Impact of central nervous system metastasis after complete resection of lung adenocarcinomas harboring common EGFR mutation - A real-world database study in Japan: The CReGYT-01 EGFR study.

OBJECTIVES: To clarify the impact of central nervous system (CNS) metastasis on performance status (PS) at relapse, on subsequent treatment(s), and on survival of patients with lung adenocarcinoma harboring common epidermal growth factor receptor (EGFR) mutation. METHODS: We conducted the multicenter real-world database study for patients with radical resections for lung adenocarcinomas between 2015 and 2018 at 21 centers in Japan. EGFR mutational status was examined at each center. RESULTS: Of 4181 patients enrolled, 1431 underwent complete anatomical resection for lung adenocarcinoma harboring common EGFR mutations. Three-hundred-and-twenty patients experienced disease relapse, and 78 (24%) had CNS metastasis. CNS metastasis was significantly more frequent in patients with conventional adjuvant chemotherapy than those without (30% vs. 20%, P = 0.036). Adjuvant chemotherapy did not significantly improve relapse-free survival at any pathological stage (adjusted hazard ratio for stage IA2-3, IB, and II-III was 1.363, 1.287, and 1.004, respectively). CNS metastasis did not affect PS at relapse. Subsequent treatment, mainly consisting of EGFR-tyrosine kinase inhibitors (TKIs), could be equally given in patients with or without CNS metastasis (96% vs. 94%). Overall survival after relapse was equivalent between patients with and without CNS metastasis. CONCLUSION: The efficacy of conventional adjuvant chemotherapy may be limited in patients with lung adenocarcinoma harboring EGFR mutations. CNS metastasis is likely to be found in practice before deterioration in PS, and may have little negative impact on compliance with subsequent EGFR-TKIs and survival after relapse. In this era of adjuvant TKI therapy, further prospective observational studies are desirable to elucidate the optimal management of CNS metastasis.

Secondary Mutations of the EGFR Gene That Confer Resistance to Mobocertinib in EGFR Exon 20 Insertion.

INTRODUCTION: Approximately 10% of mutations in the EGFR gene in NSCLC are in-frame insertions in exon 20 (X20ins). These tumors usually do not respond to conventional EGFR tyrosine kinase inhibitors (TKIs). Several novel EGFR TKIs active for X20ins are in clinical development, including mobocertinib, which was recently approved by the U.S. Food and Drug Administration. However, acquired resistance during treatment with these TKIs still occurs as in the case of EGFR TKIs of earlier generations. METHODS: We chronically exposed murine pro-B-cell line cells transduced with the five most common X20ins (A763_Y764insFQEA, V769_D770insASV, D770_N771insSVD, H773_V774insNPH and H773_V774insH) to mobocertinib in the presence of N-ethyl-N-nitrosourea and searched for secondary EGFR mutations. We evaluated the efficacies of several EGFR X20ins inhibitors, including zipalertinib and sunvozertinib, against cells with acquired resistant mutations. RESULTS: All secondary mutations resulting in acquired resistance to mobocertinib were exclusively C797S in insFQEA and insSVD. However, in the case of other X20ins (insASV, insNPH, and insH), T790M or C797S secondary mutations contributed to acquired resistance to mobocertinib. The emergence of T790M was more frequent in cells treated with lower drug concentrations. Sunvozertinib exhibited good activity against resistant cells with T790M. Cells with C797S were refractory to all EGFR TKIs, except for erlotinib, which was active for insFQEA with C797S. CONCLUSIONS: T790M or C797S, depending on the original X20ins mutations, conferred acquired resistance to mobocertinib. Sunvozertinib may be the treatment of choice for patients with tumors resistant to mobocertinib because of T790M.

Clinical Relevance of Patient-Derived Organoid of Surgically Resected Lung Cancer as an In Vitro Model for Biomarker and Drug Testing.

Introduction: Lung tumor organoids (LTOs) have attracted attention as in vitro preclinical models; however, their clinical and experimental applications have not been fully established. Methods: We attempted to establish LTOs from resected specimens of patients with lung cancer who underwent lung resection. Clinicopathologic characteristics related to the establishment of LTOs were evaluated. Histologic assessment and genetic analysis were conducted for both LTOs and their parental tumors. Organoid-derived xenografts were generated in immunocompetent mice. Drug sensitivity was assessed using cell proliferation assays. Results: We established 53 LTOs from 79 lung cancer samples, including 10 long-term culture models. The establishment rate was significantly lower in squamous cell carcinomas than in other histologic types (48% versus 75%, p = 0.034). Histologic similarities were confirmed among LTOs, the parental tumors, and organoid-derived xenografts. Seven mutations, including two EGFR L858R and one EGFR exon 20 H773delinsYNPY mutations, were detected in both LTO and parental tumors; the other four mutations were detected in either LTO or parental tumors. The extensive culture ability of LTO (passaged >10 times) correlated with poor patient prognosis. LTO9 cells harboring EGFR H773delinsYNPY were sensitive to osimertinib. The parental patient, who had new metastatic lesions, was treated with osimertinib and exhibited a remarkable response. Conclusions: The establishment and growth rates of LTOs were associated with the histologic subtype and tumor size. LTOs derived from resected specimens have become preclinical models that can be used to predict drug responses and accelerate the development of treatment strategies for patients with rare mutations.

Foretinib can overcome common on-target resistance mutations after capmatinib/tepotinib treatment in NSCLCs with MET exon 14 skipping mutation.

BACKGROUND: Capmatinib and tepotinib are guideline-recommended front-line treatments for non-small-cell lung cancer (NSCLC) patients with MET exon 14 skipping mutations (METex14). However, the emergence of acquired resistance to capmatinib/tepotinib is almost inevitable partially due to D1228X or Y1230X secondary mutations of the MET. In this study, we explored agents that are active against both D1228X and Y1230X MET to propose an ideal sequential treatment after capmatinib/tepotinib treatment failure in NSCLC patients with METex14. METHODS: The inhibitory effects of 300 drugs, including 33 MET-TKIs, were screened in Ba/F3 cells carrying METex14 plus MET D1228A/Y secondary mutations. The screen revealed four-candidate type II MET-TKIs (altiratinib, CEP-40783, foretinib and sitravatinib). Therefore, we performed further growth inhibitory assays using these four MET-TKIs plus cabozantinib and merestinib in Ba/F3 cells carrying MET D1228A/E/G/H/N/V/Y or Y1230C/D/H/N/S secondary mutations. We also performed analyses using Hs746t cell models carrying METex14 (with mutant allele amplification) with/without D1228X or Y1230X in vitro and in vivo to confirm the findings. Furthermore, molecular dynamics (MD) simulations were carried out to examine differences in binding between type II MET-TKIs. RESULTS: All 6 type II MET-TKIs were active against Y1230X secondary mutations. However, among these 6 agents, only foretinib showed potent activity against D1228X secondary mutations of the MET in the Ba/F3 cell and Hs746t in vitro model and Hs746t in vivo model, and CEP-40783 and altiratinib demonstrated some activity. MD analysis suggested that the long tail of foretinib plays an important role in binding D1228X MET through interaction with a residue at the solvent front (G1163). Tertiary G1163X mutations, together with L1195F/I and F1200I/L, occurred as acquired resistance mechanisms to the second-line treatment foretinib in Ba/F3 cell models. CONCLUSIONS: The type II MET-TKI foretinib may be an appropriate second-line treatment for NSCLCs carrying METex14 after campatinib/tepotinib treatment failure by secondary mutations at residue D1228 or Y1230.

Presence of a Ground-Glass Opacity Component Is the True Prognostic Determinant in Clinical Stage I NSCLC.

Introduction: Recent studies have suggested that including presence or absence of ground-glass opacity (GGO) may improve the tumor descriptor (T descriptor) classification in clinical stage I NSCLC. In this study, we analyzed prognostic implications of presence or absence of GGO, size of the solid component, and predominant histology to identify the true prognostic determinant for early-stage NSCLC. Methods: We retrospectively examined 384 patients with clinical stage I NSCLC (solid: 242, part solid: 142) who underwent complete resection between 2009 and 2013. Results: Survival curves of the whole cohort revealed good separation using the current TNM classification. Nevertheless, the part-solid group had a favorable prognosis irrespective of solid component size. Conversely, patients in the solid tumor group with tumors between 3 and 4 cm had a worse prognosis than patients whose tumors were less than or equal to 3 cm. Thus, we propose the following novel T descriptor classification: IA, part-solid tumors; IB, solid tumors less than or equal to 3 cm; and IC, solid tumors between 3 and 4 cm. This novel classification system stratified patient prognosis better than the current classification. On pathologic evaluation, the part-solid group always had better prognoses than the solid group in each subgroup divided by pathologic grade. Conclusions: These results suggest that presence of GGO is the true prognostic determinant of stage I NSCLC, irrespective of the size of the solid component. Our novel T descriptor classification system could more accurately predict prognoses of clinical stage I NSCLC cases.

Frequent EGFR Mutations and Better Prognosis in Positron Emission Tomography-Negative, Solid-Type Lung Cancer.

BACKGROUND: The differential diagnosis of a solitary solid-type lung nodule is diverse. 18F-fluorodeoxyglucose positron emission tomography (PET) has a high sensitivity in the diagnosis of solid-type lung cancers; however, PET-negative, solid-type lung cancers are rarely observed. In this study, we analyzed the clinical/genetic features and prognosis of PET-negative, solid-type lung cancers. PATIENTS AND METHODS: Between January 2007 and February 2020, 709 patients with solid-type lung cancers (tumor size ≥2.0 cm) underwent pulmonary resection. Clinical, genetic, and prognostic features were evaluated in 27 patients (3.8%) with tumors showing negative PET results defined as SUVmax <2.0. RESULTS: All 27 patients had lung adenocarcinoma; 23 had invasive adenocarcinomas and 4 had invasive mucinous adenocarcinomas. The PET-negative group showed high frequencies of females and never-smokers. Recurrence-free survival was significantly better in the PET-negative group compared with PET-positive counterparts extracted using propensity score matching from patients who underwent pulmonary resection during the same period (P = .0052). Furthermore, 83% of PET-negative, solid-type invasive lung adenocarcinoma patients harbored EGFR mutation, which was significantly higher than that of PET-positive, solid-type invasive lung adenocarcinoma patients (38%, n = 225) who received EGFR mutation testing in our cohort (P < .0001). PET-negative, solid-type lung adenocarcinoma patients with EGFR mutations had significantly better recurrence-free survival compared with PET-positive, solid-type lung adenocarcinoma patients with EGFR mutations extracted using propensity score matching (P = .0030). CONCLUSION: PET-negative, solid-type lung cancers are characterized with a high incidence of EGFR mutation and a better prognosis compared with PET-positive, solid-type lung cancer.

Intra-tumor and Inter-tumor Heterogeneity in MET Exon 14 Skipping Mutations and Co-mutations in Pulmonary Pleomorphic Carcinomas.

BACKGROUND: MET exon 14 skipping mutation is a driver mutation in lung cancer and is highly enriched in pulmonary pleomorphic carcinomas (PPCs). Whether there is intratumor or intertumor heterogeneity in MET exon 14 skipping status or in co-occurring genetic alterations in lung cancers driven by MET exon 14 skipping is unknown. METHODS: We analyzed tumor specimens obtained from 23 PPC patients (10 autopsied and 13 surgically resected). MET exon 14 skipping was detected by RT-PCR. For patients with MET exon 14 skipping mutation, further analyses were performed. Genomic DNA (gDNA) was extracted from various histological components for each patient who underwent surgical resection (to assess intratumor heterogeneity). In autopsied patients, gDNA and total RNA were extracted from all metastatic lesions (to assess intertumor heterogeneity). RESULTS: MET exon 14 skipping mutation was detected in 4 patients (4/23, 17.4%): two surgically resected and two autopsied patients. We found no intratumor or intertumor heterogeneity in MET exon 14 skipping mutation status in these patients. We observed intratumor and intertumor heterogeneity in the copy number variations and/or mutational status of cancer-related genes; some of these differences may have an impact on MET tyrosine kinase inhibitor (TKI) efficacy. CONCLUSION: In our exploratory analysis of four cases, we observed that MET exon 14 skipping mutations are distributed homogeneously throughout histological components and between metastatic lesions. Our results also suggest that there is marked intertumor and intratumor heterogeneity in co-occurring genetic alterations, and therapeutic implications of such heterogeneity should be evaluated in future studies.

In vitro validation study of HER2 and HER4 mutations identified in an ad hoc secondary analysis of the LUX-Lung 8 randomized clinical trial.

OBJECTIVES: The LUX-Lung 8 randomized trial (LL8) demonstrated a prolonged progression-free survival (PFS) in patients with metastatic squamous cell carcinoma (SCC) of the lung after treatment with afatinib compared with erlotinib. A secondary analysis of the LL8 reported that the presence of rare HER2/HER4 mutations may be partly responsible for this result. Patients with HER2 (hazard ratio [HR] 0.06/p-value 0.02) or HER4 (HR 0.21/p-value unreported) mutations had longer PFS after treatment with afatinib. However, the biological function of these mutations is unclear. MATERIALS AND METHODS: Ten HER2 and 13 HER4 point mutations that were detected in the secondary analysis were transduced into the mouse pro-B cell line (Ba/F3) to determine changes in interleukin-3 (IL-3) dependence and sensitivity to six EGFR or pan-HER tyrosine kinase inhibitors (TKIs), including afatinib and erlotinib. The efficacy of the six TKIs was compared using a sensitivity index, defined as the 50% inhibitory concentration divided by trough concentration of each drug at clinically recommended doses. RESULTS: Seven out of 10 Ba/F3 clones expressing HER2 mutations and all 13 Ba/F3 clones expressing HER4 mutations did not grow in the absence of IL-3, indicating these mutations were non-oncogenic. Three Ba/F3 clones expressing the HER2 mutations E395K, G815R, or R929W acquired IL-3-independent growth. The sensitivity indices for afatinib were ≤ one-fifth of those for erlotinib in all three lines. Other second/third-generation (2G/3G) TKIs showed high efficacy against clones expressing these HER2 mutations. CONCLUSIONS: The majority of HER2/4 mutations detected in lung SCC from LL8 were not oncogenic in the Ba/F3 models, suggesting that the presence of HER2/4 mutations were not responsible for the superior outcomes of afatinib in the LL8 study. However, SCC of the lung in some patients may be driven by rare HER2 mutations, and these patients may benefit from 2G/3G pan-HER-TKI treatment.

Activity and mechanism of acquired resistance to tarloxotinib in HER2 mutant lung cancer: an in vitro study.

BACKGROUND: HER2 (ERBB2) activating mutations are present in 2-3% of lung adenocarcinomas; however, no targeted therapy is approved for HER2-altered lung cancers. A novel pan-HER inhibitor, tarloxotinib, is designed to release the active form (tarloxotinib-E) under hypoxic conditions in tumor tissues after being administered as a prodrug. Following the evaluation of the in vitro activity of tarloxotinib-E in HER2-mutant cells, we explored the mechanisms of resistance to tarloxotinib-E in these cells. METHODS: Growth inhibitory assays were performed with tarloxotinib-E and its prodrug using Ba/F3 cells expressing one of six HER2 mutations or wild-type (WT) HER2, in addition to H1781 cells with HER2 exon 20 insertions. Resistant clones were established from N-ethyl-N-nitrosourea (ENU)-treated HER2-mutant Ba/F3 cells and H1781 cells by chronic exposure to tarloxotinib-E. RESULTS: Tarloxotinib-E showed potent activity against HER2-mutant Ba/F3 cells and H1781 cells. Furthermore, the half maximal inhibitory concentration (IC50) of tarloxotinib (inactive form) for WT HER2 was 180 times higher than that of tarloxotinib-E, indicating a wide therapeutic window of tarloxotinib. We established 30 resistant clones with secondary mutations of HER2 by ENU mutagenesis, all of which harbored C805S in exon 20. In the analysis of H1781 cells that acquired resistance to tarloxotinib-E, we found that increased HER3 expression was the molecular mechanism of tarloxotinib-E resistance. CONCLUSIONS: Tarloxotinib-E exhibited potent activity against cell line models with HER2 mutations. We identified a secondary C805S HER2 mutation and HER3 overexpression as the mechanisms of acquired resistance to tarloxotinib-E.

KRAS Secondary Mutations That Confer Acquired Resistance to KRAS G12C Inhibitors, Sotorasib and Adagrasib, and Overcoming Strategies: Insights From In Vitro Experiments.

INTRODUCTION: KRAS mutations have been recognized as undruggable for many years. Recently, novel KRAS G12C inhibitors, such as sotorasib and adagrasib, are being developed in clinical trials and have revealed promising results in metastatic NSCLC. Nevertheless, it is strongly anticipated that acquired resistance will limit their clinical use. In this study, we developed in vitro models of the KRAS G12C cancer, derived from resistant clones against sotorasib and adagrasib, and searched for secondary KRAS mutations as on-target resistance mechanisms to develop possible strategies to overcome such resistance. METHODS: We chronically exposed Ba/F3 cells transduced with KRASG12C to sotorasib or adagrasib in the presence of N-ethyl-N-nitrosourea and searched for secondary KRAS mutations. Strategies to overcome resistance were also investigated. RESULTS: We generated 142 Ba/F3 clones resistant to either sotorasib or adagrasib, of which 124 (87%) harbored secondary KRAS mutations. There were 12 different secondary KRAS mutations. Y96D and Y96S were resistant to both inhibitors. A combination of novel SOS1 inhibitor, BI-3406, and trametinib had potent activity against this resistance. Although G13D, R68M, A59S and A59T, which were highly resistant to sotorasib, remained sensitive to adagrasib, Q99L was resistant to adagrasib but sensitive to sotorasib. CONCLUSIONS: We identified many secondary KRAS mutations causing resistance to sotorasib, adagrasib, or both, in vitro. The differential activities of these two inhibitors depending on the secondary mutations suggest sequential use in some cases. In addition, switching to BI-3406 plus trametinib might be a useful strategy to overcome acquired resistance owing to the secondary Y96D and Y96S mutations.

Activity of tarloxotinib-E in cells with EGFR exon-20 insertion mutations and mechanisms of acquired resistance.

BACKGROUND: Approximately 10% of non-small cell lung cancers (NSCLCs) that harbor epidermal growth factor receptor (EGFR) gene mutations have in-frame insertions in exon 20 of the EGFR gene. These tumors do not usually respond to currently available EGFR-tyrosine kinase inhibitors (TKIs). Tarloxotinib is a novel hypoxia-activated prodrug that releases a potent, irreversible pan-ERBB TKI (tarloxotinib-E) under solid tumor hypoxia. METHODS: We examined the efficacy of tarloxotinib-E against several types of Ba/F3 cells with introduced EGFR exon 20 mutations (EGFR A763insFQEA, V769insASV, D770insSVD, H773insH and H773insNPH mutations). We assayed growth inhibition for tarloxotinib (prodrug), tarloxotinib-E (active form), poziotinib, afatinib, and osimertinib in Ba/F3 cells with each EGFR exon 20 mutation. We also explored acquired resistance mechanisms to tarloxotinib-E by establishing cells with resistance to tarloxotinib-E via chronic drug exposure after N-ethyl-N-nitrosourea mutagenesis treatment. RESULTS: Among all tested Ba/F3 cell lines, IC50 was ≥72.1 times higher for tarloxotinib than for tarloxotinib-E, which implies a wide therapeutic window with this prodrug strategy. Tarloxotinib-E was efficacious against all tested Ba/F3 cells except for H773insH, which was less sensitive to all tested EGFR-TKIs. As acquired resistance mechanisms to tarloxotinib-E, we identified either T790M or C797S secondary mutations, depending on the original EGFR exon 20 mutation. CONCLUSIONS: These findings indicate that tarloxotinib-E could be effective for NSCLC with EGFR exon 20 mutations. Our results also show that T790M or C797S mutations can confer acquired resistance to tarloxotinib-E; and suggest that resistance mechanisms are influenced by the baseline EGFR exon 20 mutations.

The prevalence and risk factors associated with preoperative deep venous thrombosis in lung cancer surgery.

PURPOSE: Few studies have so far focused on the preoperative presence of venous thromboembolism (VTE) in lung cancer patients undergoing surgery. In this study, we investigated the prevalence and risk factors for preoperative deep venous thrombosis (DVT) in patients scheduled to undergo lung cancer surgery. METHODS: Between June 2013 and December 2018, 948 consecutive patients underwent lung cancer surgery in Kindai University Hospital. Four patients did not undergo screening for DVT; thus, 944 patients were enrolled in this study. Preoperatively, venous ultrasonography of the lower extremities was performed in patients deemed at risk for DVT, and the prevalence and risk factors for preoperative DVT were examined. RESULTS: Ninety-one patients (9.6%) were diagnosed with preoperative DVT, and postoperative symptomatic pulmonary thromboembolism occurred in one patient (0.11%). A multivariable logistic regression analysis demonstrated that female sex, age ≥ 72 years, history of VTE, a Wells score ≥ 2 points, chronic obstructive pulmonary disease (COPD), and lower hemoglobin levels were significantly associated with preoperative DVT. CONCLUSION: Female sex, age ≥ 72 years, history of VTE, Wells score ≥ 2 points, COPD, and lower hemoglobin levels were identified to be independent risk factors for preoperative DVT. Monitoring for these risk factors and management considering them should help improve the outcomes after lung cancer surgery.

Cell Line Models for Acquired Resistance to First-Line Osimertinib in Lung Cancers-Applications and Limitations.

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are first-line drugs for lung cancers with activating EGFR mutations. Although first- and second-generation EGFR-TKIs were standard first-line treatments, acquired resistance (AR) to these drugs is almost inevitable. Cell line models have been widely used to explore the molecular mechanisms of AR to first- and second-generation EGFR-TKIs. Many research groups, including ours, have established AR cell lines that harbor the EGFR T790M secondary mutation, MET gene amplification, or epithelial-mesenchymal transition (EMT) features, which are all found in clinical specimens obtained from TKI-refractory lesions. Currently, many oncologists prescribe osimertinib, a third-generation EGFR-TKI that can overcome T790M-mediated resistance, as a first-line TKI. Although few clinical data are available about AR mechanisms that arise when osimertinib is used as a first-line therapy, many research groups have established cell lines with AR to osimertinib and have reported on their AR mechanisms. In this review, we summarize the findings on AR mechanisms against first-line osimertinib obtained from analyses of cell line models.

Dose-dependence in acquisition of drug tolerant phenotype and high RYK expression as a mechanism of osimertinib tolerance in lung cancer.

OBJECTIVE: Emergence of acquired resistance is almost inevitable during EGFR-tyrosine kinase inhibitor therapy for non-small-cell lung cancer (NSCLC) harboring EGFR mutations. Drug tolerance, a reversible state of drug insensitivity in the early phases of tyrosine kinase inhibitor therapy, is considered to serve as the basis of recurrent disease. Therefore, it is important to elucidate the molecular mechanisms of drug tolerance. MATERIALS AND METHODS: Five EGFR-mutated NSCLC cell lines were used in this study. We established drug-tolerant cells (DTCs) via 72 h treatment with osimertinib (600 nM) or afatinib (60 nM). Acquisition of drug tolerance was evaluated by growth inhibitory assay, and the molecular mechanisms of drug tolerance were analyzed by phospho-RTK array. RESULTS: DTCs were successfully induced in PC9, HCC4006, and H1975 cells against osimertinib and in PC9 cells against afatinib. We observed that a high drug concentration was required to induce DTCs, and HCC4006 cells become tolerant when a higher dose of afatinib (>180 nM) was used. In the analysis of HCC4006 DTCs against osimertinib, we observed increased receptor-like tyrosine kinase (RYK) expression, and siRNA-mediated RYK knockdown inhibited the proliferation of DTCs. CONCLUSIONS: These results suggest that induction of DTCs is dose-dependent, and increased RYK expression was the mechanism of drug tolerance in HCC4006 cells against osimertinib.

Inter- and Intratumor Heterogeneity of EGFR Compound Mutations in Non-Small Cell Lung Cancers: Analysis of Five Cases.

BACKGROUND: Several clinical and preclinical studies suggest that non-small cell lung cancers (NSCLCs) with EGFR compound mutations were associated with lower efficacies of first-generation EGFR inhibitors than tumors with single EGFR mutation. Some researchers hypothesize that EGFR mutation status is heterogeneous in such tumors and that second-generation EGFR inhibitors may eliminate cancer cells with uncommon EGFR mutations from tumors with EGFR compound mutations. However, this hypothesis is currently unproven; therefore, we performed the current study to determine if tumor cells with EGFR compound mutations are present in heterogeneous or homogeneous manners. PATIENTS AND METHODS: Multiregion analysis was performed for surgically resected primary NSCLC tumors with EGFR compound mutations to examine the intratumor heterogeneity of EGFR compound mutations. In addition, we evaluated the intertumor heterogeneity of EGFR compound mutations using 2 pleural disseminations obtained from a patient with NSCLC at exploratory thoracotomy and 9 primary or metastatic lesions obtained from 2 autopsied NSCLC patients. Digital polymerase chain reaction, target sequencing, or direct sequencing were used to detect EGFR mutations. RESULTS: This study included 5 NSCLC cases; their compound mutations were L858R+S768I, G719X+S768I, G719A+R776H, L858R+E709G, and L858R+I759M. Noncancerous pulmonary tissues from each patient did not harbor EGFR mutations, which revealed that all mutations were somatic. We did not detect any intra- or intertumor heterogeneity in these EGFR compound mutations. CONCLUSION: No intra- or intertumor heterogeneity was observed for EGFR compound mutations. Our results indicate that both EGFR mutations were truncal and selective elimination of cancer cells with uncommon EGFR mutations is unrealistic.

Prognostic implications of preoperative versus postoperative circulating tumor DNA in surgically resected lung cancer patients: a pilot study.

BACKGROUND: Recent studies of advanced lung cancer patients have shown that circulating tumor DNA (ctDNA) analysis is useful for molecular profiling, monitoring tumor burden, and predicting therapeutic efficacies and disease progression. However, the usefulness of ctDNA analysis in surgically resected lung cancers is unclear. METHODS: This study included 20 lung cancer patients with clinical stage IIA-IIIA disease. Preoperative and postoperative (3-12 days) plasma samples were collected for ctDNA analysis. Cancer personalized profiling by deep sequencing, which can detect mutations in 197 cancer-related genes, was used for ctDNA detection. The cohort consisted of 18 men and 2 women with a median age of 69 (range, 37-88) years. Sixteen patients (80%) had a history of smoking. Histologically, there were four squamous cell carcinomas, 13 adenocarcinomas, two adenosquamous cell carcinomas, and one small cell carcinoma. RESULTS: At the time of data analysis, the 20 patients had been monitored for a median follow-up of 12 months. Eight patients (40%) were positive for preoperative ctDNA, and this was significantly correlated with tumor size (≥5 vs. <5 cm, P=0.018). Four patients (20%) were positive for postoperative ctDNA, and this was significantly correlated with histological grade (3 vs. 1 or 2, P=0.032). Postoperative positivity for ctDNA also predicted shorter recurrence-free survival (RFS) (P=0.015), while pre- and post-operative carcinoembryonic antigen levels (P=0.150 and P=0.533, respectively) and preoperative positivity for ctDNA (P=0.132) were not correlated with RFS. CONCLUSIONS: Detecting ctDNA postoperatively was a poor prognostic factor in surgically resected lung cancer patients that may suggest there is minimal residual disease (MRD).

Spatial heterogeneity of acquired resistance mechanisms to 1st/2nd generation EGFR tyrosine kinase inhibitors in lung cancer.

BACKGROUND: Overcoming acquired resistance against targeted therapies to improve outcomes of lung cancer patients harboring driver mutations is a critical issue. While drug therapy oriented to a resistance mechanism appears attractive, spatial heterogeneity of resistance mechanisms in each patient will diminish treatment efficacy. However, the frequency, clinical backgrounds, clinical implications, and patterns of spatial heterogeneity in resistance mechanisms to EGFR tyrosine kinase inhibitors (TKIs) are largely unknown. PATIENTS AND METHODS: This study included 128 specimens from 24 autopsied patients with lung adenocarcinoma harboring EGFR mutation. Acquired resistance mechanisms reported as relatively frequent in lung cancer, e.g., T790 M and other secondary EGFR mutations, MET and ERBB2 gene amplification, and histological transformation, were retrospectively examined. All patients had received 1st/2nd generation EGFR-TKI and showed acquired resistance to the drug before death. No patient received osimertinib. RESULTS: No resistance mechanism was identified in two patients. T790M mutation was detected in 20 patients (83 %); however, nine of these patients also had lesions without T790M mutation. Among 22 patients whose resistance mechanisms were identified, ten had spatial heterogeneity of resistance mechanisms (45 %), and these patients had significantly shorter time-to-treatment failure compared with those without heterogeneity (median 4.7 months vs. 14.7 months, p = 0.0004). CONCLUSION: We observed significant spatial heterogeneity of acquired resistance mechanisms to EGFR-TKIs in lung adenocarcinoma. Our results also indicate that the incidence of resistance mechanisms may vary based on the biopsied tumor locations.

Prognostic value of plasma fibrinogen and D-dimer levels in patients with surgically resected non-small cell lung cancer.

PURPOSE: A high plasma level of either fibrinogen or D-dimer has been shown to correlate with a poor prognosis in patients with surgically resected non-small-cell lung cancer (NSCLC). The present study aimed to identify whether or not both markers combined had a superior prognostic value to either alone. METHODS: Of the 1344 patients who underwent surgical resection for NSCLC at our institution between January 2007 and December 2016, 1065 had preoperative plasma fibrinogen and D-dimer data available and were included in the analysis. RESULTS: The recurrence-free survival (RFS) and overall survival (OS) rates were similar for patients with high plasma levels of either or both fibrinogen (> 4.0 g/L) or D-dimer (> 1.0 µg/mL); therefore, these three groups were combined for a further analysis into a single group with high plasma levels of either or both proteins. The high-level group had significantly lower 5-year RFS (53% vs. 68%, p < 0.001) and 5-year OS (65% vs. 80%, p < 0.001) rates than patients with normal plasma levels of fibrinogen and D-dimer (control group). CONCLUSIONS: Our results suggest that preoperative tests for both plasma fibrinogen and D-dimer are necessary to identify patients with surgically resected NSCLC likely to have a poor RFS and OS.

Sensitivity and Resistance of MET Exon 14 Mutations in Lung Cancer to Eight MET Tyrosine Kinase Inhibitors In Vitro.

BACKGROUND: MNNG HOS transforming gene (MET) exon 14 mutations in lung cancer, including exon 14 skipping and point mutations, have been attracting the attention of thoracic oncologists as new therapeutic targets. Tumors with these mutations almost always acquire resistance, which also occurs in other oncogene-addicted lung cancers. However, the resistance mechanisms and treatment strategies are not fully understood. METHODS: We generated Ba/F3 cells expressing MET exon 14 mutations by retroviral gene transfer. The sensitivities of these cells to eight MET-tyrosine kinase inhibitors (TKIs) were determined using a colorimetric assay. In addition, using N-ethyl-N-nitrosourea mutagenesis, we generated resistant clones, searched for secondary MET mutations, and then examined the sensitivities of these resistant cells to different TKIs. RESULTS: Ba/F3 cells transfected with MET mutations grew in the absence of interleukin-3, indicating their oncogenic activity. These cells were sensitive to all MET-TKIs except tivantinib. We identified a variety of secondary mutations. D1228 and Y1230 were common sites for resistance mutations for type I TKIs, which bind the active form of MET, whereas L1195 and F1200 were common sites for type II TKIs, which bind the inactive form. In general, resistance mutations against type I were sensitive to type II, and vice versa. CONCLUSIONS: MET-TKIs inhibited the growth of cells with MET exon 14 mutations. We also identified mutation sites specific for TKI types as resistance mechanisms and complementary activities between type I and type II inhibitors against those mutations. These finding should provide relevant clinical implication for treating patients with lung cancer harboring MET exon 14 mutations.

Primary pulmonary mucosa-associated lymphoid tissue lymphoma with amyloid light chain-type amyloidosis.

BACKGROUND: A total of 75% of patients with Sjögren's syndrome are complicated with pulmonary lesions, of which 12% are lymphoma and 6% are amyloid nodules; the coexistence of both is considered to be rare. CASE PRESENTATION: A 67-year-old female with Sjögren's syndrome presented with multiple pulmonary nodules on chest computed tomography. Since a definitive diagnosis by transbronchial biopsy was not obtained, wedge resection of the nodules was performed. Pathologic diagnosis revealed eosinophilic deposition that stained positive with Congo red. In addition, lymphoepithelial lesions and lymphocytic infiltration were observed. Lymphocytes with monoclonal proliferation predominantly had κ chain. Based on these findings, the nodules were diagnosed as mucosa-associated lymphoid tissue (MALT) lymphoma with amyloid deposition. CONCLUSIONS: The combination of these diseases is very rare, and this is the sixth resected case to the best of our knowledge.