Evaluation of next-generation sequencing versus next-generation flow cytometry for minimal-residual-disease detection in Chinese patients with multiple myeloma.

PURPOSE: To evaluate the efficacy of next-generation sequencing (NGS) in minimal-residual-disease (MRD) monitoring in Chinese patients with multiple myeloma (MM). METHODS: This study analyzed 60 Chinese MM patients. During MRD monitoring in these patients' post-therapy, clonal immunoglobulin heavy chain (IGH) rearrangements were detected via NGS using LymphoTrack assays. MRD monitoring was performed using NGS or next-generation flow cytometry (NGF), and the results were compared. Additionally, the sensitivity and reproducibility of the NGS method were assessed. RESULTS: The MRD detection range of the NGS method was 10-6-10-1, which suggested good linearity, with a Pearson correlation coefficient of 0.985 and a limit of detection of 10-6. Intra- and inter-assay reproducibility analyses showed that NGS exhibited 100% reproducibility with low variability in clonal cells. At diagnosis, unique clones were found in 42 patients (70.0%) with clonal IGH rearrangements, which were used as clonality markers for MRD monitoring post-therapy. Comparison of NGS and NGF for MRD monitoring showed 79.1% concordance. No samples that tested MRD-positive via NGF were found negative via NGS, indicating the higher sensitivity of NGS. MRD could be detected using NGS in 6 of 7 samples before autologous hematopoietic stem-cell transplantation, and 5 of them tested negative post-transplantation. In contrast, the NGF method could detect MRD in only 1 sample pre-transplantation. CONCLUSION: Compared with NGF, NGS exhibits higher sensitivity and reproducibility in MRD detection and can be an effective strategy for MRD monitoring in Chinese MM patients.

The prognostic significance of circulating plasma cells in newly diagnosed multiple myeloma patients.

Objective: Multiple myeloma (MM) is a highly characteristic tumor that is influenced by numerous factors that determine its prognosis. Studies indicate that the presence of circulating plasma cells (cPCs) is a detrimental factor that significantly impacts the prognosis of patients with MM. Methods: This study retrospectively analyzed the prognostic value of cPCs quantified by 10-color flow cytometry in 145 newly diagnosed MM (NDMM) cases in the First Affiliated Hospital of Soochow University from November 2018 to February 2021. The study was approved by the Ethics Committee of the hospital (2021 No. 93). Results: Of the 145 patients, 99 (68.2%) were detected cPCs. Through receiver operating characteristics (ROC) analysis, an optimal threshold of 0.165% was identified as a predictor for overall survival (OS). The median progression-free survival (PFS) was 33 months in patients with cPCs ≥0.165%, whereas those with cPCs <0.165% had a PFS of <33 months (p=0.001). The median OS was not reached for two groups; the 3-year OS for patients with cPCs ≥0.165% was 71% compared with 87% for those with cPCs <0.165% (p=0.003). In transplant patients, cPCs ≥0.165% also predicted worse prognosis. Similarly, when considering cytogenetic risk factors in conjunction with cPC levels, comparable results were obtained. To evaluate whether the Revised International Staging System (R-ISS) groups could be further stratified based on different prognostic factors related to cPCs, our study revealed similar median PFS and OS rates in R-ISS II stage patients with cPCs ≥0.165% compared to those in the III stage (p=0.659 and 0.249, respectively). Conclusion: This study demonstrates that a high ratio of cPCs serves as a reliable indicator for predicting a poorer prognosis in MM cases. Furthermore, incorporating the R-ISS system and cytogenetic risk factors alongside the level of cPCs enhances the accuracy of prognostic predictions for patients with MM.

Autologous stem cell transplantation in multiple myeloma patients with renal impairment.

Renal impairment (RI) used to exclude multiple myeloma (MM) patients from autologous stem cell transplantation (ASCT) for safety concerns. Here, we retrospectively reviewed 34 consecutively transplanted patients with creatinine clearance < 60 ml/min at ASCT in recent 5 years at our institution. Busulfan/cyclophosphamide and high-dose melphalan were both employed as conditioning regimens. We found 62% grade 1-2 oral mucositis, 12% grade 3 oral mucositis, 48% grade 3 infection, 8% grade ≥ 4 infection, 50% grade 1 transient creatinine increase, 15% cardiac adverse events, and 12% engraftment syndrome. One case of secondary platelet graft failure and 1 case of transplantation-related mortality were observed. Interleukin-6 concentration was elevated among patients with increased body temperature and/or N-terminal pro-brain natriuretic peptide during engraftment, and close monitoring of these markers may help to predict susceptibility to cardiac events and engraftment syndrome. Adverse events occurred frequently, but the majority were manageable in this cohort. ASCT would further deepen the anti-myeloma efficacy and slightly ameliorated renal function. With a median follow-up of 26.2 months post transplantation (range: 1.6-74.8 months), the median progression-free survival (PFS) and overall survival (OS) post-transplantation of patients undergoing first-line transplantation were not reached; the median PFS post-transplantation of patients undergoing rescue transplantation was 19.2 months and the median OS was not reached.

The independent adverse prognostic significance of 1q21 gain/amplification in newly diagnosed multiple myeloma patients.

Objective: 1q21 gain/amplification (1q21+) is a common abnormal karyotype in multiple myeloma, and its proportion in Chinese patients is much higher. If 1q21+ is included as one of the poor prognostic factors, it will greatly increase the proportion of high-risk patients in newly diagnosed multiple myelome (NDMM) patients. Therefore, the poor prognostic significance of 1q21+ is still controversial. This study mainly analyzed the clinical characteristics, treatment response and prognostic significance of 1q21+ in NDMM patients. Methods: 248 NDMM patients admitted in The First Affiliated Hospital of Soochow University from September 01, 2018 to August 31, 2021 of a VRD registration study, were retrospectively analyzed. 135 cases (54.4%) had 1q21+ by CD38-sorted fluorescence in situ hybridization (FISH). The clinical characteristics, treatment response and prognosis of the general population and subgroups were analyzed, among which 153 patients were compared for the involved genes by CytoScan. Results: Compared with negative patients, 1q21+ patients were more likely to have anemia, hypoalbuminemia, renal insufficiency, high lactate dehydrogenase and high proportion of R-ISS-III stage. The patients with 1q21+ involving CKS1B detected by Cytoscan had a higher proportion of complex karyotypes and abnormal CNVs, and all at middle-risk or high-risk groups defined by Prognostic Index. Multivariate analysis showed that 1q21+ was an independent adverse prognostic factor (PFS HR=2.358, 95%CI 1.286-4.324, P=0.006; OS HR=2.598, 95%CI 1.050-6.425, P=0.039). 1q21+ subgroup had an inferior outcome (PFS P=0.0133, OS P=0.0293). Furthermore 1q21 amplification had a shorter PFS than 1q21 gain (24 months vs not reached, P=0.0403), but the OS difference was not clinically significant. The proportion of 1q had no effects on prognosis. In addition, 1q21+ in main clone rather than subclone was an adverse factor affecting the prognosis (PFS P=0.0172, OS P=0.1260). Autologous stem cell transplantation can effectively improve the survival of 1q21+ patients (P<0.05). Conclusion: Patients with 1q21+ have clinically significant end-stage organ damage and higher tumor burden, more likely to combine 13q14-, t(4;14), 1p32- and other cytogenetic abnormalities. 1q21+ is an independent high-risk cytogenetic factor for poor prognosis in NDMM patients, of which 4 or more copy numbers and main clone position significantly associated with prognosis results.

In Situ Bioorthogonal Conjugation of Delivered Bacteria with Gut Inhabitants for Enhancing Probiotics Colonization.

Clinical treatment efficacy of oral bacterial therapy has been largely limited by insufficient gut retention of probiotics. Here, we developed a bioorthogonal-mediated bacterial delivery strategy for enhancing probiotics colonization by modulating bacterial adhesion between probiotics and gut inhabitants. Metabolic amino acid engineering was applied to metabolically incorporate azido-decorated d-alanine into peptidoglycans of gut inhabitants, which could enable in situ bioorthogonal conjugation with dibenzocyclooctyne (DBCO)-modified probiotics. Both in vitro and in vivo studies demonstrated that the occurrence of the bioorthogonal reaction between azido- and DBCO-modified bacteria could result in obvious bacterial adhesion even in a complex physiological environment. DBCO-modified Clostridium butyricum (C. butyricum) also showed more efficient reservation in the gut and led to obvious disease relief in dextran sodium sulfate-induced colitis mice. This strategy highlights metabolically modified gut inhabitants as artificial reaction sites to bind with DBCO-decorated probiotics via bioorthogonal reactions, which shows great potential for enhancing bacterial colonization.

Soil Microbes Drive the Flourishing Growth of Plants From Leucocalocybe mongolica Fairy Ring.

Fairy ring is a natural phenomenon in which fungal fruiting bodies occur as a ring on a spot. This ring is produced due to spore ejection by Basidiomycetous fungi and forms a lush growing plant belt. However, the drivers for such formations and the potential plant growth-promoting rhizobacteria in fairy ring soils remain unknown. Fairy rings formed by Leucocalocybe mongolica were selected in this study. Soil characteristics and microbial (bacteria and fungi) community structures between beneath and outside the fairy rings were compared through high-throughput sequencing. Beneficial bacterial resources were excavated using dependent culturable methods. Soil electrical conductivity and available potassium were higher in the soil beneath the ring than outside it. These parameters were positively correlated with the dominant microbial community, but microbial diversity was lower. In the soil beneath the fairy ring, Bacteroidetes and Basidiomycota were more abundant, whereas Verrucomicrobia was less prevalent. Bacillus pumilus (strain BG-5) was isolated from the soil beneath the ring. Strain BG-5 can solubilize phosphorus and produce indole-3-acetic acid, NH4 +, and siderophores. Furthermore, strain BG-5 enhanced salt tolerance and promoted the growth of Arabidopsis thaliana, wheat (Triticum aestivum), and cotton (Gossypium hirsutum) seedlings. This study indicated the presence of abundant beneficial microbes driving the flourishing growth of plants in the fairy ring soil and provided bio-resources for agricultural growth-promoting agents.

[The Effects of Chidamide Combined with Anti-myeloma Drugs on the Proliferation and Apoptosis of Myeloma Cells].

OBJECTIVE: To investigate the effects of chidamide combined with anti-myeloma drugs on the proliferation and apoptosis of myeloma cells. METHODS: The proliferation inhibition of the cells was detected by CCK-8 method, and flow cytometry was used to detected the apoptosis of the cells. RESULTS: Chidamide could inhibit the proliferation of myeloma cells and promote the apoptosis of primary myeloma plasma cells in a time- and dose-dependent manner (P<0.05). In NCI-H929 cell line, chidamide combined with low-dose bortezomib and lenalidomide showed synergistic effect, while combined with dexamethasone and pomalidomide showed additive effect. In MM.1s cell line, chidamide combined with bortezomib, dexamethasone, lenalidomide and pomalidomide all showed synergistic effects. CONCLUSION: Chidamide inhibits proliferation of myeloma cells in a time- and dose-dependent manner and promotes apoptosis of primary myeloma plasma cells. Furthermore, it can enhance the inhibitory effect of anti-myeloma drugs.

Sequential CD19 and BCMA-specific CAR T-cell treatment elicits sustained remission of relapsed and/or refractory myeloma.

The low rate of durable response against relapsed and/or refractory multiple myeloma (RRMM) in recent studies indicates that chimeric antigen receptor T-cell (CART) treatment is yet to be optimized. This study aims to investigate the safety and efficacy of sequential infusion of CD19-CART and B-cell maturation antigen (BCMA)-CARTs for RRMM with a similar 3 + 3 dose escalation combined with a toxicity sentinel design. We enrolled 10 patients, among whom 7 received autologous infusion and 3 received allogeneic infusion. The median follow-up time was 20 months. The most common grade 3/4 treatment-emergent toxicities were hematological toxicities. Cytokine-release syndrome (CRS) adverse reactions were grade 1/2 in 9 out of 10 subjects. No dose-limited toxicity (DLT) was observed for BCMA-CAR-positive T cells ≤5 × 107 /kg), while two patients with dose-levels of 5-6.5 × 107 /kg experienced DLTs. The overall response rate was 90% (five partial responses and four stringent complete responses). Three out of four patients with stringent complete responses to autologous CART had progression-free survival for over 2 years. The three patients with allogeneic CART experienced disease progression within 2 months. These results evidence the sequential infusion's preliminarily tolerability and efficacy in RRMM, and present a simple and safe design applicable for the establishment of multiple CART therapy.

[Determination of sulfonamides, quinolones, benzimidazoles and the metabolites of benzimidazoles in chicken livers by dispersive solid-phase extraction and ultra performance liquid chromatography-tandem mass spectrometry].

A method for the determination of 12 sulfonamides, 19 quinolones and 8 benzimidazoles and the metabolites of benzimidazoles in chicken livers by quick, easy, cheap, effective, rugged and safe (QuEChERS) extraction and ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) has been developed. The samples were extracted with 1% acetic acid-acetonitrile solution, cleaned up with amine (NH2) sorbent and defatted with n-hexane. The identification and quantification were achieved by using electrospray ionization in positive ion mode (ESI+) with multiple reaction monitoring (MRM). The matrix-matched internal standard calibration curves were used for quantitative determination. The linear range was from 5 to 100 microg/kg. The average recoveries and relative standard deviations were 72% - 121% and 1.5% -23.4% respectively in the spiked range of 10 -50 microg/kg. The limits of detection were 5 microg/kg and the limits of quantification were 10 microg/kg for the 39 drugs. The method is simple, rapid, sensitive and accurate. It is suitable for the quantitative determination and confirmation of 12 sulfonamides, 19 quinolones, 8 benzimidazoles and the metabolites of benzimidazoles.

[Rapid determination of patulin in apple juice by tert-butyldimethylsilyl derivatization-gas chromatography-mass spectrometry].

A gas chromatography-mass spectrometry (GC-MS) method for the quantitative determination and confirmation of patulin in apple juice by tert-butyldimethylsilyl (TBDMS) derivatization was established. The sample was extracted with ethyl acetate-hexane and an aliquot of the supernatant was cleaned up using mixed-mode solid phase extraction (SPE) cartridge containing C18 and graphitized carbon black (GCB), evaporated to dryness under nitrogen gas and the residue was converted to tert-butyldimethylsilyl derivative which was determined with GC-MS in selected ion monitoring (SIM) mode and external standard method was used for quantitative determination. The linear range was from 0.01 to 1 mg/L. The average recoveries were 88% - 98% and relative standard deviations were 5.3% - 13.6% in the spiked range of 2 - 50 microg/kg. The limit of detection was 0.5 microg/kg and the limit of quantification and confirmation was 2 microg/kg. The method is rapid, highly sensitive, accurate, specific, rugged and suitable for the quantitative determination and confirmation of patulin in apple juice.

[Determination and confirmation of cyanuric acid in infant formula using mixed-mode solid-phase extraction cartridge clean up-gas chromatography-mass spectrometry].

A method is presented for the quantitative determination and confirmation of cyanuric acid in infant formula by mixed-mode solid-phase extraction cartridge clean up-gas chromatography-mass spectrometry (GC-MS). Cyanuric acid was extracted from infant formula with an acetic acid solution at 84 degrees C. An aliquot of the supernatant was cleaned up using mixed-mode solid-phase extraction cartridge containing C18 and graphitized carbon black (GCB), and evaporated to dryness under nitrogen. The residues were converted to trimethylsilyl derivatives, then determined by GC-MS in selected ion monitoring (SIM) mode. The linear range was from 0.01 -2 mg/L. The recoveries were 80% - 103% and the relative standard deviations were 7.7% - 14.5% in the spiked range of 0.25 -2.5 mg/kg. The limit of detection was 0.10 mg/kg and the limit of quantification was 0.25 mg/kg. The method is rapid, sensitive, accurate, specific, and rugged. It is suitable for the quantitative determination and confirmation of cyanuric acid in infant formula.

[Determination of melamine and cyanuric acid in milk by gas chromatography-mass spectrometry].

A gas chromatography-mass spectrometry method was developed for the quantitative determination and confirmation of melamine and cyanuric acid in milk. The milk sample was extracted with diethylamine-acetonitrile-water solution. The extract was evaporated to dryness and derivatized with N, O-bis (trimethylsilyl) trifluoroacetamide (BSTFA) and chlorotrimethylsilane (TMCS), then analyzed using gas chromatography-mass spectrometry (GC-MS) in selected ion monitoring (SIM) mode. The external standards were used for the quantitative determination. The linear range was from 0.025 to 2 mg/kg. The average recoveries were 84%-87% for melamine and 75%-102% for cyanuric acid, and the relative standard deviations were 5.7%-11.7% for melamine and 4.9%-7.8% for cyanuric acid in the spiked levels at 0.5, 1.0 and 2.5 mg/kg. The limits of detection of melamine and cyanuric acid were 0.05 mg/kg and 0.10 mg/kg, respectively. The method is suitable for the quantitative determination and confirmation of melainine and cyanuric acid residues in milk.