Beta vulgaris L.). Food waste, leaf and yard waste, and animal dung were collected and composted in a heap-composting method. The produced organic amendments were applied to soil at an application rate of 5, 10, and 15 t/ha, respectively. Two harvesting times were considered, and at each leaf harvesting time, plant growth parameters  were analyzed; after the second harvesting time, soil properties were analyzed. Results indicated that increasing rates of FWC significantly (p < 0.001) increased the plant height, leaf area, and fresh yield of Swiss chard. The application of 15 t/ha of FWC also increased SOC, TN, available P, and CEC. Food waste was recycled through composting as a soil amendment to improve soil properties and the yield of Swiss chard.In Dilla town and the university compound, huge amounts of biodegradable solid waste (BDSW), which include food and farm and yard wastes, are generated from student and staff cafeterias and animal farms. Improper treatment and disposal of this waste resulted in contamination of surface water and soil, air pollution, and spreading of diseases. On the other hand, soil fertility of most arable lands of Dilla Zuria woreda is highly depleted due to low levels of soil fertility management practices and inorganic-based farming. These factors make a considerable contribution to the degradation of fertile soil and reduction of yield. Thus, the management of BDSW through composting is important to minimize environmental problems and improve the soil fertility of arable lands. However, the effects of BSWC compost on plant growth and crop yield are highly variable in different studies. This study aimed to evaluate the effect of food waste compost (FWC) and leaf yard compost (LYC) at different application rates on the soil properties, growth, and yield responses of Swiss chard ( Every year in the world, about 1.7–1.9 billion metric tons of municipal solid waste are generated from offices, markets, and industries . The depIn Dilla town and university compounds, huge amounts of food and farm and yard wastes are generated from student and staff cafeterias and agriculture farms and deposited without any management system. Improper treatment and disposal of this waste resulted in contamination of surface water and soil, air pollution, and spreading of diseases . IntensiComposting BSW converts potentially toxic materials into a steady state, which can improve soil quality for plant growth . CompostBeta vulgaris L.).Several studies have been conducted to examine the effect of BSW compost on cereals, legumes , 19, andBeta vulgaris L.).Therefore, this study aimed to investigate the effect of application rates of FWC and LYC on selected soil physicochemical properties and yields of Swiss chard  was used as a test crop. It is a leafy vegetable and is extremely consumed by people who are living in Gedeo zone [The study site has an annual rainfall of 1001–1800 mm and a temperature range of 12–25°C [2 level within the heap. Heap moisture was kept under control by adding enough water to keep the moisture content at or above 50%. By increasing the population of aerobic microorganisms, this aeration speeds up the composting process.Food waste compost (FWC) is any inedible part of food removed from the food supply chain to be composted with animal manure and water with the help of microorganisms. Leaf yard waste compost (LYC) is defined as compost prepared from leaves and other organic materials, such as grass clippings, with animal manure and water with the help of microorganisms. Food waste  was collected from Dilla University students' cafeterias, local markets, and households. Leaf and yard waste, as well as animal manure, was collected from agricultural farms. The nonbiodegradable fraction was manually separated from the organic fraction, which was then shredded and composted in a heap method. The waste mixtures were composted in heaps in the size of 1.5 m high, 2 m wide, and 2 m long. This size is advantageous to turn the heap, improve the supply of oxygen, and prepare a large amount of compost . Heap teSeeds of yellow Swiss chard were obtained from the Research Center of the Ethiopian Institute of Agricultural Research Center. Swiss chard seeds were raised on a well-prepared seed bed. The seeds were drilled in rows with ten-centimeter row spacing and covered with dried grass. Weeding, watering, and protection of seedlings from insects and fungal diseases were carried out in order to produce healthy seedlings.2), were used for the experiment. Management of planted seedlings  was practiced until the second harvesting period.Before transplanting seedlings into the experiment field, the land was plowed by the hand, the bed was leveled, smoothed, and loosened uniformly, and then one week before transplanting seedlings, compost was mixed on the surface of plots with rakes to bare soil. It was attempted to maintain a uniform distribution of compost over the surface during application, and it was further mixed to an approximate soil depth of 10 cm. Finally, strong and healthy Swiss chard seedlings were transplanted in the experimental field with a spacing of 30 cm between plants and 45 cm between rows. A total of 21 plots, each measuring 3 m × 1.8 m (5.4 mT1 = control (no compost application); T2 = 5 t/ha (2.7 kg); FWC, T3 = 10 t/ha (5.4 kg); FWC, T4 = 15 t/ha (8.1 kg); FWC, T5 = 5 t/ha (2.7 kg); LYC, T6 = 10 t/ha (5.4 kg); and LYC, T7 = 15 t/ha (8.1 kg) LYC treatment.During growing seasons, the experiment was conducted using a randomized complete block design (RCBD) consisting of two types of compost: food waste (FWC) and leaf and yard waste (LYC) in the presence of animal manure with a ratio of 3 : 1 by using three application rates  with three replications. The experiment was designed with twenty-one plots having 2O (soil-H2O) at a 1 : 2.5 soil/compost-to-solution ratio using a pH meter, as described by Carter and Gregorich [3) as the extraction solution [4OAc of pH 7) extraction method was used to determine the exchangeable bases (Ca2+ and Mg2+) in soil. In this procedure, Ca2+ and Mg2+ in the extracts were determined using an atomic absorption spectrophotometer, while the contents of exchangeable K were determined using a flame photometer, as described by Rowell [Before the application of compost treatment, one kilogram of composite soil samples was collected from the top 20 cm depth. The collected sample was separated from tree roots, leaves, and other unwanted materials and then air-dried and prepared for analysis of physical and chemical properties. Moreover, before the application of compost treatments, different compost samples were taken from different layers to determine the following selected physical and chemical properties: pH was determined in Hregorich . Electriregorich . Organicregorich . Total Nregorich  using thsolution . The ammy Rowell . The cat2+ and Mg2+), and cation exchange capacity (CEC) by following the standard procedure.After the second harvesting period, soil samples were collected from each treatment plot to analyze the physical and chemical properties of soil, such as pH, electrical conductivity (EC), soil organic carbon (OC), total nitrogen (TN), available phosphorous, available potassium, exchangeable bases  was applied.The experiment was subjected to analysis of variance in the randomized complete block design, and data were analyzed using the R program (version 4.11.2021). To determine the significant difference between treatment means, Fisher's range test at a 5% significance level (+2), and high magnesium (Mg+2).According to Murphy  ratings,According to Murphy 's reportThe soil organic carbon (SOC) content of composted food waste and leaf and yard waste was 2.92% and 2.4%, respectively . The resThe percentage of total nitrogen (TN) content in the compost produced ranged from 1.4 to 1.7 percent . The amoThe observed available phosphorous values in FWC and LYC were 1.60 and 1.35 ppm, respectively . This stp < 0.001), application rates (p < 0.001), and the interaction of amendments and application rates (p < 0.05). Among amendments, FWC showed the highest PH value both at the first and second harvesting time , followed by the plants treated with LYC , and the lowest value of PH was registered in the control treatment . The PH in each treatment was higher when 15 t/ha organic amendment was applied in the treatment plots than at other applicable rates. Considering the interaction effect, the maximum values of plant height  were observed in 15 t/ha FWC, while the least value was observed in the control treatment (The measured plant height (PH) at the first and second harvesting times was significantly different between amendments , application rates (p < 0.001), and interaction of amendments and application rates (p < 0.001) in the first harvesting time. The highest average value of LN was recorded on plants treated with FWC (17.34 ± 4.33), followed by LYC (15.07 ± 2.68), while the lowest value was recorded from the control treatment (12.65 ± 0.19). Moreover, adding organic amendments of FWC and LYC increased leaf numbers by 27.05% and 20.24%, respectively, in comparison to the control treatment. Considering the application rates, the maximum value of LN was observed in plants treated with 15 t/ha, followed by 10 t/ha, while the minimum value was registered in the control and 5 t/ha treatments. The interaction effect of amendments and application rates indicated that the highest value of LN was observed with soil treated with 15 t/ha FWC (22.78 ± 2.32). Furthermore, plants treated with 15 t/ha FWC and 15 t/ha LYC increased LN by 44.47% and 31.84%, respectively, compared to the control treatment. At the second harvesting time, LN significantly differed among organic amendments (p < 0.05) and application rate (p < 0.001) (< 0.001) . The aveLN results were generally higher when the plants were treated with an application rate of 15 t/ha, followed by 10 t/ha, and the least was observed in the control treatment. Furthermore, the LN value was higher when 15 t/ha organic amendment was applied in the treatment plots than at other application rates. This finding was consistent with those by Masarirambi et al. , who repp < 0.001), application rate (p < 0.001), and the interaction effect (p < 0.05) in the first harvesting time. The soil treated with FWC and LYC had significantly higher values of LA than that with other treatments. On average, adding amendments of FWC and LYC increased LA by (30.32%) in FWC and 23.71% in LYC, respectively, compared to the control treatment. The interaction effect of amendments and application rates indicated that the highest value of LA was recorded with soil treated with 15 t/ha FWC (221.33 ± 4.04). In addition, plants treated with 15 t/ha FWC and 15 t/ha LYC increased LA by 42.32% and 34.53%, respectively, compared with the control treatment. At the second harvesting time, LA significantly differed among organic amendments (p < 0.05) and the application rate (p < 0.001) (The leaf area (LA) value  was alwa< 0.001) . The aveThe study's findings were in agreement with those of the report of Bharadwaj and Nainawat , who menp < 0.001), application rates (p < 0.001), and their interaction (p < 0.001), both at the first and second harvesting times (The fresh weight of the Swiss chard plant was significantly influenced by organic amendments (ng times . At the ng times . This rep < 0.001), application rates (p < 0.001), and their interaction (p < 0.001) after harvesting the Swiss chard crop , application rates (p < 0.001), and interaction effects (p < 0.001) after harvesting the Swiss chard crop (SOC was significantly influenced by organic amendments (ard crop . The soiard crop . The finp < 0.001), application rates (p < 0.001), and interaction of amendment and application rates (p < 0.001) after harvesting the Swiss chard crop. The highest value of soil total N was observed in plants treated with FWC amendments (0.164 ± 0.03), followed by LYC (0.153 ± 0.01), while the lowest value was recorded from the control treatment (0.10 ± 0.01). In view of the interaction effect of amendments and application rates, the maximum value of soil total N was observed in plants treated with 15 t/ha FWC (0.197 ± 0.01), followed by 15 t/ha LYC (0.161 ± 0.01), while the minimum value was recorded in the control (0.10 ± 0.01). Furthermore, plants treated with 15 t/ha FWC and 15 t/ha LYC increased soil total N by 49% and 38%, respectively, compared to the control treatment , application rates (p < 0.001), and interaction of amendments and application rates (p < 0.001). The maximum value of soil available P was registered from plots treated with FWC (3.64 ± 0.42), followed by LYC (3.32 ± 0.55), and the lowest value (2.21 ± 0.06) was observed in the control treatment. Available P in the soil was higher when 15 t/ha organic amendment was applied in the treatment plots than at other application rates. Moreover, the interaction effect of amendment and application rates was recorded from a minimum value of 2.21 ± 0.06 in the control treatment and to a maximum value of 4.13 ± 0.02 in 15 t/ha FWC, respectively. The type of feedstock used for compost production may account for the increase in available P in 15 t/ha FWC. The result findings were consistent with those of [Available phosphorus in soil was significantly affected by amendments  influenced by organic amendments (p < 0.001), application rates (p < 0.001), and interaction of amendments and application rates (p < 0.01). The application of FWC and LYC amendments significantly increased the cation exchange capacity (Soil CEC was significantly (capacity  of the tcapacity . The higcapacity . Moreovep < 0.001) affected by organic amendments, application rates (p < 0.001), and interaction effects (p < 0.001) (+3 ions to form insoluble Al(OH)3 and water. On the other hand, when the exchangeable acidity of the soil is high, resulting in low pH, it affects the soil condition and many soil processes. Furthermore, the bioavailability of iron, aluminum, or manganese could be very high, reaching toxic levels at lower pH [Exchangeable acidity was also significantly (< 0.001) . The res< 0.001) . The higlower pH .This research was conducted with the purpose of investigating the effects of different rates of FWC and LYC on selected soil properties and the yield of a Swiss chard crop under irrigation. Field experimental results showed that Swiss chard responded positively to the application of organic amendments. Particularly when the maximum application rate of FWC is added to the soil, it significantly increases soil pH by 10%, SOC by 30%, TN by 49%, AP by 47%, and CEC by 39% and reduces soil exchangeable acidity by 64% compared to the control. Moreover, the highest application rate of FWC improved the plant height, leaf number, leaf area, and fresh weight of Swiss chard when compared with the control treatment. Therefore, based on the present study findings, the application of 15 t/ha FWC could be recommended as a potential fertilizer to improve soil properties and yields of Swiss chard.


Sampling and testing for SARS‐CoV‐2 is a widely recognized method for identifying patients with COVID‐19. However, there is limited research available on the stability of nucleic acids in viral storage solutions.This paper investigates the components that provide better protection for virus and nucleic acid detection. The study utilized real‐time quantitative fluorescent PCR to detect SARS‐CoV‐2 and evaluate the preservation effect and stability of SARS‐CoV‐2 viral storage solution under various conditions, including different guanidinium salts, brands, and storage conditions.All brands of inactivated virus preservation solutions demonstrated effective preservation and stability. However, 0.5 mol/L guanidine hydrochloride and guanidine isothiocyanate solutions exhibited poor antiseptic effects. Additionally, refrigerated storage showed better preservation compared to room temperature storage.We recommend using inactivated virus collection solution to preserve and transport samples and testing preferably within 6 hours to reduce false negatives of NAT results. We found that the fire‐extinguishing sampling tube  had different effects on the preservation and detection of the virus due to different components, so it was necessary to verify the virus preservation solution. There are two types of virus preservation solutions: noninactivated and inactivated. The noninactivated preservation solution mainly consists of Hank's buffer without lysis solution, which helps enhance the survival and stability of the virus during infection. Additionally, ingredients such as gentamicin, antifungal drugs, cryoprotectants, amino acids, bovine serum albumin, and HEPES are usually added to prevent the viral protein shell from easily decomposing and to maintain the virus's activity across a wide temperature range.Currently, there are numerous brands of virus collection tubes available in China. However, it is important to note that these collection tubes and virus preservation solutions are not regulated as medical devices. Additionally, the quality control requirements for virus preservation solutions may vary among manufacturers. Surprisingly, there is a lack of studies on the effect of preservation solutions on virus preservation or the specific characteristics of virus preservation solutions in NAT research. The storage condition for commercial sample tubes is 1 year at room temperature, but the storage time after sampling is not specified. Additionally, it is important to note that due to distance and operator limitations, the virus sample tubes may be placed for more than 6 h before detection. The preservation stability of virus sampling tubes at room temperature and in cold storage is a significant area of study. This research aims to investigate how the tubes maintain their quality over time. Understanding the stability of these tubes is crucial to ensure their reliability and effectiveness in virus sampling. Additionally, this research allows us to determine the appropriate storage conditions for the tubes, which is essential for preserving the integrity of collected samples. The research findings will offer users a solid research foundation, providing valuable insights into the selection and preservation of viral preservation solution ingredients and brands.22.12.1.1The nucleic acid extraction and purification kit  and the SARS‐CoV‐2 nucleic acid detection kit  were used. Additionally, the SARS‐CoV‐2 pseudovirus  was included in the experiment. Commercial virus preservation solutions were tested in two groups: the inactivated group (IA) and the noninactivated group (NIA). The inactivated group included products from the following companies: Guangzhou Daan Gene (Lot: 2021009), Hangzhou Heshuo Biological Technology (Lot: 20210911), Yocon Biological Technology (Lot: 26210815), Hangzhou Ciping Medical Instrument (Lot: 26210815), Wuhan Easy Diagnosis Biomedicine (Lot: 210202‐05M), Zhejiang Orient Gene Biotech (Lot: S2101051), USTAR Biotechnologies (Lot: 20210820), and Zhejiang Junshan Gene Science (Lot: QY21K01). The noninactivated group included products from Guangzhou Daan Gene (Lot: 2021004), Hangzhou Heshuo Biological Technology (Lot: 20210911), Yocon Biological Technology (Beijing) (Lot: 01210215), Hangzhou Ciping Medical Instrument (Lot: 202108004), and Wuhan Easy Diagnosis Biomedicine (Lot: 210101‐02).2.1.2The storage conditions and validity period of commercial sample tubes containing viral storage solution are 2–40°C storage for 12 months. The State Council of China's regulations on the storage of virus sample tubes2.1.3The automatic fluorescent PCR analyzer used in this study was the Cobas Z480 , while the automatic nucleic acid extraction was performed using the NAE‐96 system from Easy Diagnosis. The experiments were conducted in a biological safety cabinet from Heal Force and a refrigerated centrifuge from Thermo Fisher Scientific, with an oscillator from KylinBell used for mixing. The pipettes used were from Eppendorf, and the tips used were 10, 200, and 1000 μL needle tips with filters from AXYGEN. The LightCycler 480 Multiwell Plate 96 in white  was also utilized.2.22.2.1The self‐prepared inactivated virus preservation solution group (self‐prepared group): Guanidine isothiocyanate . Guanidine hydrochloride . Sterile normal saline water for preparation and dilution of virus preservation solution . the glass tube for configuring sample preservation solution . Prior to the experiment, the glass tube, sterile normal saline water, and experimental consumables underwent autoclaving. Additionally, guanidine isothiocyanate and guanidine hydrochloride were prepared and transformed into guanidinium isothiocyanate(GITC)and Guanidine hydrochloride (GuHCl) solutions with concentrations of 0.5 mol/L, 1.0 mol/L, 2.0 mol/L, 3.0 mol/L, and 4.0 mol/L. These solutions were stored at room temperature (22 ± 2°C) and in a refrigerated environment (6 ± 2°C).2.2.2n = 20) were collected and centrifuged at 1006 g for 10 min. Epithelial cells were obtained and added into self‐made GITC and GuHCl virus storage tubes with 20 equal parts of high‐concentration epithelial cell suspension, each 50 μL, and 140 μL of the SARS‐CoV‐2 pseudovirus. The mixture was then diluted with normal saline to a total volume of 3 mL, mixed evenly, stored at room temperature, and refrigerated for 60 days, respectively. The concentration of simulated SARS‐CoV‐2 positive samples was 8.07 E+2 copies/mL after the initial addition.The negative samples (2.2.3Nucleic acid testing (NAT) was conducted on samples stored for various time intervals ranging from 0 h to 2 months . The testing method employed multiplex PCR‐fluorescence probe technology and targeted the 2019‐nCoV ORF1ab and N genes. A dual‐target gene detection primer probe was designed for the RT‐PCR reaction system. Two target genes, including open reading frame 1ab (ORF1ab) and nucleocapsid protein (N), were simultaneously amplified and tested during the real‐time RT‐PCR assay. Target 1 (ORF1ab): forward primer GGTATGTGGAAAGGTTATGGCTGTA; reverse primer ACGATTGTGCATCAGCTGACTG; and the probe 5′‐VIC‐CCCCAGCGCTTCAGCGTTCTTC‐BHQ1‐3′. Target 2 (N): forward primer GATTACAAACATTGGCCGCAAA; reverse primer TGCCAATGCGCGACATTC; and the probe 5′‐FAM‐ATCAACTCCGCGAACCCATGC‐TAMRA‐3′. The results were evaluated by analyzing the threshold cycle (Ct) value of each channel. Additionally, the kit included the human housekeeping gene RNaseP as an internal standard to monitor the sampling, extraction, sample addition, and amplification processes. Prior to use, all reagents were thawed, mixed thoroughly, and centrifuged at 3000 rpm for a few seconds. RT‐qPCR reaction system referred to 20 μL PCR reaction mixture, 5 μL RNA of samples to be tested. Reaction conditions referred to reverse transcription at 50°C for 15 min, predenaturation at 95°C for 30 s, 95°C for 3 s and 60°C for 40 s (40 cycles).2.2.4R2 ≥ 0.95) was plotted using the above 5 points, forming a standard curve that could be used to calculate the concentration of SARS‐CoV‐2 nucleic acid in the sample. In this study, the ORF1ab gene was selected as the target gene to determine the virus concentration. The logarithm of the virus concentration was used as the final data for analysis and plotting. The calculation formula was as follows:The specific genes detected by Easy Diagnosis Biomedicine amplification reagent were ORF1ab and N gene. S1, S2, S3, S4, and S5 standards were prepared with concentrations of 1.73E+4, 8.65E+3, 4.33E+3, 2.16E+3, and 1.08E+3 copies/mL, respectively. These standards were created by doubling dilution with BDS Biological Technology peak value sample (concentration of 1.73E+4 copies/mL) four times. The diluent used was sterile normal saline water. A standard curve  were conducted. The preservation difference between different brands in the noninactivated virus preservation solution was compared using K Related samples tests in Nonparametric Tests. Additionally, the Wilcoxon test was performed to compare the efficacy and stability of the same concentration of GITC and GuHCl groups for refrigerated and room‐temperature storage of the SARS‐CoV‐2. The Friedman test was performed to compare the stability of SARS‐CoV‐2 preservation among different concentrations of GuHCl solution, different concentrations of guanidine isothiocyanate solution, and different brands of virus preservation solution. The Mann–Whitney 33.1t = 39.07, p < 0.001). According to Figure χ2 = 5.806, p = 0.214).We stored two aliquots of virus preservation solution, of the same volume, from multiple brands of inactivated (IA) and noninactivated (NIA) solutions for different periods of time, as described in Section 3.2p all >0.05, see Table Z = ‐2.295, p = 0.022, see Table Eight types of inactivated virus preservation solution were stored at room temperature 22 ± 2°C) and refrigerated (6 ± 2°C). Nucleic acid extraction and viral nucleic acid detection were performed at various time intervals ranging from 0 h to 2 months. We compared the preservation effect of the same brand of inactivated virus preservation solution at room temperature and in a refrigerated environment. The results showed that there was no significant difference in the preservation effect between different brands of virus preservation solutions at room temperature and in a refrigerated environment. Additionally, we noticed a clear downward trend in the guanidine content of 0.5 mol/L in both GITC and GuHCl groups  recommends using qRT‐PCR as the standard method for confirming SARS‐CoV‐2 detection.In this study, we prepared guanidinium salt virus preservation solution with different concentrations to investigate their preservation stability against SARS‐CoV‐2, finding that the use of GITC preservation solution could reduce the probability of false negative results.Since the samples were unable to meet the required refrigeration conditions of 2–8°C prior to testing, this study aimed to compare the effects of storage under room temperature (20–24°C) and refrigeration (2–8°C) on the preservation solution for the virus. The results showed no significant difference between the virus preservation solution stored at room temperature and refrigeration. This suggests that the buffer solution and other components added to the ready‐made IA have a better ability to resist interference from temperature, pH changes, and temperature fluctuations. The aforementioned results align with a previous study that utilized Innomed VTM001 virus collection tubes to compare the impact of two distinct storage temperatures  on 30 positive samples by RT‐PCR analysis for SARS‐CoV‐2, finding no false negative results within a span of 6 days for all samples.In conclusion, we highly recommended to use inactivated virus collection solution to preserve and transport samples in order to reduce the false negative in NAT results, improve the accuracy of NAT, ensure virus preservation stability, and minimize the risk of virus transmission. Additionally, refrigerated preservation is more suitable for identifying new coronavirus nucleic acid. If it is not possible to perform performance verification of the virus preservation solution before the experiment, NAT should be conducted in the laboratory as soon as possible, preferably within 6 h.Xiuzhi Duan, Letao Huang, and Zhihua Tao contributed to bibliographic research, drafting of the paper, critical revision, and final approval of the submitted version. Xuchu Wang, Yiyi Xie, and Weiwei Liu contributed to critical revision and final approval of the submitted version. Ying Ping and Pan Yu contributed to study conception and design, critical revision, and final approval of the submitted version. All authors read and approved the final manuscript.None.All patients were informed and agree to publish.


IRF4) as a melanoma‐associated dependency. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.Melanoma is a heterogenous malignancy with an unpredictable clinical course. Most patients who present in the clinic are diagnosed with primary melanoma, yet large‐scale sequencing efforts have focused primarily on metastatic disease. In this study we sequence‐profiled 524 American Joint Committee on Cancer Stage I–III primary tumours. Our analysis of these data reveals recurrent driver mutations, mutually exclusive genetic interactions, where two genes were never or rarely co‐mutated, and an absence of co‐occurring genetic events. Further, we intersected copy number calls from our primary melanoma data with whole‐genome CRISPR screening data to identify the transcription factor interferon regulatory factor 4 ( MC1R [TERT, POT1) and cell cycle control/senescence  have also been implicated [Melanoma remains one of the most curable malignancies when diagnosed at early stages yet is difficult to treat in patients who develop metastases. In European‐descent populations, the most common type is non‐acral cutaneous melanoma, which can be further subdivided into superficial spreading, nodular and lentigo maligna melanoma , 2. ThesMC1R , 5, 6, wplicated , 10, 11.plicated , 13. Melplicated . Notablyplicated , 16, 17.BRAF, NRAS and NF1 defining around three‐quarters of non‐acral cutaneous melanoma cases [CDKN2A, regulators of cell growth such as genes in the PI3‐kinase pathway (PTEN) and telomere maintenance genes (TERT), are also frequently observed. Intriguingly, although some of these core pathways are also altered in acral and mucosal melanoma, this may be via different constellations of driver genes and genetic events [The aforementioned sequencing efforts have revealed many of the key driver mutations that promote tumour development. For example, it is well established that activation of the mitogen‐activated protein kinase (MAPK) pathway is critical for melanoma development, with the mutation of genes such as ma cases . Somaticc events , 18, agaThe eighth edition of the American Joint Committee on Cancer (AJCC) guidelines for melanoma classifies the disease into prognostic stages ranging from I to IV, with associated substages ranging from A to D, based on tumour thickness, level of ulceration and presence/absence of metastasis , 20. MosNotably, as mentioned earlier, large‐scale sequencing efforts of melanoma have revolutionized our understanding of the disease, yet few primary melanomas have been analysed. Primary melanomas, particularly those from thin Stage I cases, may be less than 1 mm thick. Therefore, the analysis of these lesions has historically been technically challenging. In this study, we analyse 524 primary melanoma cases and present a comprehensive evaluation of the somatic alterations found in this disease so as to define the genetic landscape of melanoma at its earliest stages. We reveal a heterogenous tumour architecture alongside the discovery of novel candidate driver genes, new hotspot mutations and promoter variants, as well as mutually exclusive genetic interactions, that help refine our understanding of disease development.The Leeds Melanoma Cohort (LMC) Study is a prospective cohort recruited from a geographically defined area of the UK in the period 2000–2012, Research Ethics Committee (REC) Reference No. 01/3/057  and NIHRet al. [Formalin‐fixed, paraffin‐embedded (FFPE) tumour blocks were sampled horizontally using a 0.6‐mm microarray needle from the invasive tumour. The sample site was selected by a single observer to consistently collect material from the least inflamed, least necrotic area of the tumour, as previously described by Nsengimana et al. . Cores wWe classified tumours as non‐acral cutaneous, acral, mucosal or rare sites, which included ano‐uro‐genital cases, such as those arising on vulval, vaginal, anal and penile locations but not defined as being of mucosal origin  SureSelectXT probes using a custom design . Only known hotspot variants in BRAF, NRAS and TERT were considered. A 5% FDR threshold was applied, and gene pairs were excluded prior to analysis if co‐occurring mutations were found in ≥15% of tumours.Non‐synonymous SNV and indels were analysed using dNdScv version 0.0.0.9  to identon 0.9.2  was usedTPTE2) in melanoma cell lines, we performed RT‐PCR  portal  and repru et al. , except u et al. .IRF4), ERH  or with a non‐targeting pool (negative control) according to the manufacturer's instructions. Cells were retransfected after 3 or 6 days and harvested for analysis after 10 days. Cells were stained with Annexin V‐PE  and DAPI  and analysed by flow cytometry using a BD Fortessa II and FlowJo version 10 . Successful gene knock‐down was confirmed by western blot analysis using an IRF4 antibody . GAPDH  or vinculin  were used as loading controls. Cells were also analysed for c‐MYC protein expression using antibody clone Y69 .Two cell lines defined as lethal by CRISPR screening (RVH421 and WM1799) and two non‐lethal (WM983B and HT144) melanoma cell lines were transfected with siRNAs. These siRNAs were ON‐TARGETplus SMARTpools  designed against interferon regulatory factor 4 (P < 0.001).We designed capture probes to sequence established cancer genes and genes implicated in melanoma development, as well as promoter regions. As part of this experiment, we also captured genomic regions to study copy number changes, genes implicated in the immune regulation of cancer and a collection of melanoma predisposition genes  were identified using dNdSCv , which has not been described as being associated with melanoma development previously but was identified in a pan‐cancer analysis of TCGA data [TPTE is poorly expressed in normal tissue, with expression mainly confined to the testis; however, expression in melanomas and other cancers has been reported [TPTE pseudogenes appear to be localized to other chromosomes, including 13 and 22 [TPTE genes were reported to be highly mutated in a recent pan‐cancer analysis that used long read technology for mutation confirmation [PTEN and TPTE/TPTE2 appear to be mutually exclusive  and in younger patients . Although tumours with BRAF V600K mutations showed a slightly higher mutational load compared to BRAF V600E tumours , when compared to the BRAF wild‐type tumours, BRAF V600K tumours did not have significantly more somatic mutations   (GRCh37 chr1: 247095271) affecting 2% of melanomas  were mutually exclusive from high‐level amplifications of TERT (n = 15), except for a single high‐mutation‐load tumour   Figure  but no c pathway , 53 and  of EGFR , whereas of EGFR  , this gene pair was not statistically significantly mutually exclusive because three samples in this collection had co‐occurring mutations. Since the TCGA SKCM dataset was smaller than our study, the DISCOVER analysis of the TCGA cohort had less power. PRDM2 is a histone/protein methyltransferase that is capable of binding to the retinoblastoma protein [CDKN2A. Notably, genetic alterations of PRDM2 were largely mutually exclusive from alterations of downstream components of the CDKN2A pathway  , being more frequent in acral , mucosal  and rare  subtypes compared to non‐acral cutaneous tumours . TERT was found amplified in 15 samples (4%), and IRF4, located on chromosome 6p, was amplified in 11 (3%) samples. Deletion of CDKN2A was the most common copy number event in primary melanoma: 63% of samples showed copy number losses affecting CDKN2A, with 13% showing homozygous deletions across the entire gene.At the gene level, ) Figure . AmplifiP value <0.01). Among these eight genes were important melanoma genes such as BRAF, MITF, MDM2 and SOX10 (Figure IRF4, located in a highly amplified region of chromosome 6p, to be essential in cell lines that expressed high levels of the gene (Figure IRF4 in melanoma tumours compared to non‐cancerous tissues from a range of tissue sites (Figure IRF4 amplification could play a role in melanoma development. Other cancers, such as multiple myeloma, have been shown to be dependent on IRF4, and in this context depletion of IRF4 or treatment with an IRF4 inhibitor has been shown to induce lethality [IRF4 and develop a dependency on this transcription factor such that it may represent a vulnerability that could be exploited therapeutically. We next validated the requirement of melanoma cells for IRF4 using the orthogonal technology of siRNA knockdown and flow cytometry to assess viability. Analysis in this way revealed, as observed with CRISPR, that depletion of IRF4 results in reduced cancer cell fitness associated with cell death and apoptosis (Figure We next used Broad DepMap data where CRISPR‐Cas9 screening had been used to identify essential genes in 342 cancer cell lines, 29 of which were melanoma lines. Following correction for copy number–associated artefacts using CRISPRCleanR , analysi0 Figure . Notablye Figure . These te Figure . Additios Figure , suggestethality . We thers Figure . Notablys Figure , an estaFAM58A, RQCD1 and MSR1 as statistically significant driver genes (Figure FAM58A is a cyclin family member, implicated in the regulation of the actin network and ciliogenesis [RQCD1 and MSR1 are poorly studied genes implicated in the CCR‐NOT and PI3K/AKT pathways, respectively [TPTE, a gene with multiple related pseudogenes, as another candidate, although further studies will be required to validate this observation. Using DISCOVER analysis [BRAF and two with CDKN2A (Figure BRAF or NRAS mutation, but chance explains most co‐occurrences. The gene PRDM2 showed a mutation pattern of mutual exclusivity with CDKN2A and is of particular interest since PRDM2 is a binding partner of RB1, a well‐established melanoma driver [IRF4, a result with implications for our understanding of melanoma biology.In this study, we profiled the genomic landscape of primary melanomas as a complement to previous large‐scale efforts that primarily focused on metastatic disease. Understanding primary melanoma is critically important for clinical practice because the vast majority of patients present at this stage and understanding the aetiology of these cancers may help identify those patients who would benefit most from adjuvant therapies. Our analysis revealed 15 driver genes that were significant following dNdScv analysis. Our large study was able to confirm ogenesis , whereasectively , 61. Ouranalysis  we identa driver , 63. FinThe majority of the computational analysis was performed by SB with assistance from MR, JPL, IM, KH, VI, UM, CDRE, PVL, MLK and DTB. DTB and J N‐B led the Leeds Melanoma Cohort Study with assistance from MH and TM. Experiments with cell lines were performed by SB, LSASM, PAP and SSME. The experiments were designed by SB and DJA who wrote the paper, which was approved by all authors.Figure S1. Analysis of known gene mutationsFigure S2. Analysis of TPTE gene familyFigure S3. Analysis of genetic landscape of melanomaFigure S4. MAPK pathway mutual exclusivity and analysis of copy number landscape in this cohortFigure S5. Functional analysis of IRF4 loss in melanoma cell linesClick here for additional data file.Table S1. Clinical characteristics of patients in Leeds melanoma cohort, divided into respective subtypesClick here for additional data file.Table S2. Targeted capture bait design regionsClick here for additional data file.Table S3. Summary of all mutation calls made in this study including those in non‐coding regionsClick here for additional data file.Table S4. Summary of all mutation calls made in this study that were protein changing and in melanoma genesClick here for additional data file.Table S5. Summary of all indel calls made in this studyClick here for additional data file.Table S6. Summary of copy number events that alter whole cancer/melanoma genesClick here for additional data file.


Lactiplantibacillus plantarum TWK10 (TWK10) has beneficial probiotic effects, improves exercise endurance performance, regulates body composition, and mitigates aging‐related problems in mice and humans. Here, we investigated the effects of heat‐killed TWK10 on exercise endurance performance, muscle weight and strength, fatigue, and body composition in a double‐blind, placebo‐controlled clinical trial. Thirty healthy males aged 20–40 years were assigned to the Control group or heat‐killed TWK10 group (TWK10‐HK) in a balanced order according to each individual's initial maximal oxygen uptake. After 6‐week administration, the exercise endurance time in the TWK10‐HK was significantly increased (p = 0.0028) compared with that in the Control group. The grip strength on the right and left hands of the subjects was significantly increased  in the TWK10‐HK compared with that in the Control group. Administration of heat‐killed TWK10 resulted in a significant increase (p = 0.0275) in muscle weight. After 6‐week administration, serum lactate, and ammonia levels were significantly lower in the TWK10‐HK group than in the Control group during the exercise and recovery periods. These findings demonstrate that heat‐killed TWK10 has significant potential to be used as a postbiotic for humans.Consumption of  Heat‐killed TWK10 cells were prepared by heating the liquid bacterial culture at 70°C for 60 min followed by centrifugation to collect bacterial cell pellets and spray‐drying pellets for capsule preparation. The ingredients of the placebo capsule were the same as those of the heat‐killed TWK10 capsule except for the omission of TWK10. The absence of viable bacteria in the heat‐killed TWK10 and placebo capsules was confirmed by plating the contents of the capsules on de Man, Rogosa, and Sharp  agar plates followed by anaerobic incubation at 37°C for 48 h.TWK10 was isolated from Taiwanese pickled cabbage, “Pao‐tsai”  without professional exercise training were recruited in this study. Subjects were excluded from this study if they had heart/cardiopulmonary disease, diabetes, thyroid‐related disease, hypogonadism, hepatorenal disease, musculoskeletal disorder, neuromuscular/neurological disease, autoimmune disease, cancer, peptic ulcers, ulcerative colitis, Crohn's disease, or a body mass index (BMI) > 27. During the experimental period, all participants were prohibited from taking probiotics, prebiotics, fermented products (yogurt or other foods), vitamins, minerals, herbal extracts, dietary supplements for exercise and athletic performance, and antibiotics to avoid their interference. Subjects who agreed to follow the study protocol and voluntarily signed an informed consent form were included in this study. The study was reviewed and approved by the Institutional Review Board of Landseed International Hospital . The subjects were recommended to maintain their usual lifestyle, physical activities, and diet.2.3n = 15) or TWK10‐HK  groups in a balanced order based on their basal maximal oxygen consumption (VO2max). After 2 weeks of wash‐out period, the subjects were required to take two placebos or TWK10‐HK capsules daily after meal for 6 weeks, and then assessed for exercise endurance performance, grip strength, fatigue‐related parameters, and body composition. Dietary information of the subjects was recorded before and after administration, prior to the exercise endurance performance test. The intake of carbohydrates, proteins, fats, and total calories was analyzed by professional nutritionists as reference values. A description of the experimental procedure is shown in Figure This was a double‐blind, placebo‐controlled trial, with a 2‐week wash‐out period, and a 6‐week intervention period. Before the trial, body height, body weight (BW), BMI, body composition, blood biochemical parameters, and exercise endurance capacity of the participants were measured. The subjects were assigned to Control  was higher than 1.10 and reached the maximum HR (maximum HR = 220 − age). The three highest VO2max peaks were averaged to obtain the VO2max values of individual subjects. The individual basal VO2max during the pre‐test was used as a reference to adjust the appropriate exercise intensity individually to measure physiological adaptation (60% VO2max) and exhaustive endurance (85% VO2max). The subjects performed the maximum endurance test using a treadmill, warmed up at 60% VO2max intensity for 5 min, and then started the endurance running test at an 85% VO2max workload. To assess the maximum exercise tolerance of the subjects, we monitored their physical conditions every 5 min using HR and Borg's rating of perceived exertion (RPE) and continued to record until subjects experienced exhaustion. The sustained exercise duration was recorded as an endurance index. The detailed formula for intensity adjustment was based on that used in a previous study  and an automatic breathing analyzer . A polar heart rate device was used to monitor the heart rate (HR). According to Bruce's protocol, the treadmill speed started at 7.2 km/h and was increased by 1.8 km/h every 2 min until subjects experienced fatigue . Before the formal test, the subjects were required to squeeze the gripper with minimal force to confirm their understanding of the operating procedure and the gripping distance. The researchers randomly designated the dominant or non‐dominant hand to start the test. During the test, the subjects were asked to squeeze the gripper with one hand with maximum effort, maintain squeezing for at least 5 s, and repeat the test by changing hands at 60 s intervals to prevent fatigue. The subjects repeated the exchange method three times, and the individual maximum grip strengths of the two hands were recorded . Blood samples were collected using an arm venous catheter at the indicated time points during the exercise and recovery periods, including baseline (0), 15 (E15), and 30 (E30) min during the exercise phase, and 20 (R20) and 60 (R60) min in the recovery phase. Serum levels of lactate, ammonia, glucose, and creatinine kinase (CK) were assessed to monitor physiological adaptation. All biochemical indicators were determined using an autoanalyzer (Hitachi 7060). Complete blood count (CBC) profiles (Mindray BC‐2800Vet) were also determined at the 60‐min time point in the recovery phase.To assess fatigue‐related indicators, subjects fasted for at least 8 h before the fixed intensity exercise challenge (FIEC)  with an InBody 570 device  after an 8 h fast. The device was designed using a multi‐frequency principle and provided 1, 5, 50, 260, 500, and 1000 kHz frequency screening in 60 s. For measurement, the subjects stood on the foothold electrodes after the subjects' palms and soles were cleared; they held the sensing handle using both hands, and the subjects kept their arms open and away from their body at a 30° angle without speaking and moving during the measurement period.2.8To evaluate the impact of the administration of heat‐killed TWK10 on the physical features of the subjects, that is, 30 subjects in the Control and TWK10‐HK groups were analyzed before and after administration on the basis of seven physical features  using principal component analysis (PCA). GraphPad Prism 9.3.1 (GraphPad) was used to generate PCA plots using the first two principal components according to their group.2.9t‐test for parametric comparisons, and the Wilcoxon signed‐rank test was used for non‐parametric paired comparisons, including those for BMI and fat mass. Intergroup differences (Control group vs. TWK10‐HK group) were analyzed using the unpaired Student's t‐test for parametric comparison, and the Mann–Whitney U‐test was used for non‐parametric comparisons, including the neutrophil, lymphocyte, monocyte, eosinophil, basophil, mean corpuscular hemoglobin concentration (MCHC), the ratio of the neutrophil count to the lymphocyte count (NLR), the ratio of platelets to lymphocytes (PLR), BMI, and fat mass. The statistical significance of exercise endurance performance and grip strength . Within‐group differences (before administration vs. after administration) were analyzed using the paired Student's h Figure  were ana33.12max workload, and no significant difference was observed between the Control and TWK10‐HK groups. After 6 weeks of administration, the exhaustion time in the TWK10‐HK group was significantly increased by 1.24‐fold (p = 0.0028) compared to that in the Control group. Furthermore, after 6 weeks of administration, the mean exhaustion time in the TWK10‐HK group was significantly increased by 1.26‐fold (p < 0.0001) compared with the baseline values of subjects, whereas no significant increase was observed in the Control group  and 1.09‐fold (p = 0.0140) in the right and left hands, respectively, compared to that seen in the Control group. When compared with baseline values in test subjects, grip strength in the TWK10‐HK group significantly increased after 6 weeks of administration  , and recovery phase (time point: R20 [p = 0.0015]), respectively, was observed in the TWK10‐HK group compared with those of the Control group   and recovery period (time point R60 [p = 0.0152]) when compared with that in the Control group  Figure . The levp Figure . There wp Figure  and creap Figure  levels b3.32max]) The CBC profiles of the subjects before and after administration were obtained, and NLR and PLR were analyzed. There were no significant differences in WBC, neutrophil, lymphocyte, monocyte, eosinophil, basophil, platelet, and red blood cell (RBC) counts, hemoglobin (Hb) levels, hematocrit (Hct), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and MCHC levels between or within groups. NLR and PLR, used as markers of exercise‐induced systemic inflammatory responses  The 3.4p = 0.0275) compared with their baseline values  of 30 subjects into two factors that explained 67.7% of the variance. Principal component 1 (PC1) was heavily loaded with grip strength (right hand) and endurance time and negatively loaded with muscle weight, BW, BMI, and fat mass. Principal component 2 (PC2) was heavily loaded with grip strength (both right and left hands), muscle weight, and endurance time and negatively loaded with fat mass. The subjects in the TWK10‐HK group after administration were strongly influenced by grip strength (right and left hands), muscle weight, and endurance time. The subjects in the TWK10‐HK group after administration were clearly differentiated from those before administration Figure .3.6Dietary details of the subjects were monitored before and after the administration of the placebo and heat‐killed TWK10 capsules. There was no significant difference in the calorific intake between the Control and TWK10‐HK groups Table .410 and 3 × 1011 CFU/day) of viable TWK10 or heat‐killed TWK10 (3 × 1011 cells/day) for 6 weeks improved exercise performance and decreased serum lactate and ammonia. Administration of viable TWK10 decreased change of fat mass and increased change of muscle weight  for 18 weeks also increased muscle mass and improved muscle strength  used in our previous trial. Exercise, a stressor to the body, can induce inflammatory responses through the production of pro‐inflammatory cytokines and reactive oxygen species from activated leukocytes, causing muscle damage and tissue injury  of viable TWK10 or heat‐killed TWK10 (3 × 1011 cells/day) reduced the serum lactate levels after exercise in humans  significantly reduced the production of serum lactate during exercise challenge  reduced ammonia production after exercise and accelerated muscle recovery  for 6 weeks significantly improved muscle weight gain , Butyricicoccaceae (butyrate), and Ruminococcaceae (butyrate), showed strong positive correlations with one another after administration of heat‐killed TWK10. In addition, subjects receiving heat‐killed TWK10 showed a significant increase in acetate levels (p < 0.05) in feces compared to the basal levels, and an increasing trend in propionate concentration was also detected in the heat‐killed TWK10 group (p = 0.0857) .Informed consent was obtained from all the subjects involved in the study. Before the trial, the researchers explained the trial process in detail, and the trial began after the subjects signed a consent form.


The retrieval of autobiographical memories is influenced by several factors, such as sensory modality and the emotional salience of memory cues. This study aimed at investigating the interaction between sensory modalities  and emotional dimensions  of imagery cues, on the frequency, quality, and age distribution of the autobiographical memories (AMs) elicited.A total of 296 females (aged between 18 and 35 years) received one out of eight brief instructions for olfactory or visual imagery. The participants were asked to create a mental image with either high arousal/positive valence, high arousal/negative valence, low arousal/positive valence, or low arousal/negative valence ; no specific stimulus was mentioned in the instruction.The approach used elicited imagery with autobiographical content in the majority of participants (78%). In terms of frequency, odor imagery, compared to visual imagery, turned out to be more effective at retrieving either unpleasant memories associated with experiences in adulthood, or pleasant childhood memories. In terms of quality, the imagery was rated as less vivid in the olfactory compared to the visual condition . Visual imagery was associated with the experience of more diverse emotions  than odor imagery, which was related primarily with disgust and happiness.Our findings indicate that nonspecific imagery induction is a useful approach in accessing AM.This approach presents promising clinical applications, such as in working with autobiographical memory narratives in psychotherapy.The online version contains supplementary material available at 10.1007/s00426-022-01701-y. Random odors can evoke autobiographical memories ; this is otherwise known as the Proust phenomenon. For instance, a certain odor may transport one suddenly back into the arms of one’s grandmother, who had used a perfume with a similar scent, or alternatively, it might remind another person of an unpleasant experience at the dentist’s.The phenomenon of odors as retrieval cues for AMs has been the subject of various studies  in terms of the emotional dimensions valence and arousal  to investigate the potential of odor imagery as a retrieval cue for AMs and b) to elucidate interactions between emotional dimensions  and sensory modalities of memory cues, on the frequency, vividness, and age distribution , of the AMs reported. We hypothesized that the novel imagery induction approach would facilitate the retrieval of odor-related AMs, compared to classical deliberate cueing methods. Therefore, we assumed that the percentage of participants who would retrieve an AM would be higher than in the previous literature on AM retrieval after presentation of olfactory imagery  with a probability of 1 – β = 0.95, α = 0.05 for the χ2-tests to analyze AM frequency and a minimum sample size of N = 206 would be necessary to detect an effect size of part.η2 = 0.06 (i.e. medium effect) with a probability of 1 – β = 0.95, α = 0.05 for the main effects and interaction effects of sensory modality, valence and arousal of the imagery induction (2 × 2 × 2 ANOVAs) on AM vividness. Since previous literature suggests that not all participants would be able to generate mental images as well as AMs  to account for possible dropouts.The participation requirements  were communicated prior to testing. Twentyeight male participants and nine participants aged > 35 filled out the survey anyway. They were excluded after testing. The participants were recruited via announcements at the university campus and through social media. 97% of the participants had a high school diploma, 86% were university students. Participants reported no mental health problems as indicated by the scores of the brief symptom inventory . Additionally, they indicated whether or not the image referred to an event personally experienced in the past ; e.g., “The imagery reminded me of the smell when I once had to puke after a roller coaster ride”). We analyzed the percentage of participants who reported an AM image. If an AM image occurred, the participants reported their age, when the event had occurred. The events were either classified as childhood memories (age: 0–10 years), memories attributed to adolescence (age: 11–17 years), or more recent memories of adulthood (age: 18–35 years). Finally, the vividness of the imagery experience was rated on a seven-point Likert scale . Reported arousal and valence for AM images were rated as a manipulation check .The groups did not differ concerning the number of participants and mean BSI score . After obtaining written informed consent, the participants were asked for demographic data . Then participants listened to the imagery instruction and named/rated the evoked AM images. Furthermore, participants filled out the BSI;  were examined via χ2-tests. .We compared the frequency of AM images  between the factors Sensory Modality , Valence (pleasant vs. unpleasant), and Arousal  of imagery induction via The effect of imagery induction , Valence (pleasant vs. unpleasant), and Arousal  on the reported vividness of AM imagery was tested using a 2 × 2 × 2 between subjects analysis of variance (ANOVA).χ2 tests.As exploratory analyses, the content of the reported AMs was classified according to five basic emotions: happiness , sadness , anger , anxiety (e.g. accident), and disgust . The emotional content of the memories was classified by two independent psychologists with sufficient interrater agreement (Cohen’s Kappa = 0.61-0.81). The frequency of basic emotion-related content of AMs was compared between the conditions via Finally, as a manipulation check, the effects for imagery induction , Valence (pleasant vs unpleasant), and Arousal  were tested on reported valence and arousal of AM imagery with a 2 × 2 × 2 ANOVA.χ2 (1) = 10.22, p = 0.001, V = 0.186). Also in the pleasant imagery condition more participants reported an AM (85%) compared to the unpleasant imagery condition  = 9.25, p = 0.002, V = 0.177). Regarding interactions between sensory modality, valence, and arousal, the results of the χ2-analyses were in line with our hypothesis and showed that in the unpleasant conditions , more AM images were elicited via olfactory instructions (85%) compared to visual instructions  = 6.39, p = 0.011, V = 0.284, χ2Low (1) = 9.70, p = 0.002, V = 0.362). Furthermore, via visual imagery less AM images were evoked in the unpleasant (56%) compared to the pleasant imagery conditions  = 9.91, p = 0.002, V = 0.361; χ2Low (1) = 5.15, p = 0.023, V = 0.271). All other comparisons were not statistically significant  reported an imagery experience with autobiographical content. Surprisingly, even more AM images were reported in the olfactory imagery condition (85%) compared to the visual imagery condition  = 14.15; p < 0.001; V = 0.307), whereas this was not the case in the visual conditions (χ2(1) = 0.003; p = 0.957; V = 0.004; VP = 9.72% vs VUP = 9.46%). In the odor conditions, childhood-related AMs were almost exclusively reported for pleasant imagery cues (20% of participants reported childhood-related AMs), whereas in the unpleasant conditions almost none of the reported AMs were childhood-related (0.01%). In the unpleasant condition more AMs related to childhood were reported in the visual (9%) compared to the odor imagery condition  = 5.18; p = 0.023; V = 0.184); whereas in the pleasant condition the percentage of childhood-related AMs did not significantly  differ between visual and odor imagery condition (χ2(1) = 2.85; p = 0.091; V = 0.141). See Fig. Regarding the frequency of childhood-related AMs, the conducted ction  = 5.68; p = 0.017; V = 0.199). All other comparisons were not significant . See Fig. In contrast to childhood-related AMs, the results on adolescence-related AMs and adulthood-related AMs are in line with our hypothesis. In the pleasant conditions, a higher percentage of participants reported AMs related to adolescence in the visual (25%) compared to the odor imagery conditions  = 8.10; p = 0.004; V = 0.230), whereas the percentage of participants who reported AMs related to adulthood did not differ between visual (53%) and odor imagery in the pleasant condition  = 0.17; p = 0.677; V = 0.035). Furthermore, the percentage of participants who reported AMs related to adulthood was lower in the visual unpleasant (37%) compared to the visual pleasant imagery condition  = 3.92; p = 0.048; V = 0.164), whereas the percentage of adulthood-related AMs in the odor condition did not significantly differ between pleasant and unpleasant imagery induction (χ2(1) = 1.57; p = 0.210; 0.102). See Fig. In the unpleasant imagery conditions, more adulthood-related AMs were reported in the odor (60%) compared to the visual imagery condition .M = 4.18, SD = 1.35) than in the olfactory imagery conditions . All other main and interaction effects were not statistically significant; p > 0.173). For statistics, see Table The ANOVA revealed a significant main effect for Sensory Modality  than AMs retrieved in the unpleasant conditions . All other effects were not significant .As expected, the ANOVA identified a significant main effect for valence of imagery instruction on the valence ratings for the AM imagery (supplementary Table 2). AMs retrieved in the pleasant imagery conditions were rated as more pleasant  = 8.67, p = 0.004, partη2 = 0.038). All other effects were nonsignificant (p > 0.345). For means and standard deviations see supplementary Fig. 1b and c; for statistics see supplementary Table 2.However, the computed ANOVA for arousal ratings was only partly in line with our instructions. The results revealed significant interactions between Sensory Modality, Valence, and Arousal of imagery instruction (see supplementary Table 2). In the visual imagery condition with unpleasant/high arousal adjectives, AMs were rated more arousing than in the visual imagery condition with unpleasant/low arousal adjectives; This study investigated the effects of sensory modalities  and emotional dimensions  of imagery cues on the frequency, vividness, and age distribution of reported AMs. As a new approach, we used imagery instructions as retrieval cues and not specific odors or pictures  were able to generate a mental image with autobiographical content. In contrast, Willander & Larsson , who useIn terms of the reported age distribution of AMs, most memories were attributed to adulthood/adolescence, whereas only a small percentage of participants (approximately 10%) retrieved childhood-related memories. This is in line with previous research on the reminiscence bump [i.e., the tendency to access more personal memories from young adulthood than from other periods of life . Our new approach of nonspecific induction of olfactory imagery to access AM might be an option as one component of exposure therapy, which so far relied on the administration of real odors . Thus, our results cannot be generalized to other groups. Future studies on sex and age differences in the imagery task are strongly recommended. Further shortcomings of the present investigation concern the imagery instruction. The valence instructions for the imagery worked well; AMs retrieved after pleasant retrieval cues were rated as more pleasant than AMs retrieved after unpleasant retrieval cues. However, descriptions of stimuli inducing high vs. low arousal did not lead to AMs that differed in experienced arousal. Similar findings regarding the arousal dimension have been reported by Schulkind and Woldorf , SheldonTo the best of our knowledge, this is the first study to investigate the effects of sensory modalities and emotional dimensions of imagery as retrieval cues on AMs. Future studies need to replicate the present findings and broaden the research scope. For example, it would be interesting to investigate odor imagery as a retrieval cue in clinical samples with diagnoses of depression, or PTSD.Supplementary file1 (PDF 127 KB)Below is the link to the electronic supplementary material.


Cancer metastasis and recurrence remain major challenges in renal carcinoma patient management. There are limited biomarkers to predict the metastatic probability of renal cancer, especially in the early‐stage subgroup. Here, our study applied robust machine‐learning algorithms to identify metastatic and recurrence‐related signatures across multiple renal cancer cohorts, which reached high accuracy in both training and testing cohorts.Clear cell renal cell carcinoma (ccRCC) patients with primary or metastatic site sequencing information from eight cohorts, including one out‐house cohort, were enrolled in this study. Three robust machine‐learning algorithms were applied to identify metastatic signatures. Then, two distinct metastatic‐related subtypes were identified and verified; matrix remodeling associated 5 (MXRA5), as a promising diagnostic and therapeutic target, was investigated in vivo and in vitro.We identified five stable metastasis‐related signatures  from multicenter cohorts. Additionally, we verified the specificity and sensibility of these signatures in external and out‐house cohorts, which displayed a satisfactory consistency. According to these metastatic signatures, patients were grouped into two distinct and heterogeneous ccRCC subtypes named metastatic cancer subtype 1 (MTCS1) and type 2 (MTCS2). MTCS2 exhibited poorer clinical outcomes and metastatic tendencies than MTCS1. In addition, MTCS2 showed higher immune cell infiltration and immune signature expression but a lower response rate to immune blockade therapy than MTCS1. The MTCS2 subgroup was more sensitive to saracatinib, sunitinib, and several molecular targeted drugs. In addition, MTCS2 displayed a higher genome mutation burden and instability. Furthermore, we constructed a prognosis model based on subtype biomarkers, which performed well in training and validation cohorts. Finally, MXRA5, as a promising biomarker, significantly suppressed malignant ability, including the cell migration and proliferation of ccRCC cell lines in vitro and in vivo.This study identified five robust metastatic signatures and proposed two metastatic probability clusters with stratified prognoses, multiomics landscapes, and treatment options. The current work not only provided new insight into the heterogeneity of renal cancer but also shed light on optimizing decision‐making in immunotherapy and chemotherapy. Multicohorts of clear cell renal cell carcinoma (ccRCC) datasets and machine learning algorithms were used to identify and verify metastatic biomarkers. Two distinctive ccRCC subgroups were uncovered and compared at multiomics level and targeting matrix remodeling associated 5 (MXRA5) could be treated as an effective approach to suppress ccRCC progression. ANO3anoctamin 3APMantigen processing machineryCCK‐8Cell Counting Kit‐8CCLECancer Cell Line EncyclopediaccRCCclear cell renal cell carcinomaCNVscopy number variationsCPTACClinical Proteomic Tumor Analysis ConsortiumDEGsdifferentially expressed genesEMTepithelial–mesenchymal transitionGEOGene Expression OmnibusGOGene OntologyGSEAgene set enrichment analysisGSVAgene set variation analysis50IChalf‐maximal inhibitory concentrationITGBL1integrin subunit beta‐like 1KEGGKyoto Encyclopedia of Genes and GenomesLASSOleast absolute shrinkage and selection operatorMEOX2mesenchyme homeobox 2MTCS1metastatic cancer subtype 1MTCS2metastatic cancer subtype 2MXRA5matrix remodeling associated 5NSCLCnonsmall cell lung cancerOSoverall survivalPACproportion of ambiguous clusteringPFSprogression‐free survivalRCrenal cancerRENreninRFrandom forestRT‐qPCRreverse transcription‐quantitative polymerase chain reactionSVMsupport vector machineTCGAThe Cancer Genome AtlasTIDEtumor immune dysfunction and exclusionTPMtranscripts per kilobase million1Renal cancer (RC) is one of the most fatal cancers in the urogenital system, with an incidence only secondary to that of prostate and bladder cancer . AccordiRecently, accumulated studies have indicated that genetic and epigenetic alterations are closely related to the prognosis and treatment of ccRCC , 9, 10. In this study, we attempted to apply machine‐learning algorithms to decipher ccRCC‐related metastatic biomarkers and construct an early risk stratification classifier of ccRCC, which might help clinical practitioners assess recurrence probability, metastasis, survival time, and drug sensitivity in ccRCC. Our work may facilitate optimized management and further improve the clinical outcomes of ccRCC patients.22.1n = 35, primary RC tissue = 9, metastatic RC tissues = 26; GSE22542: n = 68, primary cancer tissues = 27, metastatic RC tissues = 41; GSE23629: n = 32, primary cancer tissues = 16, metastatic RC tissues = 16; GSE85258: n = 30, primary cancer tissues = 15, metastatic RC tissues = 15); The Cancer Genome Atlas‐kidney renal clear cell carcinoma (TCGA‐KIRC); Cancer Cell Line Encyclopedia (CCLE); and single‐cell sequencing data from previous research [https://portal.gdc.cancer.gov/); the former was used for differential expression analysis and further transformed to log2 (TPM + 1) for subsequent analysis. All expression data were normalized before analysis. Patients were excluded if they 1) did not have prognostic information and 2) died within 30 days.Most ccRCC datasets in our study were retrospectively enrolled in public databases, including Gene Expression Omnibus . The R package “carcet” was used to divide the combined dataset into training data and testing data (ratio = 7:3). Then, three machine‐learning algorithms, including least absolute shrinkage and selection operator (LASSO) logistic, support vector machine (SVM), and random forest (RF), were introduced to identify metastatic relevant signatures of ccRCC.The R package “sva” was used to remove the batch effect from different datasets, and samples with primary or metastatic annotation were maintained for further analysis. First, differentially expressed genes (DEGs) between primary and metastatic sites were detected with the threshold 2.3k = 2 was treated as the optimal cluster number.After obtaining five metastatic signatures, we aimed to apply them to decipher the heterogeneity and metastasis tendency of ccRCC. This analysis was performed via the R package “ConsensusClusterPlus” to identify optimal cluster number k . Subsequently, we applied the consensus score matrix, cumulative distribution function curve, the proportion of ambiguous clustering (PAC) score, and Nbclust to determine the optimal cluster number. Finally, ccRCC patients were grouped into two distinct subtypes named metastatic cancer subtype 1 (MTCS1) and type 2 (MTCS2), and 2.4p < 0.01 and abstract log fold‐change >2 [https://www.gsea-msigdb.org/gsea/index.jsp) and ConsensusPathDB (http://cpdb.molgen.mpg.de/) databases [The R package “DEseq. 2” was used to identify DEGs between MTCS1 and MTCS2, and the thresholds were set to adjusted hange >2 . After change >2 . All gmtatabases , 30.2.5http://tide.dfci.harvard.edu/) algorithm was applied to illustrate the different immunotherapy sensitivities [We used multiple immune cell infiltration algorithms, including CIBERSORT, EPIC, MCPCOUNTER, QUANTISEQ, TIMER, and XCELL, to calculate immune cell enrichment and cellular component scores in ccRCC tissues and compare differences in immune components between subgroups , 34, 35.tivities .2.6https://cloud.genepattern.org/gp/pages/index.jsf) algorithm [Somatic datasets of ccRCC were analyzed and visualized via the R package “Maftools” . Using clgorithm , 40.2.750) and cross‐validate the predicted results. In addition, CellMiner (https://discover.nci.nih.gov/cellminer/home.do), CCLE (https://sites.broadinstitute.org/ccle), and Genomics of Drug Sensitivity in Cancer (GDSC) (https://www.cancerrxgene.org/) databases were introduced to compare the different sensitivities between ccRCC cell lines [The R package “pRRophetic” was used to estimate the half‐maximal inhibitory concentration  and immunohistochemical staining to validate differential matrix remodeling associated 5 (MXRA5) expression between paired tumor and normal tissues . Primer sequences for RT‐qPCR were as follows: MXRA5  and glyceraldehyde 3‐phosphate dehydrogenase . An MXRA5 antibody (cat number: PA5‐37267) was purchased from Thermo Fisher Group Ltd. The detailed procedures were described in our previous studies , 44, 45.2.10All cell lines used in our study were purchased from the American Type Culture Collection. Cell lines were cultured according to standard protocols. The plasmid for the MXRA5 knockdown was chemically synthesized by Shanghai GeneChem Co., Ltd. 786‐0 and ACHN cells were cultured in six‐well culture dishes at a 60% density and then infected with lentivirus. All transfections were performed with 4 µg/mL Polybrene  and lasted for 12 h. The screening was conducted with 2 µg/mL puromycin  for 3 days to acquire stably transfected cells. RT‐qPCR and western blotting were applied to verify the knockdown efficiency. Cell Counting Kit‐8 (CCK‐8) was used to detect cell viability between negative control and MXRA5 knockdown groups. The scratch assay and transwell migration assay were used to evaluate cell migration ability. A colony‐forming experiment was conducted to determine the ability of reproduction. A nude mouse xenograft tumor model assay was applied to investigate the effect of MXRA5 on tumor proliferation. The detailed procedure of these experiments was described in our previous study. All experiments mentioned above were carried out in three replications and repeated three times.2.11p < 0.05 was considered statistically significant.All data processing, statistical analysis and plotting were performed via R software (version 4.0.4). Kruskal–Wallis and Wilcoxon test were used to compare continuous variables. Chi‐square test was applied to compare categorical variable. R package “survival” was used to perform Cox regression and Kaplan–Meier analysis. The receiver operating characteristic curve (ROC) applied to predict binary categorical variables was implemented via R package “pROC”. The time‐dependent area under the ROC curve (AUC) for survival variables was conducted by the package “timeROC”. All statistical tests were two‐sided, and 33.1The workflow of this study was depicted in Figure 3.2In total, 530 DEGs were introduced to select signatures for classifying nonmetastatic and metastatic tissues. LASSO logistic, SVM–recursive feature elimination, and RF algorithms were applied to identify metastatic‐related genes Figure . After o3.3Because the five metastatic signatures displayed a significant role in ccRCC progression, we then systematically analyzed the regulation of these signatures at the DNA methylation and RNA modification level. As illustrated in Supporting Information: Figure 3.4Since the five metastatic signatures might play a pivotal role in the progression of ccRCC, we investigated their impact on ccRCC's heterogeneity. We first calculated the expression relationship of the five signatures and found that the expression level of ITGBL1 was significantly positively correlated with that of MXRA5 Figure . The TCG3.5The expression profiles in MTCS1 and MTCS2 were selected to construct the progression landscape of ccRCC. Consistent with the defined subtypes, most patients could be segregated into two distinct clusters and different states Figure . The loc3.6The expression profiles of ccRCC were analyzed to identify metastatic‐related DEGs, which included 39 downregulated and 926 upregulated genes Figure . Combine3.7To investigate the relationship between metastasis and tumor immunity, we first calculated the correlation between metastatic signatures and immune cell infiltration degree. Supporting Information: Figure 3.8Because transcriptional alterations between MTCS1 and MTCS2 were investigated, we then aimed to describe the disparity at the genomic level. The mutation rate was higher in MTCS1 , and the top 20 most frequently mutated genes were illustrated in Supporting Information: Figure In addition to the mutation pattern, we investigated differences in the copy number variations (CNVs) between subtypes. The GISTIC2.0 algorithm was used to determine the amplification and deletion of copy numbers on chromosomes. Compared with MTCS1, MTCS2 had a higher level of copy number alterations      were tested in three external datasets and one out‐house dataset and displayed high accuracy in each cohort. To determine the underlying mechanism of metastasis in ccRCC, we used an unsupervised clustering algorithm to stratify ccRCC and decipher the heterogeneity of MTCS1 and MTCS2 at a multiomics level. Among the five metastatic signatures, REN is a protein‐coding gene. Diseases associated with REN include tubulointerstitial kidney disease, autosomal dominant kidney disease, and renal tubular dysgenesis. Only a few studies have revealed the role of REN in cancer. Mehranfard et al.  determinAdditionally, we enrolled the TCGA–KIRC cohort to determine the correlation between metastatic traits and intertumoral heterogeneity. Unlike previous studies, we retrieved a total of five metastatic signatures to categorize ccRCC patients. Briefly, MTCS2 was treated as a malignant phenotype with poor clinical outcomes, metastatic tendency, high expression of REN and ANO3, activated fatty acid metabolism, and activated transcripts of HNF1A, HNFAB, and EPAS1, and reduced infiltration of immune cells. In contrast, MTCS1 exhibited high expression of ITGBL1 and MXRA5, improved clinical outcomes, activated EMT, activated FOXE1, TBX18, TFE3, and TP53, and high infiltration of immune cells. Interestingly, MTCS1 was characterized by high levels of HHLA2 and RAET1E, whereas MTCS2 was associated with high levels of CD28, TMEM173, tumor necrosis factor (TNF) family members, and interleukin‐related signatures. Previous studies have indicated that HHLA2 was more frequently expressed than PDCD1 in ccRCC, and HHLA2/PDCD1 co‐expression had an adverse impact on the prognoses of patients with ccRCC, indicating that combination immunotherapy with anti‐HHLA2 and PD‐L1 blockade may function as a new strategy for ccRCC patients. Interestingly, we found that MTCS1 displayed higher co‐expression with HHL2 and PDCD1. Combined with the clinical outcomes of MTCS1, we presumed that targeting HHLA2 may be a valuable approach to improving the survival of metastatic ccRCC patients. In addition, MTCS2 displayed enhanced immune activation as the immune infiltration degree and immune‐related score calculated using multiple algorithms were significantly higher in MTCS2. The immune exhaustion score (including LAG3 and TIDE scores) was also higher in MTCS2, which indicated that a different metastatic phenotype might reverse tumor immunity and induce immune exhaustion.Overall, MTCS2 was characterized by increased genomic instability and predominant mutations in TTN and BAP1, whereas MTCS1 displayed a lower mutation degree and mutations in PBRM1, VHL, and STD2. These results were consistent with previous reports that a high mutational load produced more neoantigens, which further induced the proliferation and activation of immune cells. In addition, the five metastatic signatures displayed no difference in mutations between MTCS1 and MTCS2, which indicated that the aberrant expression of these signatures might be regulated at the posttranscriptional level. Moreover, we found some promising targets for the high‐risk subgroup (or MTCS2). For example, the ROCK inhibitor GSK269962A was found to effectively inhibit the progression of melanoma cell lines , and theTo the best of our knowledge, this is the first study to identify ccRCC metastatic biomarkers and decipher metastatic tendency using multiple cohorts and multi‐omics analysis. Although the implication of the five metastatic signatures has been identified and verified, there are still some limitations. First, the detection array of different microarray platforms might differ, potentially leading to overlooking some signatures related to metastasis. Second, some molecular and clinical characteristics of public datasets were inadequate, which might have concealed the potential associations between ccRCC subtypes and certain variables. Third, the detailed role of MXRA5 in ccRCC and the TME remains unknown, and further in vivo and in vitro experiments are needed to reveal its function.5In summary, the present study identified the most pivotal signatures involved in ccRCC metastasis and identified two distinct metastatic subtypes using a multiomics approach. Our study might shed light on metastatic ccRCC and provide a significant clinical reference for risk stratification, therapeutic strategies, and prognosis prediction among ccRCC patients.Aimin Jiang: Conceptualization; methodology; formal analysis; and visualization. Qingyang Pang: Formal analysis. Xinxin Gan: Conceptualization. Anbang Wang: Methodology. Zhenjie Wu: Methodology. Bing Liu: Methodology. Peng Luo: Conceptualization. Le Qu: Supervision. Linhui Wang: Resources; supervision; and writing – review and editing.The authors declare no conflict of interest.The procedure related to human subjects was approved by the Ethics Committee of the Changhai Hospital (CHEC2022074). All animal experiments were approved by the Animal Ethics Committee of the Changhai Hospital (CHEC2021‐191).Informed consent was obtained from all subjects to use the specimens described in this study.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.


Non‐invasive breast carcinoma is considered to be localized disease and is distinguished from invasive ductal and lobular carcinomas. The local recurrence of non‐invasive carcinoma after surgery may lead to development of invasive carcinoma and promote distant metastasis, which worsens the prognosis for breast cancer mortality. The distant metastasis of non‐invasive carcinoma may involve the ductal microvasculature without invasion. The outcomes of non‐invasive breast carcinoma were examined in this retrospective cohort study.Of 872 primary breast cancers diagnosed at a single center between May 2008 and March 2022, 93 (10.6%) were found to be non‐invasive carcinomas and were examined in this study. The breast cancer recurrence and survival rates of patients with non‐invasive carcinoma were analyzed retrospectively. The median follow‐up period was 1891  days. All patients underwent surgical treatment . Postoperatively, radiation therapy was administered to 73 (78.4%) of the patients and endocrine therapy was administered to 64 (81.0%) of 79 patients with hormone‐receptor positivity. Of 26 patients who underwent partial mastectomy with SLNB, 24 (92.3%) showed isolated tumor cells in the SLNs on one‐step nucleic acid amplification. Local recurrence was observed in three (0.3%) patients; no distant metastasis was observed. One patient died of a noncancerous disease. The overall survival rate was 98.0% and the breast cancer‐specific survival rate was 100.0%.Non‐invasive breast carcinoma, like invasive breast carcinoma, causes local recurrence, but has a good prognosis without distant metastasis. The clinical significance of isolated tumor cells in the SLNs as a systemic component of non‐invasive breast carcinoma remains to be elucidated. One‐step nucleic acid amplification (OSNA)Sentinel lymph node biopsy (SLNB) is a standard procedure in breast cancer surgery.22.1This retrospective study was conducted using data from women with non‐invasive carcinoma According to patients' preferences, outpatient surgery under local anesthesia (LA) and intravenous anesthesia (IVA) and/or sedation was performed at the clinic,2.2In outpatient surgeries performed during the study period, LA with lidocaine, benzodiazepines  as sedatives, IVA with propofol, and pentazocine  and pethidine (synthetic opioid) as analgesics were used. Preoperatively, 10 ml 0.5% lidocaine was injected locally under ultrasound guidance into the retromammary tumor space when marking the tumor resection site; additional local injections were administered around the tumor before and during surgery. For the intraoperative monitoring of biological functions, each patient was fitted with a biometric monitor to measure the pulse rate, electrocardiographic data, blood pressure, respiratory rate, and oxygen saturation. All patients received oxygen at a rate of 3–5 L/min delivered by oxygen mask during surgery, with preparation for emergency mask ventilation and tracheal intubation. In inpatient surgeries, GA was induced with total IVA using propofol and synthetic opioids such as remifentanil, or volatile anesthesia with sevoflurane using synthetic opioids.2.399mTc‐phytate and indigo carmine dye. During outpatient surgeries, OSNA assays3/μL), micrometastasis , and negativity .The patients underwent SLNB and BCS with partial (Bp) or quadrant (Bq) breast resection or Bt. BCS was defined as resection of the primary tumor and partial mastectomy with a 1–1.5‐cm margin. In outpatient surgeries, axillary SLNB was performed using indigo carmine and indocyanine green dyes; in inpatient surgeries, it was performed using the radioisotope 2.4To evaluate the expression of hormone receptor (HR), estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER‐2) proteins, formalin‐embedded sections of surgical tumor specimens were immunostained using the BenchMark ULTRA automated immunohistochemical slide staining system  at Fukuyama Medical Laboratory Co., Ltd. The antibodies used were anti‐ER (SP‐1), anti‐PR (1E2), and anti–HER‐2 . A pathologist graded ER and PR expression using the Allred score,2.5Patients who underwent BCS received postoperative radiotherapy (RT) for the remaining breast, delivered at the affiliated hospital at standard doses for 3 weeks (hypofractionated dose: 40.5 Gy/15 Fr) or 4 weeks  after surgical treatment. Actual radiation doses were adjusted according to body surface areas. Patients with resection margins ≤5 mm or age ≤ 50 years were given RT boosters to the tumor bed (10.8 Gy/4 Fr or 10 Gy/5 Fr) as needed. Patients with HR positivity received endocrine therapy (ET) with tamoxifen  for 5 years.2.6All data were analyzed using Satcel 4 software . Cumulative OS and breast cancer‐specific survival (BCSS) rates and durations from the date of surgery were calculated using the Kaplan–Meier method. OS was defined as the time from surgical treatment to death from any cause, and BCSS was defined as the time from the date of surgical treatment to the date of death attributable to breast cancer.33.1n = 78), core needle biopsy , vacuum‐assisted biopsy , and other (n = 1). The most common surgery performed was partial mastectomy (Bp/Bq) with SLNB [n = 59 (63.4%)], followed by Bp/Bq and Bt with SLNB [n = 15 (16.1%) each]. Of 26 patients who underwent SLNB for whom OSNA was performed, 24 (92.3%) showed elevated negativity with isolated tumor cells; VAB was performed in 13 of these 24 patients. ET was administered to 64 (68.8%) patients. Postoperative RT was administered to 73 (78.4%) patients, and 43 of these patients received RT boosters.Of 872 patients with primary breast cancer diagnosed at our clinic, 94 10.7%) patients were diagnosed with non‐invasive carcinoma. One patient did not receive surgical treatment after diagnosis and was excluded from this study. The clinicopathological characteristics of the remaining 93 patients are shown in Table 0.7% pati3.2During a median follow‐up period of 1891  days, three patients experienced breast cancer recurrence Table . One patNo breast cancer‐related deaths occurred, but one patient died following a stroke. The cumulative OS and BCSS rates for the entire cohort were 98.0% and 100%, respectively ; data curation ; investigation ; methodology ; project administration ; resources ; visualization ; writing – original draft ; writing – review and editing . Ami Kawai: Data curation ; formal analysis ; investigation ; resources ; software ; visualization ; writing – review and editing . Megumi Wakisaka: Resources . Mika Shimoyama: Resources . Naomi Yasuda: Resources . Mitsuya Ito: Resources . Takanori Kin: Resources . Koji Arihiro: Resources .No funding was received for this study.The authors have stated explicitly that there are no conflicts of interest in connection with this article.This retrospective cohort study was performed in accordance with the Declaration of Helsinki. The Ethics Committee of Hiroshima Mark Clinic approved the study (no. HMC‐04), and all treatments were administered with the patients' informed consent.


Primary tracheal small‐cell carcinoma is rare, and is often treated using small‐cell lung cancer guidelines given that no standard treatment has been established for it. We report a patient in whom nodules appeared in the trachea and left main bronchus 11 months after surgery for pulmonary large‐cell neuroendocrine carcinoma; a biopsy revealed small‐cell carcinoma. Given the absence of malignant lesions elsewhere in the body, the lesions were diagnosed as primary tracheal small‐cell carcinoma. Respiratory failure progressed rapidly owing to airway stenosis caused by the growing lesion, and the patient required nasal high‐flow therapy. However, the lesions shrank a few days after commencing first‐line chemotherapy, and his respiratory failure resolved. Accelerated hyperfractionated radiotherapy was administered in conjunction with the third course of chemotherapy, and the patient ultimately achieved a complete response. Although the lesions were initially suspected of being postoperative recurrence of pulmonary large‐cell neuroendocrine carcinoma, the fact that the biopsy revealed them to be primary tracheal small‐cell carcinoma indicates that intra‐airway nodules that appear after lung cancer surgery may possibly be primary tracheal tumors. We report a patient in whom nodules appeared in the trachea and left main bronchus 11 months after surgery for pulmonary large‐cell neuroendocrine carcinoma; a biopsy revealed small‐cell carcinoma. Four courses of chemotherapy, plus accelerated hyperfractionated radiotherapy with the third course, resulted in a complete response. Physicians must consider that intra‐airway nodules that appear post lung cancer surgery may be primary tracheal tumors. Primary tracheal tumors are rare; among them, primary tracheal small‐cell carcinomas (SCCs) are very infrequent, and there are no large‐scale studies exploring their treatment.A 77‐year‐old man with a 30‐pack‐year smoking history visited our hospital after an abnormal chest shadow was detected during a physical examination. Computed tomography (CT) showed a nodule in the lower lobe of the right lung Figure . He undeEleven months after surgery, the patient developed bloody sputum and dyspnea on exertion, and CT revealed an 11‐mm intratracheal nodule Figure  as well 2 body surface area) were also initiated on the same day. Six days after the start of chemotherapy, CT revealed shrinkage of the lesion  for the second and subsequent courses, with AHF (45 Gray/30 fractions) introduced during the third course. After the fourth course of chemotherapy, the lesions had resolved on CT  with a gas flow rate of 50 L/min and a fraction of inspired oxygen of 0.6 was introduced. First‐line chemotherapy with carboplatin (area under the curve = 5 mg/mL/min) and etoposide (80 mg/mn Figure , and then Figure . The che Figures  and bron Figures . After aPrimary tracheal tumors account for approximately 0.2% of all respiratory tumors,Treatments administered to the few reported patients with primary tracheal SCC included surgical resection, radiotherapy, chemotherapy, and chemoradiotherapy Table .4, 5Additionally, ablation, snare, cryotherapy, laser photo resection, electro‐surgery, argon plasma coagulation, and endotracheal stenting have reportedly been performed to treat the symptoms of airway narrowing associated with primary tracheal tumors.Taken together, our patient's course suggests that physicians must carefully consider the possibility that intra‐airway nodules that appear post‐lung cancer surgery may be primary tracheal tumors.S.T. and C.S: writing – original draft. T.S., S.N., H.N., W.S., S.H., Y.K., C.M., S.K., K.S., and K.T: writing – review and editing. N.K., M.Y., M.K., and T.K: visualization and supervision. All authors approved the final version of the manuscript to be published.The authors have no conflicts of interest to declare.