Identification of targetable kinases in idiopathic pulmonary fibrosis.

BACKGROUND: Tyrosine kinase activation plays an important role in the progression of pulmonary fibrosis. In this study, we analyzed the expression of 612 kinase-coding and cancer-related genes using next-generation sequencing to identify potential therapeutic targets for idiopathic pulmonary fibrosis (IPF). METHODS: Thirteen samples from five patients with IPF (Cases 1-5) and eight samples from four patients without IPF (control) were included in this study. Six of the thirteen samples were obtained from different lung segments of a single patient who underwent bilateral pneumonectomy. Gene expression analysis of IPF lung tissue samples (n = 13) and control samples (n = 8) was performed using SureSelect RNA Human Kinome Kit. The expression of the selected genes was further confirmed at the protein level by immunohistochemistry (IHC). RESULTS: Gene expression analysis revealed a correlation between the gene expression signatures and the degree of fibrosis, as assessed by Ashcroft score. In addition, the expression analysis indicated a stronger heterogeneity among the IPF lung samples than among the control lung samples. In the integrated analysis of the 21 samples, DCLK1 and STK33 were found to be upregulated in IPF lung samples compared to control lung samples. However, the top most upregulated genes were distinct in individual cases. DCLK1, PDK4, and ERBB4 were upregulated in IPF case 1, whereas STK33, PIM2, and SYK were upregulated in IPF case 2. IHC revealed that these proteins were expressed in the epithelial layer of the fibrotic lesions. CONCLUSIONS: We performed a comprehensive kinase expression analysis to explore the potential therapeutic targets for IPF. We found that DCLK1 and STK33 may serve as potential candidate targets for molecular targeted therapy of IPF. In addition, PDK4, ERBB4, PIM2, and SYK might also serve as personalized therapeutic targets of IPF. Additional large-scale studies are warranted to develop personalized therapies for patients with IPF.

VEGFR2 blockade augments the effects of tyrosine kinase inhibitors by inhibiting angiogenesis and oncogenic signaling in oncogene-driven non-small-cell lung cancers.

Molecular agents targeting the epidermal growth factor receptor (EGFR)-, anaplastic lymphoma kinase (ALK)- or c-ros oncogene 1 (ROS1) alterations have revolutionized the treatment of oncogene-driven non-small-cell lung cancer (NSCLC). However, the emergence of acquired resistance remains a significant challenge, limiting the wider clinical success of these molecular targeted therapies. In this study, we investigated the efficacy of various molecular targeted agents, including erlotinib, alectinib, and crizotinib, combined with anti-vascular endothelial growth factor receptor (VEGFR) 2 therapy. The combination of VEGFR2 blockade with molecular targeted agents enhanced the anti-tumor effects of these agents in xenograft mouse models of EGFR-, ALK-, or ROS1-altered NSCLC. The numbers of CD31-positive blood vessels were significantly lower in the tumors of mice treated with an anti-VEGFR2 antibody combined with molecular targeted agents compared with in those of mice treated with molecular targeted agents alone, implying the antiangiogenic effects of VEGFR2 blockade. Additionally, the combination therapies exerted more potent antiproliferative effects in vitro in EGFR-, ALK-, or ROS1-altered NSCLC cells, implying that VEGFR2 inhibition also has direct anti-tumor effects on cancer cells. Furthermore, VEGFR2 expression was induced following exposure to molecular targeted agents, implying the importance of VEGFR2 signaling in NSCLC patients undergoing molecular targeted therapy. In conclusion, VEGFR2 inhibition enhanced the anti-tumor effects of molecular targeted agents in various oncogene-driven NSCLC models, not only by inhibiting tumor angiogenesis but also by exerting direct antiproliferative effects on cancer cells. Hence, combination therapy with anti-VEGFR2 antibodies and molecular targeted agents could serve as a promising treatment strategy for oncogene-driven NSCLC.

An immunocompetent case of chronic pulmonary aspergillosis caused by Aspergillusviridinutans.

We encountered an extremely rare immunocompetent case of chronic pulmonary aspergillosis (CPA) caused by Aspergillus viridinutans. A 74-year-old woman was admitted with fever and hemoptysis. Chest computed tomography revealed a nodule in the left upper lobe. Bronchoscopy was performed, and the transbronchial biopsy specimen revealed Aspergillus fungi. Treatment of the nodule was initially ineffective with voriconazole but effective with liposomal amphotericin B. The causative organism was later identified as A. viridinutans based on the gene sequence of ß-tubulin. This is the first immunocompetent case of CPA caused by A. viridinutans.

Clinical characteristics of patients treated with immune checkpoint inhibitors in EGFR-mutant non-small cell lung cancer: CS-Lung-003 prospective observational registry study.

PURPOSE: Immune checkpoint inhibitors (ICIs) are ineffective against epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer (NSCLC). This study aimed to investigate the clinical characteristics of patients who were treated or not treated with ICIs, and of those who benefit from immunotherapy in EGFR-mutant NSCLC. METHODS: We analyzed patients with unresectable stage III/IV or recurrent NSCLC harboring EGFR mutations using a prospective umbrella-type lung cancer registry (CS-Lung-003). RESULTS: A total of 303 patients who met the eligibility criteria were analyzed. The median age was 69 years; 116 patients were male, 289 had adenocarcinoma, 273 had major mutations, and 67 were treated with ICIs. The duration of EGFR-TKI treatment was longer in the Non-ICI group than in the ICI group (17.1 vs. 12.7 months, p < 0.001). Patients who received ICIs for more than 6 months were categorized into the durable clinical benefit (DCB) group (24 patients), and those who received ICIs for less than 6 months into the Non-DCB group (43 patients). The overall survival in the DCB group exhibited longer than the Non-DCB group (69.3 vs. 47.1 months), and an equivalent compared to that in the Non-ICI group (69.3 vs. 68.9 months). Multivariate analysis for time to next treatment (TTNT) of ICIs showed that a poor PS was associated with a shorter TTNT [hazard ratio (HR) 3.309; p < 0.001]. Patients who were treated with ICIs and chemotherapy combination were associated with a longer TTNT (HR 0.389; p = 0.003). In addition, minor EGFR mutation was associated with a long TTNT (HR 0.450; p = 0.046). CONCLUSION: ICIs were administered to only 22% of patients with EGFR-mutated lung cancer, and they had shorter TTNT of EGFR-TKI compared to other patients. ICI treatment should be avoided in EGFR mutated lung cancer with poor PS but can be considered for lung cancer with EGFR minor mutations. Pathological biomarker to predict long-term responders to ICI are needed.

PD-1 blockade augments CD8+ T cell dependent antitumor immunity triggered by Ad-SGE-REIC in Egfr-mutant lung cancer.

OBJECTIVES: No immunotherapeutic protocol has yet been established in never-smoking patients with lung cancer harboring driver oncogenic mutations, such as epidermal growth factor receptor (EGFR) mutations. The immunostimulatory effect of Ad-REIC, a genetically engineered adenovirus vector expressing a tumor suppressor gene, reduced expression in immortalized cells (REIC), has been investigated in clinical trials for various solid tumors. However, the immunostimulatory effect of the Ad-REIC in EGFR-mutant lung cancer with a non-inflamed tumor microenvironment (TME) has not been explored. MATERIALS AND METHODS: We used a syngeneic mouse model developed by transplanting Egfr-mutant lung cancer cells into single or double flanks of C57BL/6J mice. Ad-SGE-REIC, a 2nd-generation vector with an enhancer sequence, was injected only into the tumors from one flank, and its antitumor effects were assessed. Tumor-infiltrating cells were evaluated using immunohistochemistry or flow cytometry. The synergistic effects of Ad-SGE-REIC and PD-1 blockade were also examined. RESULTS: Injection of Ad-SGE-REIC into one side of the tumor induced not only a local antitumor effect but also a bystander abscopal effect in the non-injected tumor, located on the other flank. The number of PD-1+CD8+ T cells increased in both injected and non-injected tumors. PD-1 blockade augmented the local and abscopal antitumor effects of Ad-SGE-REIC by increasing the number of CD8+ T cells in the TME of Egfr-mutant tumors. Depletion of CD8+ cells reverted the antitumor effect, suggesting they contribute to antitumor immunity. CONCLUSION: Ad-SGE-REIC induced systemic antitumor immunity by modifying the TME status from non-inflamed to inflamed, with infiltration of CD8+ T cells. Additionally, in Egfr-mutant lung cancer, this effect was enhanced by PD-1 blockade. These findings pave the way to establish a novel combined immunotherapy strategy with Ad-SGE-REIC and anti-PD-1 antibody for lung cancer with a non-inflamed TME.

CDK4/6 signaling attenuates the effect of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors in EGFR-mutant non-small cell lung cancer.

Background: Epidermal growth factor receptor (EGFR) mutations, such as exon 19 deletion and exon 21 L858R, are driver oncogenes of non-small cell lung cancer (NSCLC), with EGFR tyrosine kinase inhibitors (TKIs) being effective against EGFR-mutant NSCLC. However, the efficacy of EGFR-TKIs is transient and eventually leads to acquired resistance. Herein, we focused on the significance of cell cycle factors as a mechanism to attenuate the effect of EGFR-TKIs in EGFR-mutant NSCLC before the emergence of acquired resistance. Methods: Using several EGFR-mutant cell lines, we investigated the significance of cell cycle factors to attenuate the effect of EGFR-TKIs in EGFR-mutant NSCLC. Results: In several EGFR-mutant cell lines, certain cancer cells continued to proliferate without EGFR signaling, and the cell cycle regulator retinoblastoma protein (RB) was not completely dephosphorylated. Further inhibition of phosphorylated RB with cyclin-dependent kinase (CDK) 4/6 inhibitors, combined with the EGFR-TKI osimertinib, enhanced G0/G1 cell cycle accumulation and growth inhibition of the EGFR-mutant NSCLC in both in vitro and in vivo models. Furthermore, residual RB phosphorylation without EGFR signaling was maintained by extracellular signal-regulated kinase (ERK) signaling, and the ERK inhibition pathway showed further RB dephosphorylation. Conclusions: Our study demonstrated that the CDK4/6-RB signal axis, maintained by the MAPK pathway, attenuates the efficacy of EGFR-TKIs in EGFR-mutant NSCLC, and targeting CDK4/6 enhances this efficacy. Thus, combining CDK4/6 inhibitors and EGFR-TKI could be a novel treatment strategy for TKI-naïve EGFR-mutant NSCLC.

CD8+ T-cell Responses Are Boosted by Dual PD-1/VEGFR2 Blockade after EGFR Inhibition in Egfr-Mutant Lung Cancer.

Epidermal growth factor receptor (EGFR) is the most frequently mutated driver oncogene in nonsmoking-related, non-small cell lung cancer (NSCLC). EGFR-mutant NSCLC has a noninflamed tumor microenvironment (TME), with low infiltration by CD8+ T cells and, thus, immune-checkpoint inhibitors, such as antiprogrammed cell death-1 (anti-PD-1), have weak antitumor effects. Here, we showed that CD8+ T-cell responses were induced by an EGFR-tyrosine kinase inhibitor (TKI) in syngeneic Egfr-mutant NSCLC tumors, which was further pronounced by the sequential dual blockade of PD-1 and vascular endothelial growth factor receptor 2 (VEGFR2). However, the simultaneous triple blockade had no such effect. The PD-1/VEGFR2 dual blockade did not exert tumor-inhibitory effects without pretreatment with the EGFR-TKI, suggesting that the treatment schedule is crucial for the efficacy of the dual blockade therapy. Pretreatment with EGFR-TKI increased the CD8+ T-cell/regulatory T-cell (Treg) ratio, while also increasing the expression of immunosuppressive chemokines and chemokine receptors, as well as increasing the number of M2-like macrophages, in the TME. Discontinuing EGFR-TKI treatment reversed the transient increase of immunosuppressive factors in the TME. The subsequent PD-1/VEGFR2 inhibition maintained increased numbers of infiltrating CD8+ T cells and CD11c+ dendritic cells. Depletion of CD8+ T cells in vivo abolished tumor growth inhibition by EGFR-TKI alone and the sequential triple therapy, suggesting that EGFR inhibition is a prerequisite for the induction of CD8+ T-cell responses. Our findings could aid in developing an alternative immunotherapy strategy in patients with cancers that have driver mutations and a noninflamed TME.

A case of interstitial pneumonia associated with systemic sclerosis and primary peritoneal serous carcinoma successfully treated with cyclophosphamide.

A 62-year-old woman with edema and color changes in her fingers underwent computed tomography (CT); slight interstitial changes were detected in the lungs with multiple tumors in the anterior and hilar region of the liver. Based on the blood test findings, she was diagnosed with interstitial pneumonia associated with systemic sclerosis. Ultrasound-guided biopsy from the hepatic hilar lymph node revealed poorly differentiated serous adenocarcinoma cells. High serum CA-125 levels suggested primary peritoneal serous carcinoma (PPSC). Owing to increased interstitial shadows on chest CT images and worsening respiratory distress, intravenous cyclophosphamide and oral prednisolone treatment was started. The skin-related symptoms, respiratory distress, and interstitial shadows improved, and the tumor size reduced. Eighteen months later, the patient has had no exacerbation of interstitial pneumonia, and the PPSC is well controlled.

Marginal Zone Lymphoma and Lung Adenocarcinoma with an EGFR Exon 19 E746-S752del Mutation in a Patient with IgG4-related Disease.

A 68-year-old man presented with a solid mass at the left renal pelvis and ureter with multiple systemic lymphadenopathies and a mass with a cavity in the right lower lobe of the lung. While a transbronchial lung biopsy revealed no malignancy, a biopsy of the renal pelvis showed marginal zone lymphoma with polyclonal IgG4-positive cells. The serum IgG4 level and presence of a bilateral orbital mass suggested Mikulicz disease. The lesions shrank following the administration of steroids. A rebiopsy confirmed lung adenocarcinoma, and its background showed IgG4-positive cells a year later. IgG4-related diseases require careful follow-up because they can be complicated by malignancy.

MAPK-pathway inhibition mediates inflammatory reprogramming and sensitizes tumors to targeted activation of innate immunity sensor RIG-I.

Kinase inhibitors suppress the growth of oncogene driven cancer but also enforce the selection of treatment resistant cells that are thought to promote tumor relapse in patients. Here, we report transcriptomic and functional genomics analyses of cells and tumors within their microenvironment across different genotypes that persist during kinase inhibitor treatment. We uncover a conserved, MAPK/IRF1-mediated inflammatory response in tumors that undergo stemness- and senescence-associated reprogramming. In these tumor cells, activation of the innate immunity sensor RIG-I via its agonist IVT4, triggers an interferon and a pro-apoptotic response that synergize with concomitant kinase inhibition. In humanized lung cancer xenografts and a syngeneic Egfr-driven lung cancer model these effects translate into reduction of exhausted CD8+ T cells and robust tumor shrinkage. Overall, the mechanistic understanding of MAPK/IRF1-mediated intratumoral reprogramming may ultimately prolong the efficacy of targeted drugs in genetically defined cancer patients.

Rapidly Progressive Acute Kidney Injury Associated with Nivolumab Treatment.

A 63-year-old man with pulmonary adenocarcinoma was treated with nivolumab. High fever developed within several hours after the first administration of nivolumab; subsequently, serum creatinine levels kept increasing daily. We diagnosed acute kidney injury (AKI) as an immune-related adverse event; the patient was initially treated with 50 mg prednisolone, and the dose was then tapered. Renal biopsy pathologically revealed tubulointerstitial inflammation with strong infiltration of only T cells that were CD3+, CD4+, and CD8+. The infiltration of CD163+ M2 macrophage was also observed. AKI within 1 week after the administration of nivolumab seems to be rare; therefore, the present case provides important findings useful in daily clinical practice.

Bilateral Testicular Metastases from Lung Adenocarcinoma Showing an Objective Response to Nivolumab: A Case Report and Review of the Literature.

A 69-year-old man who had undergone chemoradiotherapy for advanced pulmonary adenocarcinoma had bilateral testicular and adrenal gland masses on a routine follow-up examination. We performed left orchiectomy, and the histopathological examination confirmed metastatic pulmonary adenocarcinoma involving the extracted testis. He was treated for disease progression with nivolumab after unsuccessful cytotoxic chemotherapy, which resulted in regression of recurrent adrenal and right testicular tumors. We reviewed the existing literature on metastatic testicular tumors and found that testicular metastasis from lung cancer is rare and poses a chemotherapeutic challenge. Based on our experience, immune checkpoint inhibitors seem to have good efficacy for treating testicular metastasis.

Pembrolizumab-induced Autoimmune Hemolytic Anemia and Hemophagocytic Lymphohistiocytosis in Non-small Cell Lung Cancer.

We herein report a 78-year old man with squamous cell carcinoma of the lungs treated with pembrolizumab. At 10 days after the administration of pembrolizumab, he showed progressive anemia and increased levels of bilirubin. Because the findings of a direct coombs test and cold hemagglutinin were positive, we diagnosed the patient with autoimmune hemolytic anemia and treated him with prednisolone. Subsequently, he was admitted to our hospital owing to fatigue, a high fever, and jaundice. His clinical findings met the diagnostic criteria of hemophagocytic lymphohistiocytosis, and he was rescued with a high dose of glucocorticoids. Marked tumor regression was obtained and has been maintained since then.

Rapidly Progressive Multiple Cavity Formation in Necrotizing Pneumonia Caused by Community-acquired Methicillin-resistant Staphylococcus aureus Positive for the Panton-Valentine Leucocidin Gene.

A 66-year-old man was transferred to our hospital for pneumonia that was resistant to sulbactam/ampicillin and levofloxacin therapy. Chest computed tomography showed the rapidly progressive formation of multiple cavities. Methicillin-resistant Staphylococcus aureus (MRSA) was isolated, and the patient was diagnosed with necrotizing pneumonia caused by community-acquired MRSA (CA-MRSA). The MRSA strain had type IV staphylococcus cassette chromosome mec and genes encoding Panton-Valentine leucocidin (PVL). CA-MRSA necrotizing pneumonia with the PVL gene is rare; only three cases have been previously reported in Japan. We administered anti-MRSA antibiotics and the patient achieved complete clinical and radiological improvement.

Comparing the Clinical Performance of the New 19-G ViziShot FLEX and 21- or 22-G ViziShot 2 Endobronchial Ultrasound-guided Transbronchial Needle Aspiration Needles.

Objective Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is a minimally invasive procedure for pulmonary and mediastinal diseases. This study compared the performance of a novel 19-G needle with a 21- or 22-G needle. Patients and Methods Eleven patients at Okayama Medical Center were enrolled retrospectively between April and December 2017. Enlarged lymph nodes and a pulmonary nodule were sampled with both 19-G and 21- or 22-G needles in nine patients. Two patients underwent biopsies for suspected lymphoma with only the 19-G needle. We examined their medical records on the diagnosis, size of the lymph nodes and pulmonary nodule, and complications. Results The median longest diameter of the 13 lymph nodes (8 #7, 4 #4R, and 1 #11) and 1 pulmonary nodule (right segment 6) in the 11 patients was 31.6 mm (range, 10.4-45.0 mm). Definitive diagnoses were made using the 19-G needle in nine patients. EBUS-TBNA with a 19-G needle resulted in successful diagnoses of one case of retinal hemangioblastoma, one case of tuberculous lymphadenitis, and one case of lung adenocarcinoma, as well as the evaluation of the programmed death-ligand 1 (PD-L1) expression following initial negative findings after a 21- or 22-G biopsy. A small pulmonary nodule (lung squamous cell carcinoma) with negative findings after a 19-G biopsy was diagnosed with a 22-G biopsy. Two suspected lymphoma patients were diagnosed with a 19-G needle: one had lymphoma and the other sarcoidosis. Three patients were diagnosed with sarcoidosis using both the 19-G and 21- or 22-G needles. Conclusion EBUS-TBNA with a 19-G needle was useful for diagnosing retinal hemangioblastoma and tuberculous lymphadenitis as well as for PD-L1 testing after 21- and 22-G biopsies were unsuccessful.

Success of Crizotinib Combined with Whole-Brain Radiotherapy for Brain Metastases in a Patient with Anaplastic Lymphoma Kinase Rearrangement-Positive Non-Small-Cell Lung Cancer.

Although crizotinib shows marked antitumor activity in anaplastic lymphoma kinase (ALK) rearrangement-positive non-small-cell lung cancer (NSCLC) patients, all treated patients ultimately develop resistance to this drug. Isolated central nervous system failure without progression at extracranial sites is a common progression pattern in ALK rearrangement-positive NSCLC patients treated with crizotinib. Here, we report the success of crizotinib combined with whole-brain radiotherapy in an ALK rearrangement-positive NSCLC patient who developed leptomeningeal carcinomatosis and progression of multiple brain metastases. Additionally, we focused on the mechanism involved by examining the plasma and cerebrospinal fluid concentrations of crizotinib in the present case.