Integrated whole-exome and bulk transcriptome sequencing delineates the dynamic evolution from preneoplasia to invasive lung adenocarcinoma featured with ground-glass nodules.

OBJECTIVE: The genomic and molecular ecology involved in the stepwise continuum progression of lung adenocarcinoma (LUAD) from adenocarcinoma in situ (AIS) to minimally invasive adenocarcinoma (MIA) and subsequent invasive adenocarcinoma (IAC) remains unclear and requires further elucidation. We aimed to characterize gene mutations and expression landscapes, and explore the association between differentially expressed genes (DEGs) and significantly mutated genes (SMGs) during the dynamic evolution from AIS to IAC. METHODS: Thirty-five patients with ground-glass nodules (GGNs) lung adenocarcinomas were enrolled. Whole-exome sequencing (WES) and transcriptome sequencing (RNA-Seq) were conducted on all patients, encompassing both tumor samples and corresponding noncancerous tissues. Data obtained from WES and RNA-Seq were subsequently analyzed. RESULTS: The findings from WES delineated that the predominant mutations were observed in EGFR (49%) and ANKRD36C (17%). SMGs, including EGFR and RBM10, were associated with the dynamic evolution from AIS to IAC. Meanwhile, DEGs, including GPR143, CCR9, ADAMTS16, and others were associated with the entire process of invasive LUAD. We found that the signaling pathways related to cell migration and invasion were upregulated, and the signaling pathways of angiogenesis were downregulated across the pathological stages. Furthermore, we found that the messenger RNA (mRNA) levels of FAM83A, MAL2, DEPTOR, and others were significantly correlated with CNVs. Gene set enrichment analysis (GSEA) showed that heme metabolism and cholesterol homeostasis pathways were significantly upregulated in patients with EGFR/RBM10 co-mutations, and these patients may have poorer overall survival than those with EGFR mutations. Based on the six calculation methods for the immune infiltration score, NK/CD8+ T cells decreased, and Treg/B cells increased with the progression of early LUAD. CONCLUSIONS: Our findings offer valuable insights into the unique genomic and molecular features of LUAD, facilitating the identification and advancement of precision medicine strategies targeting the invasive progression of LUAD from AIS to IAC.

Ivermectin induces nonprotective autophagy by downregulating PAK1 and apoptosis in lung adenocarcinoma cells.

INTRODUCTION: LUAD (Lung adenocarcinoma), the most common subtype of lung carcinoma and one of the highest incidences and mortality cancers in the world remains still a substantial treatment challenge. Ivermectin, an avermectin derivative, has been traditionally used as an antiparasitic agent in human and veterinary medicine practice during the last few decades. Though ivermectin has been shown to be effective against a variety of cancers, however, there is few available data reporting the antitumor effects of ivermectin in LUAD. METHODS: The effect of ivermectin on cell viability and proliferative ability of LUAD cells was evaluated using CCK-8 and colony formation assay. Apoptosis rate and autophagy flux were detected using flow cytometry based on PI/Annexin V staining and confocal laser scanning microscope based on LC3-GFP/RFP puncta, respectively. Western blotting experiment was conducted to verify the results of changes in apoptosis and autophagy. LUAD-TCGA and GEO databases were used to analyse the expression and predictive value of PAK1 in LUAD patients. Xenograft model and immumohistochemical staining were used for verification of the inhibitor effect of ivermectin in vivo. RESULTS: Ivermectin treatment strikingly impeded the colony formation, and the viability of the cell, along with cell proliferation, and caused the apoptosis and enhanced autophagy flux in LUAD cells. In addition, ivermectin-induced nonprotective autophagy was confirmed by treating LUAD cells with 3-MA, an autophagy inhibitor. Mechanistically, we found that ivermectin inhibited PAK1 protein expression in LUAD cells and we confirmed that overexpression of PAK1 substantially inhibited ivermectin-induced autophagy in LUAD cells. Based on TCGA and GEO databases, PAK1 was highly expressed in LUAD tissues as compared with normal tissues. Furthermore, LUAD patients with high PAK1 level have poor overall survival. Finally, in vivo experiments revealed that ivermectin efficiently suppressed the cellular growth of LUAD among nude mice. CONCLUSION: This study not only revealed the mechanism of ivermectin inhibited the growth of LUAD but also supported an important theoretical basis for the development of ivermectin during the therapy for LUAD.

Marine reserves promote cycles in fish populations on ecological and evolutionary time scales.

Marine reserves are considered essential for sustainable fisheries, although their effectiveness compared to traditional fisheries management is debated. The effect of marine reserves is mostly studied on short ecological time scales, whereas fisheries-induced evolution is a well-established consequence of harvesting. Using a size-structured population model for an exploited fish population of which individuals spend their early life stages in a nursery habitat, we show that marine reserves will shift the mode of population regulation from low size-selective survival late in life to low, early-life survival due to strong resource competition. This shift promotes the occurrence of rapid ecological cycles driven by density-dependent recruitment as well as much slower evolutionary cycles driven by selection for the optimal body to leave the nursery grounds, especially with larger marine reserves. The evolutionary changes increase harvesting yields in terms of total biomass but cause disproportionately large decreases in yields of larger, adult fish. Our findings highlight the importance of carefully considering the size of marine reserves and the individual life history of fish when managing eco-evolutionary marine systems to ensure both population persistence as well as stable fisheries yields.

Nomogram Predicting the Prognosis of Patients with Surgically Resected Stage IA Non-small Cell Lung Cancer.

The American Joint Committee on Cancer (AJCC) 8th stage system was limited in accuracy for predicting prognosis of stage IA non-small cell lung cancer (NSCLC) patients. This study aimed to establish and validate two nomograms that predict overall survival (OS) and lung cancer-specific survival (LCSS) in surgically resected stage IA NSCLC patients. Postoperative patients with stage IA NSCLC in SEER database between 2004 and 2015 were examined. Survival and clinical information according to the inclusion and exclusion criteria were collected. All patients were randomly divided into the training cohort and validation cohort with a ratio of 7:3. Independent prognosis factors were evaluated using univariate and multivariate Cox regression analyses, and predictive nomogram was established based on these factors. Nomogram performance was measured using the C-index, calibration plots, and DCA. Patients were grouped by quartiles of nomogram scores and survival curves were plotted by Kaplan-Meier analysis. In total, 33,533 patients were included in the study. The nomogram contained 12 prognostic factors in OS and 10 prognostic factors in LCSS. In the validation set, the C-index was 0.652 for predicting OS and 0.651 for predicting LCSS. The calibration curves for the nomogram-predicted probability of OS and LCSS showed good agreement between the actual observation and nomogram prediction. DCA indicated that the clinical value of the nomograms were higher than AJCC 8th stage for predicting OS and LCSS. Nomogram scores related risk stratification revealed statistically significant difference which have better discrimination than AJCC 8th stage. The nomogram can accurately predict OS and LCSS in surgically resected patients with stage IA NSCLC. Supplementary Information: The online version contains supplementary material available at 10.1007/s13193-022-01700-w.

Self-organized mud cracking amplifies the resilience of an iconic "Red Beach" salt marsh.

Self-organized patterning, resulting from the interplay of biological and physical processes, is widespread in nature. Studies have suggested that biologically triggered self-organization can amplify ecosystem resilience. However, if purely physical forms of self-organization play a similar role remains unknown. Desiccation soil cracking is a typical physical form of self-organization in coastal salt marshes and other ecosystems. Here, we show that physically self-organized mud cracking was an important facilitating process for the establishment of seepweeds in a "Red Beach" salt marsh in China. Transient mud cracks can promote plant survivorship by trapping seeds, and enhance germination and growth by increasing water infiltration in the soil, thus facilitating the formation of a persistent salt marsh landscape. Cracks can help the salt marsh withstand more intense droughts, leading to postponed collapse and faster recovery. These are indications of enhanced resilience. Our work highlights that self-organized landscapes sculpted by physical agents can play a critical role in ecosystem dynamics and resilience to climate change.

Phase-separation physics underlies new theory for the resilience of patchy ecosystems.

Spatial self-organization of ecosystems into large-scale (from micron to meters) patterns is an important phenomenon in ecology, enabling organisms to cope with harsh environmental conditions and buffering ecosystem degradation. Scale-dependent feedbacks provide the predominant conceptual framework for self-organized spatial patterns, explaining regular patterns observed in, e.g., arid ecosystems or mussel beds. Here, we highlight an alternative mechanism for self-organized patterns, based on the aggregation of a biotic or abiotic species, such as herbivores, sediment, or nutrients. Using a generalized mathematical model, we demonstrate that ecosystems with aggregation-driven patterns have fundamentally different dynamics and resilience properties than ecosystems with patterns that formed through scale-dependent feedbacks. Building on the physics theory for phase-separation dynamics, we show that patchy ecosystems with aggregation patterns are more vulnerable than systems with patterns formed through scale-dependent feedbacks, especially at small spatial scales. This is because local disturbances can trigger large-scale redistribution of resources, amplifying local degradation. Finally, we show that insights from physics, by providing mechanistic understanding of the initiation of aggregation patterns and their tendency to coarsen, provide a new indicator framework to signal proximity to ecological tipping points and subsequent ecosystem degradation for this class of patchy ecosystems.

A novel enemy of cancer: recent investigations into protozoan anti-tumor properties.

Cancer remains a significant global health issue, despite advances in screening and treatment. While existing tumor treatment protocols such as surgery, chemotherapy, radiotherapy, targeted therapy, and immunotherapy have proven effective in enhancing the prognosis for some patients, these treatments do not benefit all patients. Consequently, certain types of cancer continue to exhibit a relatively low 5-year survival rate. Therefore, the pursuit of novel tumor intervention strategies may help improve the current effectiveness of tumor treatment. Over the past few decades, numerous species of protozoa and their components have exhibited anti-tumor potential via immune and non-immune mechanisms. This discovery introduces a new research direction for the development of new and effective cancer treatments. Through in vitro experiments and studies involving tumor-bearing mice, the anti-tumor ability of Toxoplasma gondii, Plasmodium, Trypanosoma cruzi, and other protozoa have unveiled diverse mechanisms by which protozoa combat cancer, demonstrating encouraging prospects for their application. In this review, we summarize the anti-tumor ability and anti-tumor mechanisms of various protozoa and explore the potential for their clinical development and application.

Long-distance facilitation of coastal ecosystem structure and resilience.

Biotic interactions that hierarchically organize ecosystems by driving ecological and evolutionary processes across spatial scales are ubiquitous in our biosphere. Biotic interactions have been extensively studied at local and global scales, but how long-distance, cross-ecosystem interactions at intermediate landscape scales influence the structure, function, and resilience of ecological systems remains poorly understood. We used remote sensing, modeling, and field data to test the hypothesis that the long-distance impact of an invasive species dramatically affects one of the largest tidal flat ecosystems in East Asia. We found that the invasion of exotic cordgrass Spartina alterniflora can produce long-distance effects on native species up to 10 km away, driving decadal coastal ecosystem transitions. The invasive cordgrass at low elevations facilitated the expansion of the native reed Phragmites australis at high elevations, leading to the massive loss and reduced resilience of the iconic Suaeda salsa "Red Beach" marshes at intermediate elevations, largely as a consequence of reduced soil salinity across the landscape. Our results illustrate the complex role that long-distance interactions can play in shaping landscape structure and ecosystem resilience and in bridging the gap between local and global biotic interactions.

Crack Patterns of Environmental Plastic Fragments.

Secondary microplastics usually come from the breakdown of larger plastics due to weathering and environmental stress cracking of plastic wastes. In the present study, 5013 plastic fragments were collected from coastal beaches, estuary dikes, and lake banks in China. The fragment sizes ranged from 0.2 to 17.1 cm, and the dominant polymers were polypropylene and polyethylene. Cracks were observed on the surfaces of 49-56% of the fragments. Based on the extracted crack images, we proposed a general crack pattern system including four crack types with specific definitions, abbreviations, and symbols. The two-dimensional spectral analysis of the cracks suggests that the first three patterns showed good regularity and supported the rationality of the pattern system. Some crack metrics (e.g., line density) were closely correlated with the carbonyl index and additives (e.g., phthalate esters) of fragments. For crack investigation in field, we proposed a succinct protocol, in which five crack ranks were established to directly characterize the degree of cracking based on the line density values. The system was successfully applied to distinguish the differences in crack features at two representative sites, which indicates that crack pattern is a useful tool to describe the morphological changes of plastic surfaces in the environment.

Postoperative radiotherapy for completely resected thymoma: Differing roles in masaoka stage II and stage III disease.

PURPOSE: The efficacy of radiotherapy for treating thymomas is unclear. The goal of this study was to analyze overall survival (OS) and disease-free survival (DFS) among thymoma patients to determine the impact of postoperative radiotherapy (PORT) on thymoma outcomes. METHODS: Recorded cases of thymoma at Xinqiao Hospital were retrospectively analyzed from 1991 to 2019. Data on stage II and III thymomas were extracted from medical records. This study evaluated OS and DFS and compared outcomes between surgery and surgery-plus-radiation groups. The Kaplan-Meier method and Cox regression analysis were used to compare DFS and OS for these groups. RESULTS: Of the 205 patients included in the current study, 142 (69.3%) presented with stage II disease and 63 (30.7%) presented with stage III disease. The median follow-up was 84.3 months. PORT did not statistically significantly improve OS (P = 0.613) and DFS (P = 0.445) in stage II thymoma patients (compared with surgery alone). However, our subgroup analysis showed a statistically significant difference in DFS in patients with stage III thymoma (P = 0.044). CONCLUSION: Although the routine use of postoperative radiotherapy in patients with thymoma does not appear warranted, patients with stage III thymoma may benefit from adjuvant radiation. These findings, if confirmed, will provide valuable information to guide medical decision-making for thymoma treatment.

Plasmodium Circumsporozoite Protein Enhances the Efficacy of Gefitinib in Lung Adenocarcinoma Cells by Inhibiting Autophagy via Proteasomal Degradation of LC3B.

Background: Almost all lung adenocarcinoma (LUAD) patients with EGFR mutant will develop resistance to EGFR-TKIs, which limit the long-term clinical application of these agents. Accumulating evidence shows one of the main reasons for resistance to EGFR-TKIs is induction of autophagy in tumor cells. Our previous study found that circumsporozoite protein (CSP) in Plasmodium can suppress autophagy in host hepatocytes. However, it is unknown whether CSP-mediated inhibition of autophagy could improve the anti-tumor effect of EGFR-TKIs. Methods: We constructed A549 and H1975 cell lines with stable overexpression of CSP (OE-CSP cells). CCK-8, Lactate Dehydrogenase (LDH), flow cytometry, and colony analysis were performed to observe the effect of CSP overexpression on cell viability, apoptosis rate, and colony formation ratio. The sensitizing effect of CSP on gefitinib was evaluated in vivo using a subcutaneous tumor model in nude mice and immunohistochemical assay. The role of CSP in regulation of autophagy was investigated by laser confocal microscopy assay and western blotting. A transcriptome sequencing assay and real-time polymerase chain reaction were used to determine the levels of mRNA for autophagy-related proteins. Cycloheximide (CHX), MG132, TAK-243, and immunoprecipitation assays were used to detect and confirm proteasomal degradation of LC3B. Results: OE-CSP A549 and H1975 cells were more sensitive to gefitinib, demonstrating significant amounts of apoptosis and decreased viability. In the OE-CSP group, autophagy was significantly inhibited, and there was a decrease in LC3B protein after exposure to gefitinib. Cell viability and colony formed ability were recovered when OE-CSP cells were exposed to rapamycin. In nude mice with xenografts of LUAD cells, inhibition of autophagy by CSP resulted in suppression of cell growth, and more marked apoptosis during exposure to gefitinib. CSP promoted ubiquitin-proteasome degradation of LC3B, leading to inhibition of autophagy in LUAD cells after treatment with gefitinib. When LUAD cells were treated with ubiquitin activating enzyme inhibitor TAK-243, cell viability, apoptosis, and growth were comparable between the OE-CSP group and a control group both in vivo and in vitro. Conclusion: CSP can inhibit gefitinib-induced autophagy via proteasomal degradation of LC3B, which suggests that CSP could be used as an autophagy inhibitor to sensitize EGFR-TKIs.

Utility of whole exome sequencing analysis in differentiating intrapulmonary metastatic multiple ground-glass nodules (GGNs) from multiple primary GGNs.

PURPOSE: Clinical evidence of metastasis with ground-glass nodules (GGNs) has been reported, including pulmonary metastasis and distant metastasis. However, the clonal relationships of multiple GGNs at the genetic level remain unclear. EXPERIMENTAL DESIGN: Sixty tissue specimens were obtained from 19 patients with multiple GGN lung cancer who underwent surgery in 2019. Whole exome sequencing (WES) was performed on tissue samples, and genomic profiling and clone evolution analysis were conducted to investigate the genetic characteristics and clonality of multiple GGNs. RESULTS: A total of 15,435 nonsynonymous mutations were identified by WES, and GGNs with shared nonsynonymous mutations were observed in seven patients. Copy number variant (CNV) analysis showed that GGNs in ten patients had at least one shared arm-level CNV. Mutational spectrum analysis showed that GGNs in three patients had similar six substitution profiles and GGNs in fou patients had similar 96 substitution profiles. According to the clone evolution analysis, we found that GGNs in five patients had shared clonal driver gene mutations. Taken together, we identified that 5 patients may have multiple primary GGNs without any similar genetic features, 2 patients may have intrapulmonary metastatic GGNs with ≥ 3 similar genetic features, and the other 12 patients cannot be determined due to insufficient evidences in our cohort. CONCLUSIONS: Our findings suggest that the intrapulmonary metastasis exist in multiple GGNs, but the number of GGNs was not associated with the probability of metastasis. Application of genomic profiling may prove to be important to precise management of patients with multiple GGNs.

Foraging behaviours lead to spatiotemporal self-similar dynamics in grazing ecosystems.

Biological behaviour-driven self-organized patterns have recently been confirmed to play a key role in ecosystem functioning. Here, we develop a theoretical phase-separation model to describe spatiotemporal self-similar dynamics, which is a consequence of behaviour-driven trophic interactions in short-time scales. Our framework integrates scale-dependent feedback and density-dependent movement into grazing ecosystems. This model derives six types of selective foraging behaviours that trigger pattern formation for top-down grazing ecosystems, and one of which is consistent with existing foraging theories. Self-organized patterns nucleate under moderate grazing intensity and are destroyed by overgrazing, which suggests ecosystem degradation. Theoretical results qualitatively agree with observed grazing ecosystems that display spatial heterogeneities under variable grazing intensity. Our findings potentially provide new insights into self-organized patterns as an indicator of ecosystem transitions under a stressful environment.

REPORT- Clinical outcomes of using second - versus first-Generation EGFR-tkis for the First-Line treatment of advanced NSCLC patients with EGFR mutations: A meta-analysis.

First-generation EGFR-TKIs (gefitinib/erlotinib) and second-generation EGFR-TKI (afatinib) have become the current first-line treatments for EGFR-mutated non-small cell lung cancer (NSCLC), however, the effects of using second-generation EGFR-TKIs compared to those of using first-generation EGFR-TKIs as a first-line treatment for NSCLC patients with EGFR mutations remain unknown. We conducted this meta-analysis based on 4 retrospective and 2 randomized controlled studies published between 2016 and 2018. We surveyed the effectiveness of afatinib/dacomitinib and gefitinib/erlotinib as first-line treatments for stage III-IV EGFR-mutated NSCLC patients. The combined hazard ratio (HR) for the progression free survival (PFS) of second-generation EGFR-TKI group versus that first-generation drug group was 0.64 [95% confidence interval (95% CI) 0.55-0.74; P<0.001], demonstrating a superior PFS in the second-generation group. This outcome coincided with the subgroup analyses comparing the PFS of patients with EGFR exon 19 deletion (HR = 0.68 [95% CI 0.55-0.83; P = 0.0002]) or L858R mutation (HR = 0.64 [95% CI 0.51-0.81; p=0.0002]). Meanwhile, second-generation drugs could to significantly improve the time to progression (TTFs) compared to first-generation drugs (HR = 0.81 [95% CI 0.67-0.89; P = 0.03]). Afatinib and dacomitinib may be the superior first-line treatment for advanced NSCLC patients with EGFR mutations.

An invasive species erodes the performance of coastal wetland protected areas.

The world has increasingly relied on protected areas (PAs) to rescue highly valued ecosystems from human activities, but whether PAs will fare well with bioinvasions remains unknown. By analyzing three decades of seven of the largest coastal PAs in China, including World Natural Heritage and/or Wetlands of International Importance sites, we show that, although PAs are achieving success in rescuing iconic wetlands and critical shorebird habitats from once widespread reclamation, this success is counteracted by escalating plant invasions. Plant invasions were not only more extensive in PAs than non-PA controls but also undermined PA performance by, without human intervention, irreversibly replacing expansive native wetlands (primarily mudflats) and precluding successional formation of new native marshes. Exotic species are invading PAs globally. This study across large spatiotemporal scales highlights that the consequences of bioinvasions for humanity's major conservation tool may be more profound, far reaching, and critical for management than currently recognized.

Ice needles weave patterns of stones in freezing landscapes.

Patterned ground, defined by the segregation of stones in soil according to size, is one of the most strikingly self-organized characteristics of polar and high-alpine landscapes. The presence of such patterns on Mars has been proposed as evidence for the past presence of surface liquid water. Despite their ubiquity, the dearth of quantitative field data on the patterns and their slow dynamics have hindered fundamental understanding of the pattern formation mechanisms. Here, we use laboratory experiments to show that stone transport is strongly dependent on local stone concentration and the height of ice needles, leading effectively to pattern formation driven by needle ice activity. Through numerical simulations, theory, and experiments, we show that the nonlinear amplification of long wavelength instabilities leads to self-similar dynamics that resemble phase separation patterns in binary alloys, characterized by scaling laws and spatial structure formation. Our results illustrate insights to be gained into patterns in landscapes by viewing the pattern formation through the lens of phase separation. Moreover, they may help interpret spatial structures that arise on diverse planetary landscapes, including ground patterns recently examined using the rover Curiosity on Mars.

A Comparison of the "Reduced Losses" and "Increased Production" Models for Mussel Bed Dynamics.

Self-organised regular pattern formation is one of the foremost examples of the development of complexity in ecosystems. Despite the wide array of mechanistic models that have been proposed to understand pattern formation, there is limited general understanding of the feedback processes causing pattern formation in ecosystems, and how these affect ecosystem patterning and functioning. Here we propose a generalised model for pattern formation that integrates two types of within-patch feedback: amplification of growth and reduction of losses. Both of these mechanisms have been proposed as causing pattern formation in mussel beds in intertidal regions, where dense clusters of mussels form, separated by regions of bare sediment. We investigate how a relative change from one feedback to the other affects the stability of uniform steady states and the existence of spatial patterns. We conclude that there are important differences between the patterns generated by the two mechanisms, concerning both biomass distribution in the patterns and the resilience of the ecosystems to disturbances.

Fish Ingest Microplastics Unintentionally.

Microplastics (size of plastic debris <5 mm) occur in various environments worldwide these days and cause detrimental effects on biota. However, the behavioral responses of fish to microplastics in feeding processes are not well understood. In the present study, juveniles from four fish species and two common shapes of microplastics were used to explore fish feeding responses. We found swallowing-feeding fish ingested more pellets than filtering- and sucking-feeding fish. With high-definition and high-speed observational experiments, we found that all species did not actively capture microfibers; instead, they passively sucked in microfibers while breathing. Surprisingly, fish showed a rejective behavior, which was spontaneously coughing up microfibers mixed with mucus. Nevertheless, some of the microfibers were still found in the gastrointestinal tracts and gills of fish, while abundances of ingested microfibers were increased in the presence of food. Our findings reveal a common phenomenon that fish ingest microplastics inadvertently rather than intentionally. We also provide insights into the pathways via which microplastics enter fish and potential strategies to assess future ecological risk and food safety related to microplastics.

A Noninvasive Multianalytical Approach for Lung Cancer Diagnosis of Patients with Pulmonary Nodules.

Addressing the high false-positive rate of conventional low-dose computed tomography (LDCT) for lung cancer diagnosis, the efficacy of incorporating blood-based noninvasive testing for assisting practicing clinician's decision making in diagnosis of pulmonary nodules (PNs) is investigated. In this prospective observative study, next generation sequencing- (NGS-) based cell-free DNA (cfDNA) mutation profiling, NGS-based cfDNA methylation profiling, and blood-based protein cancer biomarker testing are performed for patients with PNs, who are diagnosed as high-risk patients through LDCT and subsequently undergo surgical resections, with tissue sections pathologically examined and classified. Using pathological classification as the gold standard, statistical and machine learning methods are used to select molecular markers associated with tissue's malignant classification based on a 98-patient discovery cohort (28 benign and 70 malignant), and to construct an integrative multianalytical model for tissue malignancy prediction. Predictive models based on individual testing platforms have shown varying levels of performance, while their final integrative model produces an area under the receiver operating characteristic curve (AUC) of 0.85. The model's performance is further confirmed on a 29-patient independent validation cohort (14 benign and 15 malignant, with power > 0.90), reproducing AUC of 0.86, which translates to an overall sensitivity of 80% and specificity of 85.7%.