AKT and EZH2 inhibitors kill TNBCs by hijacking mechanisms of involution.

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype and has the highest rate of recurrence1. The predominant standard of care for advanced TNBC is systemic chemotherapy with or without immunotherapy; however, responses are typically short lived1,2. Thus, there is an urgent need to develop more effective treatments. Components of the PI3K pathway represent plausible therapeutic targets; more than 70% of TNBCs have alterations in PIK3CA, AKT1 or PTEN3-6. However, in contrast to hormone-receptor-positive tumours, it is still unclear whether or how triple-negative disease will respond to PI3K pathway inhibitors7. Here we describe a promising AKT-inhibitor-based therapeutic combination for TNBC. Specifically, we show that AKT inhibitors synergize with agents that suppress the histone methyltransferase EZH2 and promote robust tumour regression in multiple TNBC models in vivo. AKT and EZH2 inhibitors exert these effects by first cooperatively driving basal-like TNBC cells into a more differentiated, luminal-like state, which cannot be effectively induced by either agent alone. Once TNBCs are differentiated, these agents kill them by hijacking signals that normally drive mammary gland involution. Using a machine learning approach, we developed a classifier that can be used to predict sensitivity. Together, these findings identify a promising therapeutic strategy for this highly aggressive tumour type and illustrate how deregulated epigenetic enzymes can insulate tumours from oncogenic vulnerabilities. These studies also reveal how developmental tissue-specific cell death pathways may be co-opted for therapeutic benefit.

Immunostimulatory oncolytic activity of coxsackievirus A11 in human malignant pleural mesothelioma.

Malignant pleural mesothelioma (MPM) is an aggressive solid cancer with a poor prognosis, whereas coxsackievirus A11 (CVA11) is a potential oncolytic virus for cancer treatment. We here investigated the oncolytic activity of CVA11 with human MPM cell lines. CVA11 infection was cytotoxic in all six MPM cell lines examined and showed no or minimal cytotoxicity toward normal human normal cell lines. MPM cells with a higher surface level of intercellular adhesion molecule-1 (ICAM-1) expression tended to be more susceptible to CVA11-induced cytotoxicity, and a neutralizing antibody to ICAM-1 attenuated such cytotoxicity. CVA11 infection activated signaling by Akt and extracellular signal-regulated kinase (ERK) pathways, and inhibitors of such signaling also abrogated CVA11-mediated cytotoxicity. Furthermore, CVA11 infection-triggered multiple modes of tumor cell death including apoptosis, pyroptosis, and necroptosis, and such death was accompanied by the release or exposure of the proinflammatory cytokine interleukin-1ß and damage-associated molecular patterns such as calreticulin, high-mobility group box-1, annexin A1, and heat shock protein 70, which are hallmarks of immunogenic cell death. Notably, in vivo treatment of human MPM xenografts with intratumoral CVA11 injection resulted in significant suppression of tumor growth in SCID mice, and all mice infected with CVA11 showed no significant change in body weight. Our findings collectively suggest that the oncolytic activity of CVA11 for MPM is dependent on ICAM-1 as a virus receptor, as well as on Akt and ERK signaling, and that oncolytic virotherapy with CVA11 is a promising treatment modality with immunostimulatory activity for human MPM.

MicroRNA-326 negatively regulates CD155 expression in lung adenocarcinoma.

Treatment with immune checkpoint inhibitors induces a durable response in some patients with non-small-cell lung cancer, but eventually gives rise to drug resistance. Upregulation of CD155 expression is implicated as one mechanism of resistance to programmed death receptor-1 (PD-1)/PD-1 ligand (PD-L1) inhibitors, and it is therefore important to characterize the mechanisms underlying regulation of CD155 expression in tumor cells. The aim of this study was to identify microRNAs (miRNAs) that might regulate CD155 expression at the posttranscriptional level in lung cancer. Comprehensive miRNA screening with target prediction programs and a dual-luciferase reporter assay identified miR-346, miR-328-3p, miR-326, and miR-330-5p as miRNAs that bind to the 3'-UTR of CD155 mRNA. Forced expression of these miRNAs suppressed CD155 expression in lung cancer cell lines. Immunohistochemical staining of CD155 in tissue specimens from 57 patients with lung adenocarcinoma revealed the median tumor proportion score for CD155 to be 68%. The abundance of miR-326 in these specimens with a low level of CD155 expression was significantly greater than in specimens with a high level (p < 0.005). Our results thus suggest that miR-326 negatively regulates CD155 expression in lung adenocarcinoma and might therefore play a role in the development of resistance to PD-1/PD-L1 inhibitors.

[Kyushu Promotion Plan for the Platform of Human Resource Development for Cancer].

While it is necessary to provide a detailed cancer treatment according to patient's life stages which are childhood, adolescence and young adulthood, and senior years, there is shortage of human resources who can meet these needs because of few opportunities to train in clinical experience including rare cancers. In addition, since Kyushu has a lot of remote islands and remote areas, it is also important to take measures against elderly cancer patients in such places. On the other hand, the practical application of cancer genomic medicine is accelerating in Japan, however, there is an overwhelming shortage of medical staff who can appropriately apply genomic medicine clinically and who can contribute to the research development of this field. The purpose of"Kyushu Promotion Plan for the Platform of Human Resource Development for Cancer"is to promote the growth of the medical staff specialized for cancer genomic medicine, adolescent and young adult oncology, rare cancers, and measure for cancer depending on the patient's life stage. This plan is consisted with ten universities in Kyushu and we have carried out a variety of events along with these themes above by collaborating with each other for 5 years. These events include lectures, conferences, presentation of their researches, training at the medical center abroad or isolated island, the support system for trainees to get the specialized license for oncologist in Japan. We have run these events successfully and the response from the trainees belonging to this plan was satisfactory. I have confidence that the number of medical staffs who have learned cancer genomic medicine and the cancer treatments depending on the patient's life stage has increased through this project, and they will play an important role as the professional medical staff in the future. We are planning to continue these events even after the end of this plan to keep the number and quality of professional medical staff for cancer.

Longitudinal monitoring of somatic genetic alterations in circulating cell-free DNA during treatment with epidermal growth factor receptor-tyrosine kinase inhibitors.

BACKGROUND: The aim of this study was to evaluate the potential of liquid biopsy for prediction of the efficacy of epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) treatment and for assessment of the changes in genetic alterations during such treatment. METHODS: Plasma samples were prospectively collected from non-small cell lung cancer patients with EGFR-activating mutations during EGFR-TKI treatment until disease progression and were analyzed for such mutations with droplet digital polymerase chain reaction and for other somatic alterations with next-generation sequencing. RESULTS: One hundred patients, including 87 who were EGFR-TKI naïve, were enrolled. Median progression-free survival was significantly shorter for EGFR-TKI-naïve patients with EGFR-activating mutations detected in plasma at baseline than for those without them (7.9 vs 19.0 months; P < .001), with the values being significantly longer for initially positive patients who became negative for these mutations at 12 or 24 weeks than for those who remained positive. An increase in the number of alleles positive for EGFR-activating mutations in plasma during treatment was associated with disease progression, with a hazard ratio of 4.72 (95% CI, 2.07-10.79; P < .001) for EGFR-TKI-naïve patients showing an increase within 36 weeks. For 55 patients with available samples, the total number of somatic alterations (other than activating mutations or T790M of EGFR) in plasma was higher at disease progression than at baseline (33 vs 19; P = 0.04). CONCLUSION: Liquid biopsy shows potential for prediction of EGFR-TKI efficacy and elucidation of clonal tumor evolution during targeted therapy.

Genetic Profiling of Non-Small Cell Lung Cancer at Development of Resistance to First- or Second-Generation EGFR-TKIs by CAPP-Seq Analysis of Circulating Tumor DNA.

Patients with non-small cell lung cancer (NSCLC) treated with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) eventually acquire resistance to these drugs. The identification of various resistance mechanisms for determination of subsequent treatment for these patients will require a method for simultaneous detection of multiple genetic alterations with high sensitivity. We performed cancer personalized profiling by deep sequencing (CAPP-Seq) with circulating tumor DNA obtained from patients with NSCLC who acquired resistance to first- or second-generation EGFR-TKIs. Plasma samples from 27 patients were analyzed, and 24 samples underwent CAPP-Seq successfully. Original activating EGFR mutations were detected in 23 patients, with the remaining patient showing MET amplification. With regard to known mechanisms of EGFR-TKI resistance, the T790M mutation of EGFR was detected in 17 of the 24 patients, MET amplification in 9 patients (6 of whom also harbored T790M), ERBB2 amplification in 2 patients (1 of whom also harbored T790M), and EGFR amplification in 4 patients (all of whom harbored T790M). Our results thus show that CAPP-Seq is applicable to clinical samples for the identification of multiple somatic mutations in circulating tumor DNA obtained from patients with NSCLC at the time of disease progression during treatment with first- or second-generation EGFR-TKIs. Patients positive for the T790M mutation of EGFR were also found to constitute a molecularly heterogeneous population. KEY POINTS: CAPP-Seq is applicable to clinical samples for the identification of multiple somatic mutations.The T790M mutation of EGFR is associated with amplification of MET, ERBB2, or EGFR in NSCLC patients resistant to EGFR-TKIs.T790M-positive patients are molecularly heterogeneous, and genetic alterations coexisting with T790M may differ between patients treated with first-generation or second-generation EGFR-TKIs.

Interleukin-22 attenuates double-stranded RNA-induced upregulation of PD-L1 in airway epithelial cells via a STAT3-dependent mechanism.

Double-stranded RNA derived from viruses induces host immune responses. PD-L1, also known as B7-H1, is an immune-checkpoint molecule associated with the escape of viruses from host immune systems, which plays a role in the persistence of viral infection, resulting in exacerbations of underlying diseases such as asthma and chronic obstructive pulmonary disease. Interleukin (IL)-22 is produced from various immune cells and has protective properties on mucosal tissue. The binding of IL-22 to IL-22 receptor induces STAT3 activation. We investigated the effect of IL-22 on the expression in airway epithelial cells in vitro and in mouse lungs in vivo after the stimulation with an analog of viral double-stranded RNA, polyinosinic-polycytidylic acid (poly I:C). Stimulation with poly I:C upregulated PD-L1 expression on BEAS-2B cells. This upregulation of PD-L1 was attenuated by IL-22 administration. STAT3 phosphorylation was induced by IL-22 and poly I:C. Treatment of cells with STAT3 siRNA abolished the effect of IL-22 on the poly I:C-induced upregulation of PD-L1. This upregulation of PD-L1 was also attenuated by IL-11, a cytokine inducing STAT3 phosphorylation, in BEAS-2B cells. In mouse lung cells in vivo, IL-22 suppressed poly I:C-induced upregulation of PD-L1. These results suggest that IL-22 attenuates virus-induced upregulation of PD-L1 in airway epithelial cells via a STAT3-dependent mechanism.

TREX1 Inactivation Unleashes Cancer Cell STING-Interferon Signaling and Promotes Antitumor Immunity.

A substantial fraction of cancers evade immune detection by silencing Stimulator of Interferon Genes (STING)-Interferon (IFN) signaling. Therapeutic reactivation of this program via STING agonists, epigenetic, or DNA-damaging therapies can restore antitumor immunity in multiple preclinical models. Here we show that adaptive induction of three prime exonuclease 1 (TREX1) restrains STING-dependent nucleic acid sensing in cancer cells via its catalytic function in degrading cytosolic DNA. Cancer cell TREX1 expression is coordinately induced with STING by autocrine IFN and downstream STAT1, preventing signal amplification. TREX1 inactivation in cancer cells thus unleashes STING-IFN signaling, recruiting T and natural killer (NK) cells, sensitizing to NK cell-derived IFNγ, and cooperating with programmed cell death protein 1 blockade in multiple mouse tumor models to enhance immunogenicity. Targeting TREX1 may represent a complementary strategy to induce cytosolic DNA and amplify cancer cell STING-IFN signaling as a means to sensitize tumors to immune checkpoint blockade (ICB) and/or cell therapies. SIGNIFICANCE: STING-IFN signaling in cancer cells promotes tumor cell immunogenicity. Inactivation of the DNA exonuclease TREX1, which is adaptively upregulated to limit pathway activation in cancer cells, recruits immune effector cells and primes NK cell-mediated killing. Targeting TREX1 has substantial therapeutic potential to amplify cancer cell immunogenicity and overcome ICB resistance. This article is featured in Selected Articles from This Issue, p. 695.

Sensitivity and kinase activity of epidermal growth factor receptor (EGFR) exon 19 and others to EGFR-tyrosine kinase inhibitors.

The presence of epidermal growth factor receptor (EGFR) somatic mutations in non-small-cell lung cancer patients is associated with response to treatment with EGFR-tyrosine kinase inhibitors, such as gefitinib and erlotinib. More than 100 mutations in the kinase domain of EGFR have been identified. In particular there are many variations of deletion mutations in exon 19. In this study, using yellow fluorescent protein-tagged fragments of the EGFR intracellular domain, we examined the differences in sensitivity to gefitinib, erlotinib and afatinib between several exon 19 mutants and other common EGFR mutations. We also used serum of patients undergoing treatment with EGFR-tyrosine kinase inhibitors in this system. In addition, we examined the relative kinase activity of these mutants by measuring relative fluorescent intensity after immunofluorescence staining. We found that both sensitivity to EGFR-tyrosine kinase inhibitors and relative kinase activity differed among several EGFR mutations found in the same region of the kinase domain. This study underscores the importance of reporting the clinical outcome of treatment in relation to different EGFR mutations.

Regulation of PD-L1 expression in non-small cell lung cancer by interleukin-1ß.

Introduction: Programmed cell death-ligand 1 (PD-L1) is a biomarker for prediction of the clinical efficacy of immune checkpoint inhibitors in various cancer types. The role of cytokines in regulation of PD-L1 expression in tumor cells has not been fully characterized, however. Here we show that interleukin-1ß (IL-1ß) plays a key role in regulation of PD-L1 expression in non-small cell lung cancer (NSCLC). Methods: We performed comprehensive screening of cytokine gene expression in NSCLC tissue using available single-cell RNA-Sequence data. Then we examined the role of IL-1ß in vitro to elucidate its induction of PD-L1 on NSCLC cells. Results: The IL-1ß gene is highly expressed in the tumor microenvironment, particularly in macrophages. The combination of IL-1ß and interferon-γ (IFN-γ) induced a synergistic increase in PD-L1 expression in NSCLC cell lines. IL-1ß and IFN-γ also cooperatively activated mitogen-activated protein kinase (MAPK) signaling and promoted the binding of downstream transcription factors to the PD-L1 gene promoter. Furthermore, inhibitors of MAPK signaling blocked upregulation of PD-L1 by IL-1ß and IFN-γ. Discussion: Our study reports high levels of IL-1ß in the tumor microenvironment may cooperate with IFN-γ to induce maximal PD-L1 expression in tumor cells via activation of MAPK signaling, with the IL-1ß-MAPK axis being a promising therapeutic target for attenuation of PD-L1-mediated suppression of antitumor immunity.

Mutant forms of EGFR promote HER2 trafficking through efficient formation of HER2-EGFR heterodimers.

INTRODUCTION: Human epidermal growth factor receptor 2 (HER2) forms homodimers and is retained at the surface of cancer cells positive for HER2 amplification. The dimerization, internalization, and intracellular trafficking of HER2 in cancer cells without HER2 amplification have remained uncharacterized, however. MATERIALS AND METHODS: HER2 homodimers and heterodimers were detected in various cell lines with the use of an in situ proximity ligation assay. The effects of wild-type or mutant forms of epidermal growth factor receptor (EGFR) on intracellular trafficking of HER2 were examined by live-cell imaging. The sensitivity of cell lines without HER2 amplification to ado-trastuzumab emtansine (T-DM1), an anti-HER2 (trastuzumab)-cytotoxic drug conjugate (ADC) was also investigated. RESULTS: HER2 preferentially formed heterodimers with EGFR rather than homodimers and was rapidly internalized together with EGFR in cells without HER2 amplification. HER2-EGFR heterodimers were more abundant and HER2 was more efficiently transferred to lysosomes in such cells with than in those without EGFR activating mutations. T-DM1 showed a high cytotoxic efficacy in the cells with EGFR mutations, suggesting that mutant forms of EGFR promote the transfer of HER2-bound T-DM1 to lysosomes through efficient formation of HER2-EGFR heterodimers. CONCLUSION: Our findings reveal that HER2 trafficking is affected by EGFR, especially by mutant forms of the receptor, and they provide a rationale for the use of HER2-targeting ADCs in the treatment of EGFR-mutated lung cancer.

Docetaxel Plus Ramucirumab With Primary Prophylactic Pegylated Granulocyte-Colony Stimulating Factor Support for Elderly Patients With Advanced NSCLC: A Multicenter Prospective Single Arm Phase 2 Trial: DRAGON Study (WJOG9416L).

Introduction: Docetaxel plus ramucirumab could be a promising treatment for chemo-naive elderly patients with NSCLC, but high incidence of febrile neutropenia (FN) is a critical concern. We thus adopted a routine primary prophylactic pegylated-granulocyte-colony stimulating factor (PEG-G-CSF) to reduce FN and maximize the efficacy of docetaxel plus ramucirumab in elderly patients. Methods: This is a single arm phase 2 trial for chemo-naive elderly patients (aged ≥75 y) with advanced NSCLC. Docetaxel (60 mg/m2, d 1) plus ramucirumab (10 mg/kg, d 1) with PEG-G-CSF (3.6 mg, d 2) was administered every 3 weeks until progression. The primary end point was overall response rate (ORR) (expected ORR: 35%). Results: Between February 2018 and January 2021, 54 patients were enrolled. Median age was 78 (range: 75-86). A total of 21 (38.9%) partial response, 22 (40.7%) stable disease, nine (16.7%) progressive disease, and two (3.7%) not assessable were confirmed, resulting in ORR of 38.9% (90% confidence interval [CI]: 27.7%-51.0%) and disease control rate of 79.6%. Median progression-free survival and overall survival were 5.2 (95% CI: 4.2-6.9) and 12.7 (95% CI: 10.2-18.9) months, respectively. There were one (1.9%) FN, two (3.7%) bleeding grade greater than or equal to 3, and one (1.9%) treatment-related death (pneumonitis). Pneumonitis occurred in five patients (9.3%). Main adverse events grade greater than or equal to 3 were observed: four (7%) thrombocytopenia; three (5.6%) neutropenia; six (11.1%) hyposodium; five (9.3%) infection; five (9.3%) hypertension; four (7.4%) anorexia; and three (5.6%) oral mucositis. Conclusions: Docetaxel plus ramucirumab with PEG-G-CSF revealed efficacy and safety for chemo-naive elderly patients with NSCLC. Primary prophylactic PEG-G-CSF highly prevented FN.

Impact of increased plasma levels of calreticulin on prognosis of patients with advanced lung cancer undergoing combination treatment of chemotherapy and immune checkpoint inhibitors.

BACKGROUND: Damage-associated molecular pattern (DAMP)-related immunogenic cell death triggers secondary adaptive immune responses. The relationship between DAMP levels and prognosis in patients with non-small cell lung cancer (NSCLC) who undergo a combination therapy of immune checkpoint inhibitors (ICI) and chemotherapy remains unclear. METHODS: Serial plasma samples were prospectively collected from 45 patients treated with ICI combination therapy for advanced NSCLC. Plasma concentrations of high-mobility group box 1 (HMGB1), calreticulin (CRT), annexin A1, and heat shock protein 70 were measured. Associations between increases in plasma DAMP levels and the efficacy of the ICI combination therapy were evaluated. RESULTS: The maximum fold changes in plasma levels differed across individuals but demonstrated a marked increase, especially for CRT (mean ± SEM, 11.61 ± 46.15). Increased plasma DAMP levels were not clearly associated with clinical responses. There was a significant correlation between the maximum fold change in CRT levels and progression-free survival (PFS; r = 0.49, P < 0.001). Median PFS and overall survival (OS) rates were higher in patients with a ≥ 2-fold increase in plasma CRT levels than in those with a < 2-fold increase (PFS, 14.9 versus 6.0 months, hazard ratio (HR), 0.58; P = 0.17; OS, not reached versus 21.6 months, HR, 0.31, P = 0.02). CONCLUSIONS: Plasma CRT level monitoring has the potential to predict the efficacy of ICI combination therapy and shed light on the mechanisms underlying DAMP-related immunogenic cell death.

Association of thyroid transcription factor-1 (TTF-1) expression with efficacy of PD-1/PD-L1 inhibitors plus pemetrexed and platinum chemotherapy in advanced non-squamous non-small cell lung cancer.

Background: Thyroid transcription factor-1 (TTF-1) expression in advanced non-squamous non-small cell lung cancer (NSCLC) has been associated with the efficacy of pemetrexed plus platinum chemotherapy. However, the relation between TTF-1 expression and efficacy of the combination of programmed cell death 1 (PD-1)/programmed cell death ligand 1 (PD-L1) inhibitors plus pemetrexed and platinum chemotherapy, a standard first-line treatment regimen for advanced non-squamous NSCLC, has remained unclear. Methods: We retrospectively evaluated TTF-1 expression in tumor tissue of patients with advanced or recurrent non-squamous NSCLC treated with PD-1/PD-L1 inhibitors plus pemetrexed and platinum chemotherapy in the first-line setting. Clinical characteristics and pathological data for each patient were assessed, and progression-free survival (PFS) was evaluated. Bias due to patient background was minimized by application of inverse probability of treatment weighting (IPTW) analysis. Results: A total of 122 patients, 75 (61.5%) of whom were positive for TTF-1 immunostaining in tumor specimens, was included in this multicenter study. At the time of analysis, 89 (73.0%) patients had experienced progression events and 44 (36.1%) had died [median follow-up 14.6 months (range, 0.53-29.5 months)]. PFS was longer for TTF-1-positive patients than for TTF-1-negative patients [median, 12.2 vs. 6.0 months; hazard ratio (HR) =0.63 (95% CI: 0.37-1.06); log-rank P=0.028]. IPTW-adjusted PFS was significantly longer for TTF-1-positive than for TTF-1-negative patients [HR =0.62 (95% CI: 0.46-0.83); log-rank P=0.024]. Conclusions: TTF-1 expression in advanced non-squamous NSCLC can serve as a basis for prediction of PFS in patients treated with PD-1/PD-L1 inhibitors plus pemetrexed and platinum chemotherapy in the first-line setting.

Phase III Clinical Trial for the Combination of Erlotinib Plus Ramucirumab Compared With Osimertinib in Previously Untreated Advanced or Recurrent Non-Small Cell Lung Cancer Positive for the L858R Mutation of EGFR: REVOL858R (WJOG14420L).

INTRODUCTION: Osimertinib is a standard first-line treatment for non-small cell lung cancer (NSCLC) harboring mutations of the epidermal growth factor receptor gene (EGFR). However, tumors with the L858R mutation appear to be less sensitive to EGFR-tyrosine kinase inhibitors (TKIs) than those with exon-19 deletions, and subgroup analysis of the FLAURA study revealed that osimertinib did not significantly prolong overall survival (OS) compared with gefitinib or erlotinib in patients with the L858R. The RELAY study revealed a similar high efficacy of combination therapy with erlotinib plus ramucirumab (E+RAM) in patients with L858R and in those with exon-19 deletions. Patients who acquire the TKI resistance-associated T790M mutation during E+RAM treatment can also expect to receive benefit from second-line osimertinib. We have therefore planned a phase III study to evaluate the clinical efficacy of E+RAM compared with osimertinib monotherapy for untreated patients with advanced NSCLC harboring L858R. PATIENTS AND METHODS: A total of 230 patients will be enrolled. The primary end point is time to failure of strategy (TFS), which is defined for this study as the time from randomization of treatment until disease progression or death on osimertinib, or the time from randomization until first disease progression or death of the primary treatment when osimertinib is not administered in the E+RAM group. Secondary end points include OS and progression-free survival. CONCLUSION: This is the first phase III clinical trial to target only NSCLC patients with the L858R mutation. Its results may establish an optimal treatment for such individuals.

A Randomized Phase II Study Comparing Nivolumab with Carboplatin-Pemetrexed for EGFR-Mutated NSCLC with Resistance to EGFR Tyrosine Kinase Inhibitors (WJOG8515L).

PURPOSE: Although the efficacy of programmed cell death-1 (PD-1) blockade is generally poor for non-small cell lung cancer (NSCLC) with activating mutations of the epidermal growth factor receptor (EGFR) gene, EGFR tyrosine kinase inhibitors (TKIs) may improve the tumor immune microenvironment. We performed a randomized study to assess whether nivolumab improves outcome compared with chemotherapy in such patients previously treated with EGFR-TKIs. PATIENTS AND METHODS: Patients with EGFR-mutated NSCLC who acquired EGFR-TKI resistance not due to a secondary T790M mutation of EGFR were randomized 1:1 to nivolumab (n = 52) or carboplatin-pemetrexed (n = 50). The primary endpoint was progression-free survival (PFS). RESULTS: Median PFS and 1-year PFS probability were 1.7 months and 9.6% for nivolumab versus 5.6 months and 14.0% for carboplatin-pemetrexed [log-rank P < 001; hazard ratio (HR) of 1.92, with a 60% confidence interval (CI) of 1.61-2.29]. Overall survival was 20.7 and 19.9 months [HR, 0.88 (95% CI, 0.53-1.47)], and response rate was 9.6% and 36.0% for nivolumab and carboplatin-pemetrexed, respectively. No subgroup including patients with a high tumor mutation burden showed a substantially longer PFS with nivolumab than with carboplatin-pemetrexed. The T-cell-inflamed gene expression profile score (0.11 vs. -0.17, P = 0.036) and expression of genes related to cytotoxic T lymphocytes or their recruitment were higher in tumors that showed a benefit from nivolumab. CONCLUSIONS: Nivolumab did not confer a longer PFS compared with carboplatin-pemetrexed in the study patients. Gene expression profiling identified some cases with a favorable tumor immune microenvironment that was associated with nivolumab efficacy.

Quantification of HER family dimers by proximity ligation assay and its clinical evaluation in non-small cell lung cancer patients treated with osimertinib.

OBJECTIVES: The epidermal growth factor receptor (EGFR, also known as Her1) is a member of the human epidermal growth factor receptor (HER) family of proteins and a target of tyrosine kinase inhibitors (TKIs) in the treatment of non-small cell lung cancer (NSCLC) positive for activating mutations ofEGFR. Signal transduction by HER family proteins is dependent on their homo- or heterodimerization, but little is known of the relation between the relative proportions of such dimers of Her1 and sensitivity to EGFR-TKIs. We here investigated the feasibility of assessing this relation with the in situ proximity ligation assay (PLA) technique, which is able to detect the interaction of two proteins of interest when they are in close proximity. MATERIALS AND METHODS: In situ PLA was applied to detect Her1 homodimers and Her1 heterodimers in NSCLC cell lines and tissue specimens positive for EGFR activating mutations. RESULTS: In situ PLA allowed visualization and quantitative assessment of Her1 homodimers as well as of Her1 heterodimers with Her2, Her3, or Her4 not only in NSCLC cell lines but also in NSCLC tissue specimens obtained from various anatomic sites and by different collection methods. Treatment of NSCLC cell lines with EGFR-TKIs resulted in a decrease in the number of Her1 dimers, with the effect on homodimers being greater than that on heterodimers. A high ratio of Her1 heterodimers to homodimers was associated with poor progression-free survival in NSCLC patients treated with osimertinib. CONCLUSION: In situ PLA allows the detection of HER family dimers in NSCLC tissue, and quantitative assessment of Her1 homo- and heterodimers may prove informative for prediction of the response of NSCLC patients to EGFR-TKI treatment.

Cytotoxic chemotherapeutic agents and the EGFR-TKI osimertinib induce calreticulin exposure in non-small cell lung cancer.

OBJECTIVES: Synergistic anticancer efficacy of combination treatment with immune checkpoint inhibitors (ICIs) and platinum-based chemotherapy in patients with advanced non-small cell lung cancer (NSCLC) may be attributable in part to the phenomenon of immunogenic cell death (ICD), which is characterized by the release of damage-associated molecular patterns (DAMPs) from dying tumor cells. The ability of cytotoxic chemotherapeutic agents and molecularly targeted drugs such as epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) to induce DAMPs during the treatment of NSCLC has remained unclear, however. MATERIALS AND METHODS: We investigated the ability of seven cytotoxic chemotherapeutic agents and the third-generation EGFR-TKI osimertinib to induce translocation of the DAMP calreticulin to the cell surface in multiple NSCLC cell lines. The plasma concentration of soluble CRT in advanced NSCLC patients treated with cytotoxic chemotherapy or osimertinib was measured. RESULTS: Antimetabolites and microtubule inhibitors induced expression of CRT at the cell surface (ecto-CRT) to a greater extent than did platinum agents in six NSCLC cell lines, exhibiting higher up-regulation of phosphorylation of eukaryotic initiation factor-2α (eIF2α). Ecto-CRT expression was positively correlated with apoptosis induction in NSCLC cells treated with these various chemotherapeutic agents. The drug-induced up-regulation of ecto-CRT in NSCLC cells was attenuated by the pan-caspase inhibitor Z-VAD-FMK. Osimertinib similarly increased ecto-CRT expression in association with apoptosis induction in five EGFR-mutated NSCLC cell lines. Furthermore, the plasma concentration of soluble CRT in 16 NSCLC patients treated with single-agent pemetrexed or docetaxel and in nine EGFR-mutated NSCLC patients treated with osimertinib was increased after treatment onset. CONCLUSION: Our findings indicate that antimetabolites, microtubule inhibitors, and osimertinib are effective inducers both of CRT exposure in NSCLC cell lines and of soluble CRT release in patients with advanced NSCLC, suggesting that these agents might prove effective for promotion of antitumor immunity in combination immunotherapy.

Paired analysis of tumor mutation burden for lung adenocarcinoma and associated idiopathic pulmonary fibrosis.

Genetic alterations underlying the development of lung cancer in individuals with idiopathic pulmonary fibrosis (IPF) have remained unclear. To explore whether genetic alterations in IPF tissue contribute to the development of IPF-associated lung cancer, we here evaluated tumor mutation burden (TMB) and somatic variants in 14 paired IPF and tumor samples from patients with IPF-associated lung adenocarcinoma. We also determined TMB for 22 samples of lung adenocarcinoma from patients without IPF. TMB for IPF-associated lung adenocarcinoma was significantly higher than that for matched IPF tissue (median of 2.94 vs. 1.26 mutations/Mb, P = 0.002). Three and 102 somatic variants were detected in IPF and matched lung adenocarcinoma samples, respectively, with only one pair of specimens sharing one somatic variant. TMB for IPF-associated lung adenocarcinoma was similar to that for lung adenocarcinoma samples with driver mutations (median of 2.94 vs. 2.51 mutations/Mb) and lower than that for lung adenocarcinoma samples without known driver mutations (median of 2.94 vs. 5.03 mutations/Mb, P = 0.130) from patients without IPF. Our findings suggest that not only the accumulation of somatic mutations but other factors such as inflammation and oxidative stress might contribute to the development and progression of lung cancer in patients with IPF.

Increased plasma levels of damage-associated molecular patterns during systemic anticancer therapy in patients with advanced lung cancer.

BACKGROUND: Immunogenic cell death (ICD) characterized by the release of damage-associated molecular patterns (DAMPs) from dying cancer cells may contribute to the synergistic antitumor effect of cytotoxic chemotherapy combined with an immune checkpoint inhibitor. The kinetics of circulating DAMP levels in cancer patients have remained largely uncharacterized, however. METHODS: We evaluated the possible effects of various systemic anticancer therapy modalities on the kinetics of plasma DAMP concentrations in a prospective observational study of patients with advanced lung cancer. The plasma concentrations of high-mobility group box 1 (HMGB1), calreticulin (CRT), heat shock protein 70 (HSP70), annexin A1, and histone H3 were thus determined in 121 such patients at four time points during the first cycle of treatment. RESULTS: The mean of the maximum fold change in HMGB1, HSP70, or annexin A1 concentration observed during treatment was significantly greater than the corresponding baseline value (P<0.005). The maximum fold changes in HMGB1 and CRT concentrations tended to be associated with clinical response as evaluated by RECIST criteria, although the changes in the levels of these two DAMPs were not correlated, suggestive of differential induction mechanisms. Among the various treatment modalities administered, platinum-based combination or single-agent chemotherapy tended to elicit robust increases in the concentrations of HMGB1 and CRT. CONCLUSIONS: Serial monitoring of plasma revealed that systemic anticancer therapy increased the circulating levels of HMGB1 and CRT and that these changes tended to be associated with clinical response, suggesting that agents capable of releasing these DAMPs into plasma might induce ICD in advanced lung cancer patients.