglycan signals. To investigate these quandaries, we created well-defined substrata in which postnatal DRG neuron pathfinding dynamics and growth cone integrin expression were interrogated as a function of proteoglycan substrata density. DRGs responded by upregulating integrin expression in a proteoglycan dose dependent fashion and exhibited robust outgrowth over all proteoglycan densities at initial time frames. However, after prolonged proteoglycan exposure, neurons exhibited decreasing velocities associated with increasing proteoglycan densities, while neurons growing on low proteoglycan levels exhibited robust outgrowth at all time points. Additionally, DRG outgrowth over proteoglycan density step boundaries, and a brief ß1 integrin functional block proved that regeneration was integrin dependent and that DRGs exhibit delayed slowing and loss in persistence after even transient encounters with dense proteoglycan boundaries. These findings demonstrate the complexity of proteoglycan regulation on integrin expression and regenerative pathfinding.

The influence of sub-micron inhibitory clusters on growth cone substratum attachments and CD44 expression.

Proteoglycan expression patterns in the central nervous system guide neuronal pathfinding during development, but also disrupt regeneration after injuries. To deepen our understanding of the molecular level effects of proteoglycan spatial arrangements on neuronal pathfinding, we designed micropatterning stamps for the precise placement of near single molecule chondroitin sulfate proteoglycan (CSPG) clusters into regularly spaced arrays. Actin ultrastructural analysis in dorsal root ganglion neurons grown on laminin-coated substrata patterned with aggrecan cluster arrays revealed filopodial and lamellapodial edge contact avoidance of individual clusters, while growth cone lamellapodia and central domains were able to span multiple clusters over a range of cluster densities. Total internal reflection fluorescence microscopy interrogation of growth cone substratum morphology further revealed persistence of integrin mediated substratum adhesion and local out-of-plane membrane bending over clusters on the height scale of aggrecan glycosaminoglycan side chains. Direct imaging of cell adhesion molecule CD44 expression in growth cones revealed an aggrecan dose dependent upregulation in CD44 molecules. Evidence of CD44 clustering coinciding with underlying aggrecan molecules implies CSPG-CD44 interactions. The results reveal the limited local repulsive effect of CSPGs on neuronal structures and provide evidence that CD44 upregulation in neurons is affected by local CSPG expression.

Relating material surface heterogeneity to protein adsorption: the effect of annealing of micro-contact-printed OTS patterns.

We have investigated the influence of micrometer- and sub-micrometer-scale surface heterogeneities in patterned octadecyltrichlorosilane (OTS) films on human serum albumin (HSA) adsorption and its spatial distribution. 5-µm-wide OTS patterns were created on glass substrates by micro-contact printing and in some instances subsequent annealing was used to alter OTS molecule distribution within the patterns. Scanning force microscopy (SFM), advancing water contact angles and water vapor condensation figures were used to characterize the OTS films and to assess the nature of the heterogeneities within the various surface areas. High-resolution fluorescence microscopy was used to record images of fluorescently labeled albumin on OTS patterned films and fluorescence intensity was quantified and converted into the adsorbed amount. Adsorbed albumin was also characterized through SFM measurements. Combined SFM topography and lateral force microscopy (LFM) imaging revealed that micro-contact printing of OTS onto glass both replicated the stamp pattern and created small islands within the non-stamped regions between the patterns. The OTS coverage within stamped regions was not fully continuous but improved with subsequent annealing. Annealing also resulted in OTS island growth within the non-stamped regions and decreased average wettability on both the stamped and non-stamped areas. The extent of albumin adsorption was not proportional to OTS coverage, but correlated with the sub-µm distribution of OTS chains. We inferred that the surface distribution of ligands such as OTS on a sub-µm length scale determines the nature of albumin adsorption and its kinetics.

The differential influence of colocalized and segregated dual protein signals on neurite outgrowth on surfaces.

We present an in vitro micropatterning approach in which the density and spatial presentation of two separate protein layers can be independently controlled to form cell stripe assays through (1) the simultaneous application of microcontact printing (microCP) and microfluidic network (microFN) patterning to generate alternating stripes of pure single protein layers or (2) through microCP onto a pre-adsorbed homogeneous protein layer to generate alternating single and dual protein stripes. This approach enabled the creation of choice boundaries in which protein-protein interactions were limited and the effects of spatially segregated or colocalized dual protein signals on model primary neuronal behavior could be readily interrogated and compared on both glass and tissue culture polystyrene substrates. Dorsal root ganglion (DRG) cell body attachment was dictated largely by non-specific cell adhesion interactions and interactions between the guidance molecules laminin and aggrecan were insufficient to explain aggrecan inhibition on neurite outgrowth. The presentation of a specific laminin epitope stabilized by interactions with aggrecan and destabilized by microCP was a strong predictor of neurite promoting activity. These observations provide evidence that aggrecan is intrinsically inhibitory and that laminin-aggrecan interactions do not diminish laminin growth promoting properties.

How Surface Heterogeneity Affects Protein Adsorption: Annealing of OTS Patterns and Albumin Adsorption Kinetics.

Fluorescence microscopy and intensity histogram analysis techniques were used to monitor spatially-resolved albumin adsorption kinetics to model heterogeneous surfaces on sub-µm scales. Several distinct protein subpopulations were resolved, each represented by a normal distribution of adsorption densities on the adsorbent surface. Histogram analyses provided dynamic information of mean adsorption density, spread in adsorption density, and surface area coverage for each distinct protein subpopulation. A simple adsorption model is proposed in which individual protein binding events are predicted by the summation of multiple protein's surface sub-site interactions with different binding energy sub-sites on adsorbent surfaces. This model is predictive of the albumin adsorption on the patterns produced by one step µ-contact printing (µCP) of octadecyltrichlorosilane (OTS) on glass but fails to describe adsorption once the same patterns are altered by a thermal annealing step.