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.

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.

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.

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.

CTCF-mediated 3D chromatin predetermines the gene expression program in the male germline.

Spermatogenesis is a unidirectional differentiation process that generates haploid sperm, but how the gene expression program that directs this process is established is largely unknown. Here we determine the high-resolution 3D chromatin architecture of male germ cells during spermatogenesis and show that CTCF-mediated 3D chromatin predetermines the gene expression program required for spermatogenesis. In undifferentiated spermatogonia, CTCF-mediated chromatin contacts on autosomes pre-establish meiosis-specific super-enhancers (SE). These meiotic SE recruit the master transcription factor A-MYB in meiotic spermatocytes, which strengthens their 3D contacts and instructs a burst of meiotic gene expression. We also find that at the mitosis-to-meiosis transition, the germline-specific Polycomb protein SCML2 resolves chromatin loops that are specific to mitotic spermatogonia. Moreover, SCML2 and A-MYB establish the unique 3D chromatin organization of sex chromosomes during meiotic sex chromosome inactivation. We propose that CTCF-mediated 3D chromatin organization enforces epigenetic priming that directs unidirectional differentiation, thereby determining the cellular identity of the male germline.

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

H3K9 tri-methylation (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 upregulation 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.

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.

A case of cervical schwannoma with upper tracheal stenosis followed up as asthma for 4 years.

Cases of upper tracheal stenosis due to cervical schwannoma are fairly rare; therefore, no treatment has been determined. In this case, our patient had been treated for asthma for 4 years and was admitted to our hospital because of exacerbation. Computed tomography showed a tracheal stenosis lesion just below the vocal cords, and a biopsy revealed schwannoma. Conservative therapy was preferred rather than tumour resection by surgery. Follow-up for 5 years showed no changes on imaging. Conservative treatment is considered as an option if the extratracheal tumour does not grow.

Deep-Learning-Aided Detection of Mycobacteria in Pathology Specimens Increases the Sensitivity in Early Diagnosis of Pulmonary Tuberculosis Compared with Bacteriology Tests.

The histopathological diagnosis of mycobacterial infection may be improved by a comprehensive analysis using artificial intelligence. Two autopsy cases of pulmonary tuberculosis, and forty biopsy cases of undetected acid-fast bacilli (AFB) were used to train AI (convolutional neural network), and construct an AI to support AFB detection. Forty-two patients underwent bronchoscopy, and were evaluated using AI-supported pathology to detect AFB. The AI-supported pathology diagnosis was compared with bacteriology diagnosis from bronchial lavage fluid and the final definitive diagnosis of mycobacteriosis. Among the 16 patients with mycobacteriosis, bacteriology was positive in 9 patients (56%). Two patients (13%) were positive for AFB without AI assistance, whereas AI-supported pathology identified eleven positive patients (69%). When limited to tuberculosis, AI-supported pathology had significantly higher sensitivity compared with bacteriology (86% vs. 29%, p = 0.046). Seven patients diagnosed with mycobacteriosis had no consolidation or cavitary shadows in computed tomography; the sensitivity of bacteriology and AI-supported pathology was 29% and 86%, respectively (p = 0.046). The specificity of AI-supported pathology was 100% in this study. AI-supported pathology may be more sensitive than bacteriological tests for detecting AFB in samples collected via bronchoscopy.

Anti-tumor activity of the pan-RAF inhibitor TAK-580 in combination with KPT-330 (selinexor) in multiple myeloma.

RAS/RAF/MEK/ERK pathway inhibitors exhibit significant anti-tumor effects against various tumor types, including multiple myeloma (MM), and they are predicted to play a pivotal role in precision medicine. The XPO1 inhibitor KPT-330 has also exhibited promising efficacy in combination with other novel drugs in treating relapsed/refractory MM (RRMM). In this study, we explored the anti-tumor effects of a combination of the pan-RAF inhibitor TAK-580 and KPT-330. Importantly, TAK-580 enhanced KPT-330-induced cytotoxicity and apoptosis in human myeloma cell lines and primary myeloma cells from RRMM patients. Moreover, TAK-580 and KPT-330 synergistically inhibited nuclear phospho-FOXO3a and enhanced cytoplasmic phospho-FOXO3a in MM cells, leading to cytoplasmic enhanced Bim expression and finally apoptosis. This indicates that TAK-580 enhances KPT-330-induced cytotoxicity and apoptosis primarily via the FOXO3a-Bim axis. In addition, TAK-580 enhanced the cytotoxicity of KPT-330 against MM cells even in the presence of IGF-1. Taken together, our results demonstrate that a combination of pan-RAF inhibitor and XPO1 inhibitor is a potential therapeutic option in treating MM.

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.

Identification of germ cell-specific Mga variant mRNA that promotes meiosis via impediment of a non-canonical PRC1.

A non-canonical PRC1 (PRC1.6) prevents precocious meiotic onset. Germ cells alleviate its negative effect by reducing their amount of MAX, a component of PRC1.6, as a prerequisite for their bona fide meiosis. Here, we found that germ cells produced Mga variant mRNA bearing a premature termination codon (PTC) during meiosis as an additional mechanism to impede the function of PRC1.6. The variant mRNA encodes an anomalous MGA protein that lacks the bHLHZ domain and thus functions as a dominant negative regulator of PRC1.6. Notwithstanding the presence of PTC, the Mga variant mRNA are rather stably present in spermatocytes and spermatids due to their intrinsic inefficient background of nonsense-mediated mRNA decay. Thus, our data indicate that meiosis is controlled in a multi-layered manner in which both MAX and MGA, which constitute the core of PRC1.6, are at least used as targets to deteriorate the integrity of the complex to ensure progression of meiosis.

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).

Anti-tumor activities of the new oral pan-RAF inhibitor, TAK-580, used as monotherapy or in combination with novel agents in multiple myeloma.

Many RAS pathway inhibitors, including pan-RAF inhibitors, have shown significant anti-tumor activities in both solid and hematological tumors. The pan-RAF inhibitor, TAK-580, is a representative of the novel RAF inhibitors that act by disrupting RAF homo- or heterodimerization. In this study, we examined the anti-tumor effects of TAK-580 used as monotherapy or in combination with bortezomib, lenalidomide, or other novel agents in multiple myeloma (MM) cells in vitro. TAK-580 monotherapy potently targeted proteins in the RAS-RAF-MEK-ERK signaling pathway and induced potent cytotoxicity and apoptosis in MM cell lines and myeloma cells from patients with newly diagnosed and relapsed and/or refractory MM, compared with a representative RAF inhibitor, dabrafenib. Normal donor peripheral blood B lymphocytes and cord blood CD34-positive cells were not affected. Importantly, TAK-580 significantly inhibited phospho-FOXO3 and induced upregulation of BimL and BimS in a dose-dependent manner, finally leading to apoptosis in MM cells. Moreover, TAK-580 enhanced bortezomib-induced cytotoxicity and apoptosis in MM cells via the FOXO3-Bim axis and the terminal unfolded protein response. Importantly, TAK-580 also enhanced lenalidomide-induced cytotoxicity and apoptosis in MM cells. Taken together, our results provide the rationale for TAK-580 monotherapy and/or treatment in combination with novel agents to improve outcomes in patients with MM.

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.

Concordance between sequential transbronchial lung cryobiopsy and surgical lung biopsy in patients with diffuse interstitial lung disease.

BACKGROUND: Increasing evidence indicates the utility of transbronchial lung cryobiopsy (TBLC) for the diagnosis of interstitial lung disease (ILD). However, only one study has compared TBLC and surgical lung biopsy (SLB) performed on the same patients. METHODS: We identified seven patients with ILD with TBLC and SLB. We evaluated the clinical characteristics and made a pathological diagnosis based on the official ATS/ERS/JRS/ALAT clinical practice guideline of idiopathic pulmonary fibrosis with both TBLC and SLB. RESULTS: Six cases were diagnosed as Usual interstitial pneumonia (UIP) in both TBLC and SLB. One case was diagnosed as indeterminate for UIP with TBLC and probable UIP with SLB. Etiological diagnosis with TBLC and SLB were concordant in 2 cases of idiopathic pulmonary fibrosis (IPF) but discordant for other diagnoses. Major histological findings of UIP including dense fibrosis, peripheral distribution, and fibroblastic foci showed high concordance between TBLC and SLB, which implies that TBLC can reliably detect these features. In contrast, loose fibrosis, cellular infiltration, and airway disease showed poor concordance between the two methods. CONCLUSION: Our study showed that TBLC is useful for UIP diagnosis but not for other ILD. With a multidisciplinary approach, diagnosis of IPF may be determined by TBLC, whereas ILD other than IPF may require SLB.

Summary of the Japanese Respiratory Society statement for the treatment of lung cancer with comorbid interstitial pneumonia.

Dramatic progress in targeted therapy and immunotherapy has been changing clinical practices in lung cancer. With the accumulation of clinical practice, it has become clear that pre-existing interstitial pneumonia (IP) could be a risk factor for drug-induced lung injury, which has enhanced awareness regarding the difficulty in treating lung cancer with comorbid IP. Unfortunately, there is only low-grade evidence in the field of lung cancer with comorbid IP, because almost all clinical trials exclude such patients. There have been very few specialized clinical trials for patients with lung cancer and underlying IPs thus far. Therefore, it is necessary to treat such cases empirically or to give up on the treatment itself. Considering these circumstances, establishing how to treat lung cancer with comorbid IP is an urgent issue. This paper is a summary of the official statement reported by the Diffuse Lung Disease/Thoracic Oncology Assembly and the Japanese Respiratory Society (JRS) in 2017, which attempts to approach lung cancer with comorbid IP systematically.

Overcoming Tyrosine Kinase Inhibitor Resistance in Transformed Cell Harboring SEPT9-ABL1 Chimeric Fusion Protein.

Hematological malignancies harboring various ABL1 fusions are expected to be sensitive to tyrosine kinase inhibitors (TKIs), similar to those with BCR-ABL1. However, SEPT9-ABL1 exhibits TKI resistance both in vitro and in vivo. SEPT9-ABL1 has the same ABL1 region as seen in BCR-ABL1 but no point mutation in its kinase domain, which is one of the main mechanisms underlying TKI resistance in the leukemic cells harboring BCR-ABL1. The purpose of this study was to reveal the mechanism underlying TKI resistance induced by SEPT9-ABL1. We focused on the TP53 status because TKI-induced apoptosis in BCR-ABL1-positive cells is achieved through TP53. Mouse TP53 homologue TRP53 was downregulated and less phosphorylated in the cells expressing SEPT9-ABL1 than in those with BCR-ABL1, resulting in the prevention of apoptosis induced by TKIs. The CRM1 inhibitor KPT-330 accumulated nuclear TRP53 and NFKB1A (also known as IκBα), which is thought to capture TRP53 in the cytoplasm, and induced apoptosis in the hematopoietic cells expressing SEPT9-ABL1. In addition, the combination treatment of KPT-330 and imatinib, which induced the marked nuclear accumulation of PP2A and SET, reactivated PP2A through its dephosphorylation and inhibited SET expression, resulting in the effective induction of the apoptosis in the cells expressing SEPT9-ABL1. The combination treatment with KPT-330 and imatinib successfully reduced the subcutaneous masses expressing SEPT9-ABL1 and extended the survival of the mice intraperitoneally transplanted with SEPT9-ABL1-expressing cells. These results show that therapy with CRM1 inhibitors may be effective for overcoming TKI resistance induced by SEPT9-ABL1.

Association Between Formalin Fixation Time and Programmed Cell Death Ligand 1 Expression in Patients With Non-Small Cell Lung Cancer.

BACKGROUND/AIM: The expression of programmed cell death ligand 1 (PD-L1) determined by immunohistochemistry (IHC) may be associated with tissue formalin fixation time in non-small cell lung cancer (NSCLC) samples. We investigated the association between the PD-L1 expression and formalin fixation time, and clarified the optimal duration of fixation for accurate PD-L1 evaluation. MATERIALS AND METHODS: We collected 55 tumor specimens from resected NSCLC patients. The samples were halved and immediately fixed in 10% buffered formalin for 12-24 h (normal fixation), or 96-120 h (prolonged fixation). Each specimen was stained using two assay systems (22C3 and SP263) for PD-L1. RESULTS: The mean PD-L1 tumor proportion score was not significantly different between normal and prolonged fixation groups for either 22C3 or SP263 (normal fixation: 18.8%; prolonged fixation: 16.3%, p=0.277; normal fixation: 16.2%; prolonged fixation: 17.6%, p=0.560, respectively). CONCLUSION: Formalin fixation duration for up to 120 h does not affect PD-L1 IHC expression. PD-L1 tumor proportion score of tumor specimens can be evaluated by IHC even if these have been fixed in formalin outside the recommended duration in clinical practice.