ATF7IP2/MCAF2 directs H3K9 methylation and meiotic gene regulation in the male germline.

H3K9 trimethylation (H3K9me3) plays emerging roles in gene regulation, beyond its accumulation on pericentric constitutive heterochromatin. It remains a mystery why and how H3K9me3 undergoes dynamic regulation in male meiosis. Here, we identify a novel, critical regulator of H3K9 methylation and spermatogenic heterochromatin organization: the germline-specific protein ATF7IP2 (MCAF2). We show that in male meiosis, ATF7IP2 amasses on autosomal and X-pericentric heterochromatin, spreads through the entirety of the sex chromosomes, and accumulates on thousands of autosomal promoters and retrotransposon loci. On the sex chromosomes, which undergo meiotic sex chromosome inactivation (MSCI), the DNA damage response pathway recruits ATF7IP2 to X-pericentric heterochromatin, where it facilitates the recruitment of SETDB1, a histone methyltransferase that catalyzes H3K9me3. In the absence of ATF7IP2, male germ cells are arrested in meiotic prophase I. Analyses of ATF7IP2-deficient meiosis reveal the protein's essential roles in the maintenance of MSCI, suppression of retrotransposons, and global up-regulation of autosomal genes. We propose that ATF7IP2 is a downstream effector of the DDR pathway in meiosis that coordinates the organization of heterochromatin and gene regulation through the spatial regulation of SETDB1-mediated H3K9me3 deposition.

Chromatin remodeler CHD8 is required for spermatogonial proliferation and early meiotic progression.

Meiosis is a key step during germ cell differentiation, accompanied by the activation of thousands of genes through germline-specific chromatin reorganization. The chromatin remodeling mechanisms underpinning early meiotic stages remain poorly understood. Here we focus on the function of one of the major autism genes, CHD8, in spermatogenesis, based on the epidemiological association between autism and low fertility rates. Specific ablation of Chd8 in germ cells results in gradual depletion of undifferentiated spermatogonia and the failure of meiotic double-strand break (DSB) formation, leading to meiotic prophase I arrest and cell death. Transcriptional analyses demonstrate that CHD8 is required for extensive activation of spermatogenic genes in spermatogonia, necessary for spermatogonial proliferation and meiosis. CHD8 directly binds and regulates genes crucial for meiosis, including H3K4me3 histone methyltransferase genes, meiotic cohesin genes, HORMA domain-containing genes, synaptonemal complex genes, and DNA damage response genes. We infer that CHD8 contributes to meiotic DSB formation and subsequent meiotic progression through combined regulation of these meiosis-related genes. Our study uncovers an essential role of CHD8 in the proliferation of undifferentiated spermatogonia and the successful progression of meiotic prophase I.

Standardized Classification of Lung Adenocarcinoma Subtypes and Improvement of Grading Assessment Through Deep Learning.

The histopathologic distinction of lung adenocarcinoma (LADC) subtypes is subject to high interobserver variability, which can compromise the optimal assessment of patient prognosis. Therefore, this study developed convolutional neural networks capable of distinguishing LADC subtypes and predicting disease-specific survival, according to the recently established LADC tumor grades. Consensus LADC histopathologic images were obtained from 17 expert pulmonary pathologists and one pathologist in training. Two deep learning models (AI-1 and AI-2) were trained to predict eight different LADC classes. Furthermore, the trained models were tested on an independent cohort of 133 patients. The models achieved high precision, recall, and F1 scores exceeding 0.90 for most of the LADC classes. Clear stratification of the three LADC grades was reached in predicting the disease-specific survival by the two models, with both Kaplan-Meier curves showing significance (P = 0.0017 and 0.0003). Moreover, both trained models showed high stability in the segmentation of each pair of predicted grades with low variation in the hazard ratio across 200 bootstrapped samples. These findings indicate that the trained convolutional neural networks improve the diagnostic accuracy of the pathologist and refine LADC grade assessment. Thus, the trained models are promising tools that may assist in the routine evaluation of LADC subtypes and grades in clinical practice.

Alternative splicing for germ cell-specific Mga transcript can be eliminated without compromising mouse viability or fertility.

The stimulated by retinoic acid gene 8 (STRA8)/MEIOSIN complex and polycomb repressive complex (PRC) 1.6, a PRC1 subtype, are believed to be positive and negative regulators of meiotic onset, respectively. During meiotic initiation, the transcription repressive activity of PRC1.6 must be attenuated so that meiosis-related genes can be effectively activated by the STRA8/MEIOSIN complex. However, the molecular mechanisms that control the impairment of PRC1.6 function remain unclear. We recently demonstrated that the Mga gene, which encodes a scaffolding component of PRC1.6, produces variant mRNA by alternative splicing specifically during meiosis. Furthermore, the anomalous MGA protein encoded by the variant mRNA bears an intrinsic ability to function as a dominant negative regulator against the construction of PRC1.6 and is therefore assumed to be, at least in part, involved in impairment of the complex. Therefore, to unequivocally evaluate the physiological significance of Mga variant mRNA production in gametogenesis, we examined the consequences of a genetic manipulation that renders mice unable to produce Mga variant mRNA. Our data revealed that mutant mice were equivalent to wild-type mice in terms of viability and fertility. Our detailed examination of spermatogenesis also revealed that this genetic alteration is not associated with any apparent abnormalities in testis size, spermatogenic cycle, timing of meiotic onset, or marker gene expression of spermatogonia and spermatocytes. Taken together, these data indicate that the production of germ cell-specific Mga variant mRNA is dispensable not only for viability but also for gametogenesis.

Two DNA binding domains of MGA act in combination to suppress ectopic activation of meiosis-related genes in mouse embryonic stem cells.

Although the physiological meaning of the high potential of mouse embryonic stem cells (ESCs) for meiotic entry is not understood, a rigid safeguarding system is required to prevent ectopic onset of meiosis. PRC1.6, a non-canonical PRC1, is known for its suppression of precocious and ectopic meiotic onset in germ cells and ESCs, respectively. MGA, a scaffolding component of PRC1.6, bears two distinct DNA-binding domains termed bHLHZ and T-box. However, it is unclear how this feature contributes to the functions of PRC1.6. Here, we demonstrated that both domains repress distinct sets of genes in murine ESCs, but substantial numbers of meiosis-related genes are included in both gene sets. In addition, our data demonstrated that bHLHZ is crucially involved in repressing the expression of Meiosin, which plays essential roles in meiotic entry with Stra8, revealing at least part of the molecular mechanisms that link negative and positive regulation of meiotic onset.

CD24, not CD47, negatively impacts upon response to PD-1/L1 inhibitors in non-small-cell lung cancer with PD-L1 tumor proportion score < 50.

CD24, a heavily glycosylated glycosylphosphatidylinositol-anchored surface protein, inhibits phagocytosis as potently as CD47. The relationship between such anti-phagocytic factors and the immune response with immune-checkpoint inhibitors (ICI) remains unexplored. We evaluated CD24 and CD47 tumor proportion scores (TPS) in 68 of the 106 patients with advanced non-small-cell lung cancer who participated in a prospective observational study of ICI treatment. We also explored the impact of CD24 TPS and CD47 TPS on ICI efficacy and serum cytokine changes. CD24 positivity (TPS ≥ 1) was negatively associated with progression-free survival (PFS) of ICI when PD-L1 TPS was < 50 (median PFS; 37 vs 127 d, P = .033), but there was no association when PD-L1 TPS was ≥ 50 (median PFS; 494 vs 144 d, P = .168). CD24 positivity was also related to significantly higher increase of CCL2 from baseline to 4-6 wk later, and such increase was notably observed only when PD-L1 TPS < 50 (P = .0004). CCL2 increase after ICI initiation was negatively predictive for survival after initiation of ICI (median survival time; not reached vs 233 d; P = .028). CD47 TPS high (≥60) significantly suppressed the increase in vascular endothelial growth factor (VEGF)-A, D and PDGF-AB/BB after ICI initiation. There was no association, however, between CD47 tumor expression and the efficacy of ICI. In conclusion, CD24, not CD47, is a candidate negative predictive marker of ICI in advanced, non-small-cell lung cancer with PD-L1 TPS < 50. Tumor expression of both CD24 and CD47 was associated with changes in factors related to monocytes and angiogenesis after ICI initiation (UMIN000024414).

Tumour content ratio matters for detecting epidermal growth factor receptor mutation by cobas test in small biopsies; a retrospective study.

BACKGROUND: Recent studies indicate the benefit of treatment with osimertinib over that with conventional epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI) for untreated EGFR-mutated non-small cell lung cancer (NSCLC). Cobas ver2 is the only companion diagnostic method for detecting EGFR mutations with osimertinib treatment. We clinically experience false negative cases with this test, but its actual sensitivity is unknown. Moreover, no study has suggested the importance of tumour dissection, and most facilities do not routinely perform them on small biopsies. The purpose of this study was to evaluate the sensitivity of cobas in clinical practice and clarify the role of dissection as a component of the cobas testing. METHODS: We examined 132 patients with EGFR-mutated NSCLC diagnosed by bronchoscopy and confirmed with PCR clamp. Patients were tested with cobas and the EGFR-positive rate was calculated. Samples with undetected EGFR mutations were retested after tumour dissection and the rate of samples whose EGFR mutation was corrected to positive was assessed. To evaluate tumour cellularity, the tumour content ratio was assessed by calculating tumour cell count over the total cell count on the slide. RESULTS: The positive rate of EGFR mutation identification was 76% with cobas, although EGFR mutation-negative patients retained responses to TKI therapy equivalent to positive patients did; however, the tumour content ratio of negative samples was significantly lower than that of positive samples. Twenty-nine negative samples underwent dissection and 24% were corrected to positive. Moreover, 53% of the samples with a tumour content ratio below 10% was negative for cobas, but 33% of these turned positive after dissection. CONCLUSIONS: Cobas had a high false negative rate in clinical practice, and tumour content ratio might be associated with this rate. Dissection could improve the sensitivity of cobas, especially in samples with low tumour cellularity.

Expression of phosphatase and tensin homolog and programmed cell death ligand 1 in adenosquamous carcinoma of the lung.

BACKGROUND: Lung adenosquamous carcinoma (ASC) is a rare variant of non-small cell lung cancer (NSCLC) with poor prognosis. Certain biological differences may exist between these tumors and other common histological types of NSCLC, including adenocarcinoma (ADC) and squamous cell carcinoma (SCC). The phosphoinositide 3-kinase (PI3K) pathway, which links oncogenes and multiple receptor classes to essential cellular functions, is activated by phosphatase and tensin homolog (PTEN) loss. The PTEN loss has been suggested to induce programmed cell death ligand 1 (PD-L1) expression in various cancer types. OBJECTIVE: Here, we sought to determine the relationships between the expression of PTEN and PD-L1 in each component of ASC with ADC and SCC, and clinical parameters. MATERIAL AND METHODS: Tissue microarrays of 148 cases of surgically resected lung ADC and 102 cases of SCC, as well as full sections from 28 ASC cases, were analyzed immunohistochemically for the expression of PTEN and PD-L1. RESULTS: PD-L1 expression was similar between the adenocarcinoma component of ASC vs. lung ADC and between the squamous component of ASC vs. lung SCC. PTEN loss was higher in lung ADC than in the adenocarcinoma component of ASC and significantly higher in lung SCC than in the squamous component of ASC. PD-L1 expression was higher in the squamous component than in the glandular component of the 28 ASC cases, but PTEN loss was similar. Overall, PTEN loss was higher in lung SCC than in lung ADC and both components of ASC. In lung SCC and glandular portions of ASC, PD-L1 expression levels were significantly associated with those of PTEN. The loss of PTEN correlated with smoking status in patients with lung ADC. CONCLUSIONS: Our results implied that both squamous and glandular components of ASC may share the same oncogenic driver pathway for carcinogenesis. However, the squamous cell components of ASC likely escape the immune surveillance better than the glandular components due to higher PD-L1 expression.

Long-term culture of undifferentiated spermatogonia isolated from immature and adult bovine testes.

Undifferentiated spermatogonia eventually differentiate in the testis to produce haploid sperm. Within this cell population, there is a small number of spermatogonial stem cells (SSCs). SSCs are rare cells in the testis, and their cellular characteristics are poorly understood. Establishment of undifferentiated cell line would provide an indispensable tool for studying their biological nature and spermiogenesis/spermatogenesis in vitro. However, there have been few reports on the long-term culture of undifferentiated spermatogonia in species other than rodents. Here, we report the derivation and long-term in vitro culture of undifferentiated spermatogonia cell lines from immature and adult bovine testes. Cell lines from immature testes were maintained in serum-free culture conditions in the presence of glial-cell-line-derived neurotropic factor (GDNF) and bovine leukemia inhibitory factor (bLIF). These cell lines have embryonic stem (ES)-like cell morphology, express pluripotent-stem-cell-specific and germ-cell-specific markers at the protein and mRNA levels, and contributed to the inner cell mass (ICM) of embryos in the blastocyst stage. Meanwhile, cell lines established from adult testes were maintained in low-serum media in the presence of 6-bromoindirubin-3'-oxime (BIO). These cell lines have characteristics resembling those of previously reported male mouse germ cell lines as confirmed by their botryoidally aggregated morphology, as well as the expression of germ-cell-specific markers and pluripotent stem cell markers. These findings could be useful for the development of long-term culture of undifferentiated spermatogonia, which could aid in conservation of species and improvement of livestock production through genome editing technology.

Negative effects of G-protein-coupled free fatty acid receptor GPR40 on cell migration and invasion in fibrosarcoma HT1080 cells.

G-protein-coupled receptor 40 (GPR40) and GPR120 mediate a variety of biological functions by the binding of long and medium chain free fatty acids. In the present study, we investigated a role of GPR40 in the pathogenesis of fibrosarcoma HT1080 cells. The GPR40 gene expression was detected in HT1080 cells, but not the GPR120 gene. The cell motile and invasive activities were markedly enhanced by GPR40 knockdown, compared with control cells. To evaluate whether GPR40 is involved in the cellular functions of HT1080 cells during anticancer drug treatment, HT1080 cells were maintained in condition medium containing cisplatin (CDDP) (0.01-1.0 µM) for 6 mo. The expression levels of the GPR40 gene was elevated by the long-term CDDP treatment in HT1080 cells, while the GPR120 gene expression remained unchanged. The cell motile and invasive activities of HT1080 cells treated with CDDP were significantly lower than those of untreated cells. In gelatin zymography, the activities of matrix metalloproteinase-2 (MMP-2) and MMP-9 of HT1080 cells were enhanced by the long-term CDDP treatment. In addition, GW9508 which is an agonist of GPR40 and GPR120 suppressed the cell motile and invasive activities of HT1080 cells treated with CDDP as well as the MMP activation. These results suggest that GPR40 negatively regulates the tumor progression of fibrosarcoma cells. © 2015 Wiley Periodicals, Inc.

Management of unexpected intraoperative bleeding during thoracoscopic pulmonary resection: a single institutional experience.

PURPOSE: Unexpected intraoperative bleeding during thoracoscopic surgery, necessitating emergency conversion to thoracotomy, is gradually being reported. We reviewed our experience of encountering unexpected bleeding during thoracoscopic surgery. METHODS: We defined "unexpected intraoperative bleeding" as the need for hemostatic procedures with angiorrhaphy, with or without a sealant. The location, cause, and management of injured vessels, and perioperative outcomes were investigated and compared with those for patients without injured vessels. RESULTS: Between 2007 and 2014, a total of 241 thoracoscopic anatomical pulmonary resections were performed at our hospital. Twenty (8.3 %) of these patients required hemostatic procedures with angiorrhaphy, with or without a sealant. The main injured vessels were the pulmonary artery (n = 13) and vein (n = 3) and the main causes of injury were related to technical issues with energy devices and staplers. There were no morbidities related to intraoperative bleeding. The operation time and blood loss were significantly greater in the patients with vessel injury than in those without vessel injury, but perioperative morbidities and the duration of chest tube insertion (4.5 vs. 3.5 days, average, p = 0.20) and postoperative hospital stay (12.7 vs. 11.0 days, average, p = 0.08) were not significantly different. CONCLUSIONS: The frequency of unexpected bleeding was relatively high in this series, but its management and outcomes were satisfactory in terms of safety.

Changes in quadriceps muscle thickness in acute non-ambulatory stroke survivors.

BACKGROUND: Loss of skeletal muscle mass is one of the main reasons for disability in patients with stroke. However, lower leg muscle wasting has not been studied in acute stroke patients. OBJECTIVE: To investigate the changes in quadriceps muscle thickness in acute non-ambulatory stroke survivors. METHODS: A total of 16 consecutive acute non-ambulatory stroke survivors who were in acute inpatient rehabilitation, with a mean age of 72.1 years, were included in the study. Quadriceps muscle thickness was examined in their paretic and non-paretic limbs within the first week from admission (first week), 1 week after the first examination (second week), and 1 week after the second week examination (third week) using ultrasonography. RESULTS: Quadriceps muscle thickness in the paretic limb decreased every week (mean% difference between the first and second weeks, 12.8, 95% confidence interval (CI) 5.3-20.2%; mean% difference between the second and third weeks, 10.1, 95% CI 5.2-14.9%). Quadriceps muscle thickness in the non-paretic limb was lower in the second and third weeks than the first week, but there was no difference between the second and third weeks (mean% difference between the first and second weeks, 9.3, 95% CI 2.5-16.1%; mean% difference between the second and third weeks, 5.3, 95% CI - 1.6 to 12.1%). CONCLUSION: Quadriceps muscle thickness decreased in acute non-ambulatory stroke survivors not only in the paretic limb but also in the non-paretic limb, particularly during the period from admission to the second week.

Changes in Quadriceps Muscle Thickness, Disease Severity, Nutritional Status, and C-Reactive Protein after Acute Stroke.

BACKGROUND: Lower leg muscle wasting is common in stroke patients; however, patient characteristics in the acute phase are rarely studied. This study aimed to examine the relationship between changes in quadriceps muscle thickness and disease severity, nutritional status, and C-reactive protein (CRP) levels after acute stroke. METHODS: Thirty-one consecutive patients with acute intracerebral hemorrhage or ischemic stroke had quadriceps muscle thickness measured in the paretic and nonparetic limbs within 1 week after admission (first week) and 2 weeks after the first examination (last week) using ultrasonography. We also determined the relationship between the percentage change in muscle thickness and disease severity, nutritional status, and CRP levels on admission. RESULTS: There was a significant correlation between changes in muscle thickness for both paretic and nonparetic sides and National Institutes of Health Stroke Scale (NIHSS) scores (paretic limb: r = -.46, P = .01; nonparetic limb: r = -.54, P = .002, respectively); however, there was no significant correlation with nutritional status on admission. Quadriceps muscle thickness was reduced more in the CRP-positive (≥.3 mg/dL) patients than in the CRP-negative (<.3 mg/dL) patients in the nonparetic limb (positive: -21.4 ± 12.1, negative: -11.4 ± 16.4%; P = .039), but not in the paretic limb (positive: -23.4 ± 9.0, negative: -19.1 ± 15.7; P = .27). CONCLUSIONS: A high NIHSS score and a positive CRP on admission were both significantly correlated with decreased quadriceps muscle thickness after acute stroke. Nutritional status on admission was not correlated with changes in quadriceps muscle thickness for these patients.

Role of GPR120 in cell motile activity induced by 12-O-tetradecanoylphorbol-13-acetate in liver epithelial WB-F344 cells.

G-protein-coupled receptor 120 (GPR120) is identified as a G-protein-coupled receptor for unsaturated long-chain free fatty acids that mediates insulin signaling and anti-inflammatory effects. Recently, it has been reported that GPR120 promotes the cell motile activity and angiogenesis in cancer cells. In this study, we assessed the role of GPR120 in the cell motile activity induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in rat liver epithelial WB-F344 cells. Cells were treated with TPA at a concentration of 5 nM for 72 h. The expression level of the Gpr120 gene was measured by quantitative real-time RT-PCR analysis. Cells treated with TPA showed the elevated Gpr120 expression, in comparison with untreated cells. In cell motility assays, the cell motile activity of cells treated with TPA was significantly higher than that of untreated cells. To confirm whether GPR120 is involved in the cell motile activity mediated by TPA, we generated GPR120 knockdown cells from WB-F344 cells. The cell motile activity induced by TPA was significantly suppressed by GPR120 knockdown. These results suggest that GPR120 plays an important role in the cell motile activity induced by TPA in WB-F344 cells.

[Recurrence of Anaplastic Lymphoma Kinase Positive Lung Cancer Nineteen-year after the Primary Surgery].

A 77-years-old man who underwent middle lobectomy for lung adenocarcinoma in our hospital 19 years ago. p-Stage IA was pointed out multiple nodules in bilateral lung fields on a medical examination. Computed tomography scan revealed a tumor measuring 34×34 mm in size in the right lower lung and other 40 small pulmonary nodules. Characteristic pattern of metastatic adenocarcinoma from the previous lung cancer was pathologically demonstrated. Immunostaining revealed anaplastic lymphoma kinase (ALK) positivity of the both specimens, which determined the diagnosis of recurrence. Long-team postoperative follow-up for ALK positive lung cancer patients is considered to be necessary especially for younger patients.

KRAS mutation confers resistance to antibody-dependent cellular cytotoxicity of cetuximab against human colorectal cancer cells.

Cetuximab is a chimeric IgG1 monoclonal antibody (mAb) that targets the extracellular domain of epidermal growth factor receptor (EGFR). Oncogenic KRAS mutations in tumors have been shown to be a negative predictor of the response of colorectal cancer (CRC) to cetuximab treatment. Cetuximab exerts its therapeutic effects through several mechanisms including antibody-dependent cellular cytotoxicity (ADCC). However, the influence of KRAS mutations on cetuximab-mediated ADCC is not fully understood. Here, we investigated cetuximab-mediated ADCC in two pairs of isogenic CRC cells with or without a KRAS mutation. Peripheral blood mononuclear cells (PBMCs) from healthy volunteers and NK92, a natural killer (NK) cell line that exogenously expresses FcγRIIIa (CD16a), were used as effector cells. In an ADCC assay, perforin-dependent target cell lysis was not affected by the KRAS mutation status. On the other hand, perforin-independent ADCC was observed only in CRC cells with wild-type KRAS, but not in cells with mutant KRAS. Neutralizing experiments revealed that the Fas-Fas ligand (FasL) interaction was responsible for the induction of apoptosis and perforin-independent ADCC. Furthermore, the presence of effector cells clearly enhanced the growth-inhibitory effect of cetuximab only in CRC cells with wild-type KRAS, but not in those with mutant KRAS. These findings suggest that ADCC is an important mode of action of cetuximab and that KRAS mutation impairs the therapeutic effect exerted by cetuximab-mediated ADCC.

Pulmonary carcinosarcoma successfully resected using the rib-cross thoracotomy approach: report of a case.

Pulmonary carcinosarcoma is extremely rare and disease prognosis is very poor. A solid large tumor occupying the left thorax was detected in a 66-year-old female. Rib-cross thoracotomy was performed to excise the tumor; the 5th and 6th ribs and intercostal muscles and vessels were cut along the mid-axillary line, and the thorax was entered posteriorly at the 4th intercostal space and anteriorly at the 6th intercostal space, providing wide exposure of the entire thorax. Left pneumonectomy combined with chest wall resection was successfully performed, followed by chest reconstruction to achieve complete resection. Histopathologically, adenocarcinoma and spindle cell sarcoma containing rhabdomyosarcoma components were identified; the patient was diagnosed with pT3N1M0 stage IIIA true pulmonary carcinosarcoma. Postoperative adjuvant chemotherapy containing cisplatin and vinorelbine was administered. There was no recurrence of the disease 20 months after surgery. Aggressive excision may result in favorable outcomes for pulmonary carcinosarcoma.

Epigenetic priming in the male germline.

Epigenetic priming presets chromatin states that allow the rapid induction of gene expression programs in response to differentiation cues. In the germline, it provides the blueprint for sexually dimorphic unidirectional differentiation. In this review, we focus on epigenetic priming in the mammalian male germline and discuss how cellular memories are regulated and inherited to the next generation. During spermatogenesis, epigenetic priming predetermines cellular memories that ensure the lifelong maintenance of spermatogonial stem cells and their subsequent commitment to meiosis and to the production of haploid sperm. The paternal chromatin state is also essential for the recovery of totipotency after fertilization and contributes to paternal epigenetic inheritance. Thus, epigenetic priming establishes stable but reversible chromatin states during spermatogenesis and enables epigenetic inheritance and reprogramming in the next generation.

Machine learning analysis of pathological images to predict 1-year progression-free survival of immunotherapy in patients with small-cell lung cancer.

BACKGROUND: In small-cell lung cancer (SCLC), the tumor immune microenvironment (TIME) could be a promising biomarker for immunotherapy, but objectively evaluating TIME remains challenging. Hence, we aimed to develop a predictive biomarker of immunotherapy efficacy through a machine learning analysis of the TIME. METHODS: We conducted a biomarker analysis in a prospective study of patients with extensive-stage SCLC who received chemoimmunotherapy as the first-line treatment. We trained a model to predict 1-year progression-free survival (PFS) using pathological images (H&E, programmed cell death-ligand 1 (PD-L1), and double immunohistochemical assay (cluster of differentiation 8 (CD8) and forkhead box P3 (FoxP3)) and patient information. The primary outcome was the mean area under the curve (AUC) of machine learning models in predicting the 1-year PFS. RESULTS: We analyzed 100,544 patches of pathological images from 78 patients. The mean AUC values of patient information, pathological image, and combined models were 0.789 (range 0.571-0.982), 0.782 (range 0.750-0.911), and 0.868 (range 0.786-0.929), respectively. The PFS was longer in the high efficacy group than in the low efficacy group in all three models (patient information model, HR 0.468, 95% CI 0.287 to 0.762; pathological image model, HR 0.334, 95% CI 0.117 to 0.628; combined model, HR 0.353, 95% CI 0.195 to 0.637). The machine learning analysis of the TIME had better accuracy than the human count evaluations (AUC of human count, CD8-positive lymphocyte: 0.681, FoxP3-positive lymphocytes: 0.626, PD-L1 score: 0.567). CONCLUSIONS: The spatial analysis of the TIME using machine learning predicted the immunotherapy efficacy in patients with SCLC, thus supporting its role as an immunotherapy biomarker.

Epidermal growth factor receptor variant type III markedly accelerates angiogenesis and tumor growth via inducing c-myc mediated angiopoietin-like 4 expression in malignant glioma.

BACKGROUND: Expression of the constitutively activated mutant EGFR variant III (EGFRvIII), the most common mutation in glioblastoma multiforme (GBMs), has been clinically correlated with tumor proliferation, invasion, and angiogenesis. In this study, we examined the role of EGFRvIII on the tumor microenvironment, especially on angiogenesis. METHODS: To study the role of EGFRvIII in tumor angiogenesis, we prepared LN229 glioblastoma transfected with enhanced green fluorescent protein (EGFP), wild-type EGFR, or EGFRvIII (LN229-WT or -vIII), and examined tumor growth and microvessel density in the tumors. Additionally, the potential angiogenic factors were identified by real-time PCR analysis, and the functions in LN229-vIII cells were examined. RESULTS: LN229-vIII cells showed more aggressive tumor growth and higher vascularity as compared to LN229-WT cells in vivo, although there was no significant difference in the cell growth rates in vitro. We next investigated the expression of 60 angiogenesis-related factors to clarify the mechanisms underlying the difference in vascularity between tumor xenografts of LN229-vIII and LN229-WT. We found that the mRNA and protein expressions of angiopoietin-like 4 (Angptl4), a secreted protein involved in angiogenesis and metabolism regulation, were significantly induced by EGFRvIII overexpression, both in vitro and in vivo. Constitutive knockdown of Angptl4 in LN229-vIII using shRNA significantly decreased the microvessel density in the tumor xenografts and suppressed tumor growth. To clarify the regulatory mechanisms of Angptl4 by EGFRvIII, we analyzed the signaling pathways and transcription factors by pharmacological inhibition and RNA interference. U0126, an ERK signal inhibitor dramatically suppressed Angptl4 expression. The transcription factor c-Myc, which is regulated by ERK, was activated in the LN229-vIII cells and knockdown of c-Myc using siRNA also attenuated Angptl4 expression in the LN229-vIII cells. Furthermore, chromatin immunoprecipitation (ChIP) assay revealed increased recruitment of c-Myc to the promoter region of Angptl4 in the LN229-vIII cells. CONCLUSIONS: In summary, we demonstrated that EGFRvIII induces Angptl4 expression through the ERK/c-Myc pathway and promotes tumor angiogenesis in malignant gliomas.