A Preliminary Study For The Difference Of Gastrointestinal Microbiota In Patients With Newly Diagnosed Acute Myeloid Leukemia Before And After Chemotherapy

Objectives:Acute myeloid leukemia(AML)is a malignant clonal disease originating from hematopoietic stem cells/progenitor cells.It is the most common type of adult leukemia.Studies have shown that it is related to chemotherapy drugs,ionizing radiation,genetic factors,viral infection,immune dysfunction,environment and other factors.gastrointestinal microbiota is the largest and most complex community of human microorganisms,which is closely related to the normal life activities,internal environment stability and immune system of human body.In recent decades,more and more scholars have focused on the study of gastrointestinal microbiota,finding that gastrointestinal microbiota can affect the therapeutic effect of tumor by regulating the inflammatory response of host.In recent years,more and more scholars have focused on the study of the relationship between gastrointestinal microbiota and leukemia.Some studies have shown that there are obvious differences between the gastrointestinal microbiota of leukemia patients and normal people.Studies have also shown that in childhood acute lymphoblastic leukemia,gastrointestinal microbiota can predict the risk of infection after chemotherapy.The use of chemotherapy and antibiotics is an important part of the treatment of acute myeloid leukemia.The use of chemotherapy and antibiotics will lead to the change of gastrointestinal microbiota,which may lead to the colonization and proliferation of opportunistic pathogens and then cause some diseases and affect the normal immune function of the body.Little is known about the composition of gastrointestinal microbiota in patients with acute myeloid leukemia,the mechanism of action,and the effect of chemotherapy on it.Therefore,by studying the composition of gastrointestinal microbiota in newly diagnosed patients with AML and the changes before and after chemotherapy,we explored its significance in the treatment process of patients with AML,so as to provide help for the diagnosis and treatment of AML.Methods:1.Subjects’ recruitment and sample collectionPatients who were first diagnosed with AML according to WHO criteria in 2016 were selected as the experimental object,and stool samples from patients in the experimental group were collected as the experimental group(a total of 47cases).According to the use of antibiotics in recent 3 months and whether they received induced remission chemotherapy,the experimental group was divided into4 subgroups including antibiotics before chemotherapy(AML-Y1 group,n=23),no antibiotic treatment before chemotherapy(AML-N1 group,n=11),antibiotic treatment before and after chemotherapy(AML-Y2 group,n=9),and no antibiotics before chemotherapy and antibiotics after chemotherapy(AML-N2,n=4).The fecal samples of 21 healthy family members and 8 healthy adults from Nanchang City were used as healthy control group(CON group,n=21).A total of 38 patients in the experimental group who received induction chemotherapy in our hospital were followed up.According to the efficacy of chemotherapy,they were divided into complete response group(HLQ-CR group,n=18)and incomplete response group(HLQ-NR group,n=20).2.Experimental steps(1)Sample collection:The fecal samples of the patients in the experimental group were collected when they were the first day of chemotherapy before and after chemotherapy regimen.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 two week before the test.Collected the samples were stored in a-80 ° C refrigerator.(2)Genomic DNA extraction and sequencing: Genomic DNA was extracted、amplified and purified by PCR;The V3-V4 region of bacteria was sequenced using 16 S r RNA high-throughput sequencing technology on Illumina-Misq platform and analysis biological information.(3)Comparison of differences:1.Comparison of gastrointestinal microbiota of newly diagnosed AML patients before and after chemotherapy;2.Comparison of gastrointestinal microbiota at initial diagnosis between complete response group and incomplete response group after induction remission chemotherapy.(4)Statistical analysis: Statistical analysis: SPSS 23.0 statistical software was used in this study to analyze and process the experimental data.Kruskal Wallis was used to examine the gender and age distribution differences between the study groups.The mean is used to represent the measurement data.Wilcoxon test was used to compare the α diversity index between the two groups of samples and species differences between the two groups.Kruskal Wallis test was used to compare the differences between samples larger than the two groups.(P < 0.05 was statistically significant)Results:1.it’s dominant with Bacteroidetes,Firmicutes,Proteobacteria,Actinobacteria and Fusobacteria,Bacteroidetes and Firmicutes account for the majority in the patients with newly diagnosed AML and healthy controls.2.Observed＿species,Shannon,PD＿Whole＿tree and Simpson indexes were used to evaluate the α diversity of gastrointestinal microbiota in this study.The results of Observed＿species,PD＿whole＿tree was follows : AML-Y1 group < CON group(P <0.001);AML-Y1 group < AML-N1 group(P < 0.001);AML-N1 group < CON group(P <0.001);AML-Y1 group < AML-Y2 group(P < 0.001);AML-N1 group was < AML-N2group(P < 0.05);AML-Y2 group < CON group(P > 0.05);AML-N2 group < CON group(P > 0.05).The results of Shannon and Simpson index was follows : AML-Y1 group <CON group(P < 0.001);AML-Y1 group < AML-N1 group(P < 0.001);AML-N1 group <CON group(P > 0.05);AML-Y1 group < AML-Y2 group(P < 0.001);AML-N1 group <AML-N2 group(P > 0.05);AML-Y2 group < CON group(P > 0.05);AML-N2 group < CON group(P > 0.05).3.UPGMA analysis and PCOA analysis were used to evaluate the β diversity of gastrointestinal microbiota between groups.there was no significant difference inβ-diversity of gastrointestinal microbiota between AML-Y1 and AML-N1 groups,but there was significant difference in β-diversity between CON and the other two groups in the comparison of AML-Y1,AML-N1 and CON groups.Compared with AML-Y2 group,AML-Y1 group and CON group,AML-Y2 group had significant difference in β-diversity compared with AML-Y1 group,but had no significant difference compared with CON group.there was no significant difference inβ-diversity between AML-N2 group and CON group,but there was significant difference between AML-N1 group and CON group in the comparison of AML-N2 group,AML-N1 group and CON group.4.Adonis analysis was used for the difference between groups.we find that the differences between the AML-N1 group and the CON group(P=0.001),the AML-Y1 group and the AML-N1 group(P=0.001),the AML-Y1 group and the CON group(P=0.001),the AML-Y1 group and the AML-Y2 group(P=0.001),the AML-N1 group and the AML-N2 group(P=0.003)were statistically significant.5.LEFSE analysis was used to detect species with differences between groups.In the comparison between the AML-N1 group and the Con group,the high relatively abundant species of the AML-N1 group were Coreobacteriaceae and Collinsella.The high relatively abundant species of the CON group were Fusobacteria,Acidobacteria,Actinobacteria,Fusobacteria,Fusobacteriales,Betaproteobacteriales,Proteobacteria,V eillonellaceae,Bacteroidaceae,Leptotrichiaceae,Bacteroides,Fusobacterium,Prevotell a7,Erysipelotrichaceae＿UCG＿003,Lepotrichia.In the comparison between AML-Y1 group and CON group,the high relatively abundant species of the CON group were Fusobacteria,Fusobacteriia,Fusobacteriales,Prevotellaceae,and Lachnospiraceae.In the comparison of AML-Y1,AML-N1 and CON groups,the high relatively abundant species of the AML-Y1 group were Bacteroidaceae,Tannerellaceae,Bacteroides,Parabacteroides.the high relatively abundant species of the AML-N1 group were Prevotellaceae,Lachnospiraceae,Roseburia,Eubacterium＿＿coprostanoligenes＿group,Prevotellaceae,Lachnospiraceae.the high relatively abundant species of the CON group were Fusobacteria,Fusobacteriia,Fusobacteriales.In the comparison between AML-Y1 group and AML-Y2 group,the high relatively abundant species of AML-Y2 group was Actinobacteria,Fusobacteria,Fusobacteria,Actinobacteria,Bacilli,Betaproteobacteriales,Aromonadales,Lactobacillales,Fus Obacteriales,Streptococcaceae,Prevotellaceae,Streptococcus,etc.In the comparison between AML-N1 group and AML-N2 group,the high relatively abundant species of AML-N2 group were Epsilonbacteraeota,Campylobacteria,Alphaproteobacteriac,SAR11＿clade,Campylobacterales,Lactobacillaceae,Enterococcaceae,LActobacillus,Uncultured,Uncultured＿bacterium,etc.,the relative abundant specificity of the AML-N1 group is Eubacterium＿＿hallii＿group.6.Kruskal＿Wallis test was used to detect species with different relative abundance of TOP10 among AML-Y1 group,AML-N1 group and CON group.The species with differences among groups were Bacteroides,Parabacteroides,Fusobacterium,Roseburia,Blautia,Agathobacter,Clostridium＿Sensu＿Stricto＿1,Romboutsia,Lachnospira,Bacteroides＿the Taiotaomicron,Parabacteroides＿Johnsonii＿Cl02T12C29,Streptococcus＿pneumoniae,Dorea＿formicigenerans＿ATCC＿27755,Ruminococcus＿sp.＿unk.MGS-30,Porites＿Australiensis,Lactobacillus＿mucosae,Porphyromonas＿endodontalis,Clostridiu M＿perfringens,Dialister＿pneumosintes.Compared with AML-Y2 group and AML-Y1 group,the species with differences among groups were Sutterella,Streptococcus,Alloprevotella,Fusobacterium,Lactobacillus,Bar Nesiella,Prevotella＿7,Erysipelatoclostridium,Leptotrichia,Haemophil us,Streptococcus＿salivarius＿subsp.＿thermophilus,Streptococcus＿pneumoniae,Lacto bacillus＿fermentum,Porites＿Australiensis,Porphyromonas＿endodontalis,Actinomyce s＿Graevenitzii＿F0530,Lactobacillus＿Murinus,Clostridiales＿Bac Terium＿Canine＿Oral＿T axon＿100,Dialister＿Pneumosintes,and AML-Y1 species with higher relative richness were Des＿Johnsonii＿Cl02T12C29.Comparison of species differences between AML-N1 group and AML-N2 group at the genus level,Bifidobacterium,Parabacteroides,Lactobacillus,Rikenellaceae＿RC9＿gut＿group,Enterococcus,Clade＿IB,Oscillibacter and Lachnospiraceae＿UCG-006 were relatively abundant species in AML-N1 group.At the species level,Streptomyces＿sp,Lactobacillus＿gasseri,ALactobacillus＿rhamnosus,and Lactobacillus＿iners＿AB-1 were relatively rich species in AML-N2 group,Lactobacillus＿murinus,Pseudomonas＿poae,and ospiraceae＿bacte28-4 rium in the AML-N1 group.7.Picrust microbial function prediction was used to analyze the differences between groups.In the comparison of AML-Y1 group,AML-N1 group and CON group,significant differences in 8 major gene functional pathways were found among the three groups at Level 1(P < 0.05).Comparison of differences between groups showed that there were significant differences in 41 gene functional pathways among the three groups at Level 2(P < 0.05).The CON group had more metabolic pathways in the circulatory system,cardiovascular disease,cell proliferation and apoptosis,cell monitoring,energy metabolism,amino acid metabolism,lipid metabolism and other metabolic pathways.The functional pathways in the circulatory system,cardiovascular disease,neurodegenerative disease,signaling molecules and interactions,cell communication,polysaccharide biosynthesis and metabolism,sensory system,transport and catabolism in the AML-N1 group were fewer than those in the AML-Y1 group,and the other 33 functional metabolic pathways were more than those in the AML-Y1 group.At Level 1,there was only significant difference in genetic information between AML-Y1 group and AML-Y2 group(P=0.047).There were significant differences between AML-N1 group and AML-N2 group in three aspects of genetic information,biological system and non-function(P< 0.05).There were 12 different gene functional pathways between AML-Y1 group and AML-Y2 group(P < 0.05),and AML-Y2 group had more metabolic pathways in cardiovascular disease,circulatory system,digestion and absorption,cell proliferation and apoptosis,cell transfer and excretion,etc.AML-N1 group and AML-N2 group have 13 different gene functional pathways,and AML-N2 group has more metabolic pathways in cardiovascular disease,circulatory system,cell transfer,excretion and other metabolic pathways.8.In the comparison between HLQ-CR group,HLQ-NR group,and CON group,it was found that AML-CR group > AML-NR group(P>0.05),CON group > AML-CR group,CON group > AML-NR group(P <0.05)in the α diversity,it illustrates that there was no significant difference in GI bacteria alpha diversity between AML-CR group and AML-NR group and there was significant difference between CON and the other two groups.The PCo A analysis and UPGMA analysis were used to evaluate the beta diversity of GI bacteria,and it was found that there was no significant difference inβ-diversity between HLQ-CR group and HLQ-N group,but there was significant difference between CON group and the other two groups.Adonis analysis showed no statistically significant difference between HLQ-CR and HLQ-NR.LEFSE analysis showed that the species with high abundance and relative specificity in the AML-CR group were Aeromonas,Prevotella＿7,Insominimonas,and Aeromonadaceae.species with high abundance and relative specificity in the AML-CR group were SAR11＿CLADE,Microscillaceae,Clade＿III,Acetobacter,Prevotellaceae＿ucg＿003,Lachnospiraceae＿ucg＿010,Agathobac ter,Parasutterella.In the PICRUSt microbial function prediction analysis,we found no statistical difference between the HLQ-CR group and the HLQ-NR group at Level 1,Level 2 and levels.Conclusion:1.The gastrointestinal microbiota of human is mainly composed of five species: Bacteroidetes,Firmicutes,Proteobacteria,Actinobacteria and Fusobacteria.,Bacteroidetes and Firmicutes account for the majority.2.There were significant differences in the composition of gastrointestinal microbiota between newly diagnosed AML patients and healthy people.The αdiversity of gastrointestinal microbiota in newly diagnosed AML patients was lower than healthy people and the β diversity was significantly different with healthy people.3.The use of antibiotics will affect the gastrointestinal microbiota,which caused the alpha diversity of gastrointestinal microbiota to decrease,and there are no significant differences in beta diversity.4.The use of antibiotics and leukemia can lead to changes in the composition and functional pathways of gastrointestinal microbiota and the number of pathways is significantly reduced in newly diagnosed AML patients5.In the gastrointestinal microbiota of newly diagnosed AML patients receiving induction remission chemotherapy,the α diversity in the AML-Y2 group was significantly higher than AML-Y1 group,but had no significant difference compared with that of the CON group,and the diversity of β was significantly higher than that of the AML-Y1 group,but had no significant difference compared with CON group.The α diversity in AML-N2 group was significantly higher than that in AML-N1 group,but not significantly different from that in CON group.The β diversity in AML-N1 group was not significantly different from that in CON group,but was significantly different from that in AML-N1 group.6.Induction remission chemotherapy can lead to changes in gastrointestinal microbiota composition and functional pathways in newly diagnosed AML patients,and functional pathways are significantly increased after chemotherapy compared with before chemotherapy.7.There was no statistically significant difference in α and β diversity of gastrointestinal microbiota between the remission group and the non-remission group after induction remission chemotherapy at the time of initial diagnosis.The diversity of gastrointestinal microbiota before chemotherapy cannot be used to evaluate the efficacy of induced remission chemotherapy.8.there were differences in the abundance of some species of gastrointestinal microbiota between the complete response group and the incomplete response group after induced remission chemotherapy at initial diagnosis,and there was no significant difference in functional pathways.