Clinical Features of Patients with an Epidermal Growth Factor Receptor T790M Mutation Detected in Circulating Tumor DNA.

BACKGROUND: Osimertinib, a third-generation epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI), is effective against EGFR-mutated non-small cell lung carcinoma resistant to first- or second-generation EGFR-TKIs in patients in whom an EGFR T790M mutation has been detected. Detection of the T790M mutation using circulating tumor DNA (ctDNA) is less invasive than a tissue re-biopsy, including a transbronchial lung biopsy; however, the prognostic implications of the T790M mutation in ctDNA have not been fully elucidated. METHODS: We retrospectively reviewed the clinical features of non-small cell lung carcinoma patients in whom an EGFR T790M mutation had been detected at our hospital and assessed the clinical outcomes of osimertinib for these patients in terms of detection sites. RESULTS: An EGFR T790M mutation was detected in 32 non-small cell lung carcinoma patients, of whom 21 (65.6%) underwent osimertinib treatment after detection of the mutation. The mutation was detected using plasma samples in 10 patients (47.6%; liquid biopsy group), while it was detected using tissue samples in 11 patients (52.4%; tissue biopsy group). Liver and bone metastases were more frequently observed in patients in the liquid biopsy group than in the tissue biopsy group (30.0 vs. 0% and 60.0 vs. 18.2%, respectively). The median progression-free survival time was significantly shorter in the liquid biopsy group (132.0 days) than in the tissue biopsy group (682.0 days). The median overall survival time in the liquid biopsy group was 376.0 days, whereas that in the tissue biopsy group was not reached during our observation period. CONCLUSIONS: Non-small cell lung carcinoma patients in whom an EGFR T790M mutation was detected in plasma samples demonstrated a poorer response to osimertinib than those in whom the mutation was detected in tissue specimens.

Altered editing level of microRNAs is a potential biomarker in lung adenocarcinoma.

Adenosine-to-inosine (A-to-I) microRNA editing is associated with tumor phenotypes in various cancer types. Recent analyses of The Cancer Genome Atlas (TCGA) dataset have shown several microRNAs that undergo A-to-I editing in human cancers, some of which have been reported to be associated with prognosis. Herein, we examined published small RNA deep sequencing data of 74 cases of lung adenocarcinoma (AD) and the corresponding normal counterpart (NC) specimen in silico in order to identify A-to-I microRNA editing events. Editing levels of miR-379-5p, miR-99a-5p, and miR-497-5p were lower in AD than in NC and, in a large number of cases, the editing level of miR-200b-3p was higher in AD than in NC. Difference in the editing level between AD and NC was largest for miR-99a-5p. Then, we examined the editing level of miR-99a-5p in 50 surgically resected lung adenocarcinoma cases at our institution by a conventional sequence-based method, and its association with clinical outcomes. The editing level of miR-99a-5p was significantly lower in 19 cases of AD (38%) than in corresponding NC. These cases showed a shorter overall survival as assessed using the log-rank test (P = .047). This trend was consistent with previous analyses of TCGA dataset. The altered editing level of microRNAs in lung adenocarcinoma could serve as a potential biomarker.

Glutamate-cysteine ligase catalytic subunit is associated with cisplatin resistance in lung adenocarcinoma.

BACKGROUND: Cisplatin is a key drug for treating lung adenocarcinoma, and its sensitivity to cisplatin is directly related to prognosis. We aimed to reveal the roles of genes related to glutathione synthesis (glutamate-cysteine ligase catalytic subunit, GCLC) and cystine uptake (cystine/glutamate transporter, xCT and CD44v8-10) in cisplatin resistance and prognosis in lung adenocarcinoma. METHODS: We established cell lines stably expressing GCLC, xCT, standard isoform of CD44, and CD44v8-10, and investigated their sensitivities to cisplatin. We also measured mRNA expression levels of these genes in the tumor tissues from 92 lung adenocarcinoma patients. Patients were divided into high-expression (upper quartile, N = 23) and low-expression groups (remaining patients, N = 69). Recurrence-free survival, overall survival (N = 92), and post-recurrence survival (N = 22) were selected as endpoints. RESULTS: Compared with the control green fluorescent protein-expressing cell line (inhibitory concentration 50:6.9 µM), all the stable cell lines were more resistant to cisplatin (12.9 µM, P = 0.025; 13.9 µM, P = 0.028; 26.7 µM, P = 0.001; 17.7 µM, P = 0.008, respectively). In contrast, there was no significant difference in recurrence-free or overall survival between the high- and low-expression groups for any of the genes. However, high expression of GCLC was a risk factor for poorer post-recurrence survival (hazard ratio, 6.26; 95% confidence interval, 1.37-28.7; P = 0.018). CONCLUSION: High expression levels of genes related to glutathione synthesis and cystine uptake promote cisplatin resistance in lung adenocarcinoma cell lines. High expression of GCLC in tumor tissue may be a potential predictor of treatment failure.

High expression of IRE1 in lung adenocarcinoma is associated with a lower rate of recurrence.

OBJECTIVE: Recent reports have shown that endoplasmic reticulum stress is associated with cancer. However, the impacts of endoplasmic reticulum stress on the prognosis of lung cancer are unknown. Therefore, in this study, we sought to reveal the relationship between the expression of endoplasmic reticulum stress-related genes (endoplasmic reticulum oxidoreductase 1L, protein kinase RNA-like endoplasmic reticulum kinase, activating transcription factor 6 and inositol-requiring kinase 1) and the outcome of lung adenocarcinoma. METHODS: One hundred and twenty-six patients with surgically resected lung adenocarcinomas were subjected to an endoplasmic reticulum stress-related mRNA expression analysis using quantitative RT-PCR. The following parameters were analyzed for all the study patients: age, sex, disease stage, smoking status, lymph node invasion (ly), vascular invasion (v) and EGFR mutation status. We assigned patients to either a high-expression group or a low-expression group according to the expression levels of endoplasmic reticulum stress-related genes. RESULTS: High expressions of endoplasmic reticulum stress-related genes were observed in patients with lower stages of lung adenocarcinoma and minimal vascular invasion. A Kaplan-Meier analysis showed significant differences in recurrence-free survival and overall survival between high-expression group and low-expression group. High inositol-requiring kinase 1 expression was an independent predictor of recurrence-free survival among patients with lung adenocarcinoma (hazard ratio, 0.396; 95% confidence interval, 0.188-0.834; P = 0.015). CONCLUSIONS: Inositol-requiring kinase 1 may be a useful biomarker to predict recurrence in surgically resected lung adenocarcinoma patients.

Oncogenic TPM3-ALK activation requires dimerization through the coiled-coil structure of TPM3.

Inflammatory myofibroblastic tumor (IMT) is a mesenchymal tumor that can arise from anywhere in the body. Anaplastic lymphoma kinase (ALK) gene rearrangements, most often resulting in the tropomyosin 3 (TPM3)-ALK fusion gene, are the main causes of IMT. However, the mechanism of malignant transformation in IMT has yet to be elucidated. The purpose of this study was to clarify the role of the TPM3 region in the transformation of IMT via TPM3-ALK. Lentivirus vectors containing a TPM3-ALK fusion gene lacking various lengths of TPM3 were constructed and expressed in HEK293T and NIH3T3 cell lines. Focus formation assay revealed loss of contact inhibition in NIH3T3 cells transfected with full-length TPM3-ALK, but not with ALK alone. Blue-native polyacrylamide gel electrophoresis (BN-PAGE) revealed that TPM3-ALK dimerization increased in proportion to the length of TPM3. Western blot showed phosphorylation of ALK, ERK1/2, and STAT3 in HEK293T cells transfected with TPM3-ALK. Thus, the coiled-coil structure of TPM3 contributes to the transforming ability of the TPM3-ALK fusion protein, and longer TPM3 region leads to higher dimer formation.

Plasma homocysteine levels and hematological toxicity in NSCLC patients after the first cycle of pemetrexed under folate supplementation.

Although baseline plasma homocysteine levels are related to pemetrexed toxicities in patients treated without folate supplementation, the relationship between these parameters in patients treated with folate supplementation is not well understood. The pretreatment plasma homocysteine levels were measured in non-small-cell lung cancer patients treated with pemetrexed alone under folate supplementation. Pemetrexed (500 mg/m) was administered every 3 weeks. As folate supplementation, folic acid (0.5 mg) was orally administered daily and vitamin B12 (1 mg) was injected intramuscularly every 9 weeks starting at least 1 week before treatment. The rate of toxicities during the first cycle of pemetrexed treatment with folate supplementations was evaluated and the relationship between the plasma homocysteine levels and toxicities was examined. Between June 2009 and November 2010, 58 patients were enrolled in this study. The median pretreatment plasma homocysteine level was 7.7 µmol/ml (3.5-34.6 µmol/ml). The pretreatment plasma homocysteine levels were above 11.5 µmol/ml in nine patients (15.5%). The pretreatment plasma homocysteine level correlated significantly with the nadir of the absolute counts of leukocytes, neutrophils, and thrombocytes (r = -0.374, P = 0.004; r = -0.286, P = 0.028; r = -0.324, P = 0.012, respectively). In addition, the rates of decrease in leukocytes, neutrophils, and thrombocytes correlated significantly with the pretreatment plasma homocysteine level (r = +0.378, P = 0.003; r = +0.335, P = 0.009; r = +0.363, P = 0.005, respectively). The plasma homocysteine level is associated with hematological toxicities in patients receiving pemetrexed with folate supplementation.

Detection of epidermal growth factor receptor T790M mutation in plasma DNA from patients refractory to epidermal growth factor receptor tyrosine kinase inhibitor.

A secondary epidermal growth factor receptor (EGFR) mutation, the substitution of threonine 790 with methionine (T790M), leads to acquired resistance to reversible EGFR-tyrosine kinase inhibitors (EGFR-TKIs). A non-invasive method for detecting T790M mutation would be desirable to direct patient treatment strategy. Plasma DNA samples were obtained after discontinuation of gefitinib or erlotinib in 75 patients with non-small cell lung cancer (NSCLC). T790M mutation was amplified using the SABER (single allele base extension reaction) technique and analyzed using the Sequenom MassARRAY platform. We examined the T790M mutation status in plasma samples obtained after treatment with an EGFR-TKI. The SABER assay sensitivity using mixed oligonucleotides was determined to be 0.3%. The T790M mutation was detected in 21 of the 75 plasma samples (28%). The presence of the T790M mutation was confirmed by subcloning into sequencing vectors and sequencing in 14 of the 21 samples (66.6%). In this cohort of 75 patients, the median progression-free survival (PFS) of the patients with the T790M mutation (n = 21) was not statistically different from that of the patients without the mutation (n = 54, P = 0.94). When patients under 65 years of age who had a partial response were grouped according to their plasma T790M mutation status, the PFS of the T790M-positive patients (n = 11) was significantly shorter than that of the T790M-negative patients (n = 29, P = 0.03). The SABER method is a feasible means of determining the plasma T790M mutation status and could potentially be used to monitor EGFR-TKI therapy.

Subsequent treatment for locally advanced non-small-cell lung cancer that progressed after definitive chemoradiotherapy and consolidation therapy with durvalumab: a multicenter retrospective analysis (TOPGAN 2021-02).

PURPOSE: For patients with locally advanced non-small-cell lung cancer (LA-NSCLC) that progressed after definitive chemoradiotherapy (CRT) and durvalumab consolidation therapy, no subsequent standard treatment exists. The type of treatment selected for each timing of disease progression and its efficacy have not been investigated. METHODS: We retrospectively enrolled patients with LA-NSCLC or inoperable NSCLC that progressed after definitive CRT and durvalumab consolidation therapy at 15 Japanese institutions. Patients were classified into the following: Early Discontinuation group (disease progression within 6 months after durvalumab initiation), Late Discontinuation group (disease progression from 7 to 12 months after durvalumab initiation), and Accomplishment group (disease progression from 12 months after durvalumab initiation). RESULTS: Altogether, 127 patients were analyzed, including 50 (39.4%), 42 (33.1%) and 35 (27.5%) patients from the Early Discontinuation, Late Discontinuation, and Accomplishment groups, respectively. Subsequent treatments were Platinum plus immune checkpoint inhibitors (ICI) in 18 (14.2%), ICI in 7 (5.5%), Platinum in 59 (46.4%), Non-Platinum in 35 (27.6%), and tyrosine kinase inhibitor in 8 (6.3%) patients. In the Early Discontinuation, Late Discontinuation, and Accomplishment groups, 4 (8.0%), 7 (16.7%), and 7 (20.0%) patients were receiving Platinum plus ICI; 21 (42.0%), 22 (52.4%), and 16 (45.7%) were receiving Platinum, and 20 (40.0%), 8 (19.0%), and 7 (20.0%) were receiving Non-Platinum, respectively. No significant difference in progression-free survival was observed in the timing of disease progression. CONCLUSION: In patients with LA-NSCLC hat progressed after definitive CRT and durvalumab consolidation therapy, subsequent treatment may change depending on the timing of disease progression.

Clinical efficacy of amrubicin in patients with small cell lung cancer relapse after first-line treatment including immune checkpoint inhibitors: A retrospective multicenter study (TOPGAN 2021-01).

BACKGROUND: The therapeutic efficacy of cytotoxic anticancer drugs has been reported to be enhanced after immune checkpoint inhibitors (ICI) in non-small cell lung cancer; however, it is unclear whether the same is applicable for small cell lung cancer (SCLC). We evaluated the efficacy of second-line amrubicin (AMR) following first-line platinum-based chemotherapy and ICI combination therapy (chemo-ICI) in SCLC. PATIENTS AND METHODS: We retrospectively enrolled consecutive patients with SCLC treated with AMR as a second-line following chemo-ICI as first-line between July 2019 and April 2021 from 16 institutions throughout Japan. We investigated the therapeutic effectiveness, safety, and efficacy-enhancing variables of AMR. RESULTS: Overall, 89 patients treated with AMR after first-line chemo-ICI were analyzed. The overall response rate (ORR) was 29.2% (95% confidence intervals [CI], 20.1-39.8) and median PFS (m PFS) was 2.99 months (95% CI, 2.27-3.65). Patients who relapsed more than 90 days after receiving first-line platinum combination therapy (sensitive relapse) exhibited greater ORR (58.3% vs. 24.7%, p = 0.035) and m PFS (5.03 vs. 2.56 months, p = 0.019) than patients who relapsed in <90 days (refractory relapse). Grade 3 or higher adverse events were mainly hematological toxicity. CONCLUSIONS: Our study suggested that the therapeutic effect of AMR was not enhanced after ICI on SCLC. However, AMR may be effective in cases of sensitive relapse after chemo-ICI. There was no increase in severe toxicity associated with AMR after ICI.

Autoimmune pulmonary alveolar proteinosis with features similar to nonspecific interstitial pneumonia.

A 58-year-old woman with cough and dyspnea who was suspected of having idiopathic interstitial pneumonia had been treated with corticosteroids and cyclosporine, but the symptoms had worsened. There were no findings to suspect pulmonary alveolar proteinosis (PAP) in the bronchoalveolar lavage fluid, 17 months after the start of treatment. The transbronchial lung biopsy specimens showed eosinophilic bodies that strongly stained with periodic acid-Schiff staining. Anti-granulocyte macrophage colony-stimulating factor (anti-GM-CSF) antibodies were detected in her serum. We diagnosed the patient with autoimmune PAP. Thus, we present a rare case of PAP presenting atypical radiological images and bronchoalveolar lavage fluid findings.

Successful control of intestinal bleeding from metastasis of pulmonary pleomorphic carcinoma with pembrolizumab: A case report.

RATIONALE: Pulmonary pleomorphic carcinoma is a rare tumor with a poor prognosis and has no standard chemotherapy. We herein report a case of small intestinal metastasis of pulmonary pleomorphic carcinoma that resulted in intestinal bleeding and was successfully treated with pembrolizumab monotherapy. PATIENT CONCERNS: A 54-year-old man with a history of pulmonary pleomorphic carcinoma resection was referred to our hospital due to a 1-month history of a fever and general fatigue. DIAGNOSIS: Laboratory investigation revealed microcytic anemia. Hematochezia was also noted after admission. Computed tomography (CT) and positron emission tomography (PET)/CT at the time of this admission revealed intraperitoneal masses alongside the small intestine with no significant ascites. INTERVENTIONS: Pembrolizumab (400 mg/body) was introduced as the first-line chemotherapy. OUTCOMES: By the 15th day after the initial pembrolizumab administration, the fever had disappeared, and the intraperitoneal masses were markedly reduced. Hematochezia had also disappeared, and he no longer needed to receive blood transfusions. LESSONS: To our knowledge, this is the first report in which small intestinal metastasis of pulmonary pleomorphic carcinoma was successfully controlled by pembrolizumab monotherapy. Immune checkpoint inhibitors may be promising therapeutic agents against pulmonary pleomorphic carcinoma.

Multiple Cranial Neuropathies Similar to Orbital Apex Syndrome Associated with Pembrolizumab: A Case Report.

Neurotoxicity is one of the more serious immune-related adverse events (irAEs) linked to immune checkpoint inhibitors and calls for prompt diagnosis and treatment. We describe a case of posttreatment anti-programmed death-1 immune checkpoint inhibitor pembrolizumab-induced oculomotor, optic, and trigeminal neuropathy in an 84-year-old female patient with recurrent pulmonary adenocarcinoma. After she received 13 cycles of pembrolizumab, she experienced hyponatremia, anorexia, and right ptosis. There were signs of the suspected irAEs of pembrolizumab, including trigeminal neuropathy, optic neuropathy, and oculomotor neuropathy. Steroid pulse therapy had good results for her neurological findings. We reported this case despite reports of pembrolizumab-induced mononeuropathy of the oculomotor and optic nerves because multiple cranial neuropathies like orbital apex syndrome are thought to be uncommon.

Anaplastic Lymphoma Kinase-Positive Lung Cancer with Mucoepidermoid Carcinoma Differentiation: A Case Report.

Mucoepidermoid carcinoma (MEC) of the lung is an extremely rare tumor, and a standard chemotherapy has not been established. Furthermore, little work has been conducted on the genetic characteristics of MEC. We herein report the case of a 42-year-old nonsmoking male patient who was referred to our hospital due to cough. Chest computed tomography demonstrated infiltration and atelectasis in the right lower lobe. He was eventually diagnosed with non-small cell lung cancer (NSCLC) with MEC differentiation corresponding to clinical stage IVA (cT4N2M1a[PLE]). Genetic testing for EGFR mutations was negative, but positive for anaplastic lymphoma kinase (ALK) fusion gene. After 2 weeks of first-line treatment with alectinib, the tumor decreased in size and his symptoms improved. Advanced MEC is a rare tumor, and reports on the treatment of ALK-positive NSCLC with MEC differentiation are rare.

Cisplatin plus pemetrexed therapy and subsequent immune checkpoint inhibitor administration for malignant peritoneal mesothelioma without pleural lesions: Case report.

RATIONALE: Malignant peritoneal mesothelioma is a rare tumor with a poor prognosis and has no recommended therapy after first-line pemetrexed and platinum-based chemotherapy. Moreover, effects of immune checkpoint inhibitors on peritoneal mesothelioma remains to be elucidated. We herein report the case of a 75-year-old man with peritoneal mesothelioma treated with cisplatin plus pemetrexed and subsequent nivolumab. PATIENT CONCERNS: A 75-year-old man was referred to our hospital due to lower abdominal pain. DIAGNOSIS: Positron emission tomography-computed tomography (CT) showed the accumulation of fluorodeoxyglucose in an intraperitoneal mass. A histological examination of a laparoscopic biopsy specimen revealed malignant peritoneal mesothelioma. INTERVENTIONS: After 4 cycles of cisplatin plus pemetrexed and 13 subsequent cycles of pemetrexed maintenance therapy showed beneficial responses until CT revealed liver metastasis. Nivolumab was then administered as the second-line therapy. OUTCOMES: After 3 cycles of biweekly nivolumab administration, he developed severe abdominal distention. CT revealed an intraperitoneal mass growing much more rapidly than ever, indicating hyperprogressive disease after nivolumab treatment. He ultimately died 51 days after the initial nivolumab administration. LESSONS: To our knowledge, this is the first report of hyperprogressive disease in a case of peritoneal mesothelioma after nivolumab treatment. While immune checkpoint inhibitors may be promising therapeutic strategies for treating malignant peritoneal mesothelioma, careful monitoring must be practiced with their application.

Lung spindle cell carcinoma harbouring a constitutively active epidermal growth factor receptor mutation.

Lung spindle cell carcinoma is a rare lung tumour with a poor prognosis, and its standard therapy has not been established. Furthermore, little work has been conducted on the genetic characteristics of lung spindle cell carcinomas. Here, we report an 82-year-old woman who was referred to our hospital due to a fever and dyspnoea. Chest computed tomography demonstrated a 75-mm mass surrounded by infiltrates and atelectasis in the right upper lobe. She was eventually diagnosed with lung spindle cell carcinoma corresponding to clinical stage IVB (cT4N2M1c(ADR)). A genetic study indicated that epidermal growth factor receptor (EGFR) exon 19 was deleted in the tumour cells. She received gefitinib as first-line therapy. However, no significant effect was observed, and she died of respiratory failure 89 days after the initial admission. To our knowledge, this is the first case of spindle cell carcinoma of the lung in which a sensitizing EGFR mutation is detected.

Successful Treatment of Afatinib-Refractory Non-Small Cell Lung Cancer with Uncommon Complex EGFR Mutations Using Pembrolizumab: A Case Report.

Although there has been significant progress in immune-checkpoint inhibitor (ICI) treatment, it remains controversial whether they should be used in the treatment of epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer (NSCLC). We herein report the case of an NSCLC patient with uncommon complex EGFR mutations (G719S and L861Q) who was refractory to afatinib treatment but who showed a good response to pembrolizumab treatment. A 65-year-old female ex-smoker was diagnosed with right upper lobe NSCLC (clinical stage IVB; cT2bN3M1c). She had received afatinib for two months, but her disease showed rapid progression. Pembrolizumab treatment was initiated because more than 75% of her tumor cells expressed PD-L1. Her tumor responded well to pembrolizumab treatment and it remained effective for more than 1 year. Our case suggests that pembrolizumab treatment is a treatment option for NSCLC patients with uncommon EGFR mutations and high PD-L1 expression levels who are refractory to EGFR-TKI treatment.

Brain Metastasis Mimicking Brain Abscess in ALK-Positive Non-Small-Cell Lung Cancer.

Brain metastasis frequently develops in non-small-cell lung cancer (NSCLC). Here, we report a patient who developed brain metastasis from ALK-positive NSCLC which mimicked brain abscess. He was admitted for suspected obstructive pneumonia nine months after curative lung resection. Head magnetic resonance imaging revealed a cavitary lesion, which was compatible with brain abscess but rare in brain metastasis. However, after treatment with antibiotics, the brain lesion increased in size. Aspiration of the liquid content of the brain lesion revealed cancer cells. When a brain lesion suggestive of abscess develops in a patient with ALK-positive NSCLC, aspiration may be necessary to differentiate metastasis from abscess.

Phase II study of docetaxel-plus-bevacizumab combination therapy in patients previously treated for advanced non-squamous non-small cell lung cancer.

AIM: This phase II study was conducted to evaluate the efficacy and safety of docetaxel and bevacizumab combination therapy in patients with previously-treated non-squamous non-small cell lung cancer (Nsq NSCLC). PATIENTS AND METHODS: Patients with histologically- or cytologically-confirmed Nsq NSCLC, 20-74 years of age, who had performance status 0-2, and had undergone at least one prior chemotherapy course were eligible for the study. Patients were treated with docetaxel (60 mg/m(2)) and bevacizumab (15 mg/kg) on day 1, which was repeated every three weeks until progressive disease or unacceptable toxicity occurred. The primary end-point was the response rate (RR) and the planned sample size was 28 patients. RESULTS: Between May 2010 and July 2011, 28 patients were enrolled (16 males, 12 females; median age=65 years; performance status 0/1: 19/9; adenocarcinoma/other: 22/6; number of prior chemotherapy courses 1/2/3 or more: 16/5/7). Twenty-eight patients were included in the toxicity analysis, out of whom 27 were evaluable for response. Objective response was observed in 18 patients (partial response in 18, stable disease in 8, progressive disease in 1); the RR and disease control rates were 66.7% and 96.0%, respectively. The median follow-up was 23.9 months, median progression-free survival was 7.2 months, and median overall survival was 21.6 months. The main toxicity associated with this regimen was myelosuppression (grade 3/4 neutropenia: 82.1%; febrile neutropenia: 21%). Mild non-hematological toxicity was observed but there was no severe bleeding. CONCLUSION: The combination regimen of docetaxel-plus-bevacizumab is very active in patients with previously-treated Nsq NSCLC and warrants further research.