BACKGROUND: Several studies have demonstrated the occurrence of secondary tumors as a rare but significant complication of chimeric antigen receptor T (CAR-T) cell therapy, underscoring the need for a detailed investigation. Given the limited variety of secondary tumor types reported to date, a comprehensive characterization of the various secondary tumors arising after CAR-T therapy is essential to understand the associated risks and to define the role of the immune microenvironment in malignant transformation. This study aims to characterize the immune microenvironment of a newly identified secondary tumor post-CAR-T therapy, to clarify its pathogenesis and potential therapeutic targets. METHODS: In this study, the bone marrow (BM) samples were collected by aspiration from the primary and secondary tumors before and after CD19 CAR-T treatment. The CD45 + BM cells were enriched with human CD45 microbeads. The CD45 + cells were then sent for 10× genomics single-cell RNA sequencing (scRNA-seq) to identify cell populations. The Cell Ranger pipeline and CellChat were used for detailed analysis. RESULTS: In this study, a rare type of secondary chronic myelomonocytic leukemia (CMML) were reported in a patient with diffuse large B-cell lymphoma (DLBCL) who had previously received CD19 CAR-T therapy. The scRNA-seq analysis revealed increased inflammatory cytokines, chemokines, and an immunosuppressive state of monocytes/macrophages, which may impair cytotoxic activity in both T and natural killer (NK) cells in secondary CMML before treatment. In contrast, their cytotoxicity was restored in secondary CMML after treatment. CONCLUSIONS This finding delineates a previously unrecognized type of secondary tumor, CMML, after CAR-T therapy and provide a framework for defining the immune microenvironment of secondary tumor occurrence after CAR-T therapy. In addition, the results provide a rationale for targeting macrophages to improve treatment strategies for CMML treatment.
Humans ; Lymphoma, Large B-Cell, Diffuse/therapy* ; Tumor Microenvironment/genetics* ; Antigens, CD19/metabolism* ; Leukemia, Myelomonocytic, Chronic/genetics* ; Immunotherapy, Adoptive/adverse effects* ; Male ; Single-Cell Analysis/methods* ; Female ; Sequence Analysis, RNA/methods* ; Receptors, Chimeric Antigen ; Middle Aged

ObjectiveTo investigate the differences in clinical features and mortality rate between native patients with chronic liver failure （CHF） and migrated patients with CHF after treatment with double plasma molecular adsorption system （DPMAS） in high-altitude areas. MethodsA total of 63 patients with CHF who received DPMAS treatment in the intensive care unit of General Hospital of Tibet Military Command from January 2016 to December 2021 were enrolled， and according to their history of residence in high-altitude areas， they were divided into native group with 29 patients and migrated group with 34 patients. The two groups were compared in terms of baseline data and clinical features before and after DPMAS treatment. The independent-samples t test was used for comparison of normally distributed continuous data between groups， and the paired t-test was used for comparison before and after treatment within each group； the Mann-Whitney U test was used for comparison of non-normally distributed continuous data between groups， and the Wilcoxon signed rank sum test was used for comparison before and after treatment within each group； the chi-square test was used for comparison of categorical data between groups. The Kaplan-Meier method was used to plot survival curves， and the Log-rank test was used for comparison of the risk of death. ResultsCompared with the native group， the migrated group had a significantly higher proportion of Chinese Han patients （χ²=41.729， P<0.001）， and compared with the migrated group， the native group had a significantly longer duration of the most recent continuous residence in high-altitude areas （Z=3.364， P<0.001）. Compared with the native group， the migrated group had significantly higher MELD score and incidence rates of hepatic encephalopathy， hepatorenal syndrome， and gastrointestinal bleeding （Z=2.318， χ²=6.903， 5.154， and 6.262， all P<0.05）. Both groups had significant changes in platelet count （PLT）， hemoglobin count （HGB）， alanine aminotransferase （ALT）， aspartate aminotransferase （AST）， albumin， total bilirubin （TBil）， direct bilirubin （DBil）， lactate dehydrogenase （LDH）， creatinine （Cr）， and international normalized ratio （INR） after DPMAS treatment （all P<0.05）. Before DPMAS treatment， compared with the native group， the migrated group had significantly higher levels of ALT， AST， TBil， DBil， LDH， Cr， BUN， and INR （all P<0.05） and a significantly lower level of HGB （P<0.05）； after DPMAS treatment， compared with the native group， the migrated group had significantly greater reductions in PLT and HGB （both P<0.05） and still significantly higher levels of ALT， AST， TBil， DBil， LDH， BUN， and INR （all P<0.05）. The 60-day mortality rate of patients after DPMAS treatment was 52.5% （95% confidence interval ［CI］： 41.7 — 63.8） in the native group and 81.3% （95%CI： 77.9 — 85.6） in the migrated group. Compared with the native group （hazard ratio ［HR］=0.47， 95%CI： 0.23 — 0.95）， the migrated group had a significant increase in the risk of death on day 60 （HR=2.14， 95%CI： 1.06 — 4.32， P=0.039）. ConclusionCompared with the native patients with CHF in high-altitude areas， migrated patients have a higher degree of liver impairment， a lower degree of improvement in liver function after DPMAS treatment， and a higher mortality rate. Clinical medical staff need to pay more attention to migrated patients with CHF， so as to improve their survival rates.

Purpose To analyze the precursor lesions,clinicopathological features and prognosis of mucinous carcinoma(MC).Methods A total of 303 MC cases diagnosed by surgi-cal specimens were included,including 193 pure mucinous car-cinomas(PMC)and 110 mixed mucinous carcinomas(MMC).PMC included 163 cases of type A(hypocellular type)and 30 cases of type B(hypercellular type).The histomorphological characteristics,surrounding breast tissue morphology,and im-mune markers were evaluated to analyze the clinicopathological features related to MC precursor lesions and prognosis.Results The median age at diagnosis of MC was 50 years,and the me-dian tumor size was 2.5 cm.Compared to PMC,MMC had more lymph node(LN)involvement,more grade Ⅲ-Ⅳ and Ki67＞20％tumors(P＜0.05).There were no significant differences in tumor size,LN involvement and clinical stage between type A and type B PMC.55 cases of MC(49 PMC and 6 MMC)with precursor lesions of mucocoele-like lesions(MLL)were all grade 1 to 2,most of them(54/55,98.2％)were T1-T2,and the proportions of MC component in all 6 MMCs were all ≥ 50％.There were 119 cases of MC whose precursor lesions were ductal carcinoma in situ(DCIS),of which 28.6％of cases(34/119)had LN involvement,and 16.8％(20/119)of cases showed high proliferative activity(Ki67＞20％).The precursor lesions of MC in 16 cases were solid papillary carcinoma(SPC)in situ,inclu-ding 7 cases of type BPMC and 9 cases of MMC.The median age of these patients was 67 years and no recurrence or metastasis was observed during follow-up.The disease-free survival(DFS)and overall survival(OS)of the 303 cases of MC was 93.5％and 98.6％,respectively.PMC had a better prognosis than MMC(DFS:95.2％vs.90.6％;OS:99.5％vs.97.2％),but there was no significant difference between type A and type B PMC.The prognosis of patients with LN involvement and clinical stage Ⅲ-Ⅳ was worse(P＜0.05).Conclusion MC of the breast is a kind of heterogeneous malignant tumors.Our findings support that MC with specific precursor lesions have different ev-olutionary pathways,that MC with precursor lesions of MLL and SPC have a good prognosis,and MC associated with high-grade DCIS are more aggressive.LN involvement,higher clinical grade,and younger age are associated with poor prognosis.Focu-sing on precursor lesions and high risk clinicopathological fea-tures can contribute to providing more effective treatments for these patients.

Purpose To analyze the precursor lesions,clinicopathological features and prognosis of mucinous carcinoma(MC).Methods A total of 303 MC cases diagnosed by surgi-cal specimens were included,including 193 pure mucinous car-cinomas(PMC)and 110 mixed mucinous carcinomas(MMC).PMC included 163 cases of type A(hypocellular type)and 30 cases of type B(hypercellular type).The histomorphological characteristics,surrounding breast tissue morphology,and im-mune markers were evaluated to analyze the clinicopathological features related to MC precursor lesions and prognosis.Results The median age at diagnosis of MC was 50 years,and the me-dian tumor size was 2.5 cm.Compared to PMC,MMC had more lymph node(LN)involvement,more grade Ⅲ-Ⅳ and Ki67＞20％tumors(P＜0.05).There were no significant differences in tumor size,LN involvement and clinical stage between type A and type B PMC.55 cases of MC(49 PMC and 6 MMC)with precursor lesions of mucocoele-like lesions(MLL)were all grade 1 to 2,most of them(54/55,98.2％)were T1-T2,and the proportions of MC component in all 6 MMCs were all ≥ 50％.There were 119 cases of MC whose precursor lesions were ductal carcinoma in situ(DCIS),of which 28.6％of cases(34/119)had LN involvement,and 16.8％(20/119)of cases showed high proliferative activity(Ki67＞20％).The precursor lesions of MC in 16 cases were solid papillary carcinoma(SPC)in situ,inclu-ding 7 cases of type BPMC and 9 cases of MMC.The median age of these patients was 67 years and no recurrence or metastasis was observed during follow-up.The disease-free survival(DFS)and overall survival(OS)of the 303 cases of MC was 93.5％and 98.6％,respectively.PMC had a better prognosis than MMC(DFS:95.2％vs.90.6％;OS:99.5％vs.97.2％),but there was no significant difference between type A and type B PMC.The prognosis of patients with LN involvement and clinical stage Ⅲ-Ⅳ was worse(P＜0.05).Conclusion MC of the breast is a kind of heterogeneous malignant tumors.Our findings support that MC with specific precursor lesions have different ev-olutionary pathways,that MC with precursor lesions of MLL and SPC have a good prognosis,and MC associated with high-grade DCIS are more aggressive.LN involvement,higher clinical grade,and younger age are associated with poor prognosis.Focu-sing on precursor lesions and high risk clinicopathological fea-tures can contribute to providing more effective treatments for these patients.

Malnutrition is highly prevalent in patients with inflammatory bowel disease, which can be life-threatening when refeeding syndrome occurs. This article reports a patient with Crohn′s disease admitted to the Second Affiliated Hospital of Zhejiang University School of Medicine who developed refeeding syndrome due to long-term malnutrition and complicating lymph node tuberculosis. After the discussion, diagnosis and treatment of multidisciplinary team including Departments of Gastroenterology, Nutrition, Colorectal Surgery and Oncology, and Pathology, the patient was improved significantly.

Malnutrition is highly prevalent in patients with inflammatory bowel disease, which can be life-threatening when refeeding syndrome occurs. This article reports a patient with Crohn′s disease admitted to the Second Affiliated Hospital of Zhejiang University School of Medicine who developed refeeding syndrome due to long-term malnutrition and complicating lymph node tuberculosis. After the discussion, diagnosis and treatment of multidisciplinary team including Departments of Gastroenterology, Nutrition, Colorectal Surgery and Oncology, and Pathology, the patient was improved significantly.

Objective:To identify important prognostic molecular markers of triple negative breast cancer (TNBC) using high throughput sequencing technology and to explore the correlation of spindle checkpoint protein BUB1B and clinicopathological features with patients′ prognosis.Methods:The clinicopathological data and prognostic information of TNBC diagnosed at the West China Hospital of Sichuan University from 2009 to 2017 were collected. Forty-seven fresh tumor samples and 139 formalin fixed paraffin-embedded samples were selected. The fresh tumor samples were subject to RNA sequencing (RNA-seq). The enrichment analysis and protein-protein interaction (PPI) analysis were performed after intersection of difference analysis between RNAseq and GEO (Gene Expression Omnibus) datasets GSE38959 and GSE65194. Kaplan-Meier plotter database was used to analyze the relationship between expression of BUB1B and prognosis. Immunohistochemical staining was used to verify its expression in TNBC and correlation with clinicopathological features and prognosis.Results:Using edgeR to perform differential expression analysis between 47 TNBC tumor tissues and 12 normal tissues, 1 559 up-regulated genes and 1 376 down-regulated genes were identified, while only 131 differentially expressed genes were overlapping with those in GSE38959 and GSE65194. Enrichment analysis was mainly enriched in cell cycle, JAK-STAT signaling pathway and p53 signaling pathway. The top 10 genes ranked by degree of association were TOP2A, BUB1B, MKI67, PLK1, RRM2, PCNA, KPNA2, SMC4, PBK and IGF1. Kaplan-Meier plotter database analysis showed that the expression of BUB1B was significantly correlated with the prognosis of TNBC [overall survival, hazard ratio (HR)=0.52, 95% CI (0.35-0.77), P=0.001; distant metastasis-free, HR=0.72, 95% CI (0.52-0.98), P=0.038]. The immunohistochemical analyses of 139 formalin fixed paraffin-embedded samples showed that the low expression of BUB1B was correlated with poor prognosis in TNBC [HR=0.41, 95% CI (0.18-0.95), P=0.024]. Conclusions:The low expression of BUB1B protein is associated with poor prognosis in TNBC patients, and the molecular mechanism related with prognosis and potential therapeutic targets need to be further studied.

Objective:To describe for the determination of contents of metabolites of benzene compounds in urine sample by high performance liquid chromatography.Methods:After acidification with hydrochloric acid, metabolites in urine were first extracted by acetonitrile and isopropanol (V∶V, 9∶1) with excessive sodium chloride, then gradient separated on a C18 column and then determined by DAD detector.Results:There were good linear relationship between peak areas and injection quality in range of 2.00-100 mg/L ( r>0.999). The detection limit and quantitative limit of this method were 4.15-70.7 μg/L and 13.8-235 μg/L respectively. The precision for the analysis of urine was1.78%-8.23% ( n =6). The average recovery of metabolites was 85.4%-105.5% at thee spiked levels in the range of 2.00-100 mg/L. Conclusion:The accuracy and reproducibility obtained make this method useful for the biological monitoring of occupational exposure to toluene, xylene, styrene and ethylbenzene.

Objective:To describe for the determination of contents of metabolites of benzene compounds in urine sample by high performance liquid chromatography.Methods:After acidification with hydrochloric acid, metabolites in urine were first extracted by acetonitrile and isopropanol (V∶V, 9∶1) with excessive sodium chloride, then gradient separated on a C18 column and then determined by DAD detector.Results:There were good linear relationship between peak areas and injection quality in range of 2.00-100 mg/L ( r>0.999). The detection limit and quantitative limit of this method were 4.15-70.7 μg/L and 13.8-235 μg/L respectively. The precision for the analysis of urine was1.78%-8.23% ( n =6). The average recovery of metabolites was 85.4%-105.5% at thee spiked levels in the range of 2.00-100 mg/L. Conclusion:The accuracy and reproducibility obtained make this method useful for the biological monitoring of occupational exposure to toluene, xylene, styrene and ethylbenzene.