Volume 101 ·  Issue 13 ·  2021

Transcriptome sequencing (RNA-seq) is one of the forms of high-throughput genome sequencing. The RNA-seq data contains high-content disease-related information, including gene fusion, expression, splicing variation, sequence variation, immune gene diversity, etc. In recent years, RNA-seq has been used more and more in hematological tumors, which has promoted precision medicine research and clinical practice in hematological malignancies.

There has consistently growing value of molecular detection to deliver clinical results for diagnosis, classification, prognosis, curative effect prediction and monitoring minimal residual disease of hematological neoplasms. Currently, some clinical laboratories are adopting hematological neoplasms-associated molecular detection in in our country. The increasing demands for clinical testing require that the particular attention need to be paid to the building of the team of professional staff. At present, it is vital to cultivate two kinds of specialized personnel: the technical personnel who are educated in clinical medicine and skilled in developing molecular tests; and the trained managers who are adept at quality control for molecular testing.

Restless legs syndrome (RLS) is a sensorimotor disorder. In recent years, new progress has been made in the pathogenesis, treatment methods and comorbidities of RLS, but the diagnosis and treatment of RLS still needs to be standardized. Therefore, in order to better guide the clinical practice of Chinese doctors, the Sleep Group of Neurologist Branch of Chinese Medical Physicians Association, the Sleep Disorder Group of Neurology Branch of Chinese Medical Association, and the Sleep Disorder Professional Committee of China Sleep Research Association have formulated the Guidelines for the Diagnosis and Treatment of Restless Leg Syndrome in China (2021 Edition) based on the current evidence-based medical evidence. This guideline recommends that: (1) when a patient's serum ferritin level<75 μ g/L and/or transferrin saturation<At 45%, iron supplementation is recommended; (2) Give priority to dopamine receptor agonists. Although α 2 δ calcium channel ligand drugs have not been approved for the treatment of RLS in China, they can still be used as alternative therapeutic drugs for dopaminergic therapy; (3) when other treatments are ineffective, opioids are recommended; (4) Proper physical exercise and physical therapy can improve the disease symptoms to varying degrees. In summary, this guide aims to further standardize the diagnosis and treatment of RLS in China by systematically evaluating a series of guidelines in the field of RLS published at home and abroad in recent years and relevant evidence-based medicine research evidence.

To classify and quantify IKZF1 mutant transcripts in B-cell acute lymphoblastic leukemia (B-ALL) by RNA sequencing (RNA-seq) and bioinformatics analysis.

A cohort of 263 B-ALL cases was enrolled at Hebei Yanda Ludaopei Hospital from September 2018 to September 2020. An integrated bioinformatics pipeline was developed to adapt the classification and quantification of IKZF1 transcripts from RNA-seq and was applied to sequencing data of these cases. The IKZF1 mutant transcripts classified by RNA-seq analysis were compared with the qualitative reverse transcription PCR (RT-PCR).

IKZF1 mutant transcripts were identified in 53 B-ALL patients by RT-PCR and Sanger sequencing, among which IK6 and IK10 transcripts accounted for 67.9% (36/53) and 28.3% (15/53) respectively. Additionally, 2 patients were double positive for IK6 and IK10. RNA-seq analysis identified 51 patients with IKZF1 mutant transcripts. Compared with the RT-PCR result, the detection sensitivity and specificity of RNA-seq analysis reached 94.3% (50/53) and 99.5% (209/210), respectively. Among the 50 patients with IKZF1 mutant transcripts both in RNA-seq and RT-PCR analysis, the ratio of mutant transcripts to total IKZF1 transcripts in 6 patients was 0.14 (0.11, 0.35), which was significantly lower than that of the other 44 patients [0.88 (0.35, 0.97), Z=-3.945,P<0.001]. IKZF1 mutations mostly occurred in Ph⁺and Ph-like B-ALL, characterized by abnormal JAK-STAT pathway, and B-ALL with PAX5 translocation.

Through the optimized bioinformatics analysis process, RNA-seq data can be used to classify and quantitatively analyze IKZF1 transcripts in B-ALL. Furthermore, the relative expression of mutant IKZF1 transcripts was found to cluster into two groups, and IKZF1 mutation was found often accompanied with PAX5 translocations.

To explore the prognosis effect of the expression of long-chain non-coding RNA (lncRNA) MBNL1-AS1 on acute myeloid leukemia (AML) patients.

One hundred and twenty-five AML patients of the Cancer Genome Atlas (TCGA) from November 2001 to March 2010 were involved, including 70 patients who received chemotherapy only and other 55 patients treated with allogeneic hematopoietic stem cell transplantation (allo-HSCT) in addition to chemotherapy. According to the median expression of lncRNA MBNL1-AS1, patients of chemotherapy group were divided into high expression sub-group(n=35) and low expression sub-group (n=35), and patients of allo-HSCT group were also divided into high expression sub-group (n=28) and low expression sub-group (n=27) for prognosis analysis. Clinical characteristics at diagnosis, including peripheral white blood cell counts (WBC), blast percentages in peripheral blood and bone marrow (BM), French-American-British (FAB) subtypes and the frequencies of common genetic mutations in AML were described. The event-free survival (EFS) rate and overall survival (OS) rate of patients in different groups were analyzed, and the influence of the clinical characteristics of patients on the prognosis of AML was analyzed by COX multivariate analysis.

In the chemotherapy group, patients with low lncRNA-MBNL1-AS1 expression had significantly lower EFS and OS (60.0%, 8.6%) than patients with high lncRNA-MBNL1-AS1 expression (68.6%, 34.3%) (χ²=7.817, 10.880, allP<0.01). However, in the alloHSCT group, no significant differences were observed in EFS and OS of patients between high and low expression groups of lncRNA-MBNL1-AS1 (all P>0.05). COX multivariate analysis confirmed that age≥60 years old (EFS:HR (95%CI): 6.934 (1.918-25.075),P=0.003;OS: HR (95%CI): 4.119 (1.812-9.364), P=0.001), and low expression of lncRNA MBNL1-AS1 (EFS: HR (95%CI): 0.354 (0.126-0.941), P=0.038; OS: HR (95%CI): 0.424 (0.231-0.778), P=0.006)were independent risk factors for EFS and OS in the chemotherapy group.

The long-chain non-coding RNA MBNL1-AS1 is related to the prognosis of AML, and its low expression is an independent poor prognostic factor in AML patients.

To analyze the possible fusion genes with high-throughput transcriptome sequencing in myeloid leukemia patients with normal karyotype.

From May 2017 to January 2019, three cases of myeloid leukemia patients with normal karyotype and negative for common fusion genes from the First Affiliated Hospital of Nanchang University were selected as the research objects. The transcriptome sequencing of bone marrow mononuclear cells was performed by high-throughput gene sequencing technology. Defuse software was used to analyze the gene fusion sequence in the transcriptome data, reverse-transcription polymerase chain reaction (RT-PCR) and Sanger sequencing were used to verify the fusion gene with clear pathological significance.

All three patients were diagnosed with myeloid leukemia by clinical manifestations, bone marrow cell morphology, immunology, and histochemical staining. Cytogenetic tests showed normal chromosome karyotypes. Fluorescence in situ hybridization and RT-PCR were used to detect BCR-ABL1, PML-RARA, and other common fusion genes. The results were all negative. Transcriptome sequencing and fusion transcripts analysis revealed that these three patients carried rare fusion genes with clear pathological significance, which included BCR-FGFR1, CPSF6-RARG, and NUP98-RARG, respectively.

Transcriptome sequencing can accurately analyze rare but pathologically significant fusion genes that may exist in myeloid leukemia patients with normal karyotypes.

To investigate parameters related to quantifying the amount of degeneration in preoperative patients to identify ideal indication of artificial cervical disc replacement (ACDR) in patient with a minimum of 10 years of follow-up data.

From January 2004 to August 2008, a total of 44 patients underwent single level Bryan cervical disk replacement performed by the same group of surgeons were involved in this retrospective study, and all of the patients in this group had at least 10 years of follow-up data. Heterotopic ossification (HO) was graded in radiographic images by using the McAfee classification. Preoperative degeneration of cervical spine was evaluated in radiographs based on a quantitative"9 points"scoring system. Univariate analysis and multifactor logistic regression were made to identify significant factors. To determine the cut-off points for the significant factors, a receiver operating characteristic (ROC) curve analysis was conducted.

The incidence of HO in study group was 61.4%. Based on univariate analysis results, there were significant differences in the scores of disc height, the presence of anterior osteophytes and endplate sclerosis between the HO group and non-HO group (all P<0.05), and the indices were included in the multivariate analysis. According to the logistic regression results, disc height and endplate sclerosis were identified as the independent risk factors for HO(OR(95%CI): 10.801(1.202-97.064), 37.870(1.581-907.237), respectively, both P<0.05). ROC analysis showed the area under the curve (AUC) of disc height and endplate sclerosis were 0.822 and 0.792, respectively. According to the scoring system, the ROC curve indicated that both the optimal cutoff points were 1.5.

The incidence of postoperative HO is relatively high among the patients who had more than 10 years follow-up, and the amount of degeneration in the target level before surgery correlated with the incidence of HO.

To investigate the safety and efficacy of percutaneous left atrial appendage closure (LAAC) in nonvalvular atrial fibrillation patients (NVAF) with left atrial spontaneous echocardiographic contrast (LA SEC).

A total of 95 NVAF patients treated in the Department of Cardiology of Ningbo First Hospital from July 2018 to June 2019 were enrolled in this study. The study population was divided into two group according to the presence or absence of LA SEC detected by transesophageal echocardiography (TEE). TEE was scheduled 45 days post-procedure to detect device-related thrombus (DRT). Stroke and bleeding events were recorded during follow-up. Periprocedural complications and follow-up results were compared between the two groups.

LA diameters were smaller in non-LA SEC group than LA SEC group ((44.0±7.4)mm vs (47.3±6.6)mm, P=0.033). Watchman device was successfully implanted into all the enrolled patients. No death, transient ischemic attack (TIA)/stroke, device embolization and major bleeding events occurred. Fifty patients (90.2%) in LA SEC group and 31 patients (91.2%) in non-LA SEC group finished TEE follow-up 45 days post-procedure. No device-related thrombus was detected in non-LA SEC group, while 2 cases (6.5%) were detected in LA SEC group, but there was no significant differences in the incidence between the two groups (P=0.127). The LA SEC group and non-LA SEC group were followed-up for (12.3±3.8) months and (12.9±3.3) months, respectively; and there was no significant differences in the incidence of death, TIA/stroke, major and minor bleeding events between two groups during the follow-up (all P>0.05).

LAAC in NVAF patients with LA SEC was safe and effective. However, the incidence of DRT was slightly higher.

To investigate the role of microRNA-139-5p (miR-139-5p) in the occurrence and development of esophageal squamous cell carcinoma (ESCC) and its effects on cell proliferation and invasion of ESCC cells and its molecular mechanisms.

Seventy-five cases of ESCC tissues and paired normal tissues were obtained from thoracic surgery of the First Affiliated Hospital of Zhengzhou University from February 2017 to March 2018. Experiment was divided into two group: ESCC (n=75) and normal esophageal tissues (n=75).GEO datasets and real-time quantitative PCR (qRT-PCR) were used to detect the expression of miR-139-5p in ESCC tissues and cells. miR-139-5p inhibitor, miR-139-5p mimic, negative control, control siRNA, T-box transcliption factor 1(TBX1) siRNA, pcDNA3.1 and pcDNA3.1-TBX1 were transfected into ESCC Eca109 and TE1 cells. qRT-PCR was used to detect the expressions of miR-139-5p and TBX1 in transfected ESCC cells. Cell counting kit 8 (CCK-8) and Transwell chamber were employed to detect cell proliferation and invasion of ESCC cells, respectively. Dual-Luciferase Reporter assay was used to analyze the interaction between miR-139-5p with TBX1. qRT-PCR, Western blot and immunohistochemistry were utilized to detect the expression of TBX1 in ESCC tissues. Western blot was used to detect the expressions of E-cadherin, N-cadherin and Vimentin after transfection.

The level of miR-139-5p in ESCC tissues was significantly lower than that in normal tissues (1.17±0.43 vs 5.16±3.62,P<0.001). Log-rank test showed that the survival rate of ESCC patients with high miR-139-5p level (n = 43) was significantly higher than that with low miR-139-5p level (n=32) (67.44% vs 25.00%, P = 0.005). The expression level of miR-139-5p in ESCC cells was significantly lower than that of normal esophageal epithelial cell Het-1A (all P<0.001). The proliferation and invasion ability of ECA109 and TE1 cells with high expression of miR-139-5p were significantly lower than those transfected with negative control (NC) (all P<0.05). Dual-Luciferase Reporter assay showed that miR-139-5p could bind to the 3′-untranslated region of TBX1. miR-139-5p mimic or inhibitor suppressed or promoted the expression of TBX1 protein in Eca109 and TE1 cells, respectively (allP<0.05). Downregulation of TBX1 significantly suppressed proliferation and invasion of ECA109 and TE1 cells, while overexpression of TBX1 significantly promoted proliferation and invasion of ECA109 and TE1 cells (all P<0.05). In addition, pcDNA3.1-TBX1 partially reversed the inhibition of miR-139-5p-mediated invasion ability (allP<0.05), while TBX1 siRNA partially reversed the enhancement of miR-139-5p inhibitor-mediated invasion ability (allP<0.05).

miR-139-5p suppressed ESCC cell proliferation and invasion by targeting TBX1.

This article reports the diagnosis and treatment of a case of TEMPI syndrome. A 47-year-old male developed secondary polycythemia with elevated erythropoietin, blood IgG κ monoclonal gamma globulin 31.75 g/L, 16% abnormal plasma cells in bone marrow, telangiectasia in face, chest and back, and small low-density shadow in left kidney on abdominal CT, which was considered as perirenal effusion. Except for the absence of intrapulmonary shunt, the patient's clinical symptoms and laboratory tests met the diagnostic criteria for TEMPI syndrome.

Circular pancreas complicated with periampullary tumors are clinically rare. A middle-aged female patient was admitted with acute pancreatitis. Abdominal enhanced CT and magnetic resonance cholangiopancreatography (MRCP) showed annular pancreas, duodenoscopy showed duodenal papillary adenoma, and duodenal papillary tumor resection was performed. Intraoperative pathology showed high-grade intraepithelial neoplasia, without excluding carcinoma, and pancreatic head duodenectomy was performed. The duodenal papillary villous tubular adenoma was confirmed by postoperative pathology. Grade B pancreatic fistula, gastrointestinal bleeding, abdominal infection and deep vein thrombosis of lower limbs appeared after operation, which improved and was discharged after symptomatic treatment.

In the targeted treatment of acute lymphocytic leukemia with BCR-ABL1 fusion gene positive with tyrosine kinase inhibitors (TKIs), gene mutation (KMs) in the tyrosine kinase domain is the most common resistance mechanism. Classical Sanger gene sequencing is the current gold standard for screening BCR-ABL1-KMs, but it has limitations such as low detection sensitivity, inability to obtain variant allele frequency (VAF), inability to identify composite mutations, and inability to analyze the composition and evolution of multiple mutant clones. In recent years, more and more laboratories have applied second-generation high-throughput gene sequencing technology (NGS) to targeted detection of BCR-ABL1-KMs. Compared with Sanger sequencing, NGS has the advantages of higher detection sensitivity, accurate calculation of VAF, and analysis of composite mutations and clonal composition. It has been recommended by international experts as the preferred screening method for drug-resistant mutations in TKIs. This paper introduces the relevant progress, and suggests that the application of NGS in the detection of BCR-ABL1-KMs should be paid attention to.

Chromosome-positive acute lymphocytic leukemia (Ph⁺ALL) patients, BCR-ABL1 kinase domain (KD) point mutations are the most common mechanism of tyrosine kinase inhibitor (TKIs) resistance. First-generation Sanger gene sequencing is currently the most commonly used method to screen for BCR-ABL1 KD mutations, but it has certain limitations, that is, the detection sensitivity is poor, and the composite mutations cannot be reliably identified. Second-generation gene sequencing (NGS) technology can overcome these problems. More and more diagnostic laboratories are using NGS detection as a routine application, and it is expected to become the preferred method for BCR-ABL1 KD mutation screening. In this article published by Cancer Medicine, expert consensus-based application recommendations are published on the potential value and indications of NGS in the detection of BCR-ABL1 KD resistance mutations before and during first-line TKIs treatment, in relapsed/refractory cases, and before and after allogeneic hematopoietic stem cell transplantation, and how the test results of NGS affect treatment decisions. This application proposal agrees and has been well documented to prove that the sensitivity of NGS to analyze BCR-ABL1 KD resistance mutations is significantly better than that of Sanger sequencing, and it can directly identify composite mutations; A set of NGS analysis methods and minimum technical and methodological requirements for results reporting are also given. This application proposal has good reference significance for guiding the practical application of NGS in the detection of BCR-ABL1 KD resistance mutation. On the other hand, the article also highlights the strong need for further prospective studies on BCR-ABL1 KD resistance mutations. The publication of this application recommendation will contribute to the promotion and rational application of NGS in the detection of BCR-ABL1 KD resistance mutations.

RUNX1 mutations are widespread in patients with myelodysplastic syndrome (MDS), and most reports show that RUNX1 mutations are associated with poor prognosis. However, differences still exist, and the results of univariate analysis cannot be confirmed by multivariate analysis in some cases. Therefore, this meta-analysis assessed the impact of RUNX1 mutations on the prognosis of patients with MDS. This meta-analysis study data was obtained from eligible studies from the PubMed, Embase, and Cochrane libraries. The hazard ratios for overall survival (OS) and leukemia-free survival (LFS) were calculated from the multivariate Cox proportional hazard model (HR) and its 95% confidence interval (CI）。 The study included a total of 16 studies with 5,422 patients, including 617 patients with a RUNX1 mutation and 4,805 patients with wild-type RUNX1. Results show that the total of OSHRwas 1.43 (95%CI=1.21~1.70，P<0.001), while the correspondence of LFSHRwas 1.88 (95%CI=1.42~2.51，P<0.001）。 These results suggest that RUNX1 mutations are associated with poor prognosis and shortened survival in patients with MDS. In addition, stem cell transplantation can alleviate the impact of poor prognostic factors in patients. Patients carrying these mutations should be given priority to aggressive treatment.

With the deepening of genomic research, more and more evidence shows that some patients with hematological tumors have clear genetic predisposition factors. In the updated WHO classification criteria for hematopoietic and lymphoid tissue tumors (2017 edition), "myeloid tumors with germline factors" have been listed as a separate disease classification. Germline susceptibility genes for childhood acute lymphoblastic leukemia (ALL) are also increasingly reported. In recent years, it has been found that the germline variation of ETV6 gene is associated with the risk of ALL, and it has been suggested as a new leukemia susceptibility syndrome. To clarify the role of ETV6 variants in the pathogenesis of ALL, the study performed a comprehensive analysis of 32 cases of childhood leukemia caused by this rare syndrome. A total of 34 non-synonymous ETV6 germline variants were detected in this study, and 22 of them were identified as causing impaired transcriptional repressive activity, loss of DNA binding function, and abnormal nuclear localization. The missense variant ETV6 protein retains the ability to form a dimer with the wild-type ETV6 protein and may exert a dominant negative effect. Whole transcriptome and whole genome sequencing analyses of this group of patients also revealed that ETV6 germline variants had a significant effect on the leukemia transcriptome, and that this group of patients had different patterns of somatic concomitant mutations. Among the cases carrying deleterious ETV6 germline variants, 70% had a hyperdiploid karyotype with characteristic NRAS, KRAS and PTPN11 mutations. The other 30% are diploid genomes with very high frequency of PAX5 and ETV6 copy number deletions, and their gene expression patterns are very similar to those of ALL with positive ETV6-RUNX1 fusion genes. One patient carrying the ETV6 R386fs variant developed ALL, followed by treatment-related acute myeloid leukemia (AML); Another family of carriers of the ETV6 R399H variant developed ALL and AML, respectively. Genomic analysis showed support for concomitant somatic variation determining the lineage of leukemia. In vitro studies have shown impaired tumor suppressor activity of variant ETV6. ETV6-mediated leukemia also shows a complex interaction between hereditary and acquired genomic variation in pathogenesis. The genetic predisposition factors of hematological tumors are more common than previously expected, and they have important clinical diagnosis and treatment significance, which should be paid attention to.

The tumor protein p53 (TP53) is the most common mutant gene in cancer. In patients with myelodysplastic syndrome (MDS), TP53 mutations have been associated with high-risk disease, rapid conversion to acute myeloid leukemia (AML), resistance to traditional therapies, and poor prognosis. Consistent with the tumor suppressor effect of TP53, patients carried both monoallelic and biallelic mutations. However, the biological and clinical implications of TP53 allele status have not been comprehensively studied in MDS or any other cancer type. The study analyzed TP53 mutations and allelic imbalances in 3 324 patients with MDS. 1/3 of the patients with TP53 mutations had single allelic mutations and 2/3 had multiple mutations (multi-site mutations), consistent with bi-allelic targeting. Established associations with complex karyotypes, few co-occurring mutations, high-risk manifestations, and poor prognosis are only for patients with multisite mutations. TP53 multi-site mutation status is independent of the revised International Prognostic Scoring System (IPSS-R) and predicts the risk of MDS death and leukemia transformation. However, there were no statistically significant differences in outcome and response to treatment between monoallelic patients and TP53 wild-type patients. This study suggests that consideration of TP53 allele status is critical for diagnostic and prognostic assessment of MDS.

Diffuse large B-cell lymphoma (DLBCL) is one of the most common non-Hodgkin's lymphomas with significant clinical prognostic differences and molecular heterogeneity. In recent years, a number of different molecular typing suggestions for DLBCL have been put forward based on the data of gene expression profiling and exome studies, which increasingly show the guiding significance of accurate typing based on gene indicators for the diagnosis and treatment of DLBCL. However, at present, the typing standard that is unanimously recognized by experts and easy to implement has not been reached. In order to establish a more systematic molecular typing system that is easy to implement clinically, the British Hematology and Oncology Research Network reported a population-based patient cohort study. The project conducted targeted sequencing studies of 293 hematological tumor-related genes on tumor DNA samples from 928 DLBCL patients with complete follow-up data from 14 hospitals covering a population of 4 million UK. The study employed Bernoulli mixed models for molecular typing clustering and analyzed their correlation with clinical features and prognosis. A total of five DLBCL molecular typing were identified, namely MYD88, BCL2, SOCS1/SGK1, TET2/SGK1 and NOTCH2, and an unclassified group. Among them, subtypes characterized by BCL2, NOTCH2, and MYD88 gene abnormalities suggest good, moderate, and poor prognosis, respectively, consistent with other research reports. The biological characteristics of the SOCS1/SGK1 subtype overlap with those of primary mediastinal B-cell lymphoma and have a favorable prognosis. NOTCH1 mutations, although not identified as molecular typing indicators, are associated with poor prognosis. The effects of TP53 mutations vary by molecular subtype, with no effect on prognosis in the NOTCH2 subtype and poor prognosis in the MYD88 subtype. This study further confirms the clinical prognostic significance of genetic testing and molecular typing of patients with DLBCL, as well as the importance of reaching a classification consensus.

Tyrosine kinase inhibitors (TKIs) targeting the BCR-ABL1 oncoprotein have revolutionized the treatment of leukemias carrying this fusion gene. However, after the application of TKIs, the phenomenon of BCR-ABL1 mutation resistance, mainly kinase domain mutation, was quickly discovered. BCR-ABL1 mutation resistance mostly occurs after TKIs are treated, and it can also be manifested as primary resistance, and the dynamic evolution of multiple resistant clones can occur during the treatment process (including the replacement of different TKIs). Punatinib is currently the only third-generation TKIs approved for marketing that are effective against almost all single BCR-ABL1 kinase domain mutations, including the T315I mutation that is extensively resistant to all first-and second-generation TKIs. However, with the increase of application, the phenomenon of punatinib resistance also occurs from time to time. Patients with punatinib resistance have used multiple TKIs sequentially, with a long course of disease and complex drug-resistant mutations (including compound mutations, polyclonal mutations, dynamic evolution of mutant clones, etc.), which bring challenges to detection and research. In the past, the first generation Sanger gene sequencing method was mostly used to detect BCR-ABL1 kinase domain mutations, which had limited detection sensitivity, could not be quantitatively analyzed, and it was difficult to distinguish between polyclonal mutations and composite mutations. The BCR-ABL1 mutation detection based on second-generation gene sequencing (NGS) technology reported in this study can not only directly identify polyclonal mutations and composite mutations, but also have the advantages of high detection sensitivity, quantitative analysis, and clonal evolution analysis. The results of this study show that the drug-resistant mutations of punatinib are mostly compound mutations involving the T315 codon of ABL1 gene, which occur sequentially and occur at multiple sites; Patients who are resistant to prenatinib all experience an active process of dynamic evolution and accumulation of mutations. This further emphasizes the importance of using NGS technology to monitor the dynamic evolution of drug resistance mutations, composite mutations and mutant clones when applying TKIs such as punatinib. The clonal evolution and composite mutation of drug resistance mutations have become a serious problem in the treatment of sequential TKIs. It is necessary to deeply understand the drug resistance mutation rules of various TKIs to optimize the treatment plan. The improvement of detection technology provides a powerful tool for medical research and clinical application.