Utility of the Ba/F3 cell system for exploring on-target mechanisms of resistance to targeted therapies for lung cancer.

Molecular targeted therapies are the standard of care for front-line treatment of metastatic non-small-cell lung cancers (NSCLCs) harboring driver gene mutations. However, despite the initial dramatic responses, the emergence of acquired resistance is inevitable. Acquisition of secondary mutations in the target gene (on-target resistance) is one of the major mechanisms of resistance. The mouse pro-B cell line Ba/F3 is dependent on interleukin-3 for survival and proliferation. Upon transduction of a driver gene, Ba/F3 cells become independent of interleukin-3 but dependent on the transduced driver gene. Therefore, the Ba/F3 cell line has been a popular system to generate models with oncogene dependence and vulnerability to specific targeted therapies. These models have been used to estimate oncogenicity of driver mutations or efficacies of molecularly targeted drugs. In addition, Ba/F3 models, together with N-ethyl-N-nitrosourea mutagenesis, have been used to derive acquired resistant cells to investigate on-target resistance mechanisms. Here, we reviewed studies that used Ba/F3 models with EGFR mutations, ALK/ROS1/NTRK/RET fusions, MET exon 14 skipping mutations, or KRAS G12C mutations to investigate secondary/tertiary drug resistant mutations. We determined that 68% of resistance mutations reproducibly detected in clinical cases were also found in Ba/F3 models. In addition, sensitivity data generated with Ba/F3 models correlated well with clinical responses to each drug. Ba/F3 models are useful to comprehensively identify potential mutations that induce resistance to molecularly targeted drugs and to explore drugs to overcome the resistance.

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.

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.

Life-threatening complications after pulmonary resection for lung cancer in patients on chronic hemodialysis.

PURPOSE: The morbidity and mortality associated with lung cancer surgery in patients on chronic hemodialysis (CHD) is high; however, the relationship between the severity of postoperative complications and clinicopathological features is unclear. METHODS: Among 1214 consecutive patients who underwent pulmonary resection for primary lung cancer in our institute between 2004 and 2015, we identified 21 patients on CHD, who were the subjects of this study. Life-threatening postoperative complications were defined as grade 4 and 5 per the Clavien-Dindo classification. RESULTS: Fourteen (67%) of these 21 patients suffered postoperative complications, which were life threatening in 5. There was a higher frequency of interstitial pneumonia (IP) in the patients with life-threatening postoperative complications than in those with complications that were not life threatening (p = 0.032). The rates of acute exacerbation and 90-day mortality in the patients with IP were 50% and 75%, respectively. The overall survival (OS) rate of the patients with life-threatening postoperative complications was significantly lower than that of those with complications that were not life threatening (1- and 3-year OS rates: 40% and 0% vs. 80% and 57%, respectively, p = 0.001). CONCLUSIONS: Postoperative mortality and morbidity were high in patients on CHD who underwent pulmonary resection, especially if they had coexisting IP. Although IP is not a contraindication to pulmonary resection, the surgical strategy for CHD patients with IP should be considered carefully.

[A Case of Resected Superior Sulcus Tumor with Pathological Complete Response after Trimodality Therapy].

The current case was 73-year-oldwoman. She was referredto our hospital for an abnormal shadow of chest X-ray in the upper right lung field. Chest CT showed 3.5 cm of tumor located at the apex of right lobe with invasion of the chest wall. The tumor was diagnosed as squamous cell carcinoma using CT guided needle biopsy(superior sulcus tumor, clinical T3N0M0, Stage II B). The neoadjuvant therapy, 4 courses of chemotherapy(CBDCA plus PTX)andconcurrent radiotherapy(45 Gy/25 Fr)was performed. Chest CT revealed that tumor size was decreased to 2.3 cm in a diameter, and therapeutic effect was decided as partial response(34%). Upper right lobectomy combinedwith the chest wall(1th to 3th ribs)andmed iastinal lymph node dissection were performed. The pathological specimens showed no residual cancer cells(Ef3, pathological complete response[pCR]). She discharged without complications at 10 days after surgery. It is important to collect cases which obtainedpCR for development of more effective preoperative therapy.

Novel approach to pleurodesis with 50 % glucose for air leakage after lung resection or pneumothorax.

PURPOSE: Pleurodesis is performed in patients demonstrating air leakage after lung resection and in those with pneumothorax who must avoid surgery. However, there have so far been very few reports of pleurodesis with 50 % glucose. We herein examined the feasibility and effectiveness of this novel pleurodesis technique. METHODS: Thirty-five patients after lung resection and 11 pneumothorax patients without surgery were treated with pleurodesis using 50 % glucose. Approximately, 200 mL of 50 % glucose solution was injected into the pleural space and repeated until the air leakage stopped. Cases in which the air leakage did not stop after three injections were considered to be unsuccessful and subsequently treated with conventional pleurodesis using OK-432. RESULTS: Thirty-nine patients were successfully treated with 50 % glucose, although 7 patients required further treatment with OK-432. The unsuccessful group had some pulmonary comorbidities (P < 0.001), and the pleural effusion volume after pleurodesis was less than that in the successful group (P < 0.001). Although the air leakage did not stop in unsuccessful patients, the amount of air leakage markedly decreased. A temporary elevation of the blood sugar level was observed in 20 patients, but no other side effects had appeared. CONCLUSIONS: Pleurodesis with 50 % glucose is an easy, safe, and effective treatment modality. It is therefore considered to be a useful alternative method for pleurodesis.

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 and pathological predictors of engraftment for patient-derived xenografts in lung adenocarcinoma.

Patient-derived xenografts (PDXs) are increasingly utilized in preclinical drug efficacy studies due to their ability to retain the molecular, histological, and drug response characteristics of patient tumors. This study aimed to investigate the factors influencing the successful engraftment of PDXs. Lung adenocarcinoma PDXs were established using freshly resected tumor tissues obtained through surgery. Radiological data of pulmonary nodules from this PDX cohort were analyzed, categorizing them into solid tumors and tumors with ground-glass opacity (GGO) based on preoperative CT images. Gene mutation status was obtained from next generation sequencing data and MassARRAY panel. A total of 254 resected primary lung adenocarcinomas were utilized for PDX establishment, with successful initial engraftment in 58 cases (22.8 %); stable engraftment defined as at least three serial passages was observed in 43 cases (16.9 %). The stable engraftment rates of PDXs from solid tumors and tumors with GGO were 22.1 % (42 of 190 cases) and 1.6 % (1 of 64 cases), respectively (P < 0.001). Adenocarcinomas with advanced stage, poor differentiation, solid histologic subtype, and KRAS or TP53 gene mutations were associated with stable PDX engraftment. Avoiding tumors with GGO features could enhance the cost-effectiveness of establishing PDX models from early-stage resected lung adenocarcinomas.

An Update on Clinicopathological, Imaging, and Genetic Features of Angioleiomyoma.

Angioleiomyoma is a benign, pericytic (perivascular) neoplasm that primarily occurs in the subcutis or dermis of the extremities. The lesion typically presents as a small, firm, slow-growing, painful nodule. Magnetic resonance imaging reveals the lesion to be a well-defined, round to oval mass with signal intensity similar to or slightly hyperintense to that of skeletal muscle on T1-weightwed sequences. A dark reticular sign on T2-weighted sequences appears to be a characteristic feature of angioleiomyoma. Prominent enhancement is usually seen after intravenous contrast. Histologically, the lesion consists of well-differentiated smooth muscle cells with many vascular channels. Based on vascular morphologies, angioleiomyoma is classified into three subtypes: solid, venous, and cavernous. By immunohistochemistry, angioleiomyoma is diffusely positive for smooth muscle actin and calponin and variably for h-caldesmon and desmin. Conventional cytogenetic studies have demonstrated relatively simple karyotypes characterized by one or few structural rearrangements or numerical aberrations. In addition, metaphase comparative genomic hybridization analyses have revealed recurrent loss of 22q and gain of Xq. Angioleiomyoma can be successfully treated with simple excision, with a very low recurrence rate. Knowledge of this peculiar neoplasm is important because it can mimic a variety of benign and malignant soft-tissue tumors. This review provides an updated overview of the clinical, radiological, histopathological, cytogenetic, and molecular genetic features of angioleiomyoma.

Preclinical feasibility of bronchoscopic fluorescence-guided lung sentinel lymph node mapping.

OBJECTIVE: Lung sentinel lymph node mapping, where peritumorally injected material is tracked through the lymphatics, aims to find the first potential sites of nodal metastasis. We sought to evaluate the preclinical feasibility of bronchoscopic fluorescence-guided sentinel lymph node mapping. METHODS: Healthy Yorkshire pigs were used; sentinel lymph node mapping was performed with indocyanine green. The primary fluorescence imaging method was an ultrathin composite fiberscope placed in the bronchoscope working channel. Secondary methods used a fluorescence thoracoscope placed in the trachea (rigid bronchoscopy) and pretracheal fascial plane (mediastinoscopy) to validate ultrathin composite fiberscope settings for sentinel lymph node detection. A tracheostomy was created, and the pig was placed in a lateral decubitus position. Transbronchial intraparenchymal indocyanine green injection was performed primarily in the right lower lobe. Ultrathin composite fiberscope and rigid bronchoscopy were performed with (n = 6) or without (n = 2) mediastinoscopy, with the former group guiding dose and ultrathin composite fiberscope optimization. Fluorescent targets were interrogated by endobronchial ultrasound before ultrathin composite fiberscope-guided transbronchial needle aspiration. Specimen fluorescence was documented before creating cytological smears. Pigs were killed postprocedure for nodal dissection. RESULTS: A total of 100 µL of 10 mg/mL indocyanine green generated strong transbronchial fluorescence with low risk of indocyanine green contamination. Fluorescence was detectable by 10 minutes postinjection. There was concordance among ultrathin composite fiberscope, rigid bronchoscopy, and mediastinoscopy. Except for 1 pig with airway contamination, ultrathin composite fiberscope-guided endobronchial ultrasound transbronchial needle aspiration obtained fluorescent material in all pigs. Specimen fluorescence was associated with specimen adequacy. CONCLUSIONS: Bronchoscopic fluorescence-guided sentinel lymph node mapping was feasible, with specimen fluorescence providing real-time feedback on sentinel lymph node biopsy success. If translated to clinical practice, attention must be paid to minimizing indocyanine green leakage.

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.

Repeated photodynamic therapy mediates the abscopal effect through multiple innate and adaptive immune responses with and without immune checkpoint therapy.

In combination with immune checkpoint inhibitors, photodynamic therapy can induce robust immune responses capable of preventing local tumor recurrence and delaying the growth of distant, untreated disease (ie. the abscopal effect). Previously, we found that repeated photodynamic therapy (R-PDT) using porphyrin lipoprotein (PLP) as a photosensitizer, without the addition of an immune checkpoint inhibitor, can induce the abscopal effect. To understand why PLP mediated R-PDT alone can induce the abscopal effect, and how the addition of an immune checkpoint inhibitor can further strengthen the abscopal effect, we investigated the broader immune mechanisms facilitated by R-PDT and combination R-PDT + anti-PD-1 monoclonal antibody (αPD-1) in a highly aggressive, subcutaneous AE17-OVA mesothelioma dual tumor-bearing C57BL/6 mice. We found a 46.64-fold and 61.33-fold increase in interleukin-6 (IL-6) after R-PDT and combination R-PDT + αPD-1 relative to PBS respectively, suggesting broad innate immune activation. There was a greater propensity for antigen presentation in the spleen and distal, non-irradiated tumor draining lymph nodes, as dendritic cells and macrophages had increased expression of MHC class II, CD80, and CD86, after R-PDT and combination R-PDT + αPD-1. Concurrently, there was a shift in the proportions of CD4+ T cell subsets in the spleen, and an increase in the frequency of CD8+ T cells in the distal, non-irradiated tumor draining lymph nodes. While R-PDT had an acceptable safety profile, combination R-PDT + αPD-1 induced 1.26-fold higher serum potassium and 1.33-fold phosphorus, suggestive of mild laboratory tumor lysis syndrome. Histology revealed an absence of gross inflammation in critical organs after R-PDT and combination R-PDT + αPD-1 relative to PBS-treated mice. Taken together, our findings shed light on how the abscopal effect can be induced by PDT and strengthened by combination R-PDT + αPD-1, and suggests minimal toxicities after R-PDT.

Prognostic role of preoperative fluorine-18 fluorodeoxyglucose-positron emission tomography/computed tomography with an image-based harmonization technique: A multicenter retrospective study.

Objectives: Despite the prognostic impacts of preoperative fluorine-18 fluorodeoxyglucose-positron emission tomography/computed tomography examination, fluorine-18 fluorodeoxyglucose-positron emission tomography/computed tomography-based prognosis prediction has not been used clinically because of the disparity in data between institutions. By applying an image-based harmonized approach, we evaluated the prognostic roles of fluorine-18 fluorodeoxyglucose-positron emission tomography/computed tomography parameters in clinical stage I non-small cell lung cancer. Methods: We retrospectively examined 495 patients with clinical stage I non-small cell lung cancer who underwent fluorine-18 fluorodeoxyglucose-positron emission tomography/computed tomography examinations before pulmonary resection between 2013 and 2014 at 4 institutions. Three different harmonization techniques were applied, and an image-based harmonization, which showed the best-fit results, was used in the further analyses to evaluate the prognostic roles of fluorine-18 fluorodeoxyglucose-positron emission tomography/computed tomography parameters. Results: Cutoff values of image-based harmonized fluorine-18 fluorodeoxyglucose-positron emission tomography/computed tomography parameters, maximum standardized uptake, metabolic tumor volume, and total lesion glycolysis were determined using receiver operating characteristic curves that distinguish pathologic high invasiveness of tumors. Among these parameters, only the maximum standardized uptake was an independent prognostic factor in recurrence-free and overall survivals in univariate and multivariate analyses. High image-based maximum standardized uptake value was associated with squamous histology or lung adenocarcinomas with higher pathologic grades. In subgroup analyses defined by ground-glass opacity status and histology or by clinical stages, the prognostic impact of image-based maximum standardized uptake value was always the highest compared with other fluorine-18 fluorodeoxyglucose-positron emission tomography/computed tomography parameters. Conclusions: The image-based fluorine-18 fluorodeoxyglucose-positron emission tomography/computed tomography harmonization was the best fit, and the image-based maximum standardized uptake was the most important prognostic marker in all patients and in subgroups defined by ground-glass opacity status and histology in surgically resected clinical stage I non-small cell lung cancers.

Assessment of effectiveness and safety of thrombolytic therapy to pulmonary emboli by endobronchial ultrasound-guided transbronchial needle injection.

Objective: Endobronchial ultrasound-guided transbronchial needle injection (EBUS-TBNI) may effectively treat acute pulmonary embolisms (PEs). Here, we assessed the effectiveness of clot dissolution and safety of tissue plasminogen activator (t-PA) injection using EBUS-TBNI in a 1-week survival study of a porcine PE model. Methods: Six pigs with bilateral PEs were used: 3 for t-PA injection using EBUS-TBNI (TBNI group) and 3 for systemic administration of t-PA (systemic group). Once bilateral PEs were created, each 25 mg of t-PA injection using EBUS-TBNI for bilateral PEs (a total of 50 mg t-PA) and 100 mg of t-PA systemic administration was performed on day 1. Hemodynamic parameters, blood tests, and contrast-enhanced computed tomography scans were carried out at several time points. On day 7, pigs were humanely killed to evaluate the residual clot volume in the pulmonary arteries. Results: The average of percent change of residual clot volumes was significantly lower in the TBNI group than in the systemic group (%: systemic group 36.6 ± 22.6 vs TBNI group 9.6 ± 6.1, P < .01) on day 3. Considering the elapsed time, the average decrease of clot volume per hour at pre-t-PA to post t-PA was significantly greater in the TBNI group than in the systemic group (mm3/hour: systemic 68.1 ± 68.1 vs TBNI 256.8 ± 148.1, P < .05). No hemorrhage was observed intracranially, intrathoracically, or intraperitoneally on any contrast-enhanced computed tomography images. Conclusions: This study revealed that t-PA injection using EBUS-TBNI is an effective and safe way to dissolve clots.

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.

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.

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.

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.

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.

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.