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Abstract Background Actionable tumor genomic alterations, primarily EGFR mutations, occur in nearly 70% of Japanese advanced nonsquamous non‐small cell lung cancer (NSCLC) patients. Standard assessment of tumor tissue includes rapid testing for EGFR mutations, ALK fusions and ROS1 fusions. We conducted a prospective observational study (WJOG13620L) of follow‐on next‐generation sequencing of circulating tumor DNA (ctDNA) in patients without driver alterations after EGFR testing. Methods Patients with untreated advanced (Stage IIIB–IV or relapsed) nonsquamous NSCLC without EGFR mutations according to single‐plex testing of tumor tissue, were enrolled into this study. Patients with other known driver mutations or who underwent comprehensive genomic profiling were excluded. Plasma was analyzed by Guardant360, and the primary endpoint was the proportion of patients with pathogenic gene alterations in at least one of nine genes. Results Among the 72 patients enrolled, ALK and ROS1 fusions were tested in 86.1% and 65.2%, respectively. Alterations in pre‐defined genes were detected in 21 patients (29.2%; 95% confidence interval: 19.0–41.1, p

Neutrophil extracellular traps (NETs) released from neutrophils are related to cancer progression. However, the relationship between the therapeutic effects of immune checkpoint inhibitors (ICIs) such as anti-PD-1 and anti-PD-L1 antibodies and plasma NET concentration in patients with non-small cell lung cancer (NSCLC) is poorly understood. In this study, concentrations of citrullinated histone H3 (CitH3), a surrogate marker of NETs, in plasma before/after treatment were examined in patients with advanced or recurrent NSCLC undergoing ICI treatment (n = 185). The clinical significances of NET levels before/after treatment and posttreatment changes were statistically evaluated. As a result, multivariate Cox analysis showed that high NET levels before treatment were statistically significant predictors of unfavorable overall survival (OS; p < 0.001, HR 1.702, 95% CI 1.356–2.137) and progression-free survival (PFS; p < 0.001, HR 1.566, 95% CI 1.323–1.855). The Kaplan-Meier curves showed significant separation between the high- and low-NET groups in OS (p = 0.002) and PFS (p < 0.001). Additionally, high NET levels after treatment were also significantly associated with worse OS (p < 0.001) and PFS (p < 0.001) by multivariate Cox analysis. Notably, the pretreatment NET levels were significantly correlated with the plasma levels of NET-related inflammatory cytokines, such as IL-6 and IL-8, and with NET-related gene expression and immune-suppressive profile in peripheral blood mononuclear cells. Our findings suggest that NETs released from activated neutrophils might reduce the clinical efficacy of ICIs in patients with NSCLC.

Abstract Background The presence of cachexia negatively impacts the prognosis of patients with cancer. However, the mechanisms behind the development of cachexia and its prognostic impact on immunotherapy efficacy are not fully understood. Materials and Methods We retrospectively screened patients with advanced or recurrent non‐small cell lung cancer (NSCLC) who received PD‐1/PD‐L1 inhibitor monotherapy. Among 183 patients, pre‐treatment plasma samples were available from 100 patients. We defined cancer cachexia as weight loss of at least 5% during the past 6 months or weight loss of at least 2% and BMI

Abstract Background The presence of cachexia in cancer patients negatively affects the quality of life and survival. However, the impact of cachexia on immunotherapy, such as PD‐1/L1 inhibitors, is not fully understood. Therefore, we examined whether cancer cachexia affects the prognosis of patients with non‐small cell lung cancer (NSCLC) treated with PD‐1/PD‐L1 inhibitors. Methods We retrospectively screened patients with pathologically confirmed advanced or recurrent NSCLC who were treated with PD‐1/PD‐L1 monotherapy at Kurume University Hospital. We defined cancer cachexia as weight loss of at least 5% during the past 6 months or any degree of weight loss more than 2% and BMI

Introduction: Clinical roles of plasma IL-6 levels have been reported in patients with various cancers, including non-small cell lung cancer (NSCLC), treated with immune checkpoint inhibitors (ICIs). However, the roles of other IL-6 signaling components, soluble IL-6 receptor (sIL-6R) and soluble gp130 (sgp130), in the plasma have not been elucidated.Methods: Blood was collected from 106 patients with NSCLC before initiation of ICI treatment (anti-PD-1 or anti-PD-L1 antibody). Plasma levels of IL-6, sIL-6R, sgp130, and their complexes were assessed by Cox regression hazard model to evaluate their clinical significance. The clinical role of IL-6 or IL-6R genetic polymorphisms was also analyzed.Results: Cox regression analysis showed that higher plasma IL-6 levels significantly predicted unfavorable overall survival (OS; hazard ratio [HR] 1.34, 95% confidence interval [CI] 1.05–1.68, p = 0.012) in NSCLC patients treated with ICIs. However, plasma sIL-6R and sgp130 levels showed no prognostic significance (p = 0.882 and p = 0.934, respectively). In addition, the estimated concentrations of binary IL-6:sIL-6R and ternary IL-6:sIL-6R:sgp130 complexes and their ratios (binary/ternary complex) were not significantly associated with OS (p = 0.647, p = 0.727, and p = 0.273, respectively). Furthermore, the genetic polymorphisms of IL-6 (−634G>C) and IL-6R (48892A>C) showed no clinical role by Kaplan-Meier survival analysis (p = 0.908 and p = 0.639, respectively).Discussion: These findings demonstrated the clinical significance of plasma levels of IL-6, but not of other IL-6 signaling components, sIL-6R and sgp130, suggesting that classical IL-6 signaling, but not trans-signaling, may be related to anti-tumor immune responses in cancer patients treated with ICIs.

Introduction: Pulmonary sarcomatoid carcinoma (PSC) is a rare subtype of lung cancer associated with poor prognosis and resistance to conventional chemotherapy. Immune checkpoint inhibitors (ICIs), alone or in combination with chemotherapy, were found to have clinical benefits in PSC in recent studies. Nevertheless, because these studies included a small number of patients owing to disease rarity, larger studies are needed to evaluate the effectiveness and safety of ICI-based therapy for PSC. Methods: This multicenter retrospective study evaluated patients with ICI-naive advanced or metastatic PSC who were treated with ICI-based therapy at 25 hospitals in Japan. Results: A total of 124 patients were evaluated. The overall response rate, median progression-free survival (PFS), and median overall survival (OS) were 59.0%, 10.5 months, and 32.8 months, respectively. The PFS and OS rates at 24 months were 35.3% and 51.5%, respectively. Programmed death-ligand 1 expression, concomitant chemotherapy, and the treatment line were not significantly associated with PFS or OS. Immune-related adverse events (irAEs) were observed in 70 patients (56.5%), including 30 (24.2%) with grade 3 to 5 events. Patients with mild irAEs (grades 1–2) had longer PFS and OS than did those with severe (grades 3–5) or no irAEs. In a multivariate analysis, any-grade irAEs and the absence of liver metastases were independently associated with PFS, whereas any-grade irAEs and Eastern Cooperative Oncology Group performance status less than or equal to 1 were independently associated with OS. Conclusions: ICI-based therapy was found to have promising effectiveness in patients with advanced or metastatic PSC, regardless of programmed death-ligand 1 expression, concomitant chemotherapy, or treatment line.

Abstract Background Interstitial lung disease is frequently comorbid with dermatomyositis and has a poor prognosis, especially in patients with the anti-melanoma differentiation-associated gene 5 (MDA5) autoantibody. However, the pathogenesis of dermatomyositis-related interstitial lung disease remains unclear. Methods We examined 18 and 19 patients with dermatomyositis-related interstitial lung disease and idiopathic pulmonary fibrosis (control), respectively. Lung tissues obtained from these patients were semi-quantitatively evaluated by immunohistochemical staining with in-house anti-human MDA5 monoclonal antibodies, as well as anti-human immunoglobulin (Ig) G, IgM, IgA, and complement component 3(C3) antibodies. We established human MDA5 transgenic mice and treated them with rabbit anti-human MDA5 polyclonal antibodies, and evaluated lung injury and Ig and C3 expression. Results MDA5 was moderately or strongly expressed in the lungs of patients in both groups, with no significant differences between the groups. However, patients with dermatomyositis-related interstitial lung disease showed significantly stronger expression of C3 (p

Abstract Background Conquering acquired resistance to osimertinib remains a major challenge in treating patients with epidermal growth factor receptor (EGFR) mutation-positive non-small-cell lung cancer (NSCLC). Thus, we aimed to determine the safety and efficacy of combination treatment with osimertinib and afatinib for patients with acquired resistance to osimertinib. Methods This open-label phase I study was a feasibility study of the combination of afatinib and osimertinib for patients with advanced EGFR-positive NSCLC who had progressive disease after receiving osimertinib. The primary endpoint was to determine the maximum tolerated dose (MTD). We enrolled patients who received afatinib at three different dose levels (level 1, 20 mg; level 2, 30 mg; level 3, 40 mg) combined with osimertinib at a standard dose of 80 mg once per day. Results Thirteen patients were enrolled in this study. The MTD was defined as 30 mg afatinib when combined with daily oral administration of osimertinib (80 mg). The most frequent adverse events were diarrhea (76.9%), anemia (76.9%), and rash (69.2%). Considering the toxicity profiles during all treatment periods, the recommended oral dose of afatinib was determined as 20 mg daily, with an osimertinib dose of 80 mg. For all evaluable patients (n = 12), the response rate was 7.7% and the disease-control rate was 46.2%. Conclusion Combination therapy with osimertinib and afatinib was tolerable; however, the synergistic effect of afatinib with osimertinib may be limited in osimertinib-resistant patients. Trial registration Japan Registry of Clinical Trials ID: jRCTs051180008, registered date: 08/11/2018.

Abstract Background Interstitial lung abnormalities (ILAs) are known to be a risk of drug‐induced pneumonitis. However, there are few reports on the relationship between ILAs and immune checkpoint inhibitor‐related interstitial lung disease (ICI‐ILD). We retrospectively investigated the clinical significance of ILAs in patients with non‐small cell lung cancer (NSCLC) receiving ICIs. Methods We defined ILAs as nondependent abnormalities affecting more than 5% of any lung zone, including ground‐glass or diffuse centrilobular nodularities, traction bronchiectasis, honeycombing, and nonemphysematous cysts. Early‐onset ICI‐ILD was defined as developing within 3 months after the initiation of ICI administration. Results Of 264 patients with advanced NSCLC, 57 patients (21.6%) had ILAs (43 fibrotic and 14 nonfibrotic ILAs). The difference between the incidence of ICI‐ILD in patients with or without ILAs was not significant. Of 193 patients treated by ICI monotherapy, 18 (9.3%) developed early‐onset ICI‐ILD. Among patients receiving ICI monotherapy, the incidence of early‐onset ICI‐ILD was significantly higher in patients with than in patients without nonfibrotic ILAs. Conclusion The presence of nonfibrotic ILAs is a significant risk for early‐onset ICI‐ILD in patients with NSCLC undergoing ICI monotherapy. Clinicians should be aware of ILAs, especially nonfibrotic ILAs, before administering ICIs to lung cancer patients.

Abstract Cytokine release syndrome (CRS) is a systemic inflammatory disease caused by a variety of factors, including infections and certain drugs. A 70‐year‐old man who was diagnosed with a postoperative recurrence of lung adenocarcinoma received nivolumab, ipilimumab, pemetrexed and carboplatin every 3 weeks for two cycles followed by nivolumab and ipilimumab, which resulted in a partial response. Four days after the dose of nivolumab, the patient returned with diarrhea and fever. The patient was diagnosed with COVID‐19 infection accompanied by severe colitis. Although intensive care was performed, the patient suddenly went into cardiopulmonary arrest. Examination revealed an abnormally high interleukin‐6 level, suggesting CRS. This is the first report of a patient with CRS accompanied with COVID‐19 infection during treatment with ICIs. Cytokine release syndrome (CRS) is a systemic inflammatory disease caused by a variety of factors, including infections and certain drugs. Here, we report a case of non‐small cell lung cancer with CRS caused by COVID‐19 infection during treatment with nivolumab and ipilimumab. Fever is a common event in cancer patients, especially in COVID‐19‐infected patients, but when fever develops during cancer immunotherapy, CRS should always be kept in mind.

Background Immune checkpoint inhibitor (ICI) therapy has substantially improved the overall survival (OS) in patients with non-small-cell lung cancer (NSCLC); however, its response rate is still modest. In this study, we developed a machine learning-based platform, namely the Cytokine-based ICI Response Index (CIRI), to predict the ICI response of patients with NSCLC based on the peripheral blood cytokine profiles.Methods We enrolled 123 and 99 patients with NSCLC who received anti-PD-1/PD-L1 monotherapy or combined chemotherapy in the training and validation cohorts, respectively. The plasma concentrations of 93 cytokines were examined in the peripheral blood obtained from patients at baseline (pre) and 6 weeks after treatment (early during treatment: edt). Ensemble learning random survival forest classifiers were developed to select feature cytokines and predict the OS of patients undergoing ICI therapy.Results Fourteen and 19 cytokines at baseline and on treatment, respectively, were selected to generate CIRI models (namely preCIRI14 and edtCIRI19), both of which successfully identified patients with worse OS in two completely independent cohorts. At the population level, the prediction accuracies of preCIRI14 and edtCIRI19, as indicated by the concordance indices (C-indices), were 0.700 and 0.751 in the validation cohort, respectively. At the individual level, patients with higher CIRI scores demonstrated worse OS [hazard ratio (HR): 0.274 and 0.163, and p

Abstract Background Recently, the addition of antiprogrammed cell death‐ligand 1 (PD‐L1) monoclonal antibodies, including durvalumab and atezolizumab to platinum‐based chemotherapy, has demonstrated clinical benefits in patients with untreated advanced small cell lung cancer (SCLC). However, these clinical trials comprised small populations of elderly patients with SCLC. Therefore, the safety of anti‐PD‐L1 immunotherapy plus platinum and etoposide in elderly patients remains unclear. Methods This prospective, multicenter, single‐arm study was designed to evaluate the safety and efficacy of durvalumab plus carboplatin and etoposide in untreated elderly patients (aged > 75) with extensive stage (ES) SCLC. A total of 40 patients were recruited. Patients received up to four cycles of durvalumab 1500 mg and carboplatin at a dose equivalent to an area under the curve of 5 on day 1, and etoposide 80 mg/m2 on days 1 to 3 every 3 weeks as induction treatment, followed by durvalumab maintenance treatment every 4 weeks. The primary endpoint was safety as measured by adverse events according to the Common Terminology Criteria for Adverse Events version 5.0, laboratory analyses, vital signs, and physical examination. Key secondary endpoints were objective response rate, median progression‐free survival, 12‐month overall survival rate, and the completion rate for four cycles of induction chemotherapy. Discussion The present study was designed to evaluate the safety of durvalumab plus carboplatin and etoposide in elderly patients with ES‐SCLC.

Abstract Osimertinib, a third-generation EGFR-TKI, has nowadays been applied to non-small cell lung cancer harboring activated EGFR mutation with or without T790M, but ultimately develop resistance to this drug. Here we report a novel mechanism of acquired resistance to osimertinib and the reversal of which could improve the clinical outcomes. In osimertinib-resistant lung cancer cell lines harboring T790M mutation that we established, expression of multiple EGFR family proteins and MET was markedly reduced, whereas expression of AXL, CDCP1 and SRC was augmented along with activation of AKT. Surprisingly, AXL or CDCP1 expression was induced by osimertinib in a time-dependent manner up to 3 months. Silencing of CDCP1 or AXL restored the sensitivity to osimertinib with reduced activation of SRC and AKT. Furthermore, silencing of both CDCP1 and AXL increased the sensitivity to osimertinib. Either silencing of SRC or dasatinib, a SRC family kinase (SFK) inhibitor, suppressed AKT phosphorylation and cell growth. Increased expression of AXL and CDCP1 was observed in refractory tumor samples from patients with lung cancer treated with osimertinib. Together, this study suggests that AXL/SFK/AKT and CDCP1/SFK/AKT signaling pathways play some roles in acquired osimertinib resistance of non-small cell lung cancer.

Introduction: The use of immune checkpoint inhibitors (ICIs) with chemotherapy has increased the survival of patients with advanced NSCLC. Nevertheless, the efficacy of ICI treatment for NSCLC with EGFR mutations is limited. Previous studies have not evaluated the efficacy of ICI treatment after osimertinib treatment in real-world settings. Methods: This study performed a retrospective analysis of the association between clinical characteristics and ICI efficacy in patients with EGFR-mutant NSCLC treated with ICIs after osimertinib treatment at 12 institutions in Japan from March 2016 to March 2021. Results: Among 80 patients with EGFR-mutant lung cancer, 42 received ICI monotherapy and 38 received chemoimmunotherapy. In the chemoimmunotherapy group, the progression-free survival (PFS) was significantly longer in the group that exhibited PFS more than 10 months with osimertinib than in the group that exhibited PFS less than or equal to 10 months with osimertinib (8.4 mo versus 3.8 mo, p = 0.026). Nevertheless, there was no significant difference in PFS in the ICI monotherapy group (1.7 mo versus 1.5 mo, p = 0.45). Regardless of the EGFR mutation subtype, PFS of osimertinib treatment was a predictor of the PFS of chemoimmunotherapy (exon 19 deletion mutation: p = 0.03 and exon 21 L858R mutation: p = 0.001). Conclusions: The PFS of osimertinib might be a predictor of PFS of chemoimmunotherapy in patients with EGFR-mutant NSCLC. Further clinical investigations on the predictors of efficacy of administering ICIs after osimertinib treatment are required.

The WJOG8815L phase II clinical study involves patients with non‐small cell lung cancer (NSCLC) that harbored the EGFR T790M mutation, which confers resistance to EGFR tyrosine kinase inhibitors (TKIs). The purpose of this study was to assess the predictive value of monitoring EGFR genomic alterations in circulating tumor DNA (ctDNA) from patients with NSCLC that undergo treatment with the third‐generation EGFR‐TKI osimertinib. Plasma samples of 52 patients harboring the EGFR T790M mutation were obtained pretreatment (Pre), on day 1 of treatment cycle 4 (C4) or cycle 9 (C9), and at diagnosis of disease progression or treatment discontinuation (PD/stop). CtDNA was screened for EGFR‐TKI‐sensitizing mutations, the EGFR T790M mutation, and other genomic alterations using the cobas EGFR Mutation Test v2 (cobas), droplet digital PCR (ddPCR), and targeted deep sequencing. Analysis of the sensitizing—and T790M—EGFR mutant fractions (MFs) was used to determine tumor mutational burden. Both MFs were found to decrease during treatment, whereas rebound of the sensitizing EGFR MF was observed at PD/stop, suggesting that osimertinib targeted both T790M mutation‐positive tumors and tumors with sensitizing EGFR mutations. Significant differences in the response rates and progression‐free survival were observed between the sensitizing EGFR MF‐high and sensitizing EGFR MF‐low groups (cutoff: median) at C4. In conclusion, ctDNA monitoring for sensitizing EGFR mutations at C4 is suitable for predicting the treatment outcomes in NSCLC patients receiving osimertinib (Clinical Trial Registration No.: UMIN000022076).

Background CD73 induces the dephosphorylation of adenosine monophosphate converting it to adenosine, enabling malignancies to escape from immune surveillance. Although CD73 overexpression has been reported to be a poor prognostic factor in several malignancies including non‐small cell lung cancer (NSCLC), its predictive relevance in NSCLC patients receiving immune checkpoint inhibitors is unknown. The present research was conducted to investigate the prognostic significance of CD73 expression in NSCLC patients receiving immune checkpoint inhibitors (ICIs). Methods We screened 91 patients with advanced or recurrent NSCLC who received immune checkpoint inhibitors. CD73 expression was evaluated immunohistochemically using tissue specimens obtained just before treatment with ICIs. Results Analysis of progression‐free survival (PFS) and overall survival (OS) in relation to several levels of CD73 expression (1%, 10%, 30%, and 50%) showed that both tended to be more favorable as expression of CD73 increased. PFS and OS were longer for patients in whom at least 50% of the tumor cells expressed CD73 than for those in whom

Background Non‐small cell lung cancer (NSCLC) positive for activating mutations of the epidermal growth factor receptor (EGFR) gene is initially sensitive to EGFR‐tyrosine kinase inhibitors (TKIs) but eventually develops resistance to these drugs. Upregulation of the receptor tyrosine kinase AXL in tumor tissue has been detected in about one‐fifth of NSCLC patients with acquired resistance to EGFR‐TKIs. However, the clinical relevance of the levels of AXL and its ligand GAS6 in plasma remains unknown. Methods Tumor tissue and plasma specimens were collected from 25 EGFR‐mutated NSCLC patients before EGFR‐TKI treatment or after treatment failure. The levels of AXL and of GAS6 mRNA in tumor tissue were evaluated by immunohistochemistry and chromogenic in situ hybridization, respectively. The plasma concentrations of AXL and GAS6 were measured with enzyme‐linked immunosorbent assays. Results AXL expression was detected in three of 12 (25%) and nine of 19 (47%) tumor specimens obtained before and after EGFR‐TKI treatment, respectively. All tumor specimens assayed were positive for GAS6 mRNA. The median values for the plasma AXL concentration before and after EGFR TKI treatment were 1 635 and 1 460 pg/mL, respectively, and those for the plasma GAS6 concentration were 4 615 and 6 390 pg./mL, respectively. There was no significant correlation between the plasma levels of AXL or GAS6 and the corresponding expression levels in tumor tissue. Conclusion Plasma concentrations of AXL and GAS6 do not reflect tumor expression levels, and their measurement is thus not a viable alternative to direct analysis of tumor tissue in EGFR‐mutated NSCLC.

Summary: Background: The NEJ026 Phase 3 study demonstrated that erlotinib and bevacizumab (BE)-treated NSCLC patients with EGFR mutations had significantly better progression-free survival (PFS) than those treated with erlotinib alone (E). This study included a prospective analysis of the relationship between the mutational status of EGFR in plasma circulating tumor DNA (ctDNA) and the efficacy of TKI monotherapy or combination therapy. We describe these results herein. Methods: Plasma samples were collected from patients enrolled in NEJ026 at the start of treatment (P0), 6 weeks after the start of treatment (P1), and upon confirmation of progressive disease (P2). Plasma ctDNA was analyzed using a modified PNA-LNA PCR clamp method. PFS and OS according to EGFR status at the time of plasma collection were evaluated. Findings: Plasma activating EGFR mutation (aEGFR) at P0 was detected in 68% of cases; patients without plasma aEGFR had longer PFS. The frequency of T790M mutation at P2 was similar in both arms: 8 (19.0%) in BE and 11 (20.8%) in E. Based on the aEGFR profiles, PFS was evaluated among three groups: type A [P0(-), P1(-)], type B [P0(+), P1(-)], and type C [P0(+), P1(+)]. This revealed that BE was more efficacious than E, and that BE was associated with improved PFS in all types. Interpretation: Pre-treatment plasma aEGFR status have a potential of early predictor of response of TKI efficacy. Monitoring plasma aEGFR mutation will contribute to selection and continuation of treatment with BE or E. Funding: Chugai Pharmaceutical.

We sought to identify peripheral blood markers associated with two immune-related factors-programmed cell death-ligand-2 (PD-L2) and indoleamine 2,3-dioxygenase-1 (IDO1)-that are expressed on tumor cells in primary lung adenocarcinoma (AD) specimens. We randomly selected 448 patients (70%) from 640 consecutive patients with resected stage I-III primary lung AD, who had been treated at that point with surgery alone. Expression of PD-L2 and IDO1 in these patients was assessed by immunohistochemistry, and evaluated with respect to peripheral blood markers measured before surgery, including white blood cells, absolute neutrophil count, absolute lymphocyte count, absolute monocyte count (AMC), absolute eosinophil count (AEC), serum C-reactive protein, and serum lactate dehydrogenase levels. Membrane PD-L2 expression and cytoplasmic IDO1 expression were defined by tumor proportion score (TPS); samples with TPS < 1% were considered negative. Logistic regression models were used to identify variables associated with the immune-related factors. Advanced stage (P = 0.0090), higher AMC (P = 0.0195), and higher AEC (P = 0.0015) were independent predictors of IDO1 expression. PD-L2 expression was not associated with any tested peripheral blood markers. Peripheral blood markers, especially AMC and AEC, could potential predict IDO1 expression in lung AD. This study should be replicated in another cohort; further efforts to explore other biomarkers that predict PD-L2 or IDO1 expression are also warranted.

Epidermal growth factor receptor (EGFR) is a prototypic cell-surface receptor belonging to the ErbB/HER onocogene family. Overexpression or somatic mutations of EGFR have been reported to play an important role in tumorigenesis in various types of epithelial cancers. Therefore, targeting of EGFR with specific blocking antibodies or inhibitors have been developing for treatment for EGFR-associated tumors. Immune responses to HER2, another molecule of the ErbB/HER onocogene family, have been well studied, but only limited information on the immune responses to EGFR in cancer has been currently available. In this review, we have summarized the available data and discussed potential clinical importance of the anti-EGFR immune responses and EGFR-mediated immune regulation in cancer. Several lines of evidence suggest that cellular and humoral immune responses to EGFR might be useful as a marker and/or target for cancer therapy against EGFR-associated tumors. In addition, recent studies suggest the critical roles of EGFR-mediated signaling in regulation of expression of an immune checkpoint molecule, programmed death-ligand 1 (PD-L1) in tumor cells. Further studies are warranted to clarify the impact of the anti-EGFR immune responses and EGFR-mediated immunomodulation for clinical application for cancer treatment.

Somatic mutations in the epidermal growth factor receptor (EGFR) gene are associated with clinical response to EGFR tyrosine kinase inhibitors (TKIs), such as gefitinib, in patients with non-small cell lung cancer (NSCLC). However, humoral immune responses to EGFR in NSCLC patients have not been well studied. In this study, we investigated the clinical significance of immunoglobulin G (IgG) responses to EGFR-derived peptides in NSCLC patients receiving gefitinib. Plasma IgG titers to each of 60 different EGFR-derived 20-mer peptides were measured by the Luminex system in 42 NSCLC patients receiving gefitinib therapy. The relationships between the peptide-specific IgG titers and presence of EGFR mutations or patient survival were evaluated statistically. IgG titers against the egfr_481-500, egfr_721-740, and egfr_741-760 peptides were significantly higher in patients with exon 21 mutation than in those without it. On the other hand, IgG titers against the egfr_841-860 and egfr_1001-1020 peptides were significantly lower and higher, respectively, in patients with deletion in exon 19. Multivariate Cox regression analysis showed that IgG responses to egfr_41_ 60, egfr_61_80 and egfr_481_500 were significantly prognostic for progression-free survival independent of other clinicopathological characteristics, whereas those to the egfr_41_60 and egfr_481_500 peptides were significantly prognostic for overall survival. Detection of IgG responses to EGFR-derived peptides may be a promising method for prognostication of NSCLC patients receiving gefitinib. Our results may provide new insight for better understanding of humoral responses to EGFR in NSCLC patients.

Tumors formed by a highly metastatic human lung cancer cell line are characterized by activated signaling via vascular endothelial growth factor (VEGF)-C through its receptor (VEGFR-3) and aggressive lymph node metastasis. In this study, we examined how these highly metastatic cancers acquired aggressive lymph node metastasis. Compared with their lower metastatic counterparts, the highly metastatic tumors formed by this cell line expressed higher amounts of interleukin (IL)-1α, with similarly augmented expression of IL-1α and IL-1β by tumor stromal cells and of VEGF-A and VEGF-C by tumor-associated macrophages. These tumor-associated macrophages were mainly of the M2 type. Administration of a macrophage-targeting drug suppressed the production of these potent angiogenic and lymphangiogenic factors, resulting in decreased tumor growth, angiogenesis, lymphangiogenesis, and lymph node metastasis. In Matrigel plug assays, the highly metastatic cells formed tumors that were extensively infiltrated by M2-type macrophages and exhibited enhanced angiogenesis and lymphangiogenesis. All of these responses were suppressed by the IL-1 receptor (IL-1R) antagonist anakinra. Thus, the IL-1α-driven inflammatory activation of angiogenesis and lymphangiogenesis seems to provide a highly metastatic tumor microenvironment favorable for lymph node metastasis through cross-talk with macrophages. Accordingly, the IL-1R/M2-type macrophage axis may be a good therapeutic target for patients with this form of lung cancer.

IL-18 plays a key role in the pathogenesis of pulmonary inflammatory diseases including pulmonary infection, pulmonary fibrosis, lung injury and chronic obstructive pulmonary disease (COPD). However, it is unknown whether IL-18 plays any role in the pathogenesis of asthma. We hypothesized that overexpression of mature IL-18 protein in the lungs may exacerbate disease activities of asthma. We established lung-specific IL-18 transgenic mice on a Balb/c genetic background. Female mice sensitized- and challenged- with antigen (ovalbumin) were used as a mouse asthma model. Pulmonary inflammation and emphysema were not observed in the lungs of naïve transgenic mice. However, airway hyperresponsiveness and airway inflammatory cells accompanied with CD4(+) T cells, CD8(+) T cells, eosinophils, neutrophils, and macrophages were significantly increased in ovalbumin-sensitized and challenged transgenic mice, as compared to wild type Balb/c mice. We also demonstrate that IL-18 induces IFN-γ, IL-13, and eotaxin in the lungs of ovalbumin-sensitized and challenged transgenic mice along with an increase in IL-13 producing CD4(+) T cells. Treatment with anti-CD4 monoclonal antibody or deletion of the IL-13 gene improves ovalbumin-induced airway hyperresponsiveness and reduces airway inflammatory cells in transgenic mice. Overexpressing the IL-18 protein in the lungs induces type 1 and type 2 cytokines and airway inflammation, and results in increasing airway hyperresponsiveness via CD4(+) T cells and IL-13 in asthma.

Treatment with epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), such as gefitinib and erlotinib, has achieved high clinical response rates in patients with non-small cell lung cancers (NSCLCs). However, over time, most tumors develop acquired resistance to EGFR-TKIs, which is associated with the secondary EGFR T790M resistance mutation in about half the cases. Currently there are no effective treatment options for patients with this resistance mutation. Here we identified two novel HLA-A*0201 (A2)-restricted T cell epitopes containing the mutated methionine residue of the EGFR T790M mutation, T790M-5 (MQLMPFGCLL) and T790M-7 (LIMQLMPFGCL), as potential targets for EGFR-TKI-resistant patients. When peripheral blood cells were repeatedly stimulated in vitro with these two peptides and assessed by antigen-specific IFN-γ secretion, T cell lines responsive to T790M-5 and T790M-7 were established in 5 of 6 (83%) and 3 of 6 (50%) healthy donors, respectively. Additionally, the T790M-5- and T790M-7-specific T cell lines displayed an MHC class I-restricted reactivity against NSCLC cell lines expressing both HLA-A2 and the T790M mutation. Interestingly, the NSCLC patients with antigen-specific T cell responses to these epitopes showed a significantly less frequency of EGFR-T790M mutation than those without them [1 of 7 (14%) vs 9 of 15 (60%); chi-squared test, p = 0.0449], indicating the negative correlation between the immune responses to the EGFR-T790M-derived epitopes and the presence of EGFR-T790M mutation in NSCLC patients. This finding could possibly be explained by the hypothesis that immune responses to the mutated neo-antigens derived from T790M might prevent the emergence of tumor cell variants with the T790M resistance mutation in NSCLC patients during EGFR-TKI treatment. Together, our results suggest that the identified T cell epitopes might provide a novel immunotherapeutic approach for prevention and/or treatment of EGFR-TKI resistance with the secondary EGFR T790M resistance mutation in NSCLC patients.

BACKGROUND: In order to improve the outcome of patients with non-small cell lung cancer (NSCLC), a biomarker that can predict the efficacy of chemotherapy is needed. The aim of this study was to assess the role of EGFR mutations and ERCC1 in predicting the efficacy of platinum-based chemotherapy and the outcome of patients with NSCLC. METHODS: We conducted a retrospective study to analyze the relationships between EGFR mutations or ERCC1 expression and progression-free survival (PFS) in patients with NSCLC who received platinum-based chemotherapy. EGFR mutation status was determined using the peptide nucleic acid-locked nucleic acid polymerase chain reaction clamp method, and immunohistochemistry was used to examine the expression of ERCC1 in tumor samples obtained from the patients. RESULTS: Among the NSCLC patients who received platinum-based chemotherapy, the median PFS was significantly better in those who had never smoked and those with exon 19 deletion, and the median overall survival (OS) was significantly better in those who had never smoked, those with exon 19 deletion, and women. Cox regression analysis revealed that exon 19 deletion and having never smoked were significantly associated with both PFS and OS. Subset analysis revealed a significant correlation between ERCC1 expression and EGFR mutation, and ERCC1-negative patients with exon 19 deletion had a longer PFS than the other patients; ERCC1-positive patients without exon 19 deletion had a shorter PFS than the other patients. CONCLUSIONS: Our results indicate that among NSCLC patients receiving platinum-based chemotherapy, those with exon 19 deletion have a longer PFS and OS. Our findings suggest that platinum-based chemotherapy is more effective against ERCC1-negative and exon 19-positive NSCLC.

Non-small-cell lung cancer harboring epidermal growth factor receptor (EGFR) mutations attains a meaningful response to EGFR-tyrosine kinase inhibitors (TKIs). However, acquired resistance to EGFR-TKIs could affect long-term outcome in almost all patients. To identify the potential mechanisms of resistance, we established cell lines resistant to EGFR-TKIs from the human lung cancer cell lines PC9 and11-18, which harbored activating EGFR mutations. One erlotinib-resistant cell line from PC9 and two erlotinib-resistant cell lines and two gefitinib-resistant cell lines from 11-18 were independently established. Almost complete loss of mutant delE746-A750 EGFR gene was observed in the erlotinib-resistant cells isolated from PC9, and partial loss of the mutant L858R EGFR gene copy was specifically observed in the erlotinib- and gefitinib-resistant cells from 11-18. However, constitutive activation of EGFR downstream signaling, PI3K/Akt, was observed even after loss of the mutated EGFR gene in all resistant cell lines even in the presence of the drug. In the erlotinib-resistant cells from PC9, constitutive PI3K/Akt activation was effectively inhibited by lapatinib (a dual TKI of EGFR and HER2) or BIBW2992 (pan-TKI of EGFR family proteins). Furthermore, erlotinib with either HER2 or HER3 knockdown by their cognate siRNAs also inhibited PI3K/Akt activation. Transfection of activating mutant EGFR complementary DNA restored drug sensitivity in the erlotinib-resistant cell line. Our study indicates that loss of addiction to mutant EGFR resulted in gain of addiction to both HER2/HER3 and PI3K/Akt signaling to acquire EGFR-TKI resistance.