[Analysis of epigenetic modification gene mutations among patients with acute myeloid leukemia].

OBJECTIVE: To investigate the carrier rate and clinical characteristics of epigenetic modification gene mutations (EMMs) among patients with acute myeloid leukemia (AML). METHODS: One hundred seventy two patients who were initially diagnosed with AML at the First People's Hospital of Lianyungang from May 2011 to February 2021 were selected as the study subjects. Next-generation sequencing was carried out to detect variants of 42 myeloid genes among these patients. Clinical and molecular characteristics of patients with EMMs and the effect of demethylation drugs (HMAs) on their survival were analyzed. RESULTS: Among the 172 AML patients, 71 (41.28%) were found to harbor the EMMs, and carrier rates were TET2 (14.53%, 25/172), DNMT3A (11.63%, 20/172), ASXL1 (9.30%, 16/172), IDH2 (9.30%, 16/172), IDH1 (8.14%, 14/172), EZH2 (0.58%, 1/172). Patients with EMMs (+) had lower peripheral hemoglobin compared with those with EMMs(-) (72 g/L vs. 88 g/L, Z = -1.985, P = 0.041). The proportion of EMMs(+) among elderly AML patients was significantly higher than that of young AML patients[71.11% (32/45) vs. 30.70% (39/127), χ² = 22.38, P < 0.001]. EMMs(+) were significantly correlated with NPM1 gene variants (r = 0.413, P < 0.001), while negatively correlated with CEPBA double variants (r = -0.219, P < 0.05). Compared with conventional chemotherapy regimens, HMAs-containing chemotherapy regimens have improved the median progression-free survival (PFS) and median overall survival (OS) among intermediate-risk AML patients with EMMs(+) (PFS: 11.5 months vs. 25.5 months, P < 0.05; 12.5 months vs. 27 months, P < 0.05). Similarly, Compared with conventional chemotherapy regimens, chemotherapy with HMAs had increased median PFS and median OS in elderly AML patients with EMMs(+) (4 months vs. 18.5 months, P < 0.05; 7 months vs. 23.5 months, P < 0.05). CONCLUSION: Patients with AML have a high rate of EMMs carriage, and HMAs-containing chemotherapy regimens can prolong the survival of elderly patients with AML with poor prognosis, which may provide a reference for individualized treatment.

[The Effect and Safety of Flumatinib in Patients with Chronic Myelogenous Leukemia Failed First-and Second-line Treatment].

OBJECTIVE: To analyze the efficacy and safety of flumatinib, a second-generation tyrosine kinase inhibitor (TKI) independently developed in China, in patients with chronic myelogenous leukemia in chronic phase (CML-CP) who falied first-line and second-line treatment. METHODS: The clinical data of 30 CML-CP patients treated with flumatinib in Lianyungang First People's Hospital from January 2020 to September 2022 were collected retrospectively. Among them, 15 patients who received imatinib first-line treatment but failed treatment were included in the second-line group, and the other 15 patients who failed second-line treatment with nilotinib or dasatinib were included in the third-line group. The hematological and molecular responses of the patients in the two groups at 3, 6 and 12 months of treatment, and the event-free survival (EFS) and adverse reactions of patients at the end of follow-up were statistical analyzed. RESULTS: At 3, 6, and 12 months of treatment, 10, 11, and 12 patients in the second line group achieved major molecular response (MMR), which was higher than that of 3, 4, and 5 patients in the third line group (P =0.010, P =0.011, P =0.010). At 3 months of treatment, 12 and 13 patients achieved complete hematological response (CHR) and early molecular response (EMR) in the second-line group, which was higher than that of 9 and 13 patients in the third-line group, but the difference between the two groups was not statistically significant (P =0.232, P =1.000); At 6 and 12 months of treatment, 6 and 7 patients in the second-line group achieved MR4.5, which were higher than of 3 and 2 cases in the third-line group, but the difference was not statistically significant (P =0.427, P =0.713). The hematological adverse reactions of patients in the second-line group during treatment the period were mainly grade 1-2 thrombocytopenia and anemia, and no grade 3-4 of adverse reactions occurred. In the third-line group, there were 2 cases of grade 1-2 thrombocytopenia, grade 1-2 anemia and white blood cell 3 cases were reduced each, 1 case of grade 3-4 anemia, 2 cases of grade 3-4 neutropenia. The non-hematological adverse reactions in the second-line group were rash (2 cases), headache (1 case), diarrhea (1 case), fatigue (1 case), limb pain (1 case). There were 1 cases of diarrhea, 1 cases of nausea, and 1 cases of edema in the third-line group. There was no statistical significance in hematological and non-hematological adverse reactions between the two groups of patients (P >0.05). At the end of follow-up, the EFS rate of patients in the second-line group was higher than that in the third-line group (100% vs 93.3%), but the difference was not statistically significant (P =0.317). CONCLUSION: The second-generation TKI flumatinib independently developed in China, has good curative effect and safety for CML-CP patients who failed first-line and second-line treatment.

[Clinical Efficacy and Safety of Ixazomib-Containing Regimens in the Treatment of Patients with Multiple Myeloma].

OBJECTIVE: To investigate the clinical efficacy and safety of ixazomib-containing regimens in the treatment of patients with multiple myeloma (MM). METHODS: A retrospective analysis was performed on the clinical efficacy and adverse reactions of 32 MM patients treated with a combined regimen containing ixazomib in the Hematology Department of the First People's Hospital of Lianyungang from January 2020 to February 2022. Among the 32 patients, 15 patients were relapsed and refractory multiple myeloma (R/RMM) (R/RMM group), 17 patients who responded to bortezomib induction therapy but converted to ixazomib-containing regimen due to adverse events (AE) or other reasons (conversion treatment group). The treatment included IPD regimen (ixazomib+pomalidomide+dexamethasone), IRD regimen (ixazomib+lenalidomide+dexamethasone), ICD regimen (ixazomib+cyclophosphamide+dexamethasone), ID regimen (ixazomib+dexamethasone). RESULTS: Of 15 R/RMM patients, overall response rate (ORR) was 53.3%(8/15), among them, 1 achieved complete response (CR), 2 achieved very good partial response (VGPR) and 5 achieved partial response (PR). The ORR of the IPD, IRD, ICD and ID regimen group were 100%（3/3）, 42.9%（3/7）, 33.3%（1/3）, 50%（1/2）， respectively， there was no statistically significant difference in ORR between four groups (χ 2=3.375, P =0.452). The ORR of patients was 50% after first-line therapy, 42.9% after second line therapy, 60% after third line therapy or more, with no statistically significant difference among them (χ2=2.164, P =0.730). In conversion treatment group, ORR was 88.2%(15/17), among them, 6 patients achieved CR, 5 patients achieved VGPR and 4 patients achieved PR. There was no statistically significant difference in ORR between the IPD（100%， 3/3）, IRD（100%， 6/6）, ICD（100%， 3/3） and ID（60%， 3/5） regimen groups (χ2=3.737，P =0.184). The median progression-free survival (PFS) time of R/RMM patients was 9 months (95% CI : 6.6-11.4 months), the median overall survival (OS) time was 18 months (95% CI : 11.8-24.4 months). The median PFS time of conversion treatment group was 15 months (95% CI : 7.3-22.7 months), the median OS time not reached. A total of 10 patients suffered grade 3- 4 adverse event (AE). The common hematological toxicities were leukocytopenia, anemia, thrombocytopenia. The common non-hematological toxicities were gastrointestinal symptoms (diarrhea, nausea and vomit), peripheral neuropathy, fatigue and infections. Grade 1-2 peripheral neurotoxicity occurred in 7 patients. CONCLUSION: The ixazomib-based chemotherapy regimens are safe and effective in R/RMM therapy, particularly for conversion patients who are effective for bortezomib therapy. The AE was manageable and safe.

[Efficacy and Safety of Decitabine Combined with Modified EIAG Regimen in the Treatment of Patients with Relapsed/Refractory Acute Myeloid Leukemia and High-risk Myelodysplastic Syndrome].

OBJECTIVE: To investigate the efficacy, prognosis and safety of decitabine combined with modified EIAG regimen in the treatment of patients with relapsed/refractory acute myeloid leukemia (AML) and high-risk myelodysplastic syndrome (MDS). METHODS: The clinical data of 44 patients with relapsed/refractory AML and high-risk MDS admitted to our hospital from January 2017 to December 2020 were analyzed retrospectively. The patients were equally divided into D-EIAG group (decitabine combined with EIAG regimen) and D-CAG group (decitabine combined with CAG regimen) according to clinical treatment regimen. The complete response (CR), CR with incomplete hematologic recover (CRi), morphologic leukemia-free state (MLFS), partial response (PR), overall response rate (ORR), modified composite complete response (mCRc), overall survival (OS) time, 1-year OS rate, myelosuppression and adverse reactions between the two groups were compared. RESULTS: In D-EIAG group, 16 patients (72.7%) achieved mCRc (CR+CRi+MLFS), 3 patients (13.6%) achieved PR, and ORR (mCRc+PR) was 86.4%. In D-CAG group, 9 patients (40.9%) achieved mCRc, 6 patients (27.3%) achieved PR, and ORR was 68.2%. Difference was observed in mCRc rate between the two groups (P=0.035), but not in ORR (P>0.05). The median OS time of D-EIAG group and D-CAG group was 20 (2-38) months and 16 (3-32) months, and 1-year OS rate was 72.7% and 59.1%, respectively. There was no significant difference in 1-year OS rate between the two groups (P>0.05). After induction chemotherapy, the median time for absolute neutrophil count recovery to 0.5×109/L in D-EIAG group and D-CAG group was 14 (10-27) d and 12 (10-26) d, for platelet count recovery to 20×109/L was 15 (11-28) d and 14 (11-24)d, the median red blood cell suspension transfusion volume was 8 (6-12) U and 6 (6-12) U, and the median apheresis platelet transfusion volume was 4 (2-8) U and 3 (2-6) U, respectively. There were no statistically significant differences in comparison of the above indicators between the two groups (P>0.05). The hematological adverse reactions of patients were mainly myelosuppression. Grade III-IV hematological adverse events occurred in both groups (100%), with no increase in the incidence of non-hematological toxicities such as gastrointestinal reactions or liver function damage. CONCLUSION: Decitabine combined with EIAG regimen in the treatment of relapsed/refractory AML and high-risk MDS can improve remission rate, provide an opportunity for subsequent therapies, and have no increase in adverse reactions compared with D-CAG regimen.

[Clinical Significance of RAS Gene Mutations in Patients with Acute Myeloid Leukemia].

OBJECTIVE: To investigate the clinical characteristics of RAS gene mutations in patients with acute myeloid leukemia (AML). METHODS: 43 myeloid gene mutations were detected using next-generation sequencing (NGS) in 180 patients with AML who were first diagnosed between May 2011 and February 2021. The molecular and clinical features of RAS gene mutations and their effects on efficacy and survival of patients were retrospectively analyzed. RESULTS: Among 180 AML patients, the proportion of mutations in RAS pathway-related genes were NRAS (14.4%), KRAS (2.2%), FLT3-ITD (13.8%), PTPN11 (7.7%), KIT (5.0%), FLT3-TKD (3.8%) and CBL (2.7%). Seventy-three (40.6%) AML patients had gene mutations associated with the RAS pathway.The number of peripheral blood white blood cells and the proportion of bone marrow primitive juvenile cells in patients with NRAS/KRAS gene mutation were higher than those of patient with RAS wild-type, the difference was statistically significant (P<0.05). NRAS/KRAS gene mutations were significantly associated with the CBL gene mutation(r=0.287). In young AML patients (age <60 years), there were no significant differences in complete response rate (CR), progression-free survival (PFS), and overall survival (OS) between patients with RAS gene mutation and those with wild-type(P>0.05). In elderly AML patients (age≥60 years), PFS and OS in RAS mutants were significantly lower than those in wild-type patients(P<0.05). CONCLUSION: In AML patients, RAS gene mutation is relatively common, and RAS gene mutation is associated with clinical characteristics and efficacy of patients, and may be a molecular marker of poor prognosis for elderly AML.

High ROBO3 expression predicts poor survival in non-M3 acute myeloid leukemia.

Roundabout guidance receptor proteins are crucial components of the SLIT/ROBO signaling pathway. This pathway is important for the nervous system and in embryonic development. Recently, increasing evidence has shown that roundabout guidance receptor proteins and the SLIT/ROBO signaling pathway also participate in tumorigenesis. Here, by analyzing transcriptome data from the TCGA and GEO databases, we found that ROBO3 is highly expressed in non-M3 acute myeloid leukemia. High ROBO3 expression was associated with increased age at diagnosis and poorer risk classification (both P < 0.01). Patients with high ROBO3 expression had higher rates of TP53 and RUNX1 mutations (both P < 0.05). Significantly worse overall survival and event-free survival were observed in high ROBO3 expression patients compared with low ROBO3 expression patients (OS: P = 0.004; EFS: P= 0.012). High ROBO3 expression was also associated with poorer overall survival and event-free survival in a subgroup of patients who received intensive chemotherapy (OS: P = 0.024; EFS: P = 0.040). Moreover, multivariate analysis indicated that high ROBO3 expression was an independent risk factor for poor overall survival in non-M3 acute myeloid leukemia patients who are younger than 60 and received intensive chemotherapy during remission induction. Bioinformatics analysis by Kyoto Encyclopedia of Genes and Genomes and Gene Ontology revealed that high ROBO3 expression significantly altered cell adhesion and extracellular matrix-related pathways (adjusted P < 0.05). Taken together, the data demonstrate that ROBO3 is upregulated in non-M3 acute myeloid leukemia and may be a potent biomarker of inferior prognosis.

GRAIL inhibits the growth, migration and invasion of lung adenocarcinoma cells by modulating STAT3/C-MYC signaling pathways.

PURPOSE: To explore the role and possible regulatory mechanisms of GRAIL in lung adenocarcinoma cells. METHODS: Online databases were used to analyze the expression and prognosis of GRAIL in patients with lung adenocarcinoma. Lung adenocarcinoma cells (A549 and H1299) were transfected with small interfering RNA or infected with lentivirus. The constructed cell models were validated by real-time fluorescence quantitative PCR. Cell proliferation ability was detected by CCK8 assay, cell migration and invasion ability were detected by scratch assay and Transwell assay. Suppressed hallmark gene sets of GRAIL in lung adenocarcinoma was performed by GeneSet Enrichment Analysis. The mRNA and protein level of C-MYC were detected by real-time fluorescence quantitative PCR and western blot. The protein level of STAT3 and phosphorylated STAT3 were detected by western blot. RESULTS: GRAIL is poorly expressed in lung adenocarcinoma tissue. Down-regulation of GRAIL is associated with poor prognosis of lung adenocarcinoma patients. GRAIL expression is negatively correlated with the proliferation, migration, and invasion of lung adenocarcinoma cells. GRAIL inhibits activation of STAT3 and transcription of C-MYC. CONCLUSIONS: GRAIL inhibits the growth, migration and invasion of lung adenocarcinoma cells. GRAIL might play an anti-cancer role through regulating STAT3/ C-MYC signaling pathways.

[IDH1 Gene Mutation and Its Clinical Significance in patients with Acute Myeloid Leukemia].

OBJECTIVE: To evaluate the incidence rate of IDH1 in acute myeloid leukemia and analyze its effect on clinical characteristics and prognosis. METHODS: Mononuclear cells in bone marrow samples were collected from 192 adult patients with newly diagnosed AML. Polymerase chain reaction (PCR) and direct sequencing were used to amplify exon 4 of IDH1 gene, the gene sequencing was used to analyze the gene mutations, at same time, the detection of NPM1, FLT3-TKD, FLT3-ITD, C-KIT, CEPBA, TET2 and JAK2V617F and MLL mutations were carried out, the follow-up was used to determine its therapeutic efficacy and outcomes of patients. The clinical and laboratory data of these cases were collected, and their clinical characteristics and prognosis were then analyzed. RESULTS: Among the 192 AML patients, 13 cases were detected with IDH1 gene mutation, the mutation rate was 6.77% [95% CI (5.70%-13.38%)]. The sequencing chart of IDH1 gene showed double peaks, the mutations were heterozygous, out of them c.G395A (p.R132H) was found in 8 cases, c.C394T was found in 4 cases (p.R132C), c.C394A (p.R132S) was found in 1 cases, R132H and R132C are common, 13 cases showed missense mutation. The median age in mutation group was 52 years old, the median age in unnutration group was 40 years, there was significant difference between them (P = 0.010). Mutation rate of IDH1 gene in M1 and M2 was significantly higher than that in other FAB subtypes. There were no significant difference in sex, newly diagnosed peripheral white blood cell count, hemoglobin, platelet count, peripheral blood and bone marrow original cell proportion of primitive cells between them. Mutation of IDH1 gene had certain correlation with NPM1 gene mutation, but no correlation with FLT3-TKD, FLT3-ITD, C-KIT, TET2 and JAK2V617F and MLL natations was found. In addition, the IDH1 mutation easily occurred in patients with normal karyotype or in patients with middle prognostic risk karyotype, IDH1 mutation occurred in 11 cases with normal karyotype, the mutation rate was 10.28%, IDH1 mutation were observed in 2 cases with abnormal karyotype, the mutation rate was 3.50%, there was significant difference. In AML patients with middle prognostic risk karyotype. The complete remission (CR) and the 3 year survival (OS) rate of IDH1 mut patients were less than that in IDH1 wt, there was significant difference (P < 0.05). CONCLUSIONS: The IDH1 mutation more easily occurr in older AML patients and mutations effect of IDH1 on clinical characteristics may represent a molecular marker for poor prognosis in AML.

[The incidence of TET2 gene mutation and its clinical significance in acute myeloid leukemia patients].

OBJECTIVE: To evaluate the prevalence of TET2 gene mutation in acute myeloid leukemia (AML) patients, and analyze their clinical characteristics and prognosis. METHODS: Polymerase chain reaction (PCR) and direct sequencing were used to sequence exon 3 to 11 of TET2 gene. RESULTS: Among 96 AML patients, TET2 gene mutation was detected in 13 (13.54%) patients (95%CI 6.70% - 20.38%). The median age was 54 years in mutated group and 41 years in unmutated group (P = 0.010). Mutated and unmutated patients did not significantly differ in gender, white blood cells (WBC) count at diagnosis, platelet count, PB and BM blast percentage and chromosome karyotype, excepting for hemoglobin level 84 (70 - 108) g/L in mutated group versus 70 (55 - 87) g/L in unmutated group (P = 0.032). TET2 gene mutation had no significant correlation with C-KIT, FLT3, JAK2V617F mutations, but did with NPM1 mutation. TET2 mutated patients had lower CR1 rate and 2-year overall survival than unmutated in non-M(3) patients (P < 0.05). CONCLUSIONS: TET2 gene mutation is more prevalent in older AML patients and has a certain correlation with clinical characteristics and outcome. It may be a molecular marker for poor prognosis in AML.