A multicenter, open-label, phase II trial of pemetrexed plus bevacizumab in elderly patients with previously untreated advanced or recurrent nonsquamous non-small cell lung cancer.

BACKGROUND: Although the incidence of lung cancer in elderly individuals has been increasing in recent years, the number of clinical trials designed specifically for elderly patients with advanced non-small cell lung cancer (NSCLC) is still limited. To fulfill this unmet medical need, we conducted a phase II study to elucidate the efficacy of pemetrexed (PEM) plus bevacizumab (Bev) combination chemotherapy in elderly patients with nonsquamous NSCLC. METHODS: A total of 29 elderly patients (≥75 years old) with nonsquamous NSCLC were enrolled in this multicenter, open-label, phase II study, and 27 patients were finally analyzed. PEM at 500 mg/m2 on day 1 plus Bev at 15 mg/kg on day 1 were administered triweekly. The primary endpoint was the investigator-assessed objective response rate. RESULTS: The median age at initiating chemotherapy was 80 years old. Almost all patients (92.6%) had adenocarcinoma histology. The median number of cycles administered was 6, and the objective response rate was 40.7%. The median progression-free survival, overall survival and 1-year survival were 8.8 months, 27.2 months and 79%, respectively. The treatment was well-tolerated, and no treatment-related death was observed. CONCLUSION: Combination chemotherapy with PEM plus Bev in elderly patients with previously untreated advanced non-squamous NSCLC exhibited favorable antitumor activity and tolerability, suggesting that a combination of PEM plus Bev might be a promising treatment option for this population.

Identification of fibrocyte cluster in tumors reveals the role in antitumor immunity by PD-L1 blockade.

Recent clinical trials revealed that immune checkpoint inhibitors and antiangiogenic reagent combination therapy improved the prognosis of various cancers. We investigated the roles of fibrocytes, collagen-producing monocyte-derived cells, in combination immunotherapy. Anti-VEGF (vascular endothelial growth factor) antibody increases tumor-infiltrating fibrocytes and enhances the antitumor effects of anti-PD-L1 (programmed death ligand 1) antibody in vivo. Single-cell RNA sequencing of tumor-infiltrating CD45+ cells identifies a distinct "fibrocyte cluster" from "macrophage clusters" in vivo and in lung adenocarcinoma patients. A sub-clustering analysis reveals a fibrocyte sub-cluster that highly expresses co-stimulatory molecules. CD8+ T cell-costimulatory activity of tumor-infiltrating CD45+CD34+ fibrocytes is enhanced by anti-PD-L1 antibody. Peritumoral implantation of fibrocytes enhances the antitumor effect of PD-L1 blockade in vivo; CD86-/- fibrocytes do not. Tumor-infiltrating fibrocytes acquire myofibroblast-like phenotypes through transforming growth factor ß (TGF-ß)/small mothers against decapentaplegic (SMAD) signaling. Thus, TGF-ßR/SMAD inhibitor enhances the antitumor effects of dual VEGF and PD-L1 blockade by regulating fibrocyte differentiation. Fibrocytes are highlighted as regulators of the response to programmed death 1 (PD-1)/PD-L1 blockade.

Proteomic identification and quantification of Clostridium perfringens enterotoxin using a stable isotope-labelled peptide via liquid chromatography-tandem mass spectrometry.

PURPOSE: Detection of Clostridium perfringens enterotoxin (CPE) in human stool is critical evidence of food poisoning. However, processing patient-derived samples is difficult and very few methods exist to confirm the presence of CPE. In this study, a technique was developed using proteomic analysis to identify and quantify CPE in artificial gut fluid as an alternative. METHODS: The standard CPE was spiked into artificial gut fluids, and effective methods were developed by employing both a stable isotope-labelled internal standard peptide and liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: Proteotypic peptide EILDLAAATER formed by tryptic digestion was selected for quantitation of CPE. The peptide was identified using product ion spectra. Although the nontoxic peptides originating from CPE showed very low detectability in extraction and tryptic digestion, they could be detected with sufficient sensitivity using the method we developed. Based on a spiked recovery test at two concentrations (50 and 200 µg/kg), the recovery values were 85 and 78%, respectively. The relative standard deviations of repeatability and within-laboratory reproducibility were less than 8 and 11%, respectively. These standard deviations satisfied the criteria of the Japanese validation guidelines for residues (MHLW 2010, Director Notice, Syoku-An No. 1224-1). The limit of quantification (LOQ) was estimated to be 50 µg/kg. The combination of the product ion spectra and relative ion ratio supported CPE identification at the LOQ level. CONCLUSIONS: To the best of our knowledge, this is the first report of proteomic analysis of CPE using LC-MS/MS. The method would greatly help in assessing CPE reliably.

Efficacy and safety of second-line chemotherapy for patients with advanced non-small cell lung cancer complicated by interstitial lung disease.

BACKGROUND: Treatment of non-small cell lung cancer (NSCLC) patients with interstitial lung disease (ILD) is limited because of the risk of its acute exacerbation (AE). Furthermore, the efficacy and safety of second-line chemotherapy for these patients is unclear. METHODS: To investigate the efficacy and safety of second-line chemotherapy for NSCLC patients with ILD, we retrospectively reviewed patients who were treated at our institute between April 2010 and December 2018. RESULTS: Thirty-five patients received two or more regimens. Thirty-four patients were male and the median age at the initiation of second-line chemotherapy was 70 years. Almost all patients had a smoking history. Fourteen patients had adenocarcinoma and 15 had squamous cell carcinoma histology. Stages III and IV were observed in 20 and 11 patients, respectively. With respect to the type of ILD, 12 patients had usual interstitial pneumonia (UIP). The overall response rate and disease control rate were 11.4 and 68.6%, respectively. The median progression-free and median overall survival were 4.1 and 6.4 months, respectively. The AE of ILD was observed in eight patients, five of whom died. UIP and low percentage vital capacity were detected as significant risk factors for the AE of ILD. CONCLUSION: Second-line chemotherapy among patients with NSCLC complicated by ILD showed a certain effectiveness, but some patients experienced the AE of ILD, which may lead to death. The risk of the AE of ILD must be considered especially for patients with UIP and low percentage VC.

Efficacy of osimertinib in epidermal growth factor receptor-mutated non-small-cell lung cancer patients with pleural effusion.

BACKGROUND: Osimertinib is a standard first-line treatment for advanced non-small-cell lung cancer (NSCLC) harboring epidermal growth factor receptor (EGFR) mutations. Although malignant pleural effusion (PE) is a common clinical problem in NSCLC, information about the efficacy of osimertinib in patients with PE is limited, especially regarding its efficacy in EGFR T790M-negative patients with PE remains unclear. METHODS: We retrospectively reviewed the medical records of patients with NSCLC harboring EGFR mutations who were treated with osimertinib in our institution between May 2016 and December 2020. RESULTS: A total of 63 patients with EGFR mutated NSCLC were treated with osimertinib; 33 (12 with PE) had no EGFR T790M mutation, while 30 (12 with PE) had EGFR T790M mutation. In EGFR T790M-negative NSCLC, the progression-free survival (PFS) of the patients with PE was comparable to that of the patients without PE (median PFS 19.8 vs. 19.8 months, p = 0.693). In EGFR T790M- positive NSCLC, the PFS and overall survival (OS) of the patients with PE were significantly shorter than those of the patients without PE (median PFS 16.8 vs. 8.3 months, p = 0.003; median OS 44.9 vs. 14.2 months, p = 0.007). In the multivariate analysis, the presence of PE was independently associated with shorter PFS and OS in EGFR T790M-positive NSCLC patients, but not EGFR T790M-negative patients. CONCLUSIONS: These data suggest the efficacy of osimertinib may differ between EGFR T790M-positive and -negative NSCLC patients with PE.

Radiation therapy induces an abscopal effect and upregulates programmed death-ligand 1 expression in a patient with non-small cell lung cancer.

Radiation therapy (RT) activates the antigen presentation of dendritic cells and priming of cancer-specific cytotoxic CD8+ T cells, occasionally resulting in a systemic immune response to the tumor outside of the treatment field. The phenomenon of tumor regression at the site distant from irradiated fields is known as the abscopal effect. Several case reports have indicated a potential role of RT in overcoming primary and acquired resistance against immune checkpoint inhibitors in non-small cell lung cancer (NSCLC) and melanoma patients. We herein report an NSCLC patient who developed acquired resistance to an RT-induced abscopal effect and subsequently experienced reactivation of the systemic antitumor immune response by pembrolizumab, an antiprogrammed death 1 antibody. In this case, RT not only induced an abscopal effect but also upregulated the programmed death-ligand 1 expression outside of the irradiated field when the patient developed resistance to the abscopal effect. This case can facilitate our understanding of the mechanism underlying the RT-induced systemic immune response against cancer cells and adaptive resistance mechanism of cancer cells from immune surveillance. These findings highlight the promising results of current clinical trials combining RT and immune checkpoint inhibitors. Ongoing clinical trials will further establish evidence supporting combination therapy with RT and immune checkpoint inhibitors.

A case of pulmonary pleomorphic carcinoma with preexisting interstitial pneumonia successfully treated with pembrolizumab.

Pulmonary pleomorphic carcinoma is often refractory to chemotherapy and follows an aggressive clinical course. Immune checkpoint inhibitors (ICIs) have revolutionized the treatment of advanced lung cancer, and a few cases with pleomorphic carcinoma have been reported to show tumor shrinkage after therapy with ICIs. When treating patients with ICIs, patient selection is essential, and monitoring and management of immune-related adverse events, including pneumonitis, are needed. We herein report a case of pulmonary pleomorphic carcinoma with preexisting interstitial pneumonia treated with pembrolizumab, antiprogrammed cell death 1 antibody. Our report highlights important considerations necessary when treating advanced pleomorphic carcinoma patients complicated with interstitial pneumonia. We also review the literature regarding the use of ICIs in such patients.

Correlation between immune-related adverse events and therapeutic effects of nivolumab in patients with malignant pleural mesothelioma.

BACKGROUND: Nivolumab is used for the treatment of malignant pleural mesothelioma (MPM). However, immune-related adverse events (irAEs) occur in patients treated with nivolumab. Several studies have reported the correlation between irAEs and therapeutic effects of immune checkpoint inhibitor, but none have reported the correlation in MPM. Here we report a retrospective study which shows the correlation between irAEs and therapeutic effects of nivolumab in patients with MPM. METHODS: This study included patients treated with nivolumab at Tokushima University Hospital from February 2009 to September 2021. We retrospectively reviewed the medical records to evaluate the several clinical factors, such as the presence or absence of irAEs, their severities, progression-free survival (PFS), overall survival (OS) or objective response to the treatment. RESULTS: Eleven patients received treatment with nivolumab. Objective response rate was 18.2% and the disease control rate was 90.9%. Median PFS was 6.8 months (95% confidence interval, 1.3 to 11.9 months) and median OS was 15.2 months (95% confidence interval, 8.9 to 21.5 months). IrAEs occurred in eight patients (72.7%), and grade ≥ 2 irAEs occurred in six patients (54.5%). PFS and OS were significantly longer in the grade ≥ 2 irAEs group than in grade < 2 irAEs group (median PFS 13.6 vs. 3.8 months, p = 0.0093; median OS not reached vs. 8.6 months, p = 0.0108). CONCLUSIONS: This is the first study to report the correlation between irAEs and therapeutic effects in patients with MPM. Because the presence of irAEs may be associated with a favorable clinical outcome, early detection and appropriate management of irAEs will increase the therapeutic benefits to patients.

Programmed death (PD)-1/PD-ligand 1 blockade mediates antiangiogenic effects by tumor-derived CXCL10/11 as a potential predictive biomarker.

Immune checkpoint inhibitor (ICI) programmed death (PD)-1/PD-ligand 1 (PD-L1) blockade has been approved for various cancers. However, the underlying antitumor mechanisms mediated by ICIs and the predictive biomarkers remain unclear. We report the effects of anti-PD-L1/PD-1 Ab in tumor angiogenesis. In syngeneic mouse models, anti-PD-L1 Ab inhibited tumor angiogenesis and induces net-like hypoxia only in ICI-sensitive cell lines. In tumor tissue and serum of ICI-sensitive cell line-bearing mice, interferon-γ (IFN-γ) inducible angiostatic chemokines CXCL10/11 were upregulated by PD-L1 blockade. In vitro, CXCL10/11 gene upregulation by IFN-γ stimulation in tumor cell lines correlated with the sensitivity of PD-L1 blockade. The CXCL10/11 receptor CXCR3-neutralizing Ab or CXCL11 silencing in tumor cells inhibited the antiangiogenic effect of PD-L1 blockade in vivo. In pretreatment serum of lung carcinoma patients receiving anti-PD-1 Ab, the concentration of CXCL10/11 significantly correlated with the clinical outcome. Our results indicate the antiangiogenic function of PD-1/PD-L1 blockade and identify tumor-derived CXCL10/11 as a potential circulating biomarker of therapeutic sensitivity.

[Determination of Asulam in Livestock Products by LC-MS/MS].

An analytical method based on LC-MS/MS was developed for the determination of asulam in livestock products. Asulam in livestock products was extracted with acetone. The crude extracts were defatted by acetonitrile and n-hexane partitioning. Cleanup was carried out using a combination of ethylene diamine-N-propyl silylation silica gel (PSA) and octadecyl silylated silica gel (C18) mini columns with acidic condition. The sample solution was subjected to LC-MS/MS using an external solvent calibration curve. The average recovery (n=5) of Asulam from four types of livestock products (bovine muscle, bovine fat, bovine liver and milk) spike at the maximum residue limits (MRLs) or at a uniform limit of 0.01 mg/kg was 92.7-98.7%, with a relative standard deviation of 3.1-11.6%. The limit of quantitation of the developed method was calculated to be 0.01 mg/kg.

[Evaluation on a Simultaneous Analytical Method of Pesticide Residues in Limes].

A LC-MS/MS simultaneous analytical method for screening 191 pesticide residues in limes had been developed and validated. Pesticides were extracted with acetonitrile from samples. Then mixed salts, which were anhydrous magnesium sulfate for dehydration, sodium carbonate for adjusting pH, and sodium chloride for salting out, were added to the sample. After centrifugation, supernatant was transferred to a tube. The sample solution was cleaned up using solid phase extraction (SPE) with C18/GC/PSA for the determination by LC-MS/MS. The developed method was improved the recovery rate of thiabendazole, which had a low recovery rate by the conventional method. Validation study, which was following the guidelines of the Ministry of Health, Labor and Welfare, were carried out at 0.01 and 0.1 µg g-1 to evaluate the method. The results of 175 pesticides out of 191 were with satisfactory. A total of 19 imported lime samples sold in Tokyo was analyzed to evaluate the method, then 18 samples contained pesticide residues below MRLs. The developed method is applicable for detection of pesticide residues in lime.

Blockade of PD-1/PD-L1 Pathway Enhances the Antigen-Presenting Capacity of Fibrocytes.

Fibrocytes, a distinct population of collagen-producing, monocyte-derived cells, are involved in wound healing as well as fibrotic diseases. Recently, fibrocytes have been revealed to play a role in the tumor microenvironment, particularly under antiangiogenic therapy. In addition, combination cancer immunotherapy with immune checkpoint inhibitor and antiangiogenic agents have been developed for various cancers in the clinical setting, although the immunological background is not clear. In the current study, we aimed to determine the function of fibrocytes in tumor immunity induced by immune checkpoint inhibitor therapy. Human and murine fibrocytes were generated from PBMCs and lungs, respectively. The expression of costimulatory and inhibitory molecules on fibrocytes was examined by flow cytometry. The stimulation of CD8+ T cells by fibrocytes was examined in MLRs with a 3H-thymidine incorporation assay. Fibrocytes expressed CD80low and CD86high as a costimulatory molecule, and expressed PD-L1high, but not PD-L2, as a coinhibitory molecule. Without any stimulation, fibrocytes strongly enhanced the proliferation of CD8+ T cells in mice and humans. Treatment with anti-CD86 and -CD54 Abs inhibited the growth of CD8+ T cells induced by fibrocytes. Anti-PD-L1 Ab further enhanced the proliferation of CD8+ T cells, even in the OVA-specific MLR with OT-1Rag-/- mice. Importantly, fibrocytes derived from PBMCs of patients with lung adenocarcinoma or murine MC38 tumors augmented the proliferation of CD8+ T cells with PD-L1 blockade. These results suggest that fibrocytes infiltrating tumor sites may play a role in the antitumor immunity mediated by CD8+ T cells when the activity is further enhanced by PD-L1/PD-1 blockade.

External validation of a genitourinary cancer-specific prognostic scoring system to predict survival for patients with bone metastasis (modified B-FOM scoring model): Comparison with other scoring models in terms of accuracy.

OBJECTIVE: We previously developed genitourinary (GU) cancer-specific scoring system for prediction of survival in patients with bone metastasis (the Bone-Fujimoto-Owari-Miyake [B-FOM] scoring model) based on five prognostic factors: the type of primary tumor (prostate cancer (PCa) vs renal cell carcinoma (RCC) and PCa vs urothelial carcinoma (UC)), poor performance status (PS), visceral metastasis, high Glasgow-prognostic score (GPS), elevated neutrophil-to-lymphocyte ratio (NLR). The aim of this study was to externally validate and further improve the performance of the B-FOM score. METHODS: The external validation cohort comprised 309 patients with GU cancer with bone metastasis from multiple institutions. Clinical factors were analyzed using Kaplan-Meier method and COX regression hazard model. Performance of a modified B-FOM score was compared to that of other scoring models by the Kaplan-Meier method and the area under the curve (AUC) of receiver operating characteristic curves. RESULTS: The median follow-up period of development and validation cohort were 25 and 17 months, respectively. Kaplan-Meier curve demonstrated that the type of primary tumor (RCC and UC vs PCa), poor PS, presence of visceral metastasis, high GPS, elevated NLR were significantly associated with shorter cancer-specific survival. Risk groups were successfully stratified by the modified B-FOM score classification. Moreover, the AUC of the modified B-FOM scoring model for predicting mortality at 6, 12, and 24 months were 0.895, 0.856, and 0.815, respectively, which were the highest among evaluated models. CONCLUSIONS: The B-FOM scoring model is a simple and accurate prediction tool. By using this scoring model at the time of the diagnosis of bone metastasis in patients with GU cancers, an individualized optimal treatment strategy can be selected.

[Comparison of Validation Results Obtained by GC-MS/MS and LC-MS/MS for the Analysis of Residual Pesticides in Agricultural Products].

In the field of food analysis and regulation, different instruments are used to determine the accuracy of quantification values. This is essential, as inconsistencies in values are commonly encountered. To visualize the degree of these discrepancies in each food matrix, we compiled a validation study based on a routine method developed in our laboratory, for 121 pesticides in six agricultural products, namely the grapefruit, potato, paprika, cabbage, spinach, and brown rice. These were analyzed by GC-MS/MS and LC-MS/MS, and the results were compared mainly on the basis of trueness. According to the results of the validation study when using GC-MS/MS, of the 121 pesticides tested in each product class, the number of analytes that satisfied the criteria of the Japanese validation guidelines was 97 in grapefruit, 111 in potato, 110 in paprika, 118 in cabbage, 111 in spinach, and 63 in brown rice. In contrast, in the analysis of the same samples by using LC-MS/MS, the number of analytes that satisfied the criteria of the validation guidelines was 50 in grapefruit, 114 in potato, 103 in paprika, 112 in cabbage, 100 in spinach, and 103 in brown rice. Inconsistences in the differences of trueness were mainly attributed to matrix effects of each instrument, as well as to food matrices, of which the most diverged matrix was that of brown rice (over 20%).

Development of an alternative approach for detecting botulinum neurotoxin type A in honey: Analysis of non-toxic peptides with a reference labelled protein via liquid chromatography-tandem mass spectrometry.

In this study, we developed a reference labelled protein containing the partial amino acid sequence of botulinum neurotoxin type A (BoNTA). We also applied it as an internal standard to detect specific and non-toxic peptides originated from BoNTA in honey with the use of liquid chromatography-tandem mass spectrometry (LC-MS/MS). Original proteins in the honey sample were collected through a two-step process that included solubilisation and trichloroacetic acid (TCA) precipitation. Solubilisation by adding water enabled processing of proteins in honey. TCA precipitation collected proteins without specific binding. The combination of protein alkylation and an appropriate enzyme-to-protein ratio ensured feasibility of tryptic digestion. A desalting process eliminated a large amount of salts and other tryptic peptides in the honey sample. The use of the reference labelled protein enabled compensation for tryptic digestion efficiency and electrospray ionisation efficiency based on LC-MS/MS measurement. After the peptide selection and protein BlastP analysis, five unique peptides were chosen. The non-toxic peptides originating from BoNTA were reliably detected using LC-MS/MS based on a multiple-reaction monitoring mode. Detection of several peptides ensured screening of BoNTA in honey samples. Based on the responses, the proteotypic peptide LYGIAINPNR was selected as the quantitative peptide. Due to maintaining the relative ion ratios, the selective transition completely identified the non-toxic peptides. The intensity of the transitions established a detection limit of BoNTA estimated to be 9.4 ng mL-1. Although extraction efficiency was not evaluated using the BoNTA standard, the results suggested this method may be used for quantification of BoNTA in honey. The method was applied to 19 honey samples purchased in Tokyo; none of them was found to contain the target toxin. Overall, the method is expected to accelerate BoNTA monitoring for food safety.

Anti-PD-1 antibody combined with chemotherapy suppresses the growth of mesothelioma by reducing myeloid-derived suppressor cells.

BACKGROUND: The combination of anti-PD-1/PD-L1 antibody with chemotherapy has been approved for the first-line therapy of lung cancer. However, the effects against malignant mesothelioma (MPM) and the immunological mechanisms by which chemotherapy enhances the effect of targeting PD-1/PD-L1 in MPM are poorly understood. MATERIALS AND METHODS: We utilized syngeneic mouse models of MPM and lung cancer and assessed the therapeutic effects of anti-PD-1 antibody and its combination with cisplatin (CDDP) and pemetrexed (PEM). An immunological analysis of tumor-infiltrating cells was performed with immunohistochemistry. RESULTS: We observed significant therapeutic effects of anti-PD-1 antibody against MPM. Although the effect was associated with CD8+ and CD4+ T cells in tumors, the number of Foxp3+ cells was not reduced but rather increased. Consequently, combination with CDDP/PEM significantly enhanced the antitumor effects of anti-PD-1 antibody by decreasing numbers of intratumoral myeloid-derived suppressor cells (MDSCs) and vessels probably through suppression of VEGF expression by CDDP + PEM. CONCLUSIONS: The combination of anti-PD-1 antibody with CDDP + PEM may be a promising therapy for MPM via inhibiting the accumulation of MDSCs and vessels in tumors.

[Validation of a Method for Determining Multiple Residual Pesticides in Dried Red Pepper Using LC-MS/MS].

An analytical method has been developed and validated for determining 107 pesticide residues in dried red pepper using LC-MS/MS. LC method, the clean-up and sample dilution processes were examined to determine their impact on reducing the matrix effects. Clean up was performed using an ENVI-CarbIITM/PSA (300/600 mg, 6 mL) SPE cartridge. In the sample dilution process, eight-fold dilution was used. In the validation of the developed method at two concentrations (0.01 and 0.1 µg/g) for 107 pesticides, 96 pesticides showed recovery rates in the range of 70.1 to 112.6%, RSDs of repeatability of ≤11.5 and 3.4%, and RSDs of within-laboratory reproducibility of ≤24.3 and 19.9%. These values fulfill the criteria of the validation guidelines for pesticide residues in Japan. It is concluded that matrix effects and low recovery rates in the process of extraction are the main factors for values that do not conform to the criteria.

Cytopathological Features of SMARCA4-Deficient Thoracic Sarcoma: Report of 2 Cases and Review of the Literature.

SMARCA4-deficient thoracic sarcoma (SMARCA4-DTS) is a recently described entity of thoracic sarcomas with an undifferentiated rhabdoid morphology and SMARCA4 inactivation. Regardless of some reports about the histopathological findings so far, there have been only a few reports about the cytological features. In this article, we present the pathological features of 2 SMARCA4-DTS cases, including the cytological findings. Histopathologically, the tumor cells showed atypical loosely cohesive large epithelioid cells focally with geographic necrosis. Some cells were characterized by rhabdoid cells. Both patients showed intrathoracic masses with a history of smoking, and loss of SMARCA4 expression was confirmed with histopathological specimens. Immunohistochemically, tumor cells of both cases were at least focally positive for cytokeratin, CD34, CD99, synaptophysin, SOX2, and SALL4. In addition, tumor cells demonstrated significantly reduced expression of BRG1/SMARCA4 and SMARCA2. In conclusion, SMARCA4-DTS should be taken into consideration in the differential diagnosis of tumors with undifferentiated rhabdoid morphology involving the thoracic region.

Involvement of Spermidine in the Reduced Lifespan of Caenorhabditis elegans During Vitamin B12 Deficiency.

Vitamin B12 deficiency leads to various symptoms such as neuropathy, growth retardation, and infertility. Vitamin B12 functions as a coenzyme for two enzymes involved in amino acid metabolisms. However, there is limited information available on whether amino acid disorders caused by vitamin B12 deficiency induce such symptoms. First, free amino acid levels were determined in vitamin B12-deficient Caenorhabditis elegans to clarify the mechanisms underlying the symptoms caused by vitamin B12 deficiency. Various amino acids (valine, leucine, isoleucine, methionine, and cystathionine, among others) metabolized by vitamin B12-dependent enzymes were found to be significantly changed during conditions of B12 deficiency, which indirectly affected certain amino acids metabolized by vitamin B12-independent enzymes. For example, ornithine was significantly increased during vitamin B12 deficiency, which also significantly increased arginase activity. The accumulation of ornithine during vitamin B12 deficiency constitutes the first report. In addition, the biosynthesis of spermidine from ornithine was significantly decreased during vitamin B12 deficiency, likely due to the reduction of S-adenosylmethionine as a substrate for S-adenosylmethionine decarboxylase, which catalyzes the formation of spermidine. Moreover, vitamin B12 deficiency also demonstrated a significant reduction in worm lifespan, which was partially recovered by the addition of spermidine. Collectively, our findings suggest that decreased spermidine is one factor responsible for reduced lifespan in vitamin B12-deficient worms.

Development, validation, and comparison of gene analysis methods for detecting EGFR mutation from non-small cell lung cancer patients-derived circulating free DNA.

The feasibility and required sensitivity of circulating free DNA (cfDNA)-based detection methods in second-line epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) treatment are not well elucidated. We examined T790M and other activating mutations of EGFR by cfDNA to assess the clinical usability. In 45 non-small cell lung cancer (NSCLC) patients harboring activating EGFR mutations, cfDNAs were prepared from the plasma samples. EGFR mutations in cfDNA were detected using highly sensitive methods and originally developed assays and these results were compared to tissue-based definitive diagnoses. The specificity of each cfDNA-based method ranged 96-100% whereas the sensitivity ranged 56-67%, indicating its low pseudo-positive rate. In EGFR-TKI failure cohort, 41-46% samples were positive for T790M by each cfDNA-based method, which was comparable to re-biopsy tissue-based T790M positive rates in literature. The concordance of the results for each EGFR mutation ranged from 83-95%. In eight patients, the results of the cfDNA-based assays and re-biopsy-derived tissue-based test were compared. The observed overall agreement ranged in 50-63% in T790M, and in 63-100% in activating EGFR mutations. In this study, we have newly developed three types of assay which have enough sensitivity to detect cfDNA. We also detected T790M in 44% of patients who failed prior EGFR-TKI treatment, indicating that cfDNA-based assay has clinical relevance for detecting acquired mutations of EGFR.