RT-PCR demonstrates superior sensitivity and specificity in detecting the five neuroblastoma genes compared to the flow cytometry method for measurable residual disease.

Background: Exploring sensitive prognostic methods for patients with relapsed or refractory neuroblastoma (NB) is critical. The five NB genes (NB5) share a common trait: they are highly expressed in NB. Previous studies have identified their expression levels as markers for guiding micrometastasis. This study aimed to explore whether an improved NB5 detection method is superior to flow cytometry for predicting NB metastasis, measurable residual disease (MRD), and prognosis, and whether this result could serve as an independent factor to influence progression-free survival (PFS). Methods: We utilized reverse transcriptase polymerase chain reaction (RT-PCR) to assess the expression of NB5 (CHGA, DCX, DDC, PHOX2B, and TH) in bone marrow (BM), peripheral blood (PB), or cerebrospinal fluid (CSF) samples collected from 71 patients. The correlation between gene expression changes and clinical characteristics, as well as survival rates, based on 113 detections were analyzed. The NB5 detection results' sensitivity and specificity in all 71 patients collected from six research centers with a median follow-up of 14 months were assessed. Results: PB specimens showed 100% concordance with the BM specimens in terms of positive results. Furthermore, the BM specimens exhibited an additional 45.455% (5/11) positive results compared to the 34.091% (30/88) of PB specimens. The BM specimens were positive for NB5 assay, which was significantly higher than the positive results of flow cytometric MRD (15/88, 17.045%). NB5 was mainly expressed in newly diagnosed patients (P=0.043) and positive patients with flow cytometric MRD (P<0.001) or BM morphology (P<0.001). Positive rates of droplet digital PCR (ddPCR) were consistent with those of quantitative RT-PCR (qRT-PCR) in BM (13/18, 72.222%). However, in PB, the positive rate of ddPCR (2/5, 40.000%) was higher than that of qRT-PCR. A total of 38 specimens (BM, PB, CSF) were detected as positive under qRT-PCR. Among the positive results, the analysis revealed a significant difference between the CHGA and TH in pairwise comparisons (P=0.005). PFS analysis showed that among MRD-negative patients, the survival time of the NB5-positive group was significantly lower than that of NB5-negative group (27.408±10.791 vs. 35.961±3.084 months; P=0.034), and in the Cox regression model, risk stratification based on NB5 expression level was an independent prognostic factor for relapsed or refractory disease [95% confidence interval (CI):1.020 to 9.099, hazard ratio (HR) =3.046, P=0.046]. Combining the follow-up results, we found that the sensitivity and specificity of NB5 detection were both 100%. Conclusions: In our study, the improved NB5 detection method showed significantly higher sensitivity in assessing tumor relapse or residual disease compared to flow cytometric MRD. Moreover, it provided a more accurate assessment of treatment efficacy and prognosis. These findings support NB5 detection as an effective method for further stratification and monitoring of patients with relapsed or refractory NB.

Paspalines C-D and Paxillines B-D: New Indole Diterpenoids from Penicillium brefeldianum WZW-F-69.

Five new indole diterpenoids named paspaline C-D (1-2) and paxilline B-D (3-5), as well as eleven known analogues (6-16), were identified from fungus Penicillium brefeldianum strain WZW-F-69, which was isolated from an abalone aquaculture base in Fujian province, China. Their structures were elucidated mainly through 1D- and 2D-NMR spectra analysis and ECD comparison. Compound 1 has a 6/5/5/6/6/8 hexacyclic ring system bearing 2,2-dimethyl-1,3-dioxocane, which is rare in natural products. Compound 2 has an unusual open F-ring structure. The cytotoxic activities against 10 cancer cell lines and antimicrobial activities against model bacteria and fungi of all compounds were assayed. No compound showed antimicrobial activity, but at a concentration of 1 µM, compounds 1 and 6 exhibited the highest inhibition rates of 71.2% and 83.4% against JeKo-1 cells and U2OS cells, respectively.

Pentacyclic Cytochalasins and Their Derivatives from the Endophytic Fungus Phomopsis sp. xz-18.

Eight new cytochalasins 1-8 and ten known analogs 9-18 were isolated from the endophytic fungus Phomopsis sp. xz-18. The planar structures of the cytochalasins were determined by HR-ESI-MS and NMR analysis. Compounds 1, 2, 9 and 10 were 5/6/6/7/5-fused pentacyclic cytochalasins; compounds 3 and 4 had conjugated diene structures in the macrocycle; and compound 6 had a ß,γ-unsaturated ketone. The absolute configuration of 6 was confirmed for the first time by the octant rule. The acid-free purification process proved that the pentacyclic system was a natural biosynthetic product and not an acid-mediated intramolecular cyclized artifact. The new compounds did not exhibit activities against human cancer cell lines in cytotoxicity bioassays or antipathogenic fungal activity, but compounds 1, 3 and 4 showed moderate antibacterial activity in disk diffusion assays.