Genome-wide interaction analysis identified low-frequency variants with sex disparity in lung cancer risk.

Differences by sex in lung cancer incidence and mortality have been reported which cannot be fully explained by sex differences in smoking behavior, implying existence of genetic and molecular basis for sex disparity in lung cancer development. However, the information about sex dimorphism in lung cancer risk is quite limited despite the great success in lung cancer association studies. By adopting a stringent two-stage analysis strategy, we performed a genome-wide gene-sex interaction analysis using genotypes from a lung cancer cohort including ~ 47 000 individuals with European ancestry. Three low-frequency variants (minor allele frequency < 0.05), rs17662871 [odds ratio (OR) = 0.71, P = 4.29×10-8); rs79942605 (OR = 2.17, P = 2.81×10-8) and rs208908 (OR = 0.70, P = 4.54×10-8) were identified with different risk effect of lung cancer between men and women. Further expression quantitative trait loci and functional annotation analysis suggested rs208908 affects lung cancer risk through differential regulation of Coxsackie virus and adenovirus receptor gene expression in lung tissues between men and women. Our study is one of the first studies to provide novel insights about the genetic and molecular basis for sex disparity in lung cancer development.

First-line Treatment Patterns and Outcomes in Advanced Non-Small Cell Lung Cancer in Sweden: A Population-based Real-world Study with Focus on Immunotherapy.

BACKGROUND AND PURPOSE: The treatment landscape for patients with advanced non-small cell lung cancer (NSCLC) has evolved significantly since the introduction of immunotherapies. We here describe PD-L1 testing rates, treatment patterns, and real-world outcomes for PD-(L)1 inhibitors in Sweden. MATERIALS AND METHODS: Data were obtained from the Swedish National Lung Cancer Registry for patients with advanced NSCLC and Eastern Cooperative Oncology Group (ECOG) performance status (PS) 0-2 who initiated first-line -systemic treatment from 01 April 2017 to 30 June 2020. PD-L1 testing was available in the registry from 01 January 2018. Kaplan-Meier was used for overall survival (OS) by type treatment and histology. RESULTS: A total of 2,204 patients with pathologically confirmed unresectable stage IIIB/C or IV NSCLC initiated first-line treatment, 1,807 (82%) with nonsquamous (NSQ) and 397 (18%) with SQ. Eighty-six per cent (NSQ) or 85% (SQ) had been tested for PD-L1 expression, a proportion that increased over time. The use of platinum-based therapy as first-line treatment decreased substantially over time while there was an upward trend for PD-(L)1-based therapy. Among patients with PS 0-1 initiating a first-line PD-(L)1 inhibitor monotherapy, the median OS was 18.6 and 13.3 months for NSQ and SQ NSCLC patients, respectively, while for the PD-(L)1 inhibitor and chemotherapy combination regimen, the median OS was 24.0 months for NSQ and not evaluable for SQ patients. INTERPRETATION: The majority of advanced NSCLCs in Sweden were tested for PD-L1 expression. Real-world OS in patients with PS 0-1 receiving first-line PD-(L)1 inhibitor-based regimens was similar to what has been reported in pivotal clinical trials on PD-(L)1 inhibitors.

Genome-wide association meta-analysis identifies pleiotropic risk loci for aerodigestive squamous cell cancers.

Squamous cell carcinomas (SqCC) of the aerodigestive tract have similar etiological risk factors. Although genetic risk variants for individual cancers have been identified, an agnostic, genome-wide search for shared genetic susceptibility has not been performed. To identify novel and pleotropic SqCC risk variants, we performed a meta-analysis of GWAS data on lung SqCC (LuSqCC), oro/pharyngeal SqCC (OSqCC), laryngeal SqCC (LaSqCC) and esophageal SqCC (ESqCC) cancers, totaling 13,887 cases and 61,961 controls of European ancestry. We identified one novel genome-wide significant (Pmeta<5x10-8) aerodigestive SqCC susceptibility loci in the 2q33.1 region (rs56321285, TMEM273). Additionally, three previously unknown loci reached suggestive significance (Pmeta<5x10-7): 1q32.1 (rs12133735, near MDM4), 5q31.2 (rs13181561, TMEM173) and 19p13.11 (rs61494113, ABHD8). Multiple previously identified loci for aerodigestive SqCC also showed evidence of pleiotropy in at least another SqCC site, these include: 4q23 (ADH1B), 6p21.33 (STK19), 6p21.32 (HLA-DQB1), 9p21.33 (CDKN2B-AS1) and 13q13.1(BRCA2). Gene-based association and gene set enrichment identified a set of 48 SqCC-related genes rel to DNA damage and epigenetic regulation pathways. Our study highlights the importance of cross-cancer analyses to identify pleiotropic risk loci of histology-related cancers arising at distinct anatomical sites.

Real-world analysis of MET exon 14 mutations in non-small cell lung cancer: a retrospective study from two Swedish hospitals.

BACKGROUND: Real-World evidence on mesenchymal-epithelial transition exon 14 skipping mutations (METex14) in lung cancer remains limited. With an incidence of 3-4% across histological subtypes, METex14 is now an actionable target for MET inhibitors (METi) in advanced lung cancer, demonstrating response rates between 30-70%. Yet, its role in early stages and sensitivity to immune checkpoint inhibitors (ICIs) is still under exploration. MATERIAL AND METHODS: We conducted a retrospective analysis of the clinical data of lung cancer patients presenting with METex14 across all stages. These patients were treated at two Swedish University Hospitals: Karolinska and Skåne, between the years 2014 and 2022. RESULTS: We identified a total of 63 patients, of which 50 met the inclusion criteria. The median overall survival (OS) with corresponding 95% confidence intervals (95% CI) according to the stage was not reached (NR) for stage I, NR for stage II, 15 months (95% CI, 5.4-24.6) for stage III, and 17 months (95% CI, 9.2-NR) for stage IV. The median OS for stage IV patients who received a METi was 17 months (95% CI, 9.5-NR) vs. 10 months (95% CI, 6.2-NR) in patients without METi (p = 0.92; Hazard Ratio [HR] = 1.07). The median OS for stage IV patients who received ICIs was 18 months (95% CI, 16.5-NR) vs. 6 months (95% CI, 2.5-NR) in patients without ICIs (p = 0.15; HR = 0.47). The median OS for stage IV patients who received chemotherapy was 17 months (95% CI, 9.7-NR) vs. 10 months (95% CI, 4.5-NR) in patients without (p = 0.97; HR = 0.98). CONCLUSIONS: Our data suggest limited survival benefits from METi, ICIs, and chemotherapy for METex14 lung cancer patients. While not statistically significant, these findings underscore the need for larger trials for validation. Identifying effective treatments for this challenging lung cancer subtype remains a priority.

Comparison of immunohistochemical mesothelial biomarkers in paired biopsies and effusion cytology cell blocks from pleural mesothelioma.

OBJECTIVE: Traditionally, the diagnosis of pleural mesothelioma is based on histological material. Minimally invasive effusion cytology specimens are an alternative that, like biopsies, require ancillary analyses. Validation of immunohistochemical (IHC) analyses on cytology, including the surrogate markers for molecular alterations BAP1 and MTAP, is of interest. METHODS: IHC for eight different markers was performed on 59 paired formalin-fixed, paraffin-embedded pleural biopsies and pleural effusion cell blocks with mesothelioma. Immunoreactivity in ≥10% of tumour cells was considered positive/preserved. The concordance between histological and cytological materials was assessed. RESULTS: The overall percentage of agreement between the histological epithelioid component in 58 biopsies and paired cell blocks was 93% for calretinin, 98% for CK5, 97% for podoplanin, 90% for WT1, 86% for EMA, 100% for desmin, 91% for BAP1, and 72% for MTAP. For 11 cases with biphasic or sarcomatoid histology, the concordance between cytology and the histological sarcomatoid component was low for calretinin, CK5, and WT1 (all ≤45%). For the whole cohort, loss of both BAP1 and MTAP was seen in 40% while both markers were preserved in 11% of the biopsies for epithelioid histology. The corresponding numbers were 54% and 8%, respectively, for the paired cell blocks. CONCLUSIONS: Generally, a high concordance for IHC staining was seen between paired biopsies and pleural effusion cell blocks from mesotheliomas, but the somewhat lower agreement for WT1, EMA, and especially MTAP calls for further investigation and local quality assurance. The lower concordance for the sarcomatoid subtype for some markers may indicate biological differences.

Integration of multiomic annotation data to prioritize and characterize inflammation and immune-related risk variants in squamous cell lung cancer.

Clinical trial results have recently demonstrated that inhibiting inflammation by targeting the interleukin-1ß pathway can offer a significant reduction in lung cancer incidence and mortality, highlighting a pressing and unmet need to understand the benefits of inflammation-focused lung cancer therapies at the genetic level. While numerous genome-wide association studies (GWAS) have explored the genetic etiology of lung cancer, there remains a large gap between the type of information that may be gleaned from an association study and the depth of understanding necessary to explain and drive translational findings. Thus, in this study we jointly model and integrate extensive multiomics data sources, utilizing a total of 40 genome-wide functional annotations that augment previously published results from the International Lung Cancer Consortium (ILCCO) GWAS, to prioritize and characterize single nucleotide polymorphisms (SNPs) that increase risk of squamous cell lung cancer through the inflammatory and immune responses. Our work bridges the gap between correlative analysis and translational follow-up research, refining GWAS association measures in an interpretable and systematic manner. In particular, reanalysis of the ILCCO data highlights the impact of highly associated SNPs from nuclear factor-κB signaling pathway genes as well as major histocompatibility complex mediated variation in immune responses. One consequence of prioritizing likely functional SNPs is the pruning of variants that might be selected for follow-up work by over an order of magnitude, from potentially tens of thousands to hundreds. The strategies we introduce provide informative and interpretable approaches for incorporating extensive genome-wide annotation data in analysis of genetic association studies.

Protein Signatures of Remodeled Airways in Transplanted Lungs with Bronchiolitis Obliterans Syndrome Obtained Using Laser-Capture Microdissection.

Bronchiolitis obliterans syndrome, a common form of chronic lung allograft dysfunction, is the major limitation to long-term survival after lung transplantation. The histologic correlate is progressive, fibrotic occlusion of small airways, obliterative bronchiolitis lesions, which ultimately lead to organ failure. The molecular composition of these lesions is unknown. In this sutdy, the protein composition of the lesions in explanted lungs from four end-stage bronchiolitis obliterans syndrome patients was analyzed using laser-capture microdissection and optimized sample preparation protocols for mass spectrometry. Immunohistochemistry and immunofluorescence were used to determine the spatial distribution of commonly identified proteins on the tissue level, and protein signatures for 14 obliterative bronchiolitis lesions were established. A set of 39 proteins, identified in >75% of lesions, included distinct structural proteins (collagen types IV and VI) and cellular components (actins, vimentin, and tryptase). Each respective lesion exhibited a unique composition of proteins (on average, n = 66 proteins), thereby mirroring the morphologic variation of the lesions. Antibody-based staining confirmed these mass spectrometry-based findings. The 14 analyzed obliterative bronchiolitis lesions showed variations in their protein content, but also common features. This study provides molecular and morphologic insights into the development of chronic rejection after lung transplantation. The protein patterns in the lesions were correlated to pathways of extracellular matrix organization, tissue development, and wound healing processes.

Infiltration of NK and plasma cells is associated with a distinct immune subset in non-small cell lung cancer.

Immune cells of the tumor microenvironment are central but erratic targets for immunotherapy. The aim of this study was to characterize novel patterns of immune cell infiltration in non-small cell lung cancer (NSCLC) in relation to its molecular and clinicopathologic characteristics. Lymphocytes (CD3+, CD4+, CD8+, CD20+, FOXP3+, CD45RO+), macrophages (CD163+), plasma cells (CD138+), NK cells (NKp46+), PD1+, and PD-L1+ were annotated on a tissue microarray including 357 NSCLC cases. Somatic mutations were analyzed by targeted sequencing for 82 genes and a tumor mutational load score was estimated. Transcriptomic immune patterns were established in 197 patients based on RNA sequencing data. The immune cell infiltration was variable and showed only poor association with specific mutations. The previously defined immune phenotypic patterns, desert, inflamed, and immune excluded, comprised 30, 13, and 57% of cases, respectively. Notably, mRNA immune activation and high estimated tumor mutational load were unique only for the inflamed pattern. However, in the unsupervised cluster analysis, including all immune cell markers, these conceptual patterns were only weakly reproduced. Instead, four immune classes were identified: (1) high immune cell infiltration, (2) high immune cell infiltration with abundance of CD20+ B cells, (3) low immune cell infiltration, and (4) a phenotype with an imprint of plasma cells and NK cells. This latter class was linked to better survival despite exhibiting low expression of immune response-related genes (e.g. CXCL9, GZMB, INFG, CTLA4). This compartment-specific immune cell analysis in the context of the molecular and clinical background of NSCLC reveals two previously unrecognized immune classes. A refined immune classification, including traits of the humoral and innate immune response, is important to define the immunogenic potency of NSCLC in the era of immunotherapy. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Interventional and EBUS cytology in Sweden.

Interventional cytology was first introduced in Sweden in the late 1940ies by Sixten Franzén at the Karolinska University Hospital in Solna, Stockholm. In the early 1950ies, Nils Söderström started using the technique at the University Hospital in Lund. Cytology was successively established as common practice at the pathology departments in Sweden, and e.g. Solna and Lund today have a high rate of cytological samples. Over the years new techniques, such as endobronchial ultrasound (EBUS)-guided fine-needle aspirations, and analyses have been introduced, contributing to the maintained value of cytology as a diagnostic method. In this article, we present a brief history and the current situation of cytology in Sweden with focus on interventional and EBUS cytology.

PD-L1 Expression in Non-Small Cell Lung Cancer Specimens: Association with Clinicopathological Factors and Molecular Alterations.

Immune checkpoint inhibitors (ICI) targeting programmed cell death-1 or its ligand (PD-L1) have improved outcomes in non-small cell lung cancer (NSCLC). High tumor PD-L1 expression, detected by immunohistochemistry (IHC) typically on formalin-fixed paraffin-embedded (FFPE) histological specimens, is linked to better response. Following our previous investigation on PD-L1 in cytological samples, the aim of this study was to further explore the potential impacts of various clinicopathological and molecular factors on PD-L1 expression. Two retrospective NSCLC cohorts of 1131 and 651 specimens, respectively, were investigated for PD-L1 expression (<1%/1−49%/≥50%), sample type, sample site, histological type, and oncogenic driver status. In both cohorts, PD-L1 was positive (≥1%) in 55% of the cases. Adenocarcinomas exhibited lower PD-L1 expression than squamous cell carcinomas (p < 0.0001), while there was no difference between sample types, tumor locations, or between the two cohorts in multivariate analysis (all p ≥ 0.28). Mutational status correlated significantly with PD-L1 expression (p < 0.0001), with the highest expression for KRAS-mutated cases, the lowest for EGFR-mutated, and the KRAS/EGFR wild-type cases in between. There was no difference in PD-L1 levels between different prevalent KRAS mutations (all p ≥ 0.44), while mucinous KRAS-mutated adenocarcinomas exhibited much lower PD-L1 expression than non-mucinous (p < 0.0001). Our data indicate that cytological and histological specimens are comparable for PD-L1 evaluation. Given the impact of KRAS mutations and the mucinous growth pattern on PD-L1 expression, these factors should be further investigated in studies on ICI response.

Distinct types of plexiform lesions identified by synchrotron-based phase-contrast micro-CT.

In pulmonary arterial hypertension, plexiform lesions are associated with severe arterial obstruction and right ventricular failure. Exploring their structure and position is crucial for understanding the interplay between hemodynamics and vascular remodeling. The aim of this research was to use synchrotron-based phase-contrast micro-CT to study the three-dimensional structure of plexiform lesions. Archived paraffin-embedded tissue samples from 14 patients with pulmonary arterial hypertension (13 idiopathic, 1 with known BMPR2-mutation) were imaged. Clinical data showed high-median PVR (12.5 WU) and mPAP (68 mmHg). Vascular lesions with more than 1 lumen were defined as plexiform. Prior radiopaque dye injection in some samples facilitated 3-D rendering. Four distinct types of plexiform lesions were identified: 1) localized within or derived from monopodial branches (supernumerary arteries), often with a connection to the vasa vasorum; 2) localized between pulmonary arteries and larger airways as a tortuous transformation of intrapulmonary bronchopulmonary anastomoses; 3) as spherical structures at unexpected abrupt ends of distal pulmonary arteries; and 4) as occluded pulmonary arteries with recanalization. By appearance and localization, types 1-2 potentially relieve pressure via the bronchial circulation, as pulmonary arteries in these patients were almost invariably occluded distally. In addition, types 1-3 were often surrounded by dilated thin-walled vessels, often connected to pulmonary veins, peribronchial vessels, or the vasa vasorum. Collaterals, bypassing completely occluded pulmonary arteries, were also observed to originate within plexiform lesions. In conclusion, synchrotron-based imaging revealed significant plexiform lesion heterogeneity, resulting in a novel classification. The four types likely have different effects on hemodynamics and disease progression.

A gene expression-based single sample predictor of lung adenocarcinoma molecular subtype and prognosis.

Disease recurrence in surgically treated lung adenocarcinoma (AC) remains high. New approaches for risk stratification beyond tumor stage are needed. Gene expression-based AC subtypes such as the Cancer Genome Atlas Network (TCGA) terminal-respiratory unit (TRU), proximal-inflammatory (PI) and proximal-proliferative (PP) subtypes have been associated with prognosis, but show methodological limitations for robust clinical use. We aimed to derive a platform independent single sample predictor (SSP) for molecular subtype assignment and risk stratification that could function in a clinical setting. Two-class (TRU/nonTRU=SSP2) and three-class (TRU/PP/PI=SSP3) SSPs using the AIMS algorithm were trained in 1655 ACs (n = 9659 genes) from public repositories vs TCGA centroid subtypes. Validation and survival analysis were performed in 977 patients using overall survival (OS) and distant metastasis-free survival (DMFS) as endpoints. In the validation cohort, SSP2 and SSP3 showed accuracies of 0.85 and 0.81, respectively. SSPs captured relevant biology previously associated with the TCGA subtypes and were associated with prognosis. In survival analysis, OS and DMFS for cases discordantly classified between TCGA and SSP2 favored the SSP2 classification. In resected Stage I patients, SSP2 identified TRU-cases with better OS (hazard ratio [HR] = 0.30; 95% confidence interval [CI] = 0.18-0.49) and DMFS (TRU HR = 0.52; 95% CI = 0.33-0.83) independent of age, Stage IA/IB and gender. SSP2 was transformed into a NanoString nCounter assay and tested in 44 Stage I patients using RNA from formalin-fixed tissue, providing prognostic stratification (relapse-free interval, HR = 3.2; 95% CI = 1.2-8.8). In conclusion, gene expression-based SSPs can provide molecular subtype and independent prognostic information in early-stage lung ACs. SSPs may overcome critical limitations in the applicability of gene signatures in lung cancer.

Causal relationships between body mass index, smoking and lung cancer: Univariable and multivariable Mendelian randomization.

At the time of cancer diagnosis, body mass index (BMI) is inversely correlated with lung cancer risk, which may reflect reverse causality and confounding due to smoking behavior. We used two-sample univariable and multivariable Mendelian randomization (MR) to estimate causal relationships of BMI and smoking behaviors on lung cancer and histological subtypes based on an aggregated genome-wide association studies (GWASs) analysis of lung cancer in 29 266 cases and 56 450 controls. We observed a positive causal effect for high BMI on occurrence of small-cell lung cancer (odds ratio (OR) = 1.60, 95% confidence interval (CI) = 1.24-2.06, P = 2.70 × 10-4 ). After adjustment of smoking behaviors using multivariable Mendelian randomization (MVMR), a direct causal effect on small cell lung cancer (ORMVMR = 1.28, 95% CI = 1.06-1.55, PMVMR = .011), and an inverse effect on lung adenocarcinoma (ORMVMR = 0.86, 95% CI = 0.77-0.96, PMVMR = .008) were observed. A weak increased risk of lung squamous cell carcinoma was observed for higher BMI in univariable Mendelian randomization (UVMR) analysis (ORUVMR = 1.19, 95% CI = 1.01-1.40, PUVMR = .036), but this effect disappeared after adjustment of smoking (ORMVMR = 1.02, 95% CI = 0.90-1.16, PMVMR = .746). These results highlight the histology-specific impact of BMI on lung carcinogenesis and imply mediator role of smoking behaviors in the association between BMI and lung cancer.

PD-L1 amplification is associated with an immune cell rich phenotype in squamous cell cancer of the lung.

Gene amplification is considered to be one responsible cause for upregulation of Programmed Death Ligand-1 (PD-L1) in non-small cell lung cancer (NSCLC) and to represent a specific molecular subgroup possibly associated with immunotherapy response. Our aim was to analyze the frequency of PD-L1 amplification, its relation to PD-L1 mRNA and protein expression, and to characterize the immune microenvironment of amplified cases. The study was based on two independent NSCLC cohorts, including 354 and 349 cases, respectively. Tissue microarrays were used to evaluate PD-L1 amplification by FISH and PD-L1 protein by immunohistochemistry. Immune infiltrates were characterized immunohistochemically by a panel of immune markers (CD3, CD4, CD8, PD-1, Foxp3, CD20, CD138, CD168, CD45RO, NKp46). Mutational status was determined by targeted sequencing. RNAseq data was available for 197 patients. PD-L1 amplification was detected in 4.5% of all evaluable cases. PD-L1 amplification correlated only weakly with mRNA and protein expression. About  37% of amplified cases were negative for PD-L1 protein. PD-L1 amplification did not show any association with the mutational status. In squamous cell cancer, PD-L1 amplified cases were enriched among patients with high tumoral immune cell infiltration and showed gene expression profiles related to immune exhaustion. In conclusion, PD-L1 amplification correlates with PD-L1 expression in squamous cell cancer and was associated with an immune cell rich tumor phenotype. The correlative findings help to understand the role of PD-L1 amplification as an important immune escape mechanism in NSCLC and suggest the need to further evaluate PD-L1 amplification as predictive biomarker for checkpoint inhibitor therapy.

Clinical significance of RBM3 expression in surgically treated colorectal lung metastases and paired primary tumors.

BACKGROUND: The lungs are the second most common site of metastases in colorectal cancer (CRC). The aim of this study was to investigate prognostic factors, including RNA-binding motif protein 3 (RBM3) expression, in patients with CRC treated with pulmonary metastasectomy (PM). METHODS: The cohort included all patients treated with PM at Skåne University Hospital, Lund, Sweden, from 2000 to 2014. Clinicopathological, treatment, and survival data were collected. Immunohistochemical staining of RBM3 was evaluated on tissue microarrays with samples from all lung metastases and a subset of paired primary tumors. Kaplan-Meier analysis and Cox proportional hazards modeling were applied to examine the associations of investigative factors with overall survival (OS) and recurrence-free survival. RESULTS: In total, 216 patients with a primary tumor in the rectum (57%), left colon (34%), or right colon (9%) underwent PM. The 5-year OS rate was 56%. Age > 60 years, more than one metastasis, size of metastasis > 3 cm, disease-free interval < 24 months, low RBM3 score in the lung metastasis, and no adjuvant chemotherapy following PM were prognostic factors for shorter OS. CONCLUSIONS: Several prognostic factors, including RBM3 expression, may be of aid in selecting CRC patients with lung metastases for PM as well as adjuvant therapy.

Synchrotron-based phase-contrast micro-CT as a tool for understanding pulmonary vascular pathobiology and the 3-D microanatomy of alveolar capillary dysplasia.

This study aimed to explore the value of synchrotron-based phase-contrast microcomputed tomography (micro-CT) in pulmonary vascular pathobiology. The microanatomy of the lung is complex with intricate branching patterns. Tissue sections are therefore difficult to interpret. Recruited intrapulmonary bronchopulmonary anastomoses (IBAs) have been described in several forms of pulmonary hypertension, including alveolar capillary dysplasia with misaligned pulmonary veins (ACD/MPV). Here, we examine paraffin-embedded tissue using this nondestructive method for high-resolution three-dimensional imaging. Blocks of healthy and ACD/MPV lung tissue were used. Pulmonary and bronchial arteries in the ACD/MPV block had been preinjected with dye. One section per block was stained, and areas of interest were marked to allow precise beam-alignment during image acquisition at the X02DA TOMCAT beamline (Swiss Light Source). A ×4 magnifying objective coupled to a 20-µm thick scintillating material and a sCMOS detector yielded the best trade-off between spatial resolution and field-of-view. A phase retrieval algorithm was applied and virtual tomographic slices and video clips of the imaged volumes were produced. Dye injections generated a distinct attenuation difference between vessels and surrounding tissue, facilitating segmentation and three-dimensional rendering. Histology and immunohistochemistry post-imaging offered complementary information. IBAs were confirmed in ACD/MPV, and the MPVs were positioned like bronchial veins/venules. We demonstrate the advantages of using synchrotron-based phase-contrast micro-CT for three-dimensional characterization of pulmonary microvascular anatomy in paraffin-embedded tissue. Vascular dye injections add additional value. We confirm intrapulmonary shunting in ACD/MPV and provide support for the hypothesis that MPVs are dilated bronchial veins/venules.

Genome-wide association study of INDELs identified four novel susceptibility loci associated with lung cancer risk.

Genome-wide association studies (GWAS) have identified 45 susceptibility loci associated with lung cancer. Only less than SNPs, small insertions and deletions (INDELs) are the second most abundant genetic polymorphisms in the human genome. INDELs are highly associated with multiple human diseases, including lung cancer. However, limited studies with large-scale samples have been available to systematically evaluate the effects of INDELs on lung cancer risk. Here, we performed a large-scale meta-analysis to evaluate INDELs and their risk for lung cancer in 23,202 cases and 19,048 controls. Functional annotations were performed to further explore the potential function of lung cancer risk INDELs. Conditional analysis was used to clarify the relationship between INDELs and SNPs. Four new risk loci were identified in genome-wide INDEL analysis (1p13.2: rs5777156, Insertion, OR = 0.92, p = 9.10 × 10-8 ; 4q28.2: rs58404727, Deletion, OR = 1.19, p = 5.25 × 10-7 ; 12p13.31: rs71450133, Deletion, OR = 1.09, p = 8.83 × 10-7 ; and 14q22.3: rs34057993, Deletion, OR = 0.90, p = 7.64 × 10-8 ). The eQTL analysis and functional annotation suggested that INDELs might affect lung cancer susceptibility by regulating the expression of target genes. After conducting conditional analysis on potential causal SNPs, the INDELs in the new loci were still nominally significant. Our findings indicate that INDELs could be potentially functional genetic variants for lung cancer risk. Further functional experiments are needed to better understand INDEL mechanisms in carcinogenesis.

Transcriptome-wide association study reveals candidate causal genes for lung cancer.

We have recently completed the largest GWAS on lung cancer including 29,266 cases and 56,450 controls of European descent. The goal of our study has been to integrate the complete GWAS results with a large-scale expression quantitative trait loci (eQTL) mapping study in human lung tissues (n = 1,038) to identify candidate causal genes for lung cancer. We performed transcriptome-wide association study (TWAS) for lung cancer overall, by histology (adenocarcinoma, squamous cell carcinoma and small cell lung cancer) and smoking subgroups (never- and ever-smokers). We performed replication analysis using lung data from the Genotype-Tissue Expression (GTEx) project. DNA damage assays were performed in human lung fibroblasts for selected TWAS genes. As expected, the main TWAS signal for all histological subtypes and ever-smokers was on chromosome 15q25. The gene most strongly associated with lung cancer at this locus using the TWAS approach was IREB2 (pTWAS = 1.09E-99), where lower predicted expression increased lung cancer risk. A new lung adenocarcinoma susceptibility locus was revealed on 9p13.3 and associated with higher predicted expression of AQP3 (pTWAS = 3.72E-6). Among the 45 previously described lung cancer GWAS loci, we mapped candidate target gene for 17 of them. The association AQP3-adenocarcinoma on 9p13.3 was replicated using GTEx (pTWAS = 6.55E-5). Consistent with the effect of risk alleles on gene expression levels, IREB2 knockdown and AQP3 overproduction promote endogenous DNA damage. These findings indicate genes whose expression in lung tissue directly influences lung cancer risk.

Immune checkpoint inhibitors of the PD-1/PD-L1-axis in non-small cell lung cancer: promise, controversies and ambiguities in the novel treatment paradigm.

Immune checkpoint inhibitors (ICIs) have received much attention not least for melanoma since the award of the Nobel prize in 2018. Here, we review the current state of knowledge about the use of these monoclonal antibodies (mAbs) in non-small cell lung cancer (NSCLC). These drugs have generally been conditionally approved on limited early data and there are few long-term follow-up data from randomized clinical trials. The effect observed for NSCLC thus far is, on average, moderately better than that obtained with chemotherapy. Severe side-effects are more common than might have been expected. The drugs themselves are expensive and are associated with time-consuming histopathologic testing even though the predictive value of these tests can be discussed. In addition, monitoring for side-effects involves increased workload and budgetary expense for clinical chemistry laboratories. Here, we review and summarize the current knowledge, controversies and ambiguities of ICIs for the treatment of NSCLC.

Pre-operative plasma cell-free circulating tumor DNA and serum protein tumor markers as predictors of lung adenocarcinoma recurrence.

Background: Lung cancer patients have a risk of recurrence even after curatively intended surgery. Cell-free circulating tumor DNA (ctDNA) and circulating tumor marker measurements are easily accessible through peripheral blood and could potentially identify patients with worse prognosis. The aim of this study was to examine ctDNA in pre-operative plasma and the role of tumor markers in pre-operative serum for their predictive potential on risk of tumor recurrence. Methods: Mutation analysis by 26-gene targeted sequencing was performed on 157 lung adenocarcinomas (ACs) from patients surgically treated at the Lund University Hospital 2005-2014. Of these, 58 tumors from patients in stages I-IIIA (34 stage I, 14 stage II and 10 stage III) with mutation(s) in EGFR, BRAF or KRAS were included. ctDNA from corresponding plasma (median 1.5 ml, range 1-1.6) was analyzed for one tumor-specific mutation in either of these three oncogenes using ultrasensitive IBSAFE droplet digital PCR (ddPCR). The tumor markers cancer antigen 125 (CA 125) and carbohydrate antigen 19-9 (CA 19-9) were analyzed in corresponding serum with electrochemiluminiscence immunoassay. Results: 6/7 patients with ctDNA and 19/51 without detected ctDNA were diagnosed with recurrence (log-rank test p = .001). 8/10 patients with positive serum tumor markers and 17/47 without tumor markers were diagnosed with recurrence (log-rank test, p = .0002). Fifteen patients had positive ctDNA and/or tumor markers, 12 of these had recurrence (log-rank test, p < .0001). Conclusion: A combination of tumor markers and ctDNA single mutation detection in low-volume pre-operative blood samples is a promising prognostic test. Prediction of recurrent disease in surgically treated early stage lung cancer can likely be further improved by using larger volumes of blood.