Single-cell analysis is of significant importance in delineating the exact phylogeny of the subclonal population and in discovering subtle diversification. So far, studies of intratumor heterogeneity and clonal evolution in multiple myeloma (MM) were largely focused on the bulk tumor population level. We performed quantitative multigene fluorescence in situ hybridization (QM-FISH) in 129 longitudinal samples of 57 MM patients. All the patients had newly diagnosed and relapsed paired samples. An expanded cohort of 188 MM patients underwent conventional FISH (cFISH) to validate the cytogenetic evolution in bulk tumor level. Forty-three of 57 patients (75.4%) harbored 3 or 4 cytogenetic clones at diagnosis. We delineated the phylogeny of the subclonal tumor population and derived the evolutionary architecture in each patient. Patients with clonal stabilization had a significantly improved overall survival (OS) than those with other evolutionary patterns (median OS, 71.2 months vs 39.7 months vs 35.2 months vs 25.5 months, for stable, differential, branching, and linear patterns, respectively; P = .001). A high degree of consistency and complementarity across QM-FISH and cFISH was observed in the evaluation of cytogenetic evolution patterns in MM. Survival after relapse was greater influenced by the presence of high-risk aberrations at relapse (hazard ratio = 2.07) rather than present at diagnosis (hazard ratio = 1.55). This study shows that QM-FISH is a valuable tool to elucidate the clonal architecture at the single-cell level. Clonal evolution pattern is of prognostic significance, highlighting the need for repeated cytogenetic evaluation in relapsed MM.

BackgroundThis study investigated the protective effect of aplysin on the liver and its influence on inflammation and the gut microbiota in rats with ethanol-induced liver injury.MethodsMale Sprague-Dawley rats were randomly assigned to an alcohol-containing liquid diet, control liquid diet or treatment with aplysin for 8 weeks. Hepatic and intestinal histopathological analysis was performed, and cytokine levels and the intestinal mucosal barrier were assessed. Enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR) and 16S rDNA high-throughput sequencing were performed to provide an overview of the gut microbiota composition.ResultsChronic alcohol exposure caused liver damage in rats. Serum aspartate aminotransferase (AST), aminotransferase (ALT), alkaline phosphatase (ALP) and triglyceride (TG) activities in liver tissue were higher than in the control group. Alcohol administration elevated the levels of serum transforming growth factor-β (TGF-β) and tumor necrosis factor-α (TNF-α) and reduced interleukin-10 (IL-10) levels compared with those of control rats. In addition, the levels of plasma endotoxin, diamine oxidase (DAO), and fatty acid-binding protein 2 (FABP2) in the alcohol group were higher than in the control group. The results of ERIC-PCR indicated that aplysin treatment shifted the overall structure of the ethanol-disrupted gut microbiota toward that of the control group. One hundred twenty to 190 genera of bacteria were detected by high throughput sequencing. Alcohol-induced changes in the gut microbial composition were detected at the genus level. These alcohol-induced effects could be reversed with aplysin treatment.ConclusionsThese results suggest that aplysin exerts a protective effect on ethanol-induced hepatic injury in rats by normalizing fecal microbiota composition and repairing intestinal barrier function.

Lymphoplasmacytic lymphoma/Waldenström macroglobulinemia (LPL/WM) is a heterogeneous disease in which the role of immunoglobulin heavy chain genes (IGH) remains unknown. To determine the clinical relevance of the IGH repertoire in LPL/WM patients, we performed immunoglobulin gene rearrangement and complementarity determining region 3 (CDR3) analysis. The IGH variable gene repertoire was remarkably biased in LPL/WM. IGHV3-23, IGHV4-34, IGHV3-30, IGHV3-7, and IGHV3-74 accounted for half of the cohorts' repertoire. Most cases (97.1%) were found to carry mutated IGHV genes, based on a 98% IGHV germline homology cutoff. IGHV3-30 was associated with long heavy chain CDR3, indicating there was specific antigen selection in LPL/WM. Patients with IGHV3-7 were significantly more likely to harbor the 6q deletion (P<0.001) and an abnormal karyotype (P=0.004). The IGHV hypermutation rate in patients with the MYD88 L265P mutation was significantly higher than that of wild-type patients (P=0.050). IGHV3-23 and IGHV3-74 segments were more frequently detected in mutated MYD88 LPL/WM patients (P=0.050), while IGHV3-7 presented more frequently in MYD88 wild-type patients (P=0.042). Patients with IGHV4, especially IGHV4-34, had higher levels of lactate dehydrogenase, and IGHV4 was a predictive marker of shorter progression-free survival. These results showed for the first time that the IGHV repertoire has clinical relevance in LPL/WM.