Maria Dolores Lozano

Purpose: Lung adenocarcinomas (LUADs) harboring concurrent mutations in KRAS and STK11 define a clinically aggressive subgroup characterized by primary resistance to immune checkpoint inhibitors (ICIs). In contrast, KRAS/TP53 co-mutant tumors typically exhibit an inflamed tumor microenvironment (TME) and improved responsiveness to immunotherapy. The mechanisms underlying immune evasion in KRAS/STK11-mutant LUADs remain incompletely understood. Experimental Design: We assembled a multicenter cohort of human LUAD specimens and performed integrative genomic and immunophenotypic analyses to dissect the TME of KRAS/STK11 (KS) and KRAS/TP53 (KP) co-mutant tumors. Results: We identify neutrophil extracellular trap (NET) formation as a defining feature of KS tumors, associated with neutrophil enrichment and reduced CD8⁺ T-cell infiltration. Using immunocompetent Kras-mutant murine models, we demonstrate that Stk11 loss promotes NETosis through activation of the complement C5a-C5aR1 axis, resulting in an immunosuppressive TME and resistance to PD-1/PD-L1 blockade. Pharmacologic inhibition of C5a signaling suppresses NETosis, reduces tumor growth, and restores T-cell infiltration in KS tumors. Conclusions: Our findings uncover NETosis as a key mechanism of immune escape in KRAS/STK11-mutant LUADs and identify the C5a-C5aR1 pathway as a therapeutically actionable vulnerability to overcome immunotherapy resistance in this patient population. These findings provide a rationale for biomarker-driven clinical strategies targeting the complement pathway in patients with STK11-mutant lung adenocarcinoma.

Background: Immunohistochemistry (IHC) is essential for tumor subtyping and predictive biomarker analysis in non–small cell lung carcinoma (NSCLC). When only cytological material is available and no concurrent biopsy or cell block exists, immunocytochemistry (ICC) may need to be performed on slides prepared using diverse fixation protocols, introducing substantial pre‐analytical variability. Methods: Seven laboratories representing commonly used cytology fixation protocols were selected based on an exploratory global survey of 63 institutions. Two central laboratories prepared direct smears, cytospins and provided cells in PreservCyt from specimens with confirmed marker expression using the selected fixation protocols. Materials were distributed for immunocytochemical demonstration of TTF‐1, p40, ALK , and ROS1 according to local protocols. Digitized slides were independently evaluated using standardized four‐tier scoring criteria. Result: A total of 105 stained slides from 17 study runs were assessed. Delaunay‐fixed, Papanicolaou‐stained, and methanol‐fixed polyethylene glycol‐coated preparations demonstrated consistently superior ICC performance compared with other evaluated fixation protocols. In contrast, high rates of suboptimal staining were observed with PreservCyt fixation (81.8%) and air‐dried preparations (58.8%). Conclusions: This study demonstrates that fixation protocol significantly impacts ICC performance on non–cell block cytological preparations. The observed fixation‐dependent differences underscore the need for harmonized pre‐analytical practices, robust local validation, and external quality assessment.

Spatial transcriptomics is increasingly used to infer cellular phenotypes, molecular interactions, and functional tissue organization from RNA, although most of these biological processes are ultimately mediated by proteins. Here, we directly tested the interchangeability of spatial RNA and protein measurements using paired Xenium profiling of 21 matched targets across ∼1.8 million cells from primary lung adenocarcinoma and metastatic castration-resistant prostate cancer. After accounting for marker- and dataset-specific protein background, we quantified concordance at multiple levels, including single-cell detection, expression magnitude, cell-type distribution, patient-level abundance, and spatial organization. Agreement at single-cell level was strongly marker-dependent. Canonical lineage markers generally showed concordant RNA and protein patterns, whereas several molecules, including in our study PD-1, PD-L1, and LAG-3, showed little concordance across modalities. Importantly, spatial organization remained substantially more discordant between modalities than overall abundance, indicating that spatial biomarkers are not directly transferable across modalities. Together, our results show that spatial RNA and protein measurements provide largely overlapping information for lineage markers and overall abundance, but can diverse for specific molecules and for higher-order spatial features, where the two layers provide complementary rather than equivalent readouts. These findings provide practical guidance on when spatial transcriptomic readouts can be interpreted as proxies for protein and when orthogonal protein validation is advisable.

Objective To identify key aspects of the lung cancer care process and generate prioritised recommendations to optimise coordination, quality, and equitable access to diagnosis and treatment. Methods 24 experts from the National Advisory Committee of the Lung Ambition Alliance in Spain participated in a structured consensus process using an adapted RAND/UCLA method. The process comprised three phases: literature review (Phase I), identification of key issues (Phase II, n = 21), and prioritisation of recommendations (Phase III, n = 24). Each recommendation was scored for impact and feasibility on a 1–9 Likert scale. Agreement was defined when ≥ 66.6% of experts rated within the same range as the median; disagreement when ≥ 33.3% scored in both the 1–3 and 7–9 ranges; other cases were considered indeterminate. Results A total of 76 recommendations addressing 34 key aspects were identified (66% related to humanisation and perceived care quality and 34% to coordination and continuity). From which, 62 achieved agreement for impact and 14 were indeterminate; regarding feasibility, 10 reached agreement, 2 met disagreement criteria and 64 were indeterminate. In this regard, 17 high-priority recommendations (high impact and feasibility) were prioritised, covering prevention, access to diagnosis and treatment, standardisation of care processes and communication with patients. In addition, 29 recommendations were classified as high impact, relating to information systems, care coordination, prevention and awareness, equity in access to diagnostic tests and treatment, and patient care and safety. Conclusions Implementing these recommendations is essential to enhance equity, coordination, and quality of lung cancer care, requiring a nationwide, multi-stakeholder effort to advance towards an integrated and patient-centred model.

Objective: Formalin‐fixed, paraffin‐embedded (FFPE) cell blocks (CBs) are widely used for processing cytology specimens, but preparation methods remain variable and non‐standardised. Low cellularity is a common limitation that may compromise diagnostic utility. This study aimed to evaluate the cellularity of routinely prepared CBs across different preparation methods, laboratories and sample types. Methods: Each laboratory in this multicentre observational study retrospectively assessed 50 consecutive CBs prepared using a single method. Cellularity of Haematoxylin and Eosin (H&E)‐stained sections was semi‐quantitatively evaluated by two independent reviewers per laboratory in four categories: acellular, low (≤ 100 cells), medium (100–500 cells) and high cellular (> 500 cells). The proportion of poorly cellular CBs (acellular + low cellular) was compared across methods, laboratories and sample types using non‐parametric tests. Results: Cellularity was assessed for 1817 CBs prepared using Agar (22%), HistoGel (19%), Plasma‐Thrombin (19%), Cellient (17%), In‐house (15%) and Shandon/Epredia (8%) methods. The proportion of poorly cellular CBs ranged from 4% to 80% across laboratories (mean 27%, median 25%), with no consistent clustering by method. Median proportions by method ranged from 12% (Cellient) to 35% (Agar), but inter‐method differences were not statistically significant ( p > 0.05). Substantial variability was observed both across and within sample types and preparation methods. Conclusions: The study revealed marked variability in the proportion of poorly cellular CBs among preparation methods and laboratories, highlighting the need for improved processing and monitoring of CB adequacy.

Introduction: Low-dose computed tomography (LDCT) lung cancer screening has significantly enhanced early detection and patient survival rates in the population at risk. Current screening methods, that primarily rely on LDCT imaging, will very likely benefit from molecular biomarkers to achieve a more comprehensive, accurate, personalized and non-invasive risk assessment leveraging multimodal tools. We present a novel open access multimodal (imaging, proteomics and demographic) dataset designed to provide an available research resource on LDCT-based early lung cancer detection. The dataset includes annotated screening LDCT scans and plasma proteomics generated by proximity extension assay (Olink) platform. Methods: The dataset integrates data from control screened individuals without nodules or with benign nodules, and LDCT-diagnosed lung cancer individuals, matched by sex, age and time between image and sample collection. Both radiological and molecular signatures were collected within a six month window, providing detailed insights into disease progression. Nodules were considered as lung cancer cases if biopsy-confirmed lung cancer was diagnosed within 5 years after imaging, enabling the study of longitudinal biomarker evolution and its correlation with imaging findings. To complement the dataset, clinical and demographic data are also available in open access, providing a detailed overview of patient characteristics. The informed consent signed by the participants allows for unrestricted open access for requests directy or indirectly related to lung cancer research. Results: The dataset consists of annotated screening LDCT scans and plasma proteomics data measured with most of the Olink Target 96 platforms (1078 individual proteins across 12 panels focused on a specific area of disease or biology) for a total of 211 screening participants. There are 67 lung cancer patients, 68 matched controls with benign pulmonary nodules, 71 matched controls without nodules and 5 surgically excised false positive lesions. Experiments were performed to assess the technical quality and provide a proof-of-concept of usability of the dataset, showing the alignment with findings from previous published studies. Conclusion: This comprehensive dataset aims to facilitate research towards the development of personalized multimodal artificial intelligence models. We also aim to support the investigation of the relationship between imaging and molecular data, paving the way for more accurate understanding of early lung cancer biology. Finally, our open access dataset may help to develop or validate individualized risk prediction models that could significantly advance early lung cancer detection and intervention strategies.

NUT carcinoma (NC) is a rare and highly aggressive type of poorly differentiated carcinoma that originates from undefined anatomical sites, primarily in midline structures of the upper respiratory tract. We report the case of a male patient in his fifth decade of life who is a non‐smoker. He presented with symptoms of dyspnea and cough, and had previously been treated for an atypical infectious process. A PET scan revealed a right pulmonary hilar mass alongside pathological lymphadenopathy. An endobronchial ultrasound‐guided fine needle aspiration (EBUS‐FNA) of the subcarinal lymph node was performed, followed by a cytomorphological evaluation in situ (Rapid On‐Site Evaluation—ROSE) for sample management. The smear showed high hypercellularity with loosely cohesive cells arranged mostly in a single‐cell pattern, exhibiting a poorly differentiated appearance. Immunohistochemical studies on the cell block indicated nuclear positivity for NUT expression, confirming the diagnosis of pulmonary NC. Poorly differentiated high‐grade neoplasms in middle‐aged patients present a significant challenge when the tumor sample is limited. The cytopathologist's role is to optimize the available material for a complete and accurate diagnosis, which is essential for appropriate treatment.

Lung cancer continues to be the leading cause of cancer‐related mortality, with cytologic samples often serving as the primary diagnostic tool. However, differentiating between benign and malignant processes in lung cytology can be difficult because various non‐neoplastic conditions closely resemble malignant lesions, which may lead to misdiagnosis. This review offers nine essential tips to help cytopathologists prevent false‐positive diagnoses of malignancy in non‐neoplastic processes. By examining common non‐neoplastic entities that can mimic lung malignancies, this review emphasizes their key cytomorphologic features and distinguishing characteristics. The authors stress the importance of a thorough clinical and radiologic context, slide background evaluation, and identification of cytomorphologic features for an accurate diagnosis of these mimics. The objective of this review was to enhance diagnostic accuracy and prevent misdiagnosis, thereby improving patient management in lung cancer cytopathology.

The prevalence of pancreatic lesions has increased over the years because of an increase in accessibility to and the quality of cross‐sectional imaging. This commentary describes the common non‐neoplastic and neoplastic pancreatic lesions. The images in this commentary depict classic cross‐sectional images, sonographic findings, and the cytopathologic diagnosis of each lesion. Most common non‐neoplastic lesions include pseudocysts, autoimmune pancreatitis, and chronic pancreatitis. Most common neoplastic lesions include serous cystadenomas, intraductal papillary mucinous neoplasms, mucinous cystic neoplasms, solid pseudopapillary neoplasms, neuroendocrine tumors, pancreatic ductal adenocarcinoma, acinar cell carcinoma, and metastases to the pancreas. The aim of this Cytoimaging Correlation Series is to demonstrate the multidisciplinary involvement in the diagnosis of pancreatic pathology and to highlight main findings in the most common entities found in everyday practice.