Multi-omics and clustering analyses reveal the mechanisms underlying unmet needs for patients with lung adenocarcinoma and identify potential therapeutic targets.

BACKGROUND: The cancer genome contains several driver mutations. However, in some cases, no known drivers have been identified; these remaining areas of unmet needs, leading to limited progress in cancer therapy. Whole-genome sequencing (WGS) can identify non-coding alterations associated with the disease. Consequently, exploration of non-coding regions using WGS and other omics data such as ChIP-sequencing (ChIP-seq) to discern novel alterations and mechanisms related to tumorigenesis have been attractive these days. METHODS: Integrated multi-omics analyses, including WGS, ChIP-seq, DNA methylation, and RNA-sequencing (RNA-seq), were conducted on samples from patients with non-clinically actionable genetic alterations (non-CAGAs) in lung adenocarcinoma (LUAD). Second-level cluster analysis was performed to reinforce the correlations associated with patient survival, as identified by RNA-seq. Subsequent differential gene expression analysis was performed to identify potential druggable targets. RESULTS: Differences in H3K27ac marks in non-CAGAs LUAD were found and confirmed by analyzing RNA-seq data, in which mastermind-like transcriptional coactivator 2 (MAML2) was suppressed. The down-regulated genes whose expression was correlated to MAML2 expression were associated with patient prognosis. WGS analysis revealed somatic mutations associated with the H3K27ac marks in the MAML2 region and high levels of DNA methylation in MAML2 were observed in tumor samples. The second-level cluster analysis enabled patient stratification and subsequent analyses identified potential therapeutic target genes and treatment options. CONCLUSIONS: We overcome the persistent challenges of identifying alterations or driver mutations in coding regions related to tumorigenesis through a novel approach combining multi-omics data with clinical information to reveal the molecular mechanisms underlying non-CAGAs LUAD, stratify patients to improve patient prognosis, and identify potential therapeutic targets. This approach may be applicable to studies of other cancers with unmet needs.

Association between lorlatinib blood concentration and adverse events and clinical impact of dose modification.

OBJECTIVES: Lorlatinib, a third-generation anaplastic lymphoma kinase (ALK) inhibitor, causes distinct adverse events (AEs), including hyperlipidemia and central nervous system (CNS) disorders. Although dose modifications are recommended to manage these AEs, whether dose modifications can achieve optimal blood lorlatinib concentrations and reduce the incidence of lorlatinib-induced AEs remains unclear. Therefore, we investigated the association between lorlatinib exposure and AEs in each patient. MATERIALS AND METHODS: We retrospectively reviewed patients with advanced ALK-rearranged non-small cell lung cancer treated with lorlatinib between November 2018 and July 2022. Serum lorlatinib concentrations were assessed using high-performance liquid chromatography-tandem mass spectrometry. All AEs were evaluated using the Common Terminology Criteria for Adverse Events version 5.0. RESULTS: The median age of the 55 eligible patients was 59 years (range: 23-79 years). All patients were administered lorlatinib after first line ALK-tyrosine kinase inhibitor failure. Grade ≥ 3 AEs occurred in 25 patients (25/55, 45 %), including hyperlipidemia in 17 (17/55, 31 %), CNS disorders in 7 (7/55, 13 %), and edema in 6 (6/55, 11 %). Dose modification was required in 23 patients (23/55, 42 %). Among the 36 patients with available data on serum lorlatinib levels at day 28 (±14) and no drug dose modifications, lorlatinib serum concentrations were significantly higher in patients with grade ≥ 3 AEs than in those without AEs (median: 462 ng/mL vs. 177 ng/mL, p < 0.01). In eight patients with data on serial lorlatinib serum concentrations following dose modifications, lorlatinib serum concentrations were effectively reduced, facilitating the ongoing administration of lorlatinib. Additionally, no significant difference was observed in the landmark analysis of progression-free survival between patients with dose modification within the first 16 weeks and those without (median: 24.8 months vs. 10.1 months, p = 0.46). CONCLUSION: Dose modification of lorlatinib was associated with successful management of AEs and decreased serum concentration of lorlatinib.

Mechanism of ERBB2 gene overexpression by the formation of super-enhancer with genomic structural abnormalities in lung adenocarcinoma without clinically actionable genetic alterations.

BACKGROUND: In an extensive genomic analysis of lung adenocarcinomas (LUADs), driver mutations have been recognized as potential targets for molecular therapy. However, there remain cases where target genes are not identified. Super-enhancers and structural variants are frequently identified in several hundred loci per case. Despite this, most cancer research has approached the analysis of these data sets separately, without merging and comparing the data, and there are no examples of integrated analysis in LUAD. METHODS: We performed an integrated analysis of super-enhancers and structural variants in a cohort of 174 LUAD cases that lacked clinically actionable genetic alterations. To achieve this, we conducted both WGS and H3K27Ac ChIP-seq analyses using samples with driver gene mutations and those without, allowing for a comprehensive investigation of the potential roles of super-enhancer in LUAD cases. RESULTS: We demonstrate that most genes situated in these overlapped regions were associated with known and previously unknown driver genes and aberrant expression resulting from the formation of super-enhancers accompanied by genomic structural abnormalities. Hi-C and long-read sequencing data further corroborated this insight. When we employed CRISPR-Cas9 to induce structural abnormalities that mimicked cases with outlier ERBB2 gene expression, we observed an elevation in ERBB2 expression. These abnormalities are associated with a higher risk of recurrence after surgery, irrespective of the presence or absence of driver mutations. CONCLUSIONS: Our findings suggest that aberrant gene expression linked to structural polymorphisms can significantly impact personalized cancer treatment by facilitating the identification of driver mutations and prognostic factors, contributing to a more comprehensive understanding of LUAD pathogenesis.

Disease progression status during initial immune checkpoint inhibitor (ICI) affects the clinical outcome of ICI retreatment in advanced non-small cell lung cancer patients.

BACKGROUND: It is still unclear whether patients with advanced non-small cell lung cancer (NSCLC), with disease progression after initial immune checkpoint inhibitor (ICI) therapy, would benefit from ICIs readministration. PATIENTS AND METHODS: We retrospectively collected data from patients with advanced NSCLC who received ICI retreatment. Depending on the disease status at the discontinuation of the initial ICI therapy, the patients were divided into two groups: with disease progression (PD group) and without disease progression (Without PD group). Patients in the Without PD group were required to experience disease progression during the treatment-free period. Efficacy was assessed by measuring the objective response rate (ORR) and progression-free survival in retreatment (PFS-R), while safety was assessed using the incidence of immune-related adverse events (irAEs). RESULTS: 30 (46.7%) of 64 eligible patients were included in the PD group and 34 (53.1%) in the Without PD group. Patients in the Without PD group had better clinical outcomes than those in the PD group (ORR, 29.4% vs. 6.7%; p = 0.03, median PFS-R, 4.1 months vs. 2.2 months, hazard ratio [HR], 0.61; 95% confidence interval [CI], 0.36-1.04; p = 0.07). Multivariate Cox regression analysis showed that patients in the Without PD group had significantly longer PFS-R than those in the PD group (HR 0.42, 95% CI, 0.21-0.85; p = 0.015). In terms of safety, 28.1% of patients observed irAEs during ICI retreatment, and the incidence rate of grade 3 or higher irAEs was 7.8%. Specifically, of the 28 patients who discontinued their initial ICI treatment because of irAEs, 35.7% developed irAEs, and 28.6% experienced relapsed irAEs during ICI retreatment. CONCLUSION: Immune checkpoint inhibitor retreatment demonstrated efficacy in patients who discontinued initial ICI therapy for reasons other than disease progression. However, ICI retreatment was ineffective in patients with disease progression during the initial ICI treatment.

Incidence of serious adverse events caused by tyrosine kinase inhibitor treatment following immune checkpoint inhibitor therapy in advanced NSCLC patients with oncogenic driver alterations.

BACKGROUND: Sequential tyrosine kinase inhibitors (TKIs) following immune checkpoint inhibitors (ICIs) increases the incidence of serious adverse events (SAEs). However, the factors and the types of TKIs that affect the incidence of SAEs remain unknown. METHODS: We retrospectively reviewed advanced non-small cell lung cancer (NSCLC) patients who received sequential TKIs following ICIs between November 2015 and April 2021. All AEs were evaluated using Common Terminology Criteria for Adverse Events (CTCAE) ver 5.0. RESULTS: Among 1,638 NSCLC patients who received ICIs, 63 patients received sequential TKIs following ICIs. The types of TKIs included EGFR-TKIs in 48 patients, ALK-TKIs in 10 patients, and others in 5 patients. The median dosing interval was 57 days (range: 7-698). Eighteen (28.6%) patients developed SAEs (Grade 3/4 or hospitalized). The incidence of SAEs and withdrawal of TKIs due to AEs were significantly higher in patients (n = 40) who initiated TKI treatment within 3 months after ICIs than in patients (n = 23) who initiated TKI treatment 3 months after ICIs (SAEs, 40.0% vs. 4.3%, p < 0.01; withdrawal rate: 57.5% vs. 21.7%, p < 0.01). There was no significant difference in the incidence of SAEs and withdrawal rate due to AEs between EGFR-TKIs and other TKIs (SAE, 22.9% vs. 40.0%, p = 0.20; withdrawal rate: 41.7% vs. 53.3%, p = 0.55). CONCLUSION: The dosing interval from last ICI to the initiation of TKI treatment can affects the incidence of SAEs and the withdrawal rate due to AEs regardless of the types of TKIs.

Onco-biome in pharmacotherapy for lung cancer: a narrative review.

Background and Objective: The gut microbiota (GM) was recently recognized to play an important role in modulating systemic immune responses and is known to influence the effects or adverse events of immune checkpoint blockade (ICB) or carcinogenesis by crosstalk with regulators of cancer-related immunity, and this relationship is complex and multifactorial. Diversity in the gut microbiome and the abundance of specific bacterial species have been identified to be associated with better response and prognosis. Therefore, the purpose of the current interest in the gut microbiome is to enable modulation of the immune system in donor cancer patients by the administration of specific bacterial species and enabling their dominance. To understand this "terra incognita" is to uncover the role of the mechanisms underlying unknown organ functions, and this knowledge will lead to enhanced immunotherapy for lung cancer patients. Methods: In this article, we summarized the literature on the relationship between the microbiome and lung cancer and the potential of the microbiome as a therapeutic target. Key Content and Findings: This article is organized into the following sections: introduction, methods, microbiota and cancer development, microbiota and lung cancer treatment, future directions, and conclusion. Conclusions: The gut microbiome is currently becoming the hallmark of cancer research and has an established and critical role in regulating antitumor immunity and the response to ICB in patients with lung cancers.

[Cancer Therapy Targeting Fusion Genes in Lung Cancer].

Therapeutic landscape of lung cancer has undergone a dramatic shift due to the better understandings of disease biology and the identification of oncogenic driver alterations. Consequently, the new classification paradigm of non-small-cell lung cancer is further characterized by molecularly defined subsets, and making the therapeutic landscape increasingly complex. Driver gene mutations have been found in approximately 75% of Japanese non-squamous, non-small-cell lung cancers. Among these, the gene mutations for which molecularly-targeted therapies are being developed include EGFR mutations, ALK fusion gene, ROS1 fusion gene, BRAF V600E mutation, MET exon 14 skipping mutation, KRAS G12C mutation, RET fusion gene, and NTRK fusion gene. When limited to fusion genes, as of June 2022, crizotinib, ceritinib, alectinib, brigatinib, and lorlatinib are approved for the ALK fusion gene; crizotinib and entrectinib for the ROS1 fusion gene; selpercatinib for the RET fusion gene; entrectinib and larotrectinib for the NTRK fusion gene have been approved in Japan. This article summarizes the therapeutic development of each fusion gene mutation, as well as the therapeutic outcomes and adverse events of the approved drugs.

Correlation between body mass index and efficacy of anti-PD-1 inhibitor in patients with non-small cell lung cancer.

BACKGROUND: High body mass index (BMI) has been reported to be associated with the efficacy of immune checkpoint inhibitors in patients with advanced non-small cell lung cancer (NSCLC), but the association between BMI and efficacy of anti-PD-1 inhibitors remains controversial. The present study investigated this association in patients with advanced NSCLC. METHODS: We retrospectively reviewed patients with advanced NSCLC who received PD-1 inhibitors at the National Cancer Center Hospital between January 2016 and December 2018. The efficacy of PD-1 inhibitors (progression-free survival [PFS], overall survival [OS], and response rate) was compared between overweight (BMI ≥25 kg/m2) and non-overweight (BMI <25 kg/m2) groups. Cohort 1 included patients with high PD-L1 expression who were treated with pembrolizumab as first-line therapy; Cohort 2 included patients treated with nivolumab/pembrolizumab as second- or later-line treatment. RESULTS: A total of 324 patients were included in this study and the median BMI (IQR) was 21.4 (19.5-23.6) kg/m2. Of the 324 patients, 279 (86.1%) and 45 (13.9%) were in the non-overweight and overweight groups, respectively. No significant differences in objective response rate (ORR), PFS, or OS were found between overweight and non-overweight patients overall (n = 324; overweight vs. non-overweight: ORR, 28.9% vs. 31.9%, respectively [p = 0.68]; PFS, 7.6 vs. 5.8 months, respectively [p = 0.43]; and OS, 17.6 vs. 15.3 months, respectively [p = 0.90]), or between overweight and non-overweight patients in Cohorts 1 and 2. CONCLUSIONS: No significant differences in the efficacy of PD-1 inhibitors were observed between overweight and non-overweight patients.

Disease flare of leptomeningeal metastases without radiological and cytological findings after the discontinuation of osimertinib.

OBJECTIVES: Rapid tumor progression occurring after the discontinuation of epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) therapy is referred to as a disease flare of non-small cell lung cancer (NSCLC). The clinicopathological features of disease flares after osimertinib discontinuation remain unclear. RESULTS: We report a patient with EGFR-mutated NSCLC who experienced the progression of leptomeningeal metastases as a disease flare shortly after the discontinuation of osimertinib despite the absence of radiological or cytological findings. CONCLUSION: If CNS symptoms develop immediately after the discontinuation of osimertinib, the possibility of a CNS disease flare should be considered even if no radiological or cytological findings are present.

The utility of transbronchial rebiopsy for peripheral pulmonary lesions in patients with advanced non-squamous non-small cell lung cancer.

BACKGROUND: Patients treated for non-squamous (non-Sq) non-small cell lung cancer (NSCLC) often require repeat biopsies to determine the optimal subsequent treatment. However, the differences between rebiopsy and initial biopsy in terms of their diagnostic yields and their ability to test the molecular profiles using bronchoscopy with radial endobronchial ultrasound guidance in patients with advanced NSCLC remain unclear. Hence, we aimed to compare the diagnostic yields and ability for molecular analyses of rebiopsies with those of initial biopsies. METHODS: We investigated 301 patients with advanced non-Sq NSCLC who underwent radial endobronchial ultrasound-guided transbronchial biopsy (TBB) for peripheral pulmonary lesions (PPLs) between August 2014 and July 2017. Patients were divided into the rebiopsy and initial biopsy groups: the latter referred to the biopsy that determined the definitive diagnosis. The diagnostic yields and ability for molecular analyses were compared between the two groups, and the factors affecting the TBB diagnostic yield were identified using univariate and multivariate analyses. RESULTS: The diagnostic yields of the rebiopsy and initial biopsy groups were comparable (86.8 and 90.8%, respectively; p = 0.287). Furthermore, 93.0 and 94.0% of the patients in the rebiopsy and initial biopsy groups, respectively, had adequate specimens for gene profiling and mutational analysis (p = 0.765). The factors that increased the diagnostic yield were a positive bronchus sign (p < 0.001) and tumour location within the internal two-thirds of the lungs (p = 0.026). CONCLUSIONS: The PPL diagnostic yield of the rebiopsy group was as high as that of the initial biopsy group. Hence, TBB for PPLs is feasible for patients requiring rebiopsy as well as for those with initial diagnoses. Adequate, high-quality biopsy specimens can be obtained by transbronchial rebiopsy for molecular testing.

Remarkable Alteration of PD-L1 Expression after Immune Checkpoint Therapy in Patients with Non-Small-Cell Lung Cancer: Two Autopsy Case Reports.

Pembrolizumab is an immune checkpoint inhibitor (ICI), currently recommended as the first-line treatment for patients with advanced non-small-cell lung cancer (NSCLC) showing ≥50% expression of programmed death-ligand 1 (PD-L1). Previously it was reported that platinum-based chemotherapy may change PD-L1 expression in solid cancers. However, no reports addressing alteration of PD-L1 expression after ICI therapy in NSCLC are available so far. The patients were Japanese males 83 and 87 years old, who were diagnosed with NSCLC based on the transbronchial lung biopsies showing sarcomatoid feature with high PD-L1 expression. They received Pembrolizumab, however, passed away with disease progression on day 27 and day 9, respectively. PD-L1, PD1, and CD8 antibodies were applied to pretreatment tumor biopsies and autopsy specimens. Immunoexpression of all the markers was evaluated using Aperio ImageScope. We found that PD-L1 expression decreased significantly from 75.6% to 13.2% and from 100% to 58.8%, in patients 1 and 2, respectively. This alteration was less prominent in the perinecrotic tumor area. A considerable decrease of PD-L1 score was linked with a little effect of Pembrolizumab in our patients. This association might be one of the contributing mechanisms of resistance to ICI and needs further investigation in large-scale studies.

Pulmonary Tumor Thrombotic Microangiopathy Mimicking Inhalation Lung Injury.

Pulmonary tumor thrombotic microangiopathy (PTTM) is a complication characterized by dyspnea, pulmonary hypertension, and occasionally sudden death. We encountered a man who developed PTTM and had an inhalation history of chemical herbicides and abnormal findings on chest computed tomography, mimicking chemical inhalation lung injury. He was diagnosed with PTTM with adenocarcinoma by a transbronchial lung biopsy and received chemotherapy and anticoagulant therapy. He survived for one month. An autopsy revealed primary gastric cancer with PTTM that can have a presentation similar to diffuse pulmonary diseases, including chemical inhalation lung injury. The examination of a biopsy specimen is crucial in such patients.

Successful treatment with idarucizumab for diffuse alveolar hemorrhage induced by dabigatran etexilate: a case report.

We report the case of an 81-year-old man taking dabigatran etexilate (dabigatran) for chronic atrial fibrillation, who presented with acute-onset hemoptysis and hypoxia. Chest high-resolution computed tomography showed bilateral ground grass opacities. After admission, his respiratory failure progressed rapidly and bronchoalveolar lavage was performed immediately, which showed copious amounts of bloody fluid and hemosiderin-laden macrophages with Prussian blue staining. He was diagnosed as having diffuse alveolar hemorrhage (DAH). We therefore stopped dabigatran and initiated multimodality therapy including idarucizumab, which is a reversal agent for dabigatran. Clinical and radiological improvement was observed and he was discharged without any impairment. There has been no relapse of DAH since then. No abnormalities were detected on further investigation; finally, we concluded that his DAH was caused by dabigatran. This is the first known case of idarucizumab use for severe DAH caused by dabigatran. Our case suggested that dabigatran can cause life-threatening DAH; in such cases, administering idarucizumab could be an effective treatment option.

Uptake of dihomo-gamma-linolenic acid by murine macrophages increases series-1 prostaglandin release following lipopolysaccharide treatment.

Administration of dihomo-gamma-linolenic acid is useful for atopic dermatitis and atherosclerosis in mice; however, the metabolites of dihomo-gamma-linolenic acid have been little studied. We employed a method which enabled simultaneous analysis of nine prostaglandins using liquid chromatography-tandem mass spectrometry, and determined the concentrations of prostaglandins in the supernatants of cultures of mouse peritoneal macrophages stimulated with lipopolysaccharide after pre-incubation with dihomo-gamma-linolenic acid, arachidonic acid, or eicosapentaenoic acid. Accumulated prostaglandin concentrations from mouse macrophages with dihomo-gamma-linolenic acid uptake increased in a dihomo-gamma-linolenic acid concentration-dependent fashion. These increases were mainly due to prostaglandin D(1) and prostaglandin E(1). The order of accumulated prostaglandin concentrations was dihomo-gamma-linolenic acid>arachidonic acid>eicosapentaenoic acid in supernatants with the same concentration of polyunsaturated fatty acid. Since mouse macrophages can clearly produce series-1 prostaglandins, they must be formed in vivo. These findings suggest that the effects of dihomo-gamma-linolenic acid on diseases may be due to series-1 prostaglandins.

Anti-atherosclerotic effects of dihomo-gamma-linolenic acid in ApoE-deficient mice.

AIM: Dihomo-gamma-linolenic acid (DGLA) is an n-6 polyunsaturated fatty acid that is mainly metabolized to an anti-inflammatory eicosanoid, prostaglandin (PG) E1, via the cyclooxygenase (COX) pathway. We evaluated the effect of DGLA on atherosclerosis in apoE-deficient mice and studied the mechanism of the anti-atherosclerotic effect. METHODS: ApoE-deficient mice were fed a normal diet supplemented with 0.5% DGLA or vehicle for 6 months. ApoE-deficient mice were also fed a high-cholesterol diet supplemented with 0.5% DGLA or vehicle for 1 month. To clarify the influence of a COX inhibitor, naproxen, on the anti-atherosclerotic effect of DGLA, age-matched apoE-deficient mice fed a high-cholesterol diet supplemented with 0.5% DGLA were given oral naproxen for 1 month. RESULTS: In normal diet-fed mice, acetylcholine-induced vascular relaxation was significantly greater in the DGLA group than in the vehicle group. NADPH oxidase subunits, p22phox and gp91phox, intercellular adhesion molecule-1, and vascular cellular adhesion molecule-1 were significantly lower in the DGLA group than in the vehicle group, and DGLA significantly prevented atherosclerosis. In high-cholesterol diet-fed mice, DGLA also significantly prevented atherosclerosis, but the anti-atherosclerotic effect was attenuated by naproxen. CONCLUSION: DGLA may have an anti-atherosclerotic effect in apoE-deficient mice via PGE1 formation.

Oral supplementation with dihomo-gamma-linolenic acid-enriched oil altered serum fatty acids in healthy men.

Dihomo-gamma-linolenic acid (DGLA)-enriched oil (50 or 150 mg as free DGLA) was administered to healthy men for 4 weeks. The DGLA content in serum phospholipids dose-dependently increased and returned to the initial level after a 4-week washout. No side effects or changes in platelet aggregation were observed. These results indicate that oral supplementation with DGLA oil can safely increase serum DGLA content.