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.

BACKGROUND:More and more novel anticancer drugs have been approved for patients with hematological malignancies in recent years, but HBV reactivation (HBV-R) data in this population is very scarce. This study aimed to evaluated HBV-R risk in patients with hematological malignancies receiving novel anticancer drugs. METHODS:HBV markers and serum HBV DNA levels of patients with hematological malignancies receiving novel anticancer drugs in a tertiary cancer hospital were retrospectively collected. HBV-R risk in the whole cohort and subgroups was described. The relevant literature was reviewed to make a pooled analysis. RESULTS:Of 845 patients receiving novel anticancer drugs, 258 (30.5%) were considered at risk for HBV-R. The median duration of exposure to novel drugs was 5.6 (0.1-67.6) months. The incidence of HBV-R was 2.1% in patients with past HBV infection without prophylactic antiviral treatment (PAT) and 1.2% in all patients at risk of HBV-R. In a pooled analysis of 11 studies with 464 patients, the incidence of HBV-R was 2.4% (95% CI: 1.3-4.2) in all at-risk patients receiving novel anticancer drugs and 0.6% (95% CI: 0.03-3.5) in patients with anticancer drugs plus PAT. The incidence of death due to HBV-R was 0.4% (95% CI: 0.1-1.6) in all at-risk patients and 18.2% (95% CI: 3.2-47.7) in patients with HBV-R. CONCLUSION:Most episodes of HBV-R are preventable, and most cases with HBV-R are manageable. We recommend that novel anticancer drugs should not be intentionally avoided when treating cancer patients with HBV infection.

T cell immune dysfunction is a prominent characteristic of chronic lymphocytic leukemia (CLL) and the main cause of failure for immunotherapy and multi-drug resistance. There remains a lack of specific biomarkers for evaluating T cell immune status with outcome for CLL patients. T cell factor 1 (TCF1, encoded by the TCF7 gene) can be used as a critical determinant of successful anti-tumor immunotherapy and a prognostic indicator in some solid tumors; however, the effects of TCF1 in CLL remain unclear. Here, we first analyzed the biological processes and functions of TCF1 and co-expressing genes using the GEO and STRING databases with the online tools Venny, Circos, and Database for Annotation, Visualization, and Integrated Discovery (DAVID). Then the expression and prognostic values of TCF1 and its partner gene B cell leukemia/lymphoma 11B (BCL11B) were explored for 505 CLL patients from 6 datasets and validated with 50 CLL patients from Henan cancer hospital (HNCH). TCF1 was downregulated in CLL patients, particularly in CD8+ T cells, which was significantly correlated with poor time-to-first treatment (TTFT) and overall survival (OS) as well as short restricted mean survival time (RMST). Function and pathway enrichment analysis revealed that TCF1 was positively correlated with BCL11B, which is involved in regulating the activation and differentiation of T cells in CLL patients. Intriguingly, BCL11B was highly consistent with TCF1 in its decreased expression and prediction of poor prognosis. More importantly, the combination of TCF1 and BCL11B could more accurately assess prognosis than either alone. Additionally, decreased TCF1 and BCL11B expression serves as an independent risk factor for rapid disease progression, coinciding with high-risk indicators, including unmutated IGHV, TP53 alteration, and advanced disease. Altogether, this study demonstrates that decreased TCF1 and BCL11B expression is significantly correlated with poor prognosis, which may be due to decreased TCF1+CD8+ T cells, impairing the effector CD8+ T cell differentiation regulated by TCF1/BCL11B.

Background: Fusion genes generated from chromosomal translocation play an important role in hematological malignancies. Detection of fusion genes currently employ use of either conventional RT-PCR methods or fluorescent in situ hybridization (FISH), where both methods involve tedious methodologies and require prior characterization of chromosomal translocation events as determined by cytogenetic analysis. In this study, we describe a real-time quantitative reverse transcription PCR (qRT-PCR)-based multi-fusion gene screening method with the capacity to detect 22 fusion genes commonly found in leukemia. This method does not require pre-characterization of gene translocation events, thereby facilitating immediate diagnosis and therapeutic management.Methods: We performed fluorescent qRT-PCR (F-qRT-PCR) using a commercially-available multi-fusion gene detection kit on a patient cohort of 345 individuals comprising 108 cases diagnosed with acute myeloid leukemia (AML) for initial evaluation; remaining patients within the cohort were assayed for confirmatory diagnosis.Results obtained by F-qRT-PCR were compared alongside patient analysis by cytogenetic characterization. Results: Gene translocations detected by F-qRT-PCR in AML cases were diagnosed in 69.4% of the patient cohort, which was comparatively similar to 68.5% as diagnosed by cytogenetic analysis, thereby demonstrating 99.1% concordance. Overall gene fusion was detected in 53.7% of the overall patient population by F-qRT-PCR, 52.9% by cytogenetic prediction in leukemia, and 9.1% in non-leukemia patients by both methods. The overall concordance rate was calculated to be 99.0%. Fusion genes were detected by F-qRT-PCR in 97.3% of patients with CML, followed by 69.4% with AML, 33.3% with acute lymphoblastic leukemia (ALL), 9.1% with myelodysplastic syndromes (MDS), and 0% with chronic lymphocytic leukemia (CLL).Conclusions: We describe the use of a F-qRT-PCR-based multi-fusion gene screening method as an efficient one-step diagnostic procedure as an effective alternative to lengthy conventional diagnostic procedures requiring both cytogenetic analysis followed by targeted quantitative reverse transcription (qRT-PCR) methods, thus allowing timely patient management.

Background: BCR-ABL1 fusion proteins contain constitutively active tyrosine kinases that are potential candidates for targeted therapy with tyrosine kinase inhibitors such as imatinib in chronic myeloid leukemia (CML). However, uncharacterized BCR-ABL1 fusion genes can be missed by quantitative RT-PCR (qRT-PCR)-based routine screening methods, causing adverse effect on drug selection and treatment outcome.Case presentation: In this study, we demonstrated that the next-generation sequencing (NGS) can be employed to overcome this obstacle. Through NGS, we identified a novel BCR-ABL1 fusion gene with breakpoints in the BCR intron 14 and the ABL1 intron 2, respectively, in a rare case of CML. Its mRNA with an e14a3 junction was then detected using customized RT-PCR followed by Sanger sequencing. Subsequently, the patient received targeted medicine imatinib initially at 400 mg/day, and later 300 mg/day due to intolerance reactions. With this personalized treatment, the patient's condition was significantly improved. Interestingly, this novel fusion gene encodes a fusion protein containing a compromised SH3 domain, which is usually intact in the majority of CML cases, suggesting that dysfunctional SH3 domain may be associated with altered drug response and unique clinicopathological manifestations observed in this patient.Conclusion: We identified a novel BCR-ABL1 fusion gene using NGS in a rare case of CML while routine laboratory procedures were challenged, demonstrating the power of NGS as a diagnostic tool for detecting novel genetic mutations. Moreover, our new finding regarding the novel fusion variant will provide useful insights to improve the spectrum of the genomic abnormalities recognizable by routine molecular screening.

Chromosomal aberrations are useful in assessing treatment options and clinical outcomes of acute myeloid leukemia (AML) patients. However, 40 ∼ 50% of the AML patients showed no chromosomal abnormalities, i.e., with normal cytogenetics aka the CN-AML patients. Testing of molecular aberrations such as FLT3 or NPM1 can help to define clinical outcomes in the CN-AML patients but with various successes. Goal of this study was to test the possibility of Wilms' tumor 1 (WT1) gene overexpression as an additional molecular biomarker. A total of 103 CN-AML patients, among which 28% had overexpressed WT1, were studied over a period of 38 months. Patient's response to induction chemotherapy as measured by the complete remission (CR) rate, disease-free survival (DFS) and overall survival (OS) were measured. Our data suggested that WT1 overexpression correlated negatively with the CR rate, DFS and OS. Consistent with previous reports, CN-AML patients can be divided into three different risk subgroups based on the status of known molecular abnormalities, i.e., the favorable (NPM1(mt)/no FLT3(ITD)), the unfavorable (FLT3(ITD)) and the intermediate risk subgroups. The WT1 overexpression significantly reduced the CR, DFS and OS in both the favorable and unfavorable groups. As the results, patients with normal WT1 gene expression in the favorable risk group showed the best clinical outcomes and all survived with complete remission and disease-free survival over the 37 month study period; in contrast, patients with WT1 overexpression in the unfavorable risk group displayed the worst clinical outcomes. WT1 overexpression by itself is an independent and negative indicator for predicting CR rate, DFS and OS of the CN-AML patients; moreover, it increases the statistical power of predicting the same clinical outcomes when it is combined with the NPM1(mt) or the FLT3(ITD) genotypes that are the good or poor prognostic markers of CN-AML.

目的 研究慢性粒细胞白血病(CML)中JAK2 V617F突变与BCR-ABL210易位的关系及其与临床特征及疗效的关系.方法 对1例新诊断的CML患者行血常规、染色体、融合基因及荧光原位杂交等检查,明确诊断后给予伊马替尼治疗.结果 患者发病时白细胞及血小板数目明显增多,同时存在JAK2 V617F突变与BCR-ABL210易位阳性,伊马替尼治疗1个月后患者达到血液学完全缓解,治疗3个月时达细胞遗传学完全缓解.结论 在典型CML患者中,可同时检测到JAK2 V617F突变与BCR-ABL210易位阳性,临床医师应提高对该现象的认识。