Protective effects of 10°C preservation on donor lungs with lipopolysaccharide-induced acute lung injury.

OBJECTIVES: Hypothermic lung preservation at 10°C has recently shown to enhance quality of healthy donor lungs during ischemia. This study aims to show generalizability of the 10°C lung preservation using an endotoxin-induced lung injury with specific focus on the benefits of post-transplant lung function and mitochondrial preservation. METHODS: Lipopolysaccharide (3 mg/kg) was injected intratracheally in rats to induce lung injury. Injured lungs were flushed with preservation solution and allocated to 3 groups (n = 6 each): minimum cold storage, 6-hour storage on ice (Ice), and 6-hour storage at 10°C (10C). Left lungs were transplanted and reperfused for 2 hours. After storage, lung tissue was used to evaluate the effects of hypothermic storage on the mitochondrial function: mitochondrial membrane potential was assessed by JC-1 staining; mitochondrial oxygen consumption was assessed using high resolution respirometry. RESULTS: Two hours after reperfusion, the PO2/FiO2 ratio from the graft was significantly higher in the 10C group than in the Ice group (P = 0.015), while the wet-to-dry weight ratio was significantly lower (P = 0.041). Levels of interleukin-8 in lung tissues were significantly lower in the 10C group than in the Ice group (P = 0.004). Mechanistically, we noted higher mitochondrial membrane potential and elevated state III respiration in the 10C group than in the Ice group (P = 0.015 and P = 0.002, respectively), implying higher metabolic activities may be maintained during 10°C preservation. CONCLUSIONS: Favorable metabolism during 10°C preservation prevented ischemia-induced mitochondrial damages in injured lungs, leading to better post-transplant outcomes.

Systematic Reporting of Eosinophils in Transbronchial Biopsies After Lung Transplantation Defines a Distinct Inflammatory Response.

BACKGROUND: Descriptions of eosinophils in transbronchial biopsy (TBBx) pathology reports after lung transplantation (LTx) are associated with poor long-term outcomes. The absence of routine reporting and standardization precludes accurate assessment of this histologic predictor. A systematic reporting scheme for the presence of TBBx eosinophils after LTx was implemented. This report aims to assess this scheme by describing the presence, pattern, and gradation of TBBx eosinophils and clinical associations. METHODS: A prospective cross-sectional study of all TBBx reports was performed including all patients presenting for a surveillance or diagnostic TBBx between January 2020 and June 2023. Each TBBx was systematically reported in a blinded manner. Mixed-effects logistic regression was performed to measure the association between concurrent clinical and histologic features, and the presence of TBBx eosinophils. RESULTS: A total of 410 TBBx reports from 201 patients were systematically reported. In 43.8% recipients, any TBBx eosinophils were detected and in 17.1% recipients, higher-grade eosinophils (≥3 per high power field) were present. Adjusted analysis showed that retransplantation, A- and B-grade cellular rejection, positive bronchoalveolar lavage (BAL) bacterial microbiology, and elevated blood eosinophil count were independently associated with the presence of any TBBx eosinophils. Diagnostic "for-cause" procedures were independently associated with higher quantities of TBBx eosinophils. CONCLUSIONS: Systematic reporting demonstrates that TBBx eosinophils are a distinct inflammatory response associated with rejection, infection, and peripheral eosinophilia. Although these findings require multicenter external validation, standardized reporting for TBBx eosinophils may assist in identifying recipients at risk of poor outcomes and provides a platform for mechanistic research into their role after lung transplantation.

Spectrum of chronic lung allograft dysfunction pathology in human lung transplantation.

BACKGROUND: Long-term survival after lung transplantation (LTx) remains limited by chronic lung allograft dysfunction (CLAD), which includes 2 main phenotypes: bronchiolitis obliterans syndrome (BOS) and restrictive allograft syndrome (RAS), with possible overlap. We aimed to detail and quantify pathological features of these CLAD sub-types. METHODS: Peripheral and central paraffin-embedded explanted lung samples were obtained from 20 consecutive patients undergoing a second LTx for CLAD, from 3 lobes. Thirteen lung samples, collected from non-transplant lobectomies or donor lungs, were used as controls. Blinded semi-quantitative grading was performed to assess airway fibrotic changes, parenchymal and pleural fibrosis, and epithelial and vascular abnormalities. RESULTS: CLAD lung samples had higher scores for all airway- and lung-related parameters compared to controls. There was a notable overlap in histologic scores between BOS and RAS, with a wide range of scores in both conditions. Parenchymal and vascular fibrosis scores were significantly higher in RAS compared to BOS (p = 0.003 for both). We observed a significant positive correlation between the degree of inflammation around each airway, the severity of epithelial changes, and airway fibrosis. Immunofluorescence staining demonstrated a trend toward a lower frequency of club cells in CLAD and a higher frequency of apoptotic club cells in BOS samples (p = 0.01). CONCLUSIONS: CLAD is a spectrum of airway, parenchymal, and pleural fibrosis, as well as epithelial, vascular, and inflammatory pathologic changes, where BOS and RAS overlap significantly. Our semi-quantitative grading score showed a generally high inter-reader reliability and may be useful for future CLAD histologic assessments.

HAVOC: Small-scale histomic mapping of cancer biodiversity across large tissue distances using deep neural networks.

Intratumoral heterogeneity can wreak havoc on current precision medicine strategies because of challenges in sufficient sampling of geographically separated areas of biodiversity distributed across centimeter-scale tumor distances. To address this gap, we developed a deep learning pipeline that leverages histomorphologic fingerprints of tissue to create "Histomic Atlases of Variation Of Cancers" (HAVOC). Using a number of objective molecular readouts, we demonstrate that HAVOC can define regional cancer boundaries with distinct biology. Using larger tumor specimens, we show that HAVOC can map biodiversity even across multiple tissue sections. By guiding profiling of 19 partitions across six high-grade gliomas, HAVOC revealed that distinct differentiation states can often coexist and be regionally distributed within these tumors. Last, to highlight generalizability, we benchmark HAVOC on additional tumor types. Together, we establish HAVOC as a versatile tool to generate small-scale maps of tissue heterogeneity and guide regional deployment of molecular resources to relevant biodiverse niches.

Mediastinal Hemangioma Masquerading as a Simple Cyst.

ABSTRACT: This report presents imaging from a mediastinal mass in a patient with colon cancer. At baseline and surveillance chest computed tomography examinations, it was characterized as a pericardial cyst. However, during chemotherapy, complications arose and this mass was further characterized with a chest MRI. It was then decided to be removed, and histopathology confirmed the diagnosis of a hemangioma.

Association of Circulating Tumor DNA Testing Before Tissue Diagnosis With Time to Treatment Among Patients With Suspected Advanced Lung Cancer: The ACCELERATE Nonrandomized Clinical Trial.

Importance: Liquid biopsy has emerged as a complement to tumor tissue profiling for advanced non-small cell lung cancer (NSCLC). The optimal way to integrate liquid biopsy into the diagnostic algorithm for patients with newly diagnosed advanced NSCLC remains unclear. Objective: To evaluate the use of circulating tumor DNA (ctDNA) genotyping before tissue diagnosis among patients with suspected advanced NSCLC and its association with time to treatment. Design, Setting, and Participants: This single-group nonrandomized clinical trial was conducted among 150 patients at the Princess Margaret Cancer Centre-University Health Network (Toronto, Ontario, Canada) between July 1, 2021, and November 30, 2022. Patients referred for investigation and diagnosis of lung cancer were eligible if they had radiologic evidence of advanced lung cancer prior to a tissue diagnosis. Interventions: Patients underwent plasma ctDNA testing with a next-generation sequencing (NGS) assay before lung cancer diagnosis. Diagnostic biopsy and tissue NGS were performed per standard of care. Main Outcome and Measures: The primary end point was time from referral to treatment initiation among patients with advanced nonsquamous NSCLC using ctDNA testing before diagnosis (ACCELERATE [Accelerating Lung Cancer Diagnosis Through Liquid Biopsy] cohort). This cohort was compared with a reference cohort using standard tissue genotyping after tissue diagnosis. Results: Of the 150 patients (median age at diagnosis, 68 years [range, 33-91 years]; 80 men [53%]) enrolled, 90 (60%) had advanced nonsquamous NSCLC. The median time to treatment was 39 days (IQR, 27-52 days) for the ACCELERATE cohort vs 62 days (IQR, 44-82 days) for the reference cohort (P < .001). Among the ACCELERATE cohort, the median turnaround time from sample collection to genotyping results was 7 days (IQR, 6-9 days) for plasma and 23 days (IQR, 18-28 days) for tissue NGS (P < .001). Of the 90 patients with advanced nonsquamous NSCLC, 21 (23%) started targeted therapy before tissue NGS results were available, and 11 (12%) had actionable alterations identified only through plasma testing. Conclusions and Relevance: This nonrandomized clinical trial found that the use of plasma ctDNA genotyping before tissue diagnosis among patients with suspected advanced NSCLC was associated with accelerated time to treatment compared with a reference cohort undergoing standard tissue testing. Trial Registration: ClinicalTrials.gov Identifier: NCT04863924.

Plasma-first: accelerating lung cancer diagnosis and molecular profiling through liquid biopsy.

Introduction: Molecular profiling of tumor tissue is the gold standard for treatment decision-making in advanced non-small cell lung cancer (NSCLC). Results may be delayed or unavailable due to insufficient tissue, prolonged wait times for biopsy, pathology assessment and testing. We piloted the use of plasma testing in the initial diagnostic workup for patients with suspected advanced lung cancer. Methods: Patients with ⩽15 pack-year smoking history and suspected advanced lung cancer referred to the lung cancer rapid diagnostic program underwent plasma circulating-tumor DNA testing using a DNA-based mutation panel. Tissue testing was performed per standard of care, including comprehensive next-generation sequencing (NGS). The primary endpoint was time from diagnostic program referral to cancer treatment in stage IV NSCLC patients (Cohort A) compared to a contemporary cohort not enrolled in the study (Cohort B) and an historical pre-COVID cohort referred to the program between 2018 and 2019 (Cohort C). Results: From January to June 2021, 20 patients were enrolled in Cohort A; median age was 70.5 years (range 33-87), 70% were female, 55% Caucasian, 85% never smokers, and 75% were diagnosed with NSCLC. Seven had actionable alterations detected in plasma or tissue (4/7 concordant). Fusions, not tested in plasma, were identified by immunohistochemistry for three patients. Mean result turnaround time was 17.8 days for plasma NGS and 23.6 days for tissue (p = 0.10). Mean time from referral to treatment initiation was significantly shorter in cohort A at 32.6 days (SD 13.1) versus 62.2 days (SD 31.2) in cohort B and 61.5 days (SD 29.1) in cohort C, both p < 0.0001. Conclusion: Liquid biopsy in the initial diagnostic workup of patients with suspected advanced NSCLC can lead to faster molecular results and shorten time to treatment even with smaller DNA panels. An expansion study using comprehensive NGS plasma testing with faster turnaround time is ongoing (NCT04862924).

Upfront Next Generation Sequencing in Non-Small Cell Lung Cancer.

In advanced non-small cell lung cancer (NSCLC), patients with actionable genomic alterations may derive additional clinical benefit from targeted treatment compared to cytotoxic chemotherapy. Current guidelines recommend extensive testing with next generation sequencing (NGS) panels. We investigated the impact of using a targeted NGS panel (TruSight Tumor 15, Illumina) as reflex testing for NSCLC samples at a single institution. Molecular analysis examined 15 genes for hotspot mutation variants, including AKT1, BRAF, EGFR, ERBB2, FOXL2, GNA11, GNAQ, KIT, KRAS, MET, NRAS, PDGFRA, PIK3CA, RET and TP53 genes. Between February 2017 and October 2020, 1460 samples from 1395 patients were analyzed. 1201 patients (86.1%) had at least one variant identified, most frequently TP53 (47.5%), KRAS (32.2%) or EGFR (24.2%). Among these, 994 patients (71.3%) had clinically relevant variants eligible for treatment with approved therapies or clinical trial enrollment. The incremental cost of NGS beyond single gene testing (EGFR, ALK) was CAD $233 per case. Reflex upfront NGS identified at least one actionable variant in more than 70% of patients with NSCLC, with minimal increase in testing cost. Implementation of NGS panels remains essential as treatment paradigms continue to evolve.

PD-L1 assessment in cytology samples predicts treatment response to checkpoint inhibitors in NSCLC.

BACKGROUND: Testing for tumor programmed death ligand-1 (PD-L1) expression was initially developed with histology specimens in non-small cell lung cancer (NSCLC). However, cytology specimens are widely used for primary diagnosis and biomarker studies in clinical practice. Limited clinical data exist on the predictiveness of cytology-derived PD-L1 scores for response to immune checkpoint inhibitor (ICI) therapy. METHODS: We reviewed all NSCLC specimens clinically tested at the University Health Network (UHN) for PD-L1 with 22C3pharmDx, from 01/2013 to 04/2021. Treatment outcomes in patients treated with single agent ICI therapy were reviewed and compared according to cytology- and histology-derived PD-L1 scores. RESULTS: We identified 494 and 1942 unique patients with cytology- and histology-derived tumor proportion scores, respectively, during the study period. Informative testing rates were 95 % vs 98 % for cytology and histology, respectively. Clinical data were available for 152 patients treated with single agent ICI: 61 cytology and 91 histology. Overall response rates (ORR) were similar for cytology and histology (36 % vs 34 %; p = 0.23), as well as median progression free survival (PFS) (4.9 vs 4.2 months; p = 0.99) and overall survival (23.4 vs 19.7 months; p = 0.99). The results remained similar even after adjusting for PD-L1 expression levels and line of ICI treatment (PFS HR 1.15; 95 %CI 0.78-1.70; p = 0.47). CONCLUSIONS: Treatment outcomes to single agent ICI based on cytology-derived PD-L1 scores were comparable to histology controls. Our results support PD-L1 biomarker testing on both cytology and histology specimens.