Tumor-informed deep sequencing of ctDNA detects minimal residual disease and predicts relapse in osteosarcoma.

Background: Current surveillance modalities of osteosarcoma relapse exhibit limited sensitivity and specificity. Although circulating tumor DNA (ctDNA) has been established as a biomarker of minimal residual disease (MRD) in many solid tumors, a sensitive ctDNA detection technique has not been thoroughly explored for longitudinal MRD detection in osteosarcoma. Methods: From August 2019 to June 2023, 59 patients diagnosed with osteosarcoma at the First Affiliated Hospital of Sun Yat-sen University were evaluated in this study. Tumor-informed MRD panels were developed through whole exome sequencing (WES) of tumor tissues. Longitudinal blood samples were collected during treatment and subjected to multiplex PCR-based next-generation sequencing (NGS). Kaplan-Meier curves and Log-rank tests were used to compare outcomes, and Cox regression analysis was performed to identify prognostic factors. Findings: WES analysis of 83 patients revealed substantial mutational heterogeneity, with non-recurrent mutated genes accounting for 58.1%. Tumor-informed MRD panels were successfully obtained for 85.5% of patients (71/83). Among 59 patients with successful MRD panel customization and available blood samples, 13 patients exhibited positive ctDNA detection after surgery. Patients with negative post-operative ctDNA had better event-free survival (EFS) compared to those with positive ctDNA, at 1-6 months after surgery, after adjuvant chemotherapy, and more than 6 months after surgery (p < 0.05). In both univariate and multivariate Cox regression analysis, ctDNA results emerged as a significant predictor of EFS (p < 0.05). ctDNA detection preceded positive imaging in 5 patients, with an average lead time of 92.6 days. Thirty-nine patients remained disease-free, with ctDNA results consistently negative or turning negative during follow-up. Interpretation: Our study underscores the applicability of tumor-informed deep sequencing of ctDNA in osteosarcoma MRD surveillance and, to our knowledge, represents the largest cohort to date. ctDNA detection is a significant prognostic factor, enabling the early identification of tumor relapse and progression compared to standard imaging, thus offering valuable insights in guiding osteosarcoma patient management. Funding: The Grants of National Natural Science Foundation of China (No. 82072964, 82072965, 82203798, 82203026), the Natural Science Foundation of Guangdong (No. 2023A1515012659, 2023A1515010302), and the Regional Combination Project of Basic and Applied Basic Research Foundation of Guangdong (No. 2020A1515110010).

Chinese expert consensus on the diagnosis and treatment of malignant pleural mesothelioma.

Malignant pleural mesothelioma (MPM) is a malignant tumor originating from the pleura, and its incidence has been increasing in recent years. Due to the insidious onset and strong local invasiveness of MPM, most patients are diagnosed in the late stage and early screening and treatment for high-risk populations are crucial. The treatment of MPM mainly includes surgery, chemotherapy, and radiotherapy. Immunotherapy and electric field therapy have also been applied, leading to further improvements in patient survival. The Mesothelioma Group of the Yangtze River Delta Lung Cancer Cooperation Group (East China LUng caNcer Group, ECLUNG; Youth Committee) developed a national consensus on the clinical diagnosis and treatment of MPM based on existing clinical research evidence and the opinions of national experts. This consensus aims to promote the homogenization and standardization of MPM diagnosis and treatment in China, covering epidemiology, diagnosis, treatment, and follow-up.

Personalized circulating tumor DNA detection to monitor immunotherapy efficacy and predict outcome in locally advanced or metastatic non-small cell lung cancer.

OBJECTIVE: Immune checkpoint inhibitors (ICIs) or combined with chemotherapy exhibit substantial efficacy for the treatment of advanced non-small cell lung cancer (NSCLC). However, reliable biomarkers that can monitor response to first-line ICIs ± chemotherapy remain unclear. METHODS: A total of 16 tumor tissues and 46 matched peripheral blood samples at baseline and during treatment were retrospectively collected from 19 locally advanced or metastatic NSCLC patients. The circulating tumor DNA (ctDNA) burden by tumor-informed assay was detected to monitor and predict the therapeutic response and survival of NSCLC patients treated with first-line ICIs or plus chemotherapy. RESULTS: We found that ctDNA was only positively detected in one patient by tumor-agnostic assay with a mean variant allele fraction (VAF) of 6.40%, whereas it was positively detected in three patients by tumor-informed assay with a mean VAF of 8.83%, 0.154%, and 0.176%, respectively. Tumor-informed assays could sensitively detect ctDNA in 93.75% (15/16) of patients. Trends in the level of ctDNA from baseline to first evaluation was consistent with the radiographic changes. There was a greater decrease in ctDNA after treatment compared with baseline in patients with partial response compared to patients with stable disease/progressive disease. Patients with over a 50% reduction in ctDNA had a significant progression-free survival and overall survival benefit. CONCLUSION: The tumor-informed assay was favorable for ctDNA detection, and early dynamic changes in plasma ctDNA may be a valuable biomarker for monitoring the efficacy and predicting the outcome in advanced NSCLC patients treated with first-line ICIs ± chemotherapy.

Chinese expert consensus on the diagnosis and treatment of thymic epithelial tumors.

Thymic epithelial tumors (TETs) are a relatively rare type of thoracic tumor, accounting for less than 1% of all tumors. The incidence of TETs is about 3.93/10000 in China, slightly higher than that of European and American countries. For resectable TETs, complete surgical resection is recommended. Radiotherapy or chemotherapy may be used as postoperative adjuvant treatment. Treatment for advanced, unresectable TETs consist mainly of radiotherapy and chemotherapy, but there is a lack of standard first- and second-line treatment regimens. Recently, targeted therapies and immune checkpoint inhibitors have shown promising outcomes in TETs. Based on the currently available clinical evidences and the opinions of the national experts, the Thymic Oncology Group of Yangtze River Delta Lung Cancer Cooperation Group (East China LUng caNcer Group, ECLUNG; Youth Committee) established this Chinese expert consensus on the clinical diagnosis and treatment of TETs, covering the epidemiology, diagnosis, treatment, prognosis and follow-up of TETs.

New insights into fumonisin production and virulence of Fusarium proliferatum underlying different carbon sources.

Fusarium proliferatum is not only a dangerous plant pathogen but also produces various mycotoxins. The aim of this study was to investigate the influences of different carbon sources on fumonisins (FB1 and FB2) production and fungal virulence of F. proliferatum. The use of sucrose as the sole carbon source significantly increased the production of fumonisins in F. proliferatum in comparison to fructose. While F. proliferatum showed the stronger ability to infect banana fruit in the fructose group than the sucrose group, which was further investigated by scanning electron microscope (SEM) observations. Differentially expressed genes (DEGs) between the two groups were revealed by Illumina sequencing, including 1293 up-regulated and 907 down-regulated genes. Among them, many important genes were involved in carbohydrate metabolism, including starch and sucrose, and fructose and mannose metabolism. Additionally, the homologous FUM genes in association with fumonisins biosynthesis, including FUM1, FUM8 and TUM15, were up-regulated on sucrose compared with fructose. Furthermore, F. proliferatum cultured on fructose source compared with sucrose source showed higher virulence for colonizing green and mature bananas by up-regulating CWDE (cell wall-degrading enzyme)-related genes, which was further confirmed by qRT-PCR. Hence, the results obtained by RNA-Seq, HPLC-MS/MS and virulence experiments elucidated partially that the use of fructose as the sole carbon source can facilitate the fungal pathogenicity, but depress fumonisins production in F. proliferatum.

Complete genome sequencing of the luminescent bacterium, Vibrio qinghaiensis sp. Q67 using PacBio technology.

Vibrio qinghaiensis sp.-Q67 (Vqin-Q67) is a freshwater luminescent bacterium that continuously emits blue-green light (485 nm). The bacterium has been widely used for detecting toxic contaminants. Here, we report the complete genome sequence of Vqin-Q67, obtained using third-generation PacBio sequencing technology. Continuous long reads were attained from three PacBio sequencing runs and reads >500 bp with a quality value of >0.75 were merged together into a single dataset. This resultant highly-contiguous de novo assembly has no genome gaps, and comprises two chromosomes with substantial genetic information, including protein-coding genes, non-coding RNA, transposon and gene islands. Our dataset can be useful as a comparative genome for evolution and speciation studies, as well as for the analysis of protein-coding gene families, the pathogenicity of different Vibrio species in fish, the evolution of non-coding RNA and transposon, and the regulation of gene expression in relation to the bioluminescence of Vqin-Q67.

Rapid Assessment of the Toxicity of Fungal Compounds Using Luminescent Vibrio qinghaiensis sp. Q67.

Most tropical fruits after harvest are very perishable because of fungal infection. Since some pathogenic fungi can produce hazardous compounds such as mycotoxins, novel rapid and effective methods to assess those hazardous compounds are urgently needed. Herein we report that Vibrio qinghaiensis sp. Q67, a luminescent bacterium, can be used to rapidly assess the toxicities of mycotoxins and cultures from mycotoxin-producing pathogens. A good correlation (R² > 0.98) between concentrations of the mycotoxins (fumonisin B1, deoxynivalenol, zearalenone, ochratoxin A, patulin, and citrinin) and the luminous intensity of V. qinghaiensis sp. Q67 was obtained. Furthermore, significant correlations (R² > 0.96) between the amount of mycotoxin and the luminous intensity from the cultures of 10 major mycotoxin-producing pathogens were also observed. In addition, Fusarium proliferatum (half-maximal inhibitory concentration (IC50) = 17.49%) exhibited greater luminescence suppression than Fusarium semitectum (IC50 = 92.56%) or Fusarium oxysporum (IC50 = 28.61%), which was in agreement with the existing higher levels of fumonisin B1, fumonisin B2, and deoxynivalenol, which were measured by high-performance liquid chromatography-tandem mass spectrometry. These results suggest that V. qinghaiensis sp. Q67 is a promising alternative for the rapid evaluation of the toxicity of fungal mycotoxins.

Proteomics analysis of Fusarium proliferatum under various initial pH during fumonisin production.

Fusarium proliferatum as a fungal pathogen can produce fumonisin which causes a great threat to animal and human health. Proteomic approach was a useful tool for investigation into mycotoxin biosynthesis in fungal pathogens. In this study, we analyzed the fumonisin content and mycelium proteins of Fusarium proliferatum cultivated under the initial pH5 and 10. Fumonisin production after 10days was significantly induced in culture condition at pH10 than pH5. Ninety nine significantly differently accumulated protein spots under the two pH conditions were detected using two dimensional polyacrylamide gel electrophoresis and 89 of these proteins were successfully identified by MALDI-TOF/TOF and LC-ESI-MS/MS analysis. Among these 89 proteins, 45 were up-regulated at pH10 while 44 were up-accumulated at pH5. At pH10, these proteins were found to involve in the modification of fumonisin backbone including up-regulated polyketide synthase, cytochrome P450, S-adenosylmethionine synthase and O-methyltransferase, which might contribute to the induction of fumonisin production. At pH5, these up-regulated proteins such as l-amino-acid oxidase, isocitrate dehydrogenase and citrate lyase might inhibit the condensation of fumonisin backbone, resulting in reduced production of fumonisins. These results may help us to understand the molecular mechanism of the fumonisin synthesis in F. proliferatum. BIOLOGICAL SIGNIFICANCE: To extend our understanding of the mechanism of the fumonisin biosynthesis of F. proliferatum, we reported the fumonisin production in relation to the differential proteins of F. proliferatum mycelium under two pH culture conditions. Among these 89 identified spots, 45 were up-accumulated at pH10 while 44 were up-accumulated at pH5. Our results revealed that increased fumonisin production at pH10 might be related to the induction of fumonisin biosynthesis caused by up-regulation of polyketide synthase, cytochrome P450, S-adenosylmethionine synthase and O-methyltransferase. Meanwhile, the up-regulation of l-amino-acid oxidase, isocitrate dehydrogenase and citrate lyase at pH5 might be related to the inhibition of the condensation of fumonisin backbone, resulting in reduced production of fumonisin. These results may help us to understand better the molecular mechanism of the fumonisin synthesis in F. proliferatum and then broaden the current knowledge of the mechanism of the fumonisin biosynthesis.

Comparative Transcriptome Analysis of Penicillium citrinum Cultured with Different Carbon Sources Identifies Genes Involved in Citrinin Biosynthesis.

Citrinin is a toxic secondary metabolite of Penicillium citrinum and its contamination in many food items has been widely reported. However, research on the citrinin biosynthesis pathway and its regulation mechanism in P. citrinum is rarely reported. In this study, we investigated the effect of different carbon sources on citrinin production by P. citrinum and used transcriptome analysis to study the underlying molecular mechanism. Our results indicated that glucose, used as the sole carbon source, could significantly promote citrinin production by P. citrinum in Czapek's broth medium compared with sucrose. A total of 19,967 unigenes were annotated by BLAST in Nr, Nt, Swiss-Prot and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Transcriptome comparison between P. citrinum cultured with sucrose and glucose revealed 1085 differentially expressed unigenes. Among them, 610 were upregulated while 475 were downregulated under glucose as compared to sucrose. KEGG pathway and Gene ontology (GO) analysis indicated that many metabolic processes (e.g., carbohydrate, secondary metabolism, fatty acid and amino acid metabolism) were affected, and potentially interesting genes that encoded putative components of signal transduction, stress response and transcription factor were identified. These genes obviously had important impacts on their regulation in citrinin biosynthesis, which provides a better understanding of the molecular mechanism of citrinin biosynthesis by P. citrinum.

Novel synthesized 2, 4-DAPG analogues: antifungal activity, mechanism and toxicology.

2, 4-Diacetylphloroglucinol (2,4-DAPG), a natural phenolic compound, has been investigated in light of its biological activities against plant pathogens. To improve its potential application, fourteen 2,4-DAPG analogous were synthesized through the Friedel-Crafts reaction using acyl chlorides and phloroglucinol. Of the 2,4-DAPG derivatives, MP4 exhibited much higher antifungal activity against Penicillium digitatum and P. italicum, the major pathogenic fungi in citrus fruit, than 2, 4-DAPG in vitro, and significantly inhibited the development of decay in harvested mandarin (Citrus reticulata Blanco cv. Shatang.) fruit in vivo. It was found that MP4 resulted in the wrinkle of the hyphae in both fungi with serious folds and breakage. In addition, the expression of several cytochrome P450 (CYP) genes were also modified in both fungi by MP4, which might be associated with the disorder of cell membrane formation. Furthermore, the toxicology of MP4 by evaluating the cell proliferation effect on human normal lung epithelial (16HBE) and kidney 293 (HEK293) cells, was significantly lower than that of albesilate, a widely used fungicide in harvested citrus fruit. In summary, the synthesized MP4 has shown a great potential as a novel fungicide that might be useful for control of postharvest decay in citrus fruit.

Influence of Butylated Hydroxyanisole on the Growth, Hyphal Morphology, and the Biosynthesis of Fumonisins in Fusarium proliferatum.

Fusarium proliferatum as a common fungus pathogen in foods can produce toxic fumonisins, which can cause animal diseases and increase risks of human cancers. On contrary, butylated hydroxyanisole (BHA) as a synthetic antioxidant offers a clue for preventing growth of fungal species and inhibiting production of mycotoxins. Unfortunately, information of the inhibitory mechanism of BHA on Fusarium species is still limited. In this study, influence of BHA treatment on growth and inhibition of fumonisin production in relation to the expression of the fumonisin biosynthesis-related genes of the F. proliferatum ZYF was investigated, which revealed that BHA had a negative influence on growth and fumonisin production of F. proliferatum. To further elucidate the mechanism of BHA on the growth of F. proliferatum, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to examine the F. proliferatum hyphae. The BHA treatment induced the loss of cytoplasm and cellular constituents, as well as distortion of mycelia, but it did not directly degrade the fumonisin. Furthermore, the BHA treatment markedly inhibited the expressions of FUM1 (a polyketide synthase encoding gene) and FUM8 (an aminotransferase encoding gene) genes, which resulted in the depression of metabolic pathway of F. proliferatum. The transcriptional analyses of the FUM1 and FUM8 genes confirmed a correlation between the fumonisin production and its gene expression. This study provided some insights into mechanisms of production of fumonisin and feasible prevention to reduce fumonisin contamination in favor of human and animal health.

Inhibitory mechanism of butylated hydroxyanisole against infection of Fusarium proliferatum based on comparative proteomic analysis.

UNLABELLED: Fusarium proliferatum as a filamentous fungal pathogen can produce mycotoxins that can contaminate postharvest fruits and thus impact risks on human health. The extracellular proteomes of F. proliferatum grown in the absence and presence of butylated hydroxyanisole (BHA) were analyzed comparatively. A total of 66 significantly different expressed secreted proteins were identified by LC-ESI-MS/MS analysis. The BHA treatment suppressed the accumulation of some pathogenic factors such as aspartic protease, cell wall degradation enzymes, porin, superoxide dismutase and glyceraldehyde-3-phosphate dehydrogenase. On the contrary, the BHA treatment increased the abundances of some proteins, such as ATP binding cassette transporter substrate-binding protein and lipopolysaccharide-assembly lipoprotein, involved in the growth of F. proliferatum. These findings suggest that BHA treatment could influence the pathogenic ability of F. proliferatum via inhibiting the levels of virulence factors and cell wall degradation-associated enzymes. Moreover, the induction of the growth-related proteins after the BHA treatment suggests that the livelihood of F. proliferatum might depend on the cost of reduced pathogenic ability. This study has provided some evidence for understanding the complicated mechanisms of F. proliferatum infection in an effort to develop new targets for the control of this fungal pathogen. BIOLOGICAL SIGNIFICANCE: To better understand the inhibitory mechanism of F. proliferatum by butylated hydroxyanisole (BHA) treatment, a comprehensive proteomic analysis of the secreted proteins of F. proliferatum was firstly conducted. Among the 66 identified spots, 34 and 32 proteins were down- and up-accumulated significantly by BHA treatment, respectively. Many of the identified key protein species were involved in the pathogenic ability and the growth of F. proliferatum. This study is helpful for broadening our knowledge of the pathogenic mechanism of F. proliferatum.