6557 Background: Chimerism (CHIM) analysis assesses the ratio of donor to recipient hematopoietic cells, confirming engraftment and detecting impending graft failure or relapse, in allogeneic hematopoietic stem cell transplantation (aHSCT). However, the conventional short tandem repeat (STR) analysis, has limited sensitivity (1–5%), often missing low-level CHIM. In this retrospective study, Multiple Single Nucleotide Polymorphism sequencing (MSNPseq), an NGS-based assay targeting DNA fragments each harboring 4–8 SNPs, demonstrated superior sensitivity and specificity compared with STR analysis, enabling reclassification of STR-negative cases and improved prognostic stratification. Methods: MSNPseq, based on Ion AmpliSeq technology, amplifies 100 fragments for sequencing on the Ion Torrent PGM platform, achieving >2000× depth for 96.8% of loci. Its sensitivity and accuracy were validated in simulated chimeric and post-transplant samples against STR analysis. Calibration models used pre-transplant donor/recipient data to generate tailored correction coefficients. CHIM was assessed via MSNPseq at three 30-day time points (TP1, 2, 3) post-transplant, in 741 aHSCT patients (AML 49.9%, ALL 27.4%, MDS 13.5%, lymphoma 4.7%, others 4.5%). PFS and OS were stratified by CHIM status and its dynamics to evaluate transplant outcomes, prognostic significance, and late relapse. Results: CHIM sensitivity was assessed using BCR-ABL1–positive standards. MSNPseq achieved a 0.01% detection limit, outperforming STR (1%) with strong correlation (R² = 0.9953). CHIM status was classified by donor fraction as complete (cCHIM, 100%), microchimerism (mCHIM, 99% to <100%), subthreshold chimerism (sCHIM, 95% to <100%; below STR sensitivity), and incomplete chimerism (iCHIM, ≤95%) across three time points. While iCHIM was consistently associated with inferior PFS and OS, sCHIM and mCHIM also showed worse outcomes compared with cCHIM, defining distinct high-risk subsets. CHIM dynamics revealed decreasing sCHIM/mCHIM and increasing cCHIM/iCHIM over time. Notably, a sustained CHIM decrease >0.5% over two consecutive time points was associated with significantly poorer OS and PFS. Interestingly, sCHIM and mCHIM status, as well as a sustained CHIM decrease >0.5% over two consecutive time points, were also associated with a high risk of acute graft-versus-host disease (aGvHD). Overall, MSNPseq enables more precise CHIM stratification than STR, improving post-transplant risk assessment. Conclusions: MSNPseq's superior sensitivity enables more precise chimerism classification and detection of clinically relevant sCHIM and mCHIM subsets that STR often misses. These refined CHIM categories, along with sustained CHIM decline, correlate with poorer survival and higher aGvHD risk, supporting MSNPseq as a superior tool for post-aHSCT risk stratification and therapeutic guidance.

Abstract Early complications following allogeneic hematopoietic stem cell transplantation (allo-HSCT), including graft-versus-host disease (GVHD) and viral reactivation, remain major causes of post-transplant morbidity, but whether immune perturbations precede these events remains unclear. We performed longitudinal TCRβ repertoire profiling in 108 allo-HSCT recipients and their corresponding donors at baseline and three early post-transplant time points to characterize immune reconstitution dynamics. Reduced baseline TCR diversity was most strongly associated with subsequent Epstein–Barr virus (EBV) reactivation, whereas cytomegalovirus (CMV) reactivation was more closely linked to post-transplant repertoire remodeling characterized by clonal expansion and reduced donor–recipient repertoire similarity. Sequence-based predictive modeling demonstrated meaningful discrimination, with fusion models achieving area under the curve (AUC) values of 0.745 for CMV, 0.819 for EBV, and 0.834 for GVHD. Temporal analyses further revealed complication-specific predictive windows. These findings indicate that major post-transplant complications are preceded by detectable immune perturbations and support the potential utility of TCR repertoire monitoring for early risk stratification after transplantation.

Prenatal alcohol exposure is a leading cause of developmental abnormalities and neurobehavioral deficits, collectively known as fetal alcohol spectrum disorder (FASD). The underlying molecular mechanisms, however, are not fully elucidated, hindering the development of effective therapeutic strategies. Ferroptosis, an iron-dependent form of regulated cell death driven by lipid peroxidation, has emerged as a key pathological process in various diseases. Gastrodin (GAS), the primary bioactive component of Gastrodia elata, has demonstrated potent antioxidant and neuroprotective properties. This study aimed to investigate the protective effects of GAS against alcohol-induced developmental and neurotoxic damage and to elucidate the underlying molecular mechanisms. Using a zebrafish larval model, we found that exposure to 200 mM alcohol from 2 to 24 hours post-fertilization (hpf) induced significant developmental toxicity, including a decreased hatching rate, body length and eye diameter, and increased morphological malformations in larvae. Alcohol-exposed larvae also exhibited severe neurobehavioral deficits, characterized by a reduction in movement distance and average velocity in dark conditions. Mechanistically, alcohol exposure triggered ferroptosis, evidenced by an increase in intracellular Fe2+, malondialdehyde (MDA), and reactive oxygen species (ROS) levels, alongside a decrease in the levels of glutathione (GSH) and reduced glutathione peroxidase 4 (GPX4) and the nuclear factor erythroid 2-related factor 2 (Nrf2) activities. Co-treatment with GAS (200 mg/L) significantly ameliorated these alcohol-induced developmental and neurobehavioral defects. GAS administration effectively suppressed the hallmarks of ferroptosis by restoring the ROS level and altering the expression of genes related to oxidative stress. In addition, GAS suppressed alcohol-induced cell apoptosis, downregulated caspase3b, bax, caspase8, and upregulated bcl2 in mRNA levels. Molecular analysis revealed that GAS exerts its anti-ferroptotic effect by activating Nrf2/GPX4 signaling pathway, which was suppressed by alcohol. Our findings indicate that ferroptosis plays a key role in alcohol-induced developmental neurotoxicity, and GAS provides protection by activating the Nrf2/GPX4 axis. This suggests that GAS could be a potential therapeutic option for reducing the negative effects of prenatal alcohol exposure.

Introduction Chimerism (CHIM) analysis following allogeneic hematopoietic stem cell transplantation (aHSCT) is critical for assessing engraftment and predicting relapse risk in hematological malignancies. The current gold standard, short tandem repeat (STR) analysis, has limited sensitivity, with a detection limit of 1–5%, rendering low-level CHIM undetectable and potentially overlooking prognostic indicators. To address this limitation, we developed a novel next-generation sequencing (NGS)-based method, termed Multiple Single Nucleotide Polymorphism sequencing (MSNPseq). MSNPseq targets 100 DNA fragments (~200 bp each), each containing 4–8 single nucleotide polymorphism (SNP) loci. Our results demonstrate that MSNPseq offers superior sensitivity and specificity compared to STR, enabling the reclassification of patients with undetectable CHIM by STR and refining their CHIM status for improved prognostic accuracy. Methods MSNPseq is based on Ion AmpliSeq technology to amplify 100 fragments for sequencing on the Ion Torrent PGM platform, achieving a sequencing depth >2000× for 96.8% of loci. The sensitivity and accuracy of MSNPseq were validated using simulated chimeric samples and post-transplant clinical samples, benchmarked against STR analysis. Calibration models were developed using pre-transplant sequencing data from donors and recipients to derive correction coefficients tailored for clinical applications. CHIM was evaluated using MSNPseq in 741 patients receiving aHSCT, comprising AML (49.9%), ALL (27.4%), MDS (13.5%), lymphoma (4.7%), and other diagnoses (4.5%). Assessments were conducted at three time points (TPs), spaced 30 days apart, following aHSCT, with ethical approval from participating hospitals and informed consent from all patients. Progression-free survival (PFS) and overall survival (OS) were analyzed across patient groups based on CHIM status to evaluate transplant outcomes, predict prognosis, and detect relapse at later stages. Results The sensitivity of CHIM detection by MSNPseq and STR analysis was assessed using simulated standards containing BCR-ABL1-positive cells at varying concentrations. MSNPseq achieved a detection limit of 0.01%, compared to 1% for STR, with high correlation between methods (Pearson R² = 0.9953). In a cohort of 741 patients post-aHSCT, MSNPseq detected complete donor chimerism (cCHIM, 100% donor) in 201, 238, and 239 patients at TP 1, 2, and 3 (30-day intervals), respectively. Subthreshold donor chimerism (sCHIM, 95% to <100% donor), a range where STR may lack sufficient detection accuracy, was observed in 339, 301, and 288 patients, microchimerism (mCHIM, 99% to <100% donor) in 200, 191, and 180 patients, and incomplete donor chimerism (iCHIM, ≤95% donor) in 17, 18, and 30 patients at TP 1, 2, and 3, respectively. Notably, sCHIM and mCHIM prevalence decreased over time, while cCHIM (potential responders) and iCHIM (potential relapses) increased, reflecting dynamic CHIM shifts in the first three months post-aHSCT. High-sensitivity CHIM monitoring thus holds significant clinical potential. To evaluate its prognostic value, MSNPseq-defined CHIM status was correlated with aHSCT outcomes. Patients with iCHIM exhibited significantly worse PFS and OS compared to those with cCHIM or sCHIM at all TPs. OS was also significantly worse in the sCHIM group versus the cCHIM group at TP 2 (p = 0.001) and TP 3 (p = 0.013), with a trend at TP 1 (p = 0.06). Similarly, patients with mCHIM showed significantly worse OS than those with cCHIM at TP 2 (p < 0.001), with a trend at TP 3 (p = 0.11). Interestingly, among patients with sCHIM, those with mCHIM exhibited distinct prognostic differences compared to other patients, underscoring the need for further classification within this group. These findings highlight the critical importance of MSNPseq, as sCHIM and mCHIM are frequently misclassified as cCHIM by STR, potentially impacting risk stratification and therapeutic outcomes. Conclusions MSNPseq's superior sensitivity over STR enables precise chimerism stratification, revealing prognostic differences in sCHIM and mCHIM that are often missed by traditional methods. This enhanced detection capability can significantly improve risk assessment and guide therapeutic decisions post-aHSCT, potentially improving patient outcomes.

OBJECTIVE:To compare the efficacy and clinical value of high-throughput sequencing (HTS) and Sanger sequencing in detecting ABL kinase domain mutations in patients with chronic myeloid leukemia (CML).METHODS:A total of 198 samples of 147 CML patients from July 2017 to March 2021 in Henan Cancer Hospital were collected and underwent high-throughput sequencing and Sanger sequencing to detect the mutations in ABL kinase domain, and the relevant clinical data were collected for comparative analysis.RESULTS:The proportion of total mutations and ≥2 mutations detected by high-throughput sequencing were significantly higher than those detected by Sanger sequencing (P =0.01; P =0.046). ≥2 mutations were detected in 22 cases, of which 5 cases (22.7%) had compound mutations. High-throughput sequencing can detect low level mutations that cannot be detected by Sanger sequencing. In 198 samples, 25 (12.6%) were low level mutations, 33 (16.7%) were high level mutations and 10 (5.1%) were mixed high and low level mutations. In the analysis of related clinical factors, the total mutation rate and the low level mutation rate in the optimal period, failure period and warning period were gradually increased (total mutation rate, P =0.016; low level mutation rate, P =0.005). The mutation rate of the samples with additional chromosomal abnormalities was also significantly increased (P =0.009). The mutation rate of patients who received first- and second-line treatment was significantly lower than that of patients who received third- or higher-line treatment (P =0.006). Analysis based on variant allele frequency (VAF) of the mutation site was helpful to visually evaluate the clonal evolution status of TKI-resistance CML cells.CONCLUSION:High-throughput sequencing is more sensitive and accurate than Sanger sequencing in mutation detection, which is helpful to accurately and visually evaluate TKI treatment response and optimize treatment strategy for CML.

目的 探讨慢性粒细胞白血病(CML)伴发非ABL1激酶区(KD)突变与小分子酪氨酸激酶抑制剂(TKI)疗效及疾病进展的关系.方法 采用二代测序技术(NGS)检测2017年3月至2021年4月于河南省肿瘤医院血液科就诊的120例CML患者108种血液肿瘤相关基因,分析非ABL1 KD突变谱及其与患者临床资料和ABL1 KD突变的关系.结果 58例(48％)CML患者检出非ABL1KD突变,其中附加染色体异常患者的突变比例(63％)显著高于无附加染色体异常的患者(34％),差异有统计学意义(P=0.003).不同分期CML患者突变比例分析中,急变期(73％)突变比例高于加速期(67％)和慢性期(42％)(P=0.031);WT1突变和转录调节类相关突变在AP期CML患者的比例(WT1+:17％,P=0.027;转录调节类突变:42％,P=0.001)均高于CP期;患者TKI治疗反应分析中,最佳反应组突变比例(30％)低于警告组(52％)和耐药组(61％)(P=0.018);RUNX1突变在TKI治疗失败组(16％)的比例高于警告组(2％)(P=0.024);非ABL1 KD突变的CML患者出现ABL1 KD突变的比例(60％)高于无伴发非ABL1 KD突变患者(36％)(P=0.022);差异有统计学意义.结论 非ABL1 KD突变与TKI疗效及疾病进展密切相关,WT1和RUNX1突变可作为CML精准诊断的分子靶点.

Objective:To examine the clinical characteristics and anemia-related factors in patients with newly diagnosed multiple myeloma (NDMM), as well as the effect and mechanism of erythroblastic islands (EBIs) and EBI macrophages in NDMM patients with anemia.Methods:We collected and analyzed clinical data to find anemia-related factors. Using flow cytometry, the numbers and ratios of erythroblasts and EBI macrophages were determined. RNA sequencing (RNA-seq) was used to determine the differences of EBI macrophages in NDMM patients with or without anemia.Results:Based on the clinical characteristics of NDMM patients with anemia, MCV, abnormal levels of albumin, osteolytic lesions, and Durie-Salmon (DS) stage are risk factors for anemia. Patients with anemia have fewer erythroblasts, erythroblastic islands (EBIs), and EBI macrophages in their bone marrow than patients without anemia. RNA-seq analysis of EBI macrophages from the bone marrow of patients with and without anemia revealed that macrophages from patients with anemia are impaired and tend to promote the production of interleukin-6, which has been demonstrated to be an essential survival factor of myeloma cells and protects them from apoptosis.Conclusion:In NDMM patients with anemia, EBI macrophages are impaired, which causes anemia in those patients. Our finding highlights the significance of EBI macrophages in anemia in NDMM patients and provides a new strategy for recovery from anemia in these patients.

Copy number aberrations (CNA) are the core determinants for diagnosis, risk stratification and prognosis in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). In this study, a shallow whole-genome sequencing-based assay, LeukoPrint, was utilized to depict genomic CNA profiles from the bone marrow of 137 newly diagnosed AML/MDS patients. It demonstrated 98.1% concordance of CNA profiles with cytogenetics and/or fluorescence in situ hybridization (FISH). It is advantageous in detecting CNAs of short segments (1 Mb) and from samples with low leukemic cell content, more accurate for describing complex karyotypes and less confounded by subjective bias. LeukoPrint improved the overall diagnostic yield by redefining the risk categories for 16 patients by presenting new information. In summary, LeukoPrint provided an automated, convenient, and cost-effective approach to describe genomic CNA profiles. It brought greater diagnostic yield and risk stratification information by incorporating into the routine cytogenetics based on the CNA-related criteria of standard ELN/IPSS-R guidelines.

AbstractObjective: To investigate the sample selection, result correction and clinical application value of multi nucleotide polymorphism chimerism detection method based on Next-generation sequencing.METHODS:The chimerism samples from November 2018 to June 2020 were collected, and Pearson correlation coefficient (r) was used to analyze the consistency of bone marrow and peripheral blood results detected by MNPseq; according to the different information integrity before transplantation, the calibration model was constructed to analyze the correction value of the micro chimerism results in each model; the clinical results were retrospectively analyzed to verify the reliability and practicability of chimerism results correction and the clinical value of MNPseq method.RESULTS:The results of bone marrow and peripheral blood chimerism detected by MNPseq method were consistent with each other and showed significant correlation (r=0.985, P<0.01). The three groups of calibration models were constructed according to different pre-transplant information. For the no donor and pre-transplant patients information group, the correction value was 1%; while for the group with pre-transplant patients and without donor information, 0.61% of the chimerism rate and 13 heterotopic points were used as the correction value; 0.26% of the chimerism rate and 21.57% of the heterotopic points were used as the correction value for the group with pre-transplantation patients and donor information. After correction, the number of the patients with incomplete chimerism decreased from 276 (74.19%) to 141 (37.91%) (P<0.01). Among 18 (18/141, 12.77%) patients with incomplete chimerism, the results of MNPseq in the patients were 25-39 days earlier than those in STR and flow MRD, and the result showed statistical significance.CONCLUSION:MNPseq method can be used to monitor chimerism with peripheral blood instead of bone marrow samples, and the results can be corrected to detect the changes of graft status in vivo in a more timely manner.

e19023 Background: Genomic alternations especially copy number aberrations (CNAs) are particularly imperative for diagnostic classification and risk stratification in patients with acute myeloid leukemia (AML), myelodysplastic syndromes (MDS) and multiple myeloma (MM). With the technological advances of next generation sequencing (NGS), the current CCA test and the molecular tests are likely to be challenged by whole-genome sequencing (WGS), which is a more unbiased method to detect all types of genomic aberrations. Methods: 178 patients who met the clinical manifestations of blood malignancies were included in the study, including 89 patients with AML, 48 with MDS and 41 with MM. Bone marrow was collected and mononuclear cells were separated to harvest genomic DNA which was sequenced without capture at approximately 1x coverage depth (shallow whole-genome sequencing, sWGS). Genome-wide CNAs greater than 5 Mbp was analyzed for each sample. European Leukemia Net (ELN), Revised International Prognostic Scoring System (IPSS-R) or Revised International Staging System (R-ISS)-defining CNAs were selected and used to assign patients to a genetic risk group through the same classification systems that are used for CCA. Results: In this study, CNA events were identified in 109 (73.6%) patients via sWGS. By calculating frequencies of recurrent gains and losses at chromosome arm level, some recurrent events were detected like gains of chr 8 (observed in 12 AML and 9 MDS patients), chr 1 (observed in 18 MM patients), losses of chr 7 (observed in 9 AML and 3 MDS patients) and chr 13 (observed in 16 MM patients), which were consistent with previously reported cytogenetic events. Besides, amplifications of odd-numbered chromosomes were detected in 15(36.6%) of patients with multiple myeloma, including chromosomes 3, 5, 7, 9, 11, 15, 19 and/or 21. And the trisomy of chr 5, 7 or 9 are mainly observed with a frequency of 66.7%. Among 145 patients with CCA results, a high concordance of 97.7% (98.5%, 97.6% and 97.1% in AML, MDS and MM respectively) was achieved in CNA profiles at individual patient level with cytogenetics and/or FISH. Noteworthily, CNA events were detected in 20 of 28 (71.4%) MM patients with negative karyotyping results. Moreover, sWGS improved the overall diagnostic yield by reassign 18 patients (9 with AML and 9 with MDS) to risk categories with poorer prognosis. Nearly identical CNA profiles from the paired plasma and bone marrow suggest that peripheral blood may serve as a substitute to spare bone marrow aspiration. Conclusions: In summary, a streamlined sWGS provides an accurate, convenient and cost-effective approach to describe genomic profiles of CNAs in patients with AML, MDS and MM. Clinical trial information: ChiCTR2100052276.

Genomic alternations especially copy number aberrations (CNAs) are particularly imperative for diagnostic classification and risk stratification in patients with acute myeloid leukemia (AML) or myelodysplastic syndromes (MDS). Although conventional cytogenetic analysis (CCA) has been mandatory in clinical routine to detect recurrent genomic abnormalities in AML/MDS, it presents a few obvious shortcomings, such as time-consuming in vitro cell-culturing procedure, low resolution (no smaller than 10 Mbp), and subjective bias introduced from the visual interpretation by cytogeneticists. With the technological advances of next generation sequencing (NGS), the current CCA test and the molecular tests are likely to be challenged by whole-genome sequencing (WGS), which is a more unbiased method to detect all types of genomic aberrations not only those well-characterized clinically actionable mutations but also cryptic alternations yet to be defined and annotated. In this study a shallow whole-genome sequencing (sWGS) based assay, LeukoPrint, was utilized to depict genomic CNA profiles from the bone marrow cells of 107 newly diagnosed patients with AML (n=63, 58.9%) or MDS (n=44, 41.1%). Bone marrow was collected and mononuclear cells were separated to harvest genomic DNA which was sequenced without capture at approximately 1x coverage depth. Genome-wide CNAs was analyzed for each sample using an automated pipeline. European Leukemia Net (ELN) or Revised International Prognostic Scoring System (IPSS-R)-defining CNAs were selected and used to assign patients to a genetic risk group through the same classification systems that are used for CCA. It demonstrated 98.1% concordance of CNA profiles at individual patient level with cytogenetics and/or FISH. It is advantageous in detecting CNAs of short segments (1 Mb) and from samples with low leukemic cell content, more accurate for describing complex karyotypes and less confounded by subjective bias. LeukoPrint improved the overall diagnostic yield by redefining the risk categories for 16 patients (15%) by presenting new information. Nearly identical CNA profiles from the paired plasma and bone marrow suggest that peripheral blood may serve as a substitute to spare bone marrow aspiration. In summary, LeukoPrint provided an automated, convenient and cost-effective approach to describe genomic CNA profiles in AML/MDS. It brought greater diagnostic yield and risk stratification information by incorporating into the routine cytogenetics based on the standard ELN/IPSS-R guidelines. Citation Format: Xiaodong Lyu, Tao Li, Dandan Zhu, Mengna Zhang, Yuexin Cheng, Yan Chen, Zhenling Li, Shiyong Li, Wei Wu, Shuaipeng Geng, Jingshuai Li, Xiangxiang He, Yangwei Li, Yinyin Chang, Zunmin Zhu, Mao Mao, Yongping Song. Whole-genome sequencing as an alternative to analyze copy number abnormalities in acute myeloid leukemia and myelodysplastic syndrome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5263.

BackgroundDilated cardiomyopathy (DCM), which is a major cause of heart failure, is a primary cardiac muscle disease with high morbidity and mortality rates. DCM is a genetically heritable disease and more than 10 gene ontologies have been implicated in DCM. CDH2 encodes N-cadherin and belongs to a superfamily of transmembrane proteins that mediate cell–cell adhesion in a calcium-dependent manner. Deficiency of CDH2 is associated with arrhythmogenic right ventricular cardiomyopathy (OMIM: 618920) and agenesis of the corpus callosum, cardiac, ocular, and genital syndrome (OMIM: 618929). However, there have been no reports of isolated DCM associated with CDH2 deficiency.MethodsWe performed whole exome sequencing in a 12-year-old girl with non-syndromic DCM and her unaffected parents. Variants in both known DCM-related genes and novel candidate genes were analyzed and pathogenicity confirmation experiments were performed.ResultsNo pathogenic/likely pathogenic variant in known DCM-related genes was identified in the patient. We found a de novo variant in a candidate gene CDH2 in the patient, namely, c.474G>C/p.Lys158Asn (NM_001792.5). This variant has not been reported in the ClinVar or Human Gene Mutation Database (HGMD). CDH2 p.Lys158Asn was found in the conserved domain of N-cadherin, which is associated with the hydrolysis of the precursor segment and interference with adhesiveness. Furthermore, we tested the expression and efficiency of cell–cell adhesion while overexpressing the CDH2 Lys158Asn mutant and two previously reported variants in CDH2 as positive controls. The adhesion efficiency was considerably reduced in the presence of the mutated CDH2 protein compared with wild-type CDH2 protein, which suggested that the mutated CDH2 protein's adhesion capacity was impaired. The variant was probably pathogenic after integrating clinical manifestations, genetic analysis, and functional tests.ConclusionWe identified a CDH2 variant in DCM. We observed a new clinical symptom associated with N-cadherin deficiency and broadened the genetic spectra of DCM.

Objective: To investigate the effect of CD4-positive T lymphocyte expression rate on the pain control and prognosis of stage Ⅳ non-small cell lung cancer (NSCLC) patients with cancerous pain. Methods: The clinical data of 128 stage Ⅳ NSCLC patients with cancerous pain who were admitted to the Affiliated Cancer Hospital of Zhengzhou University from January to December 2020 were retrospectively analyzed, including 92 males and 36 females, with a male-to-female ratio of 23∶9 and an average age of (56±21) years old. The expression rate of CD4-positive T lymphocytes in peripheral blood was routinely detected on admission, and the expression rate of CD4-positive T lymphocytes ≥45% was defined as the CD4 high expression group, and<45% was defined as the low expression group. The differences in the time required for pain control, the dosage of opioids and the incidence of adverse reactions between the two groups were compared and analyzed. Survival analysis was performed by Kaplan-Meier method, and the overall survival (OS) time and progression-free survival (PFS) time of the two groups were calculated. Cox regression model was used to analyze the influencing factors of patients' OS time and PFS time. Results: The median time required for pain control in the high CD4 expression group [M (Q1, Q3)] was 18.6 (4.6, 21.5) h, which was lower than that in the low CD4 expression group [28.2 (7.1, 38.9) h] (P=0.012). The dosage of morphine in the CD4 high expression group [M (Q1, Q3)] was 88.6 (42.5, 295.0) mg, which was lower than that in the low expression group [145.8 (82.5, 442.5) mg] (P=0.010). There was no significant difference in the incidence of adverse reactions such as nausea and vomiting, constipation, urinary retention, intestinal obstruction, and respiratory depression between the two groups (all P>0.05). The OS time and PFS time in the CD4 high expression group [M (Q1, Q3)] were 12.5 (8.1, 13.8) months and 8.5(3.1, 9.4) months, respectively, which were higher than those in the CD4 low expression group [8.6 (4.1, 12.9) months and 6.5 (2.1, 7.9) months, respectively] (all P<0.01). Cox multivariate analysis showed that high expression of CD4 was a protective factor affecting OS (HR=0.876, 95%CI: 1.224-6.641, P=0.004) and PFS (HR=0.675, 95%CI: 1.742-5.930, P=0.031) Conclusion: The stage Ⅳ NSCLC patients with cancerous pain and high expression of CD4-positive T lymphocytes have shorter pain control time, less morphine dosage, and longer OS and PFS time.

Objective: This study aimed to investigate the clinical and prognostic significance of TET2 single nucleotide polymorphism I1762V in patients with acute myeloid leukemia (AML) . Methods: The high-throughput sequencing method was used to sequence 58 hematological tumor-related genes in bone marrow samples from 413 patients with AML. TET2 I1762V and other somatic mutations were annotated and compared with patients' clinical information and prognosis. Results: I1762V was found in 154 patients with AML, which was significantly different from the general population in NyuWa Chinese Population Variant Database (χ(2)=72.4, P<0.001) . I1762V was not related to sex, age, and karyotype of patients with AML (P>0.05) . Patients with I1762V had a significantly higher proportion of NPM1 and KIT gene mutations than others (P<0.001) . NPM1 and KIT mutations were mutually exclusive. The survival analysis results revealed that the overall survival (OS) and progression-free survival (PFS) of patients with AML with I1762V were significantly greater than those of wild-type patients (HR=0.57, P=0.030; HR=0.55, P=0.020) , whereas the OS and PFS in patients with AML with DNMT3A mutation (with or without I1762V mutation) were lower than those of wild-type patients (HR=1.79, P=0.030; HR=1.74, P=0.040) . Conclusion: TET2 SNP I1762V has been linked to AML. I1762V is a prognostic factor of patients with AML, which can be used to guide the treatment and evaluate the prognosis of AML.

目的 通过生物信息学分析筛选出神经母细胞瘤外周血血浆外泌体中差异表达miRNA,并对其靶基因功能进行分析预测.方法 从高通量基因表达数据库下载数据集GSE128004,分析神经母细胞瘤血浆外泌体中miRNA的差异表达;通过miRTarBase数据库筛选差异表达miRNA的靶基因;进一步通过运用clusterProfiler进行靶基因的基因本体(GO)功能富集与京都基因与基因组百科全书数据库(KEGG)通路富集.结果 经筛选发现,数据集GSE128004包含41个表达差异在2倍以上的血浆外泌体miRNA.靶基因预测显示,hsa-miR-199a-3p,hsa-miR-196b-5p,hsa-miR-127-3p,hsa-miR-410-3p和hsa-miR-487b-3p为靶基因最多的前5个差异表达miRNA.GO功能分析发现,这些靶基因大都在细胞运动正调节、细胞迁移正调节、血管生成等生物过程富集;在膜侧、细胞-基底连接、细胞-基底黏附连接、焦点黏连等细胞组成富集;在蛋白丝氨酸/苏氨酸激酶活性、蛋白异二聚体化活性、转录因子活性和RNA聚合酶Ⅱ核心启动子近端区序列特异性结合等分子功能富集.KEGG分析显示:这些差异表达miRNA的靶基因主要参与磷脂酰肌醇3激酶-蛋白激酶B信号通路、癌症相关miRNA、丝裂原活化蛋白激酶信号通路等通路富集.结论 hsa-miR-199a-3p,hsa-miR-127-3p和hsa-miR-410-3p可能作为神经母细胞瘤的潜在生物标志物或治疗靶标,进一步为该病的发病机制提供研究思路.

癌症是威胁人类生命健康的重大疾病,目前全球每年约有880万人死于癌症.地下鼠营地下封闭黑暗环境生活,具有长寿和抗癌的特性,是国际抗癌研究的良好动物模型.我国地下鼠物种资源丰富,但有关地下鼠抗癌机理的研究亟待加强.2014年以来,郑州大学生命科学学院王振龙教授带领项目团队开展了地下鼠低氧适应的分子机制及其抗癌特性研究,发现了地下鼠低氧适应与抗癌特性的关系,为人类的癌症治疗研究提供重要线索.

目的 分析慢性缺氧对胶质母细胞瘤DNA修复基因表达的影响,寻找胶质母细胞瘤潜在的治疗靶点.方法 使用limma R包对GEO芯片数据(GSE139250)进行差异计算,利用ggplot包绘制火山图,Gene Oncology与KEGG软件对差异基因的生物过程、分子功能及信号通路进行富集分析,并进一步通过String数据库分析差异表达基因的相互作用网络.结果 GSE139250数据库中180个基因通过差异筛选,7个基因出现差异表达,3个基因表达升高,4个基因表达下调.分子功能富集分析显示上调的3个基因(ABL1、DDB2和EGFR)主要与酪氨酸激酶活性、磷酸酶结合、肌动蛋白丝结合等相关,下调的4个基因(PARP1、ATR、BCL2和LIG4)富集于细胞分化、增殖、凋亡、DNA复制和细胞有丝分裂等;KEGG信号通路分析显示差异表达基因主要与P53信号通路、志贺氏菌、胃癌、结直肠癌、癌症中microRNA等通路相关.结论 胶质母细胞瘤慢性缺氧可引起酪氨酸激酶活性、蛋白质磷酸化和肌动蛋白异常,引起细胞DNA复制等过程中DNA损伤及细胞增殖与凋亡等,靶向DNA复制及蛋白质酪氨酸激酶激活和磷酸化过程等分子信号通路可为胶质母细胞瘤提供潜在的治疗靶点.

Objective:To explore the characteristics and clinical significance of clonal heterogeneity in patients with acute lymphoblastic leukemia(ALL).Methods:From January 2016 to June 2019, 170 newly diagnosed ALL patients were enrolled in the Department of Hematology, Henan Cancer Hospital, including 93 males and 77 females, with a median age of 17 (2-80) years. Fifty-two ALL-related genes were detected by high-throughput sequencing technique. The clonal heterogeneity of mutations was analyzed according to the variant allele frequency (VAF) and the results of flow cytometry. The prognostic value of mutations was also evaluated.Results:Gene mutations were detected in 121 (71.2%, 121/170) patients, of which 2 or more clones were detected in 18 (52.9%, 18/34) T-cell acute lymphoblastic leukemia patients, while only 23 (16.9%, 23/136) B-cell acute lymphoblastic leukemia patients were positive of multiple mutations ( P<0.01).Gene mutation-related clonal heterogeneity analysis showed that 2 or more clones were frequent in patients with NOTCH1 mutations (13/19 patients) ( P<0.01). Event free survival (EFS) in patients with 3 or more clones was significantly lower than other patients (χ 2=10.330, P=0.016). Child ALL patients had similar result, that multiple clones predicted lower overall survival (OS) and EFS (OS: χ 2=7.974, P=0.047; EFS: χ 2=10.860, P=0.013). Conclusion:Clonal heterogeneity in ALL patients is closely related to the different origin of lymphocyte lineages and the age of onset, which may reveal the nature of the disease and predict the clinical outcome.

Objective: This study aimed to explore the characteristics and clinical value of clonal heterogeneity in acute myeloid leukemia (AML) . Method: A high-throughput sequencing was carried out to detect 68 related genes in 465 AML patients. Clonal heterogeneity was analyzed based on variant allele frequency (VAF) and flow cytometry results combined with clinical data. Results: Gene mutations were discovered in 338 (81.4%) newly diagnosed patients, and 2 or more clones were significantly increased in patients with DNMT3A, NRAS, and RUNX1 mutations (DNMT3A, χ(2)=15.23; P<0.001; NRAS, χ(2)=19.866; P<0.001; RUNX1, χ(2)=23.647; P<0.001) . The number of clones significantly differed between groups at different ages (χ(2)=17.505, P=0.022) . The proportion of carrying 2 and ≥3 clones increased in patients aged more than 60 years old. There was a significant difference in the clonal heterogeneity between newly diagnosed patients and relapsed or secondary patients (χ(2)=11.302, P=0.010) . Moreover, the proportion of patients with clonal heterogeneity gradually increased according to their prognostic risk (χ(2)=17.505, P=0.022) . Based on the clone analysis, the proportion of primary clones of patients with RUNX1 mutation was higher (χ(2)=4.527, P=0.033) . The analysis of clonal heterogeneity and efficacy demonstrated that patients with three or more clones had significantly lower overall survival (OS) and progression-free survival (PFS) compared to other patients (OS, χ(2)=13.533; P=0.004; PFS, χ(2)=9.817; P=0.020) , while in the intermediate-risk group, patients with a significant clonal heterogeneity also exhibited a significant decrease in PFS (χ(2)=10.883, P=0.012) . Cox regression multivariate analysis revealed that carrying three or more clones was an independent factor affecting prognosis, and OS and PFS were significantly lower than those of patients without clones (OS, HR=3.296; 95% CI, 1.568-6.932; P=0.002; PFS, HR=3.241; 95% CI, 1.411-7.440; P=0.006) . Conclusion: Clonal heterogeneity may reflect the biological characteristics of a tumor, suggesting its drug resistance, refractory, and invasiveness, and further evaluate the treatment effect and prognosis of patients.

Objective: To explore the relationship between driver gene mutation (JAK2, MPL and CALR) and disease type in BCR-ABL negative myeloproliferative neoplasms (MPNs) including primary myeloid fibrosis (PMF), essential thrombocytosis (ET) and polycythemia vera (PV). Methods: A total of 32 MPN related genes were detected by high-throughput sequencing in 156 MPN patients. The relationships between disease type and patients' general performance, the characteristics of driver gene mutations, concomitant gene mutations were analyzed. Results: In the population with JAK2 V617F positive mutation, the proportion of patients over 60 years old in PMF was higher than that with ET or PV. By high-throughput sequencing, 22 concomitant gene mutations were detected in 46 patients with JAK2, MPL or CALR mutations, including 4 (8.3%) in PV, 20 (29.4%) in ET, and 22 (55.0%) in PMF. DNMT3A mutation was detected only in patients with PV, while splicing factor related genes including SF3B1, SRSF2 and U2AF1 were only accompanied by PMF. According to the variation allele frequency (VAF) value of JAK2 V617F mutation, the VAF value associated with PV was the highest (68.15%), followed by PMF (37.7%) and ET (23%). However, there were significant differences in the incidence of JAK2 V617F homozygous among 3 different diseases. In patients with JAK2 mutation, the proportion of other gene mutations in PV and ET was significantly lower than that in PMF. Conclusions: Under the condition of common driver gene mutations (JAK2, MPL and CALR), patients' age, VAF value and homozygous state, concomitant gene mutations are closely related to different disease type. These correlations help to improve clinical understanding of disease characteristics and risk assessment.