[Genetic analysis of a case of B-acute lymphoblastic leukaemia with double Philadelphia chromosomes and double derivative chromosome 9s].

OBJECTIVE: To explore the genetic basis for a rare case of acute B-lymphocytic leukemia (B-ALL) with double Philadelphia chromosomes (Ph) and double derivative chromosome 9s [der(9)]. METHODS: A patient with double Ph and double der(9) B-ALL who presented at Shanghai Zhaxin Intergrated Traditional Chinese and Western Medicine Hospital in June 2020 was selected as the subject. Bone marrow morphology, flow cytometry, G-banding karyotyping, fluorescence in situ hybridization (FISH), genetic testing and chromosomal microarray analysis (CMA) were used to analyze bone marrow samples from the patient at various stages. RESULTS: At initial diagnosis, the patient's bone marrow morphology and flow immunotyping have both supported the diagnosis of B-ALL. G-banded karyotyping of the patient indicated double Ph, in addition with hyperdiploid chromosomes involving translocations between chromosomes 9 and 22. BCR-ABL1 fusion gene was positive. Genetic testing at the time of recurrence revealed presence of a heterozyous c.944C>T variant in the kinase region of the ABL1 gene. FISH showed a signal for ABL1-BCR fusion on both chromosome 9s. CMA showed that the mosaicism homozygosity ratio of chromosome 9 was about 40%, and the mosaicism duplication ratio of chromosome 22 was about 43%. CONCLUSION: Since both der(9) homologs were seen in 40% of cells, the possible mechanism for the double der(9) in this patient may be similar to that of double Ph, which might have resulted from non-disjunction during mitosis in the Ph chromosome-positive cell clone.

A metagenome-wide association study of gut microbiota in type 2 diabetes.

Assessment and characterization of gut microbiota has become a major research area in human disease, including type 2 diabetes, the most prevalent endocrine disease worldwide. To carry out analysis on gut microbial content in patients with type 2 diabetes, we developed a protocol for a metagenome-wide association study (MGWAS) and undertook a two-stage MGWAS based on deep shotgun sequencing of the gut microbial DNA from 345 Chinese individuals. We identified and validated approximately 60,000 type-2-diabetes-associated markers and established the concept of a metagenomic linkage group, enabling taxonomic species-level analyses. MGWAS analysis showed that patients with type 2 diabetes were characterized by a moderate degree of gut microbial dysbiosis, a decrease in the abundance of some universal butyrate-producing bacteria and an increase in various opportunistic pathogens, as well as an enrichment of other microbial functions conferring sulphate reduction and oxidative stress resistance. An analysis of 23 additional individuals demonstrated that these gut microbial markers might be useful for classifying type 2 diabetes.

Metagenomic analysis of faecal microbiome as a tool towards targeted non-invasive biomarkers for colorectal cancer.

OBJECTIVE: To evaluate the potential for diagnosing colorectal cancer (CRC) from faecal metagenomes. DESIGN: We performed metagenome-wide association studies on faecal samples from 74 patients with CRC and 54 controls from China, and validated the results in 16 patients and 24 controls from Denmark. We further validated the biomarkers in two published cohorts from France and Austria. Finally, we employed targeted quantitative PCR (qPCR) assays to evaluate diagnostic potential of selected biomarkers in an independent Chinese cohort of 47 patients and 109 controls. RESULTS: Besides confirming known associations of Fusobacterium nucleatum and Peptostreptococcus stomatis with CRC, we found significant associations with several species, including Parvimonas micra and Solobacterium moorei. We identified 20 microbial gene markers that differentiated CRC and control microbiomes, and validated 4 markers in the Danish cohort. In the French and Austrian cohorts, these four genes distinguished CRC metagenomes from controls with areas under the receiver-operating curve (AUC) of 0.72 and 0.77, respectively. qPCR measurements of two of these genes accurately classified patients with CRC in the independent Chinese cohort with AUC=0.84 and OR of 23. These genes were enriched in early-stage (I-II) patient microbiomes, highlighting the potential for using faecal metagenomic biomarkers for early diagnosis of CRC. CONCLUSIONS: We present the first metagenomic profiling study of CRC faecal microbiomes to discover and validate microbial biomarkers in ethnically different cohorts, and to independently validate selected biomarkers using an affordable clinically relevant technology. Our study thus takes a step further towards affordable non-invasive early diagnostic biomarkers for CRC from faecal samples.

Involvement of miR-125a in resistance to daunorubicin by inhibiting apoptosis in leukemia cell lines.

In this study, we investigated whether miR-125a participated in the resistance of the leukemia cell lines to the chemotherapeutic agent daunorubicin. Higher expression of miR-125a is correlated with lower treatment response and shorter overall survival in acute leukemia patients. Overexpression of miR-125a induced drug resistance in HL-60, K562, and THP-1cell lines through reducing apoptosis. We also showed that miR-125a mediated daunorubicin resistance in leukemia cell lines through the decrease of GRK2 and Puma which were proved to be direct targets of miR-125a. This study may provide novel therapeutic targets for therapy and improve predictions of therapeutic responses in leukemia to daunorubicin.

Consistent signatures in the human gut microbiome of old- and young-onset colorectal cancer.

The incidence of young-onset colorectal cancer (yCRC) has been increasing in recent decades, but little is known about the gut microbiome of these patients. Most studies have focused on old-onset CRC (oCRC), and it remains unclear whether CRC signatures derived from old patients are valid in young patients. To address this, we assembled the largest yCRC gut metagenomes to date from two independent cohorts and found that the CRC microbiome had limited association with age across adulthood. Differential analysis revealed that well-known CRC-associated taxa, such as Clostridium symbiosum, Peptostreptococcus stomatis, Parvimonas micra and Hungatella hathewayi were significantly enriched (false discovery rate <0.05) in both old- and young-onset patients. Similar strain-level patterns of Fusobacterium nucleatum, Bacteroides fragilis and Escherichia coli were observed for oCRC and yCRC. Almost all oCRC-associated metagenomic pathways had directionally concordant changes in young patients. Importantly, CRC-associated virulence factors (fadA, bft) were enriched in both oCRC and yCRC compared to their respective controls. Moreover, the microbiome-based classification model had similar predication accuracy for CRC status in old- and young-onset patients, underscoring the consistency of microbial signatures across different age groups.

Mendelian randomization analyses reveal causal relationships between the human microbiome and longevity.

Although recent studies have revealed the association between the human microbiome especially gut microbiota and longevity, their causality remains unclear. Here, we assess the causal relationships between the human microbiome (gut and oral microbiota) and longevity, by leveraging bidirectional two-sample Mendelian randomization (MR) analyses based on genome-wide association studies (GWAS) summary statistics of the gut and oral microbiome from the 4D-SZ cohort and longevity from the CLHLS cohort. We found that some disease-protected gut microbiota such as Coriobacteriaceae and Oxalobacter as well as the probiotic Lactobacillus amylovorus were related to increased odds of longevity, whereas the other gut microbiota such as colorectal cancer pathogen Fusobacterium nucleatum, Coprococcus, Streptococcus, Lactobacillus, and Neisseria were negatively associated with longevity. The reverse MR analysis further revealed genetically longevous individuals tended to have higher abundances of Prevotella and Paraprevotella but lower abundances of Bacteroides and Fusobacterium species. Few overlaps of gut microbiota-longevity interactions were identified across different populations. We also identified abundant links between the oral microbiome and longevity. The additional analysis suggested that centenarians genetically had a lower gut microbial diversity, but no difference in oral microbiota. Our findings strongly implicate these bacteria to play a role in human longevity and underscore the relocation of commensal microbes among different body sites that would need to be monitored for long and healthy life.

Evolution and transmission of antibiotic resistance is driven by Beijing lineage Mycobacterium tuberculosis in Vietnam.

IMPORTANCE: Drug-resistant tuberculosis (TB) infection is a growing and potent concern, and combating it will be necessary to achieve the WHO's goal of a 95% reduction in TB deaths by 2035. While prior studies have explored the evolution and spread of drug resistance, we still lack a clear understanding of the fitness costs (if any) imposed by resistance-conferring mutations and the role that Mtb genetic lineage plays in determining the likelihood of resistance evolution. This study offers insight into these questions by assessing the dynamics of resistance evolution in a high-burden Southeast Asian setting with a diverse lineage composition. It demonstrates that there are clear lineage-specific differences in the dynamics of resistance acquisition and transmission and shows that different lineages evolve resistance via characteristic mutational pathways.

Administration of granulocyte-macrophage colony-stimulating factor enhanced chimeric antigen receptor T-cell expansion and cellular immunity recovery without inducing cytokine release syndrome.

Background: Neutropenia and cytokine release syndrome (CRS) are two major toxicities of chimeric antigen receptor (CAR)-T cell therapy. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is an ideal candidate treatment for neutropenia except for its potential aggravation of CRS. We hypothesized that the optimal timing of supplemental with GM-CSF in a shortage of host immunity and CAR T-cell was chosen as avoidance of CRS. In the study we evaluated the safety and efficacy of GM-CSF intervention post-CAR T-cell therapy while circulating CAR T-cell declined. Materials and methods: Nine patients received GM-CSF therapy who displayed moderate neutropenia with absolute neutrophil counts (ANC) < 1,500 cells/mm3 with concomitant declination of circulating CAR T-cell. Results: The median duration of GM-CSF intervention was 15 days (4-30). CAR T-cell expansion was observed in peripheral blood (PB) of seven patients (7/9). The median baseline and peak CAR T cells count in PB of the seven patients with CAR T-cell expansion were 0.85 × 106/L (0-50.9) and 6.06 × 106/L (1.43-112.55). And the peaks of CAR T-cell levels in PB appeared in day 7 (2-11) following the initiation of GM-CSF administration with increases of 2.84 × 106/L (0.38-61.65). Also, increased white blood cells in PB were observed in all patients. The median onset and duration time of WBC recovery were 9 (1-14) and 17 (3-53) days. Moreover, the increment of WBC, neutrophil, lymphocyte and CD3-CD16 + CD56 + natural killer cell in PB was observed. In addition, no CRS or fatal infection occurred during GM-CSF treatment. Conclusion: This study provides evidence for the clinical feasibility of combining CAR T-cell therapy with the GM-CSF to treat neutropenia patients with concomitant declination of circulating CAR T-cell.

Combined effects of host genetics and diet on human gut microbiota and incident disease in a single population cohort.

Human genetic variation affects the gut microbiota through a complex combination of environmental and host factors. Here we characterize genetic variations associated with microbial abundances in a single large-scale population-based cohort of 5,959 genotyped individuals with matched gut microbial metagenomes, and dietary and health records (prevalent and follow-up). We identified 567 independent SNP-taxon associations. Variants at the LCT locus associated with Bifidobacterium and other taxa, but they differed according to dairy intake. Furthermore, levels of Faecalicatena lactaris associated with ABO, and suggested preferential utilization of secreted blood antigens as energy source in the gut. Enterococcus faecalis levels associated with variants in the MED13L locus, which has been linked to colorectal cancer. Mendelian randomization analysis indicated a potential causal effect of Morganella on major depressive disorder, consistent with observational incident disease analysis. Overall, we identify and characterize the intricate nature of host-microbiota interactions and their association with disease.

Substantial Fat Loss in Physique Competitors Is Characterized by Increased Levels of Bile Acids, Very-Long Chain Fatty Acids, and Oxylipins.

Weight loss and increased physical activity may promote beneficial modulation of the metabolome, but limited evidence exists about how very low-level weight loss affects the metabolome in previously non-obese active individuals. Following a weight loss period (21.1 ± 3.1 weeks) leading to substantial fat mass loss of 52% (−7.9 ± 1.5 kg) and low body fat (12.7 ± 4.1%), the liquid chromatography-mass spectrometry-based metabolic signature of 24 previously young, healthy, and normal weight female physique athletes was investigated. We observed uniform increases (FDR < 0.05) in bile acids, very-long-chain free fatty acids (FFA), and oxylipins, together with reductions in unsaturated FFAs after weight loss. These widespread changes, especially in the bile acid profile, were most strongly explained (FDR < 0.05) by changes in android (visceral) fat mass. The reported changes did not persist, as all of them were reversed after the subsequent voluntary weight regain period (18.4 ± 2.9 weeks) and were unchanged in non-dieting controls (n = 16). Overall, we suggest that the reported changes in FFA, bile acid, and oxylipin profiles reflect metabolic adaptation to very low levels of fat mass after prolonged periods of intense exercise and low-energy availability. However, the effects of the aforementioned metabolome subclass alteration on metabolic homeostasis remain controversial, and more studies are warranted to unravel the complex physiology and potentially associated health implications. In the end, our study reinforced the view that transient weight loss seems to have little to no long-lasting molecular and physiological effects.

Metagenome-genome-wide association studies reveal human genetic impact on the oral microbiome.

The oral microbiota contains billions of microbial cells, which could contribute to diseases in many body sites. Challenged by eating, drinking, and dental hygiene on a daily basis, the oral microbiota is regarded as highly dynamic. Here, we report significant human genomic associations with the oral metagenome from more than 1915 individuals, for both the tongue dorsum (n = 2017) and saliva (n = 1915). We identified five genetic loci associated with oral microbiota at study-wide significance (p < 3.16 × 10-11). Four of the five associations were well replicated in an independent cohort of 1439 individuals: rs1196764 at APPL2 with Prevotella jejuni, Oribacterium uSGB 3339 and Solobacterium uSGB 315; rs3775944 at the serum uric acid transporter SLC2A9 with Oribacterium uSGB 1215, Oribacterium uSGB 489 and Lachnoanaerobaculum umeaense; rs4911713 near OR11H1 with species F0422 uSGB 392; and rs36186689 at LOC105371703 with Eggerthia. Further analyses confirmed 84% (386/455 for tongue dorsum) and 85% (391/466 for saliva) of host genome-microbiome associations including six genome-wide significant associations mutually validated between the two niches. As many of the oral microbiome-associated genetic variants lie near miRNA genes, we tentatively validated the potential of host miRNAs to modulate the growth of specific oral bacteria. Human genetics accounted for at least 10% of oral microbiome compositions between individuals. Machine learning models showed that polygenetic risk scores dominated over oral microbiome in predicting risk of dental diseases such as dental calculus and gingival bleeding. These findings indicate that human genetic differences are one explanation for a stable or recurrent oral microbiome in each individual.

Simultaneous suppression of multidrug resistance and antiapoptotic cellular defense induces apoptosis in chemoresistant human acute myeloid leukemia cells.

The emergence of acquired drug resistance is a major hurdle in the successful treatment of leukemia. Researches indicated that the main mechanisms of most cancers included so-called "pump" and "nonpump" resistance. We studied the mechanisms involved in the drug resistance of HL-60/ADR and found that its drug resistance was associated with the simultaneous overexpression of XIAP and MRP. We compared the reversal effects of XIAP and MRP ASO used in combination and separately. Reverse transcription-PCR and Western blot were applied to examine the changes of mRNA and protein levels, respectively. The results showed that XIAP and MRP ASO used separately could down-regulate the expression of XIAP and MRP in HL-60/ADR cells, respectively. XIAP and MRP ASO used in combination did not enhance the inhibition expression of XIAP and MRP of HL-60/ADR cells. The apoptosis of co-transfection group was significantly higher than XIAP ASO (P<0.05). The cytotoxicity was determined by MTT cell viability/proliferation assay. When used in combination the sensitivity of HL-60/ADR cells to DNR was increased significantly compared with XIAP or MRP ASO used separately (P<0.05). The results indicated that both XIAP and MRP were involved in the drug resistance mechanisms of HL-60/ADR cells. The sensitivity to DNR could be enhanced significantly when XIAP and MRP ASO used in combination.

Alterations in Gastric Microbiota After H. Pylori Eradication and in Different Histological Stages of Gastric Carcinogenesis.

The role of bacteria other than Helicobacter pylori (HP) in the stomach remains elusive. We characterized the gastric microbiota in individuals with different histological stages of gastric carcinogenesis and after receiving HP eradication therapy. Endoscopic gastric biopsies were obtained from subjects with HP gastritis, gastric intestinal metaplasia (IM), gastric cancer (GC) and HP negative controls. Gastric microbiota was characterized by Illumina MiSeq platform targeting the 16 S rDNA. Apart from dominant H. pylori, we observed other Proteobacteria including Haemophilus, Serratia, Neisseria and Stenotrophomonas as the major components of the human gastric microbiota. Although samples were largely converged according to the relative abundance of HP, a clear separation of GC and other samples was recovered. Whilst there was a strong inverse association between HP relative abundance and bacterial diversity, this association was weak in GC samples which tended to have lower bacterial diversity compared with other samples with similar HP levels. Eradication of HP resulted in an increase in bacterial diversity and restoration of the relative abundance of other bacteria to levels similar to individuals without HP. In conclusion, HP colonization results in alterations of gastric microbiota and reduction in bacterial diversity, which could be restored by antibiotic treatment.

[A modified cytogenetic study for multiple myeloma].

OBJECTIVE: To evaluate the effect of modified culture method used to cytogenetic analysis and the clinically significance of chromosomal abnormalities to multiple myeloma (MM). METHODS: Mononuclear cells were isolated from bone marrow aspirate of 20 MM patients; and then cultured for 3 days without any cytokines, and 6 days in the presence of IL-6 (10 ng/mL) and GM-CSF (30 ng/mL) before RHG banding analysis; the remained part of aspirates were treated directly. Eight cases of iron deficiency anemia were taken as control. RESULTS: The experiment was failure in 2 cases because of blood clot, and another 2 cases could be analyzed only by direct method due to inadequate cells. The karyotype abnormalities were found from 4 cases of 16 available patients. Of them, three cases had complex karyotypes. The abnormalities were detected after 6 days culture with addition of cytokines. No abnormalities were detected from those groups of directly analysis and 3 day culture. Meantime, the clinical data showed that the patients with cytogenetic abnormalities were in stage III, and had a high percentage of MM cells (25%-56%) in their bone marrow, and also poor responses to prior chemotherapy. No cytogenetic abnormalities were found from control individuals in all groups. CONCLUSION: Extended culture in the presence of cytokines could improve the efficiency of cytogenetic analysis to MM. Complex karyotype was common cytogenetic abnormalities in MM patients with poor response to chemotherapy.

[Partial deletion of chromosome 13 long arm in multiple myeloma and its clinical significance].

OBJECTIVE: To explore the specific locus deletion of the long arm of chromosome 13 and its relationship with the clinical behavior and prognosis of multiple myeloma (MM). METHODS: FISH analysis was performed on bone marrow smears from 68 patients with MM to study the deletion of Rb-1 gene and locus 13q14.3 on chromosome 13. The statistic value of its effect on clinical features were determined. RESULTS: 35 out of the 68 (51%) cases were found with deletion of chromosome 13; deletion of Rb-1 gene were found in 29 (43%) cases; deletion of locus 13q14.3 were found in 23 out of 44 (52%) cases; the analysis results were same in 66% of the cases (29/44) with the above two probes. Chi-square test showed that partial deletion of chromosome 13 was associated with clinical behavior, early chemotherapy response and 1 year survival. CONCLUSION: Deletion of Rb-1 gene and locus 13q14.3 were both common cytogenetic changes in MM patients with effect on the biological behavior of the disease.

[An analysis of leukocyte subsets chimerism following allogeneic peripheral blood stem cell transplantation].

OBJECTIVE: To analyse the relationship of T lymphocyte and granulocyte chimerism following allogeneic peripheral blood cell transplantation and the occurrence of relapse, graft failure and graft versus host disease. METHODS: 21 patients underwent allogeneic peripheral blood stem cell transplantation (allo-PBSCT). Fluorescence-activated cell sorter (FACS) sorted CD3+ T lymphocytes and CD15+ granulocyte from peripheral blood of all the patients were analyzed for short tandem repeats in 7 days interval for 1 month starting from the day of PBSCT, then 1 month interval for 6 months, and then 3 months interval to the end of one year. RESULTS: Chimerism of granulocyte was higher than T lymphocyte on day 7 posttransplant in 4 patients given myeloablative conditioning. The median donor chimerism of granulocyte and T lymphocyte was 95% and 55% respectively. The other 17 patients had higher chimerism of T lymphocyte than granulocyte on day 7, which was 60% (15%-76%) and 0% (0%-40%) respectively. 20 patients reached complete donor chimerism (CDC) on day 21 (14-102 days) for T lymphocyte and on day 14 for granulocyte, except one relapsed on day 28. Seven patients had decreasing mixed chimerism when disease relapsed or graft failure occurred. T cell donor chimerism decreased earlier than myeloid cells, however, bone marrow sample and granulocyte still remained in complete donor chimerism or stable mixed chimerism, bone marrow smear showed normal at the same time. CONCLUSION: Blood leukocyte subset chimerism analysis, especially T cell chimerism analysis may provide earlier information of engraftment, relapse and graft failure than blood and bone marrow samples, therefore immunomodulatory therapies may be given to recipients earlier and overall survival may be improved.

Chronic myelogenous leukemia-like hematological malignancy with t(8;22) in a 26-year-old pregnant woman: A case report.

t(8;22)(p11;q11) is a rare but recurrent genetic alteration in various hematological disorders. Patients with t(8;22)(p11;q11) may be misdiagnosed with chronic myelogenous leukemia (CML), due to the similar clinical features. Thus, the current study presents a patient with t(8;22)(p11;q11) who was previously misdiagnosed with CML in the chronic phase. The current patient was a 26-year-old woman who was 4-weeks pregnant and in whom an increased white blood cell count (4.0×1010/l) was found upon physical examination. The patient had no history of hematological disease. Although cytogenetics showed a normal karyotype and no breakpoint cluster region/Abelson murine leukemia viral oncogene homolog 1 (BCR/ABL) fusion gene was detected by reverse transcription-polymerase chain reaction, a diagnosis of chronic myelogenous leukemia (CML) was initially made according to the clinical and morphological features. Another 6 weeks later, t(8;22)(p11;q11) rearrangement was present in 9 out of 10 analyzed metaphases. Fluorescence in situ hybridization and reverse transcription-polymerase chain reaction indicated a negative result for the BCR/ABL fusion, but gave a positive result for the BCR-fibroblast growth factor receptor 1 fusion. A hematological diagnosis of atypical CML was again formed.

[Value of a Panel Fluorescence in Situ Hybridization in Three Kinds of Hematological Malignancies].

OBJECTIVE: To evaluate the role of a panel fluorescence in situ hybridization (Panel-FISH) for the detection of common cytogenetic abnormalities in patients with chronic lymphoblastic leukemia (CLL), multiplemyeloma (MM) and myelodysplastic syndrome (MDS). METHODS: Three panels of probes were used to perform FISH assays in 46 patients with CLL, 53 with MM and 93 with MDS. Their results were compared with that obtain by conventional cytogenetic examination. RESULTS: The panel FISH detection in CLL and MM groups showed significantly higher sensitivity in revealing chromosomal abnormalities than that in conventional cytogenetics (73.8% vs 9.5%, 70.8% vs 22.9%, respectively). There were significant differences between these 2 technologies(P<0.001, P<0.001, respectively). However, there was no difference between Panel-FISH and conventional cytogenetics in MDS group (30.4 vs 27.2%, P=0.625). CONCLUSION: Panel-FISH can increase the detection rate in CLL and MM patients while it did not in MDS patients. However, it can increase the detection rate of aberration clones in MDS cases with normal karyotypes or without enough karyotypes to be analysis.

A meta-analysis comparing clinical characteristics and outcomes in CALR-mutated and JAK2V617F essential thrombocythaemia.

A systematic review and meta-analysis were carried out to compare the clinical features and outcomes in calreticulin (CALR)-mutated and JAK2V617F patients of essential thrombocythemia (ET). Compared with JAK2V617F ET patients, CALR-mutated ET was associated with a clear increase in male predominance [OR 1.71 (95 % CI 1.28-2.28), P < 0.001, I(2)) = 51.6] and a significant decrease in thrombosis events [OR 0.40 (95 % CI 0.32-0.50), P < 0.001, I(2) = 0]. No difference was observed in hemorrhagic events [OR 0.86 (95 % CI 0.52-1.42), P = 0.558, I(2) = 0] or splenomegaly [OR 0.8 (95 % CI 0.55-1.14), P = 0.217 I (2) = 42.9]. CALR-mutated ET did not show better overall survival (OS) [HR 1.03 (95 % CI 0.74-1.44) P = 0.854, I(2) = 47.6] but showed better thrombosis-free survival (TFS) [HR 0.62 (0.44-0.87), P = 0.005, I(2) = 0] than JAK2V617F ET. In conclusion, CALR-mutated ET and JAK2V617F ET may represent two different subgroups of essential thrombocythaemia with respect to clinical features and outcomes.