Difference Of Gastrointestinal Microbiota In Newly Diagnosed Acute Myeloid Leukemia Patients And Its Relationship With Efficacy Of Chemotherapy

Objectives:Acute myeloid leukemia?AML?,as a hematological malignancy,is the most common type in adult leukemia.However,the etiology and pathogenesis of AML are still unclear.It has been reported the factors such as radiation,virus,genetic background,personal diet and lifestyle are related to its occurrence.As an important part of human microorganisms,the GI bacteria mediated metabolism,the maintenance of the integrity of the intestinal barrier,the regulation of the immune system and other aspects.Studies have shown that GI bacteria is related to the occurrence and development of various diseases.In recent years,more and more scholars have linked the GI bacteria to leukemia,some scholars have proposed that analyzing the structure of the GI bacteria can distinguish leukemia patients from health people.In mouse model,it was found that the use of antibiotics would inhibit the hematopoietic function of bone marrow,moreover,the GI bacteria played an important role in this process.Antibiotics are important factors in the diagnosis and treatment of leukemia patients,analyzing the effect of antibiotics on the human microbial community may be beneficial to the control of infection during treatment.Some studies have pointed out that the GI bacteria plays an important role in leukemia patients who have underwent hematopoietic stem cell transplantation,such as predicting the occurrence of acute GVHD and the related mortality,or assessing the risk of infection.However,there are few related studies on the aspect that using GI bacteria to assess the effect of induction chemotherapy.This study analyzes the composition of GI bacteria to evaluate the clinical significance of bacteria in the treatment of patients,which may provide important values for the treatment of leukemia patients.Methods:1.Subjects' recruitment and sample collectionCollected fecal samples from patients which diagnosed with acute myeloid leukemia by WHO criteria as the experimental group?a total of 25 cases?;according to the medication history of antibiotics for three months prior to this study,the patients in experimental group were divided into two subgroups,including the antibiotic group?AML-ABY Group,n=19?and the non-antibiotic group?AML-ABN group,n=6?.Fecal samples from 16 patient's healthy family members and 9 healthy adults from Nanchang as the control group?CON Group,n=25?.Followed up the acute myeloid leukemia patients in the experimental group,a total of 20 patients underwent induction chemotherapy in our hospital,according to their treatment effects,they were divided into complete remission group?AML-CR group,n=11?and incomplete remission group?AML-NR group,n=9?.2.Experimental steps?1?Sample collection: The fecal samples of the patients in the experimental group were collected when they were first admitted to the hospital.The difference of sample collection time between CON group and patients was less than 3 days,and the fecal samples of the local healthy adults in CON group were collected within one week before the test.After the samples were collected,they were stored in a-80 ° C refrigerator.?2?DNA extraction and sequencing: Extract genomic DNA,perform PCR amplification and product purification;based on the illumina platform,16 S rRNA high-throughput sequencing technology was used to transform the V3-V4 region of the bacteria;and analysis biological information on sequencing results.Statistical analysis based on biological information analysis results:?1?Compare the GI bacteria between the newly diagnosed adult patients with acute myeloid leukemia and healthy adults;?2?Compare the GI bacteria between the newly diagnosed AML patients with and without antibiotics;?3?Follow-up of patients who underwent induction chemotherapy,compare the GI bacteria between the complete response group and the incomplete response group.Results:1.Among the 50 patients' GI bacteria,it's dominant with Bacteroidetes,Firmicutes,Proteobacteria,Actinobacteria and Fusobacteria,Bacteroidetes and Firmicutes account for the majority.2.Observed_species,PD_whole_tree,shannon,and simpson obtained from statistical analysis were used to evaluate the alpha diversity.The results were as follows: AML-ABN group > AML-ABY group,CON group> AML-ABY group,the difference is statistically significant?P<0.05?,it means the alpha diversity of AML-ABY group is lower than CON group,and is lower than AML-ABN group;the observed_species and PD_whole_tree indexes of AML-ABN group are lower than those of CON group,but the difference between shannon and simpson index was not statistically significant.3.We used PCoA analysis and UPGMA analysis to evaluate the beta diversity of GI bacteria,and found that the distribution of samples between three groups which including AML-ABN group,AML-ABY group and CON group can be obvious distinguished,and the general trend is to combine with another group after clustering in group.Next,we analyzed the difference between groups by Adonis analysis,and it was concluded that the differences between CON group and AML-ABN group?P=0.009?,AML-ABN group and AML-ABY group?P=0.001?,CON group and AML-ABY group?P=0.001?were statistically significant.4.Using LEfSe analysis to obtain the difference between AML-ABN group and CON group.It revealed that the differential species in AML-ABN group was Escherichia_Shigella,Eubacterium_<sub>coprostanoligenes_group,Eubacterium_<sub>ruminantium_group,metagenome;and relatively abundant species in CON group were Fusobacteria,Actinobacteria,Fusobacteriia,Fusobacteriales,Fusobacteriaceae,Leptotrichiaceae,Dialister,Leptotrichia,Erysipelotrichaceae_UCG₀03,Prevotella₇,Fusobacterium.5.LEfSe was used to analyze the difference between the AML-ABN group and AML-ABY group.It revealed that 6 species in AML-ABY group had relatively increased abundance,including Bacteroidaceae?Clostridiaceae₁?Bacteroides?Klebsiella ? Clostridium_sensu_stricto₁ and Ruminococcus_<sub>gnavus_grou;23 species existed statistical differences in AML-ABN group,including Prevotellaceae,Alloprevotella,Roseburia,Prevotella₉,Eubacterium_<sub>coprostanoligenes_group,Prevotella₂,Ruminococcaceae_UCG₀02 and so on.6.Using LEfSe analysis to obtain the difference between CON group and AMLABY group.The species enriched in AML-ABY group were Parabacteroides,Klebsiella,Ruminococcus_<sub>gnavus_group,Clostridium_sensu_stricto₁,Clostridiaceae₁;the abundance of 27 species was increased in CON group,including Prevotellaceae,Prevotella₉,Lachnospiraceae,Fusobacteria,Fusobacteriales,Fusobacteriales,Fusobacteriia,Faecalibacterium,Alloprevotella,Romboutsia and so on.7.PICRUSt microbial function prediction analysis results show that,in the comparison of AML-ABN group,AML-ABY group and CON group,the level one includes Cellular processes,Human diseases,Organismal Systems,Genetic Information Processing,Metabolism and Unclassified.A total of 6 functional gene pathways within the group have differences between groups,and a total of 38 functional pathways in the level two have significant differences between groups,and this part of the metabolic transmission was enriched in CON group.8.In the comparison between AML-CR group and AML-NR group,the alpha diversity between the two groups was compared with four indices,including observed_species,PD_whole_tree,shannon and simpson,it was found that AML-CR group > AML-NR group,but P>0.05,it illustrates that there was no significant difference in GI bacteria alpha diversity between AML-CR group and AML-NR group.The PCoA analysis and UPGMA analysis were used to evaluate the beta diversity of GI bacteria,and it was found that the samples in AML-CR group and AML-NR group were mixed and could not be distinguished.Further,using Adonis analysis to compare the differences betweenAML-CR group and AML-NR group and obtain the P value?P= 0.616>0.05?,suggesting that the difference between AML-CR group and AML-NR group was not statistically significant..Using LEfSe to analyze the difference species between the groups,it was concluded that the samples in AML-CR group were predominately enriched by Bogororiellaceae,Georgenia,Negativibacillus and uncultured,AML-NR group were mainly enriched by Micrococcales,Prevotellaceae_UCG₀03,Lachnospiraceae_UCG₀01 and Coprobacillus.In the PICRUSt microbial function prediction analysis,there are 3 functional gene pathways were significantly different between the groups in level one,including Cellular processes,Genetic Information Processing and Human diseases.Besides,15 differential metabolic pathways were enriched in AML-CR group at the level two.Conclusion:1.In this study,the human GI bacteria is mainly composed of five species: Bacteroidetes,Firmicutes,Proteobacteria,Fusobacteria and Actinobacteria;besides,Bacteroidetes and Firmicutes account for the majority.2.The GI bacteria of patients with acute myeloid leukemia is different from the healthy population.The alpha diversity of the leukemia patients' GI bacteria is reduced,and there is a significant difference in the beta diversity between the two groups.3.The use of antibiotics will affect the GI bacteria,which caused the alpha diversity of GI bacteria to decrease,and there are significant differences in beta diversity.4.Leukemia and the exposure of antibiotics will change the components of GI bacteria and the metabolic pathways of bacteria,besides,most of these pathways are down-regulated in leukemia patients.5.The effect of induction remission chemotherapy has no significant correlation with the overall diversity of GI bacteria before chemotherapy,it means the diversity of GI bacteria before chemotherapy cannot be used to evaluate the effect of induction remission chemotherapy.However,it may be related to the recent exposure to antibiotics and the choice of chemotherapy,and more research is needed in this area.6.Although there is no significant difference in the overall diversity of GI bacteria between groups,there are still differences in the composition of the bacteria,it means there are changes in the abundance of some species and changes in metabolic pathways.