Major Clones Research Articles

Molecular Characterization of Methicillin Resistant Staphylococcus aureus in West Bank-Palestine.

Background: Methicillin-resistant Staphylococcus aureus (MRSA) is a public health threat and a major cause of hospital-acquired and community-acquired infections. This study aimed to investigate the genetic diversity of MRSA isolates from 2015 to 2017 and to characterize the major MRSA clones and anti-biogram trends in Palestine.Methodology: Isolates were obtained from 112 patients admitted to different hospitals of West Bank and East Jerusalem, originating from different clinical sources. Antibiotic susceptibility patterns, staphylococcal chromosomal cassette mec (SCCmec) typing, and Staphylococcus aureus protein A (spa) typing were determined. Also, a panel of toxin genes and virulence factors was studied, including: Panton-Valentine Leukocidin (PVL), ACME-arcA, Toxic Shock Syndrome Toxin-1 (TSST-1), and Exfoliative Toxin A (ETA).Results: Of the 112 confirmed MRSA isolates, 100% were resistant to all β-lactam antibiotics. Resistance rates to other non- β-lactam classes were as the following: 18.8% were resistant to trimethoprim-sulfamethoxazole, 23.2% were resistant to gentamicin, 34.8% to clindamycin, 39.3% to ciprofloxacin, and 63.4% to erythromycin. All MRSA isolates were susceptible to vancomycin (100%). Of all isolates, 32 isolates (28.6%) were multidrug- resistant (MDR). The majority of the isolates were identified as SCCmec type IV (86.6%). The molecular typing identified 29 spa types representing 12 MLST-clonal complexes (CC). The most prevalent spa types were: spa type t386 (CC1)/(12.5%), spa type t044 (CC80)/(10.7%), spa type t008 (CC8)/(10.7%), and spa type t223 (CC22)/(9.8%). PVL toxin gene was detected in (29.5%) of all isolates, while ACME-arcA gene was present in 18.8% of all isolates and 23.2% had the TSST-1 gene. The two most common spa types among the TSST-1positive isolates were the spa type t223 (CC22)/(Gaza clone) and the spa type t021 (CC30)/(South West Pacific clone). All isolates with the spa type t991 were ETA positive (5.4%). USA-300 clone (spa type t008, positive for PVL toxin gene and ACME-arcA genes) was found in nine isolates (8.0%).Conclusions: Our results provide insights into the epidemiology of MRSA strains in Palestine. We report a high diversity of MRSA strains among hospitals in Palestine, with frequent SCCmec type IV carriage. The four prominent clones detected were: t386-IV/ CC1, the European clone (t044/CC80), Gaza clone (t223/CC22), and the USA-300 clone (t008/CC8).

Characterization of a novel, type II staphylococcal cassette chromosome mec element from an endemic oxacillin-resistant Staphylococcus lugdunensis clone in a hospital setting.

Staphylococcus lugdunensis is a significant pathogen that causes community-acquired and nosocomial infections. The high prevalence of oxacillin-resistant S. lugdunensis (ORSL) is of major concern. Resistance to β-lactams is caused by acquisition of the staphylococcal cassette chromosome mec (SCCmec) element. The cassette is highly diverse, both structurally and genetically, among CoNS. Isolates carrying SCCmec II-ST6 are the major persistent clones in hospitals. To investigate the structure and evolutionary origin of a novel type II SCCmec element in an endemic ST6 S. lugdunensis clone. The structure of the SCCmec II element carried by ST6 strain CGMH-SL118 was determined by WGS and compared with those reported previously. A novel 39 kb SCCmec element, SCCmecCGMH-SL118, with a unique mosaic structure comprising 41 ORFs integrated into the 3' end of the rlmH gene, was observed. Some regions of SCCmecCGMH-SL118 were homologous to SCCmec IIa of the prototype MRSA strain N315. The structure of SCCmecCGMH-SL118 was similar to that of SCCmec IIb of the MRSA strain, JCSC3063, mainly lacking the aminoglycoside resistance determinant pUB110 in the J3 region but containing the insertion sequence IS256 in the J2 region. Notably, SCCmecCGMH-SL118 deletions in the J1 region compared with SCCmec types IIa and IIb, and a high homology to SCCmec elements of Staphylococcus aureus JCSC4610 and Staphylococcus haemolyticus strain 621 were found. The genetic diversity of the type II SCCmec element in ORSL suggests that CoNS is a potential reservoir for interspecies transfer of SCCmec to S. aureus in hospitals.

The association of pili with the emergence and replacement of the major antibiotic resistant pneumococcal clones.

The aim of this study was to evaluate the presence of pilus islet 1 (PI-1) and to determine its clade type in pneumococcal isolates with reduced susceptibility to penicillin (penicillin non-susceptible pneumococci - PNSP) and/or resistant to macrolides isolated prior to and after the introduction of pneumococcal conjugate vaccines (PCVs) in the Czech Republic. Clinical isolates of serotypes 9V (n=68) and 19A (n=89) were examined. Isolates were characterised by multilocus sequence typing (MLST). The presence of PI-1 was determined by screening for the sortase B, C, and D genes located within PI-1. In the presence of PI-1 pilus, clade types were classified by PCR. In the pre-PCV period (2000-2007), the prevalence of PNSP was 3.9% and 2.7% of isolates were resistant to erythromycin. During 2012-2015 (post-PCV period), the rates of PNSP remained stable (3.6%), but resistance to erythromycin increased to 8.3%. While in 2000-2007, resistance to antibiotics was associated mainly with serotype 9V, in 2012-2015, it was replaced by serotype 19A. PI-1 positive isolates were seen in both serotypes. All isolates (68) of serotype 9V belonged to the Spain9V-3 (CC156) clone and carried PI-1 of clade type I while 96.5% (56/58) of isolates of 19A serotype belonged to the Netherlands15B-37 (CC199) clone and carried PI-1 of clade type II. Both major antibiotic resistant clones carried PI-1, although they differ in the clade type. Thus the role of PI-1 should be evaluated in further studies and potentially considered in the spread of antibiotic resistant clones.

Characterization of antibiotic-susceptibility patterns and virulence genes of five major sequence types of Escherichia coli isolates cultured from extraintestinal specimens: a 1-year surveillance study from Iran.

ObjectivesEscherichia coli sequence types (STs) 69, 73, 95, 127, and 131 are major STs frequently causing extraintestinal infections. The prevalence of specific clones and their virulence and resistance profiles has not been described from Iran. The aim of this study was to characterize antimicrobial-susceptibility profiles and virulence traits of five major clones of E. coli recovered from human extraintestinal infections in Semnan, Iran. We compared these traits between major ST clones and also between O25b and O16 subgroups of the ST131 clone.MethodsWe characterized the five major ST clones among 335 collected E. coli isolates obtained from extraintestinal infections, and phylogenetic groups, antimicrobial susceptibility, and virulence/resistance-gene profiles of these major STs were studied.ResultsThe highest rates of the multidrug-resistance phenotype were detected among ST131 (85.7%) and ST69 (41.7%), and trimethoprim/sulfamethoxazole resistance was detected significantly among the latter clone. Of the 151 isolates belonging to major ST clones, blaOXA-48 was detected among all except the ST127 clone, while blaNDM genes were harbored by 14 (9.2%) isolates, which all belonged to the ST131 clone. Aggregate virulence scores (median) of ST131 isolates (11) were slightly higher than ST69 (8.50) strains, but were lower than ST73 (16), ST95 (16), and ST127 (12.50) isolates. Principal-coordinate analysis revealed distinct virulence profiles with the ST131 clone. ST73, ST95 and ST131 were enriched with “urovirulence” traits, including phylogroup B2 and group B2-associated accessory traits (chuA, iutA, yfcV, papGII, usp, kpsMTII and malX) and the derived variables extraintestinal pathogenic E. coli and uropathogenic E. coli. In contrast, ST69 was depleted of these traits, but enriched with phylogroups D and E.ConclusionOur data emphasize that isolates of the ST131 clone have the ability to make a balance between resistance and virulence traits to establish a wider clone in extraintestinal pathogenic E. coli.

Population Structure of Pythium ultimum from Greenhouse Floral Crops in Michigan.

Pythium ultimum causes seedling damping-off and root and crown rot in greenhouse ornamental plants. To understand the population dynamics and assess population structure of P. ultimum in Michigan floriculture crops, simple sequence repeats (SSRs) were developed using the previously published P. ultimum predicted transcriptome. A total of 166 isolates sampled from 2011 to 2013 from five, one, and three greenhouses in Kalamazoo, Kent, and Wayne Counties, respectively, were analyzed using six polymorphic and fluorescently labeled SSR markers. The average unbiased Simpson's index (λu, 0.95), evenness (E5, 0.56), and recovery of 12 major clones out of the 65 multilocus genotypes obtained, suggests that P. ultimum is not a recent introduction into Michigan greenhouses. Analyses revealed a clonal population, with limited differentiation among seasons, hosts, and counties sampled. Results also indicated the presence of common genotypes among years, suggesting that sanitation measures should be enhanced to eradicate resident P. ultimum populations. Finally, the presence of common genotypes among counties suggests that there is an exchange of infected plant material among greenhouse facilities, or that there is a common source of inoculum coming to the region. Continued monitoring of pathogen populations will enhance our understanding of population dynamics of P. ultimum in Michigan and facilitate improvement of control strategies.

Metagenome – Inferred bacterial replication rates in cystic fibrosis airways

Bacterial replication rates were determined from metagenome sequencing of nasal lavage, throat swabs and induced sputa collected from healthy subjects and individuals with COPD or cystic fibrosis. More than 90% of peak-to-trough coverage ratios of major clones were above 1.4 indicating that the most abundant bacterial species in the microbial communities were replicating in the airways including common inhabitants such as Prevotella and Streptococcus species as well as the cystic fibrosis pathogens Staphylococcus aureus and Pseudomonas aeruginosa. The populations of P. aeruginosa and S. aureus were replicating their pool of chromosomes more slowly than the populations of the common inhabitants of a healthy airway microbial flora. The assessment of growth dynamics in microbial metagenomes could become a decision-making tool for the diagnosis and management of bacterial infections in cystic fibrosis.

NOTCH1 pathway activating mutations and clonal evolution in pediatric T-cell acute lymphoblastic leukemia.

Molecular mechanisms involved in the relapse of T‐cell acute lymphoblastic leukemia (T‐ALL) are not fully understood, although activating NOTCH1 signaling due to NOTCH1/FBXW7 alterations is a major oncogenic driver. To unravel the relevance of NOTCH1/FBXW7 mutations associated with relapse, we performed whole–exome sequencing in 30 pediatric T‐ALL cases, among which 11 diagnosis‐relapse paired cases were further investigated to track the clonal evolution of relapse using amplicon–based deep sequencing. NOTCH1/FBXW7 alterations were detected in 73.3% (diagnosis) and 72.7% (relapse) of cases. Single nucleotide variations in the heterodimerization domain were the most frequent (40.0%) at diagnosis, whereas proline, glutamic acid, serine, threonine–rich (PEST) domain alterations were the most frequent at relapse (54.5%). Comparison between non–relapsed and relapsed cases at diagnosis showed a predominance of PEST alterations in relapsed cases (P = .045), although we failed to validate this in the TARGET cohort. Based on the clonal analysis of diagnosis‐relapse samples, we identified NOTCH1 “switching” characterized by different NOTCH1 mutations in a major clone between diagnosis and relapse samples in 2 out of 11 diagnosis‐relapse paired cases analyzed. We found another NOTCH1 “switching” case in a previously reported Berlin‐Frankfurt‐Münster cohort (n = 13), indicating NOTCH1 importance in both the development and progression of T‐ALL. Despite the limitations of having a small sample size and a non–minimal residual disease–based protocol, our results suggest that the presence of NOTCH1 mutations might contribute to the disease relapse of T‐ALL.

Single-cell genetic analysis of clonal dynamics in colorectal adenomas indicates CDX2 gain as a predictor of recurrence.

Colorectal adenomas are common precancerous lesions with the potential for malignant transformation to colorectal adenocarcinoma. Endoscopic polypectomy provides an opportunity for cancer prevention; however, recurrence rates are high. We collected formalin-fixed paraffin-embedded tissue of 15 primary adenomas with recurrence, 15 adenomas without recurrence, and 14 matched pair samples (primary adenoma and the corresponding recurrent adenoma). The samples were analysed by array-comparative genomic hybridisation (aCGH) and single-cell multiplex interphase fluorescence in situ hybridisation (miFISH) to understand clonal evolution, to examine the dynamics of copy number alterations (CNAs) and to identify molecular markers for recurrence prediction. The miFISH probe panel consisted of 14 colorectal carcinogenesis-relevant genes (COX2, PIK3CA, APC, CLIC1, EGFR, MYC, CCND1, CDX2, CDH1, TP53, HER2, SMAD7, SMAD4 and ZNF217), and a centromere probe (CEP10). The aCGH analysis confirmed the genetic landscape typical for colorectal tumorigenesis, that is, CNAs of chromosomes 7, 13q, 18 and 20q. Focal aberrations (≤10 Mbp) were mapped to chromosome bands 6p22.1-p21.33 (33.3%), 7q22.1 (31.4%) and 16q21 (29.4%). MiFISH detected gains of EGFR (23.6%), CDX2 (21.8%) and ZNF217 (18.2%). Most adenomas exhibited a major clone population which was accompanied by multiple smaller clone populations. Gains of CDX2 were exclusively seen in primary adenomas with recurrence (25%) compared to primary adenomas without recurrence (0%). Generation of phylogenetic trees for matched pair samples revealed four distinct patterns of clonal dynamics. In conclusion, adenoma development and recurrence are complex genetic processes driven by multiple CNAs whose evaluations by miFISH, with emphasis on CDX2, might serve as a predictor of recurrence.

Comparison of community- and healthcare-associated methicillin-resistant Staphylococcus aureus isolates at a Chinese tertiary hospital, 2012\u20132017

The transmission between community-associated (CA-) and healthcare-associated (HA-) methicillin-resistant Staphylococcus aureus (MRSA) has increased the challenge of infection control. To understand the clonal evolution and transmission of MRSA isolates, we compared the characteristics of 175 CA-MRSA and 660 HA-MRSA strains at a Chinese tertiary hospital in 2012–2017. Antibiotic susceptibility was performed on VITEK system, the genetic background of the isolates was characterized by SCCmec, spa, and MLST typing, while virulence determinants were screened using conventional PCR. Although more than 70% of the CA-MRSA isolates were erythromycin and clindamycin resistant, CA-MRSA was more susceptible than HA-MRSA to most of the antibiotics tested. ST239-MRSA-III-t030 (30%) was the most prevalent clone among HA-MRSA, while ST59-MRSA-IVa-t437 (28.8%) was the major clone among CA-MRSA. Notably, ST59-MRSA-IVa-t437 accounted for 6.7% of the chosen HA-MRSA isolates. Additionally, difference in virulence gene content was found between the CA- and HA-MRSA strains. In conclusion, epidemiological characteristics were largely different between CA- and HA-MRSA. Although ST239-MRSA-III-t030 is still the predominant clone among HA-MRSA, the community clone ST59-MRSA-IVa-t437 has the potential of becoming an essential part of HA-MRSA in the region tested.

Dynamics of Genetic Landscapes and Clonal Evolution between Patients and PDX Models in Acute Myeloid Leukemia

Introduction: Recent genome-wide studies of leukemia have revealed its clonal architecture and subclonal heterogeneity. Patient-derived xenograft (PDX) models are assumed to capture the cellular and molecular characteristics of human cancer and widely utilized for studying drug response. However, it has not been elucidated whether PDXs faithfully represent the genetic features of primary leukemia. In this study, we monitored the dynamics of somatic mutations in primary leukemia cells and PDX cells to evaluate their genetic stability and clonal selections.Methods: A total of 90 fresh bone marrow or peripheral blood samples from patients with hematological malignancies were intravenously transplanted into NOD/Shi-scid, IL-2Rγnull (NOG) mice at 0.2-15x106 cells per mouse. Targeted sequencing of 54 genes frequently identified in myeloid malignancies was performed in 40 paired genomic DNA of successfully engrafted AML patient and their PDX samples and 20 AML patients with engraftment failure. Clonal diversities were analyzed by comparing variant allele frequencies (VAF) of somatic mutations in 16 sets of serial samples from AML patients at diagnosis and subsequent relapse, and their PDX models. All patients provided written informed consent.Results: Sixty-five of 90 (72%) primary cells (AML; 60, ALL; 5) engrafted in NOG mice at the median of day 112 (29-549) post transplantation. Patient samples with successful establishment of PDX models included significantly higher number of infused cell counts (7.2 vs. 4.9 x 106 cells, P=0.002), higher percentage of blasts (56.2% vs. 38.7%, P=0.04) and harbored activating kinase gene mutations more frequently than those with engraftment failure (83% vs. 40%, P=0.001; Figure 1). Especially, FLT3 (53% vs. 10%, P=0.001), NPM1 (28% vs. 5%, P=0.04), RUNX1 (25% vs. 0%, P=0.01), IDH1 (20% vs. 0%, P=0.03) and ASXL1 (18% vs. 0%, P=0.05) mutations were significantly accumulated in successfully engrafted patients. In terms of the sensitivity to chemotherapy, patients with successful engraftment in NOG mice showed poorer 2-year overall survival than those without engraftment (36.1% vs. 100%, P=0.04; Figure 2).In 40 pairs of AML patients and their PDXs, a total of 172 genetic alterations (101 SNV, 39 frameshift, 32 inframe insertion/deletion) were identified. Mean VAF changes in PDXs compared with patients showed dominant elevation in driver gene mutations including FLT3 (+16.9%), CEBPA (+19.1%) and WT1 (+26.4%) demonstrating the expansion of clones carrying these mutations in PDXs, whereas those of ASXL1 (-2.2%), NPM1 (+4.8%) and DNMT3A (+6.0%) in PDXs were almost the same as primary patient samples, implicating that these mutations are founder events in clonal evolution. VAF of 26 pairs of patients' samples and their PDXs at relapse were more concordant than those of 12 pairs at diagnosis (r2=0.689 vs. 0.550).In 16 sets of AML patients' samples at diagnosis and relapse, and their PDX samples, clonal diversities between primary leukemia at diagnosis and their PDXs were classified into 2 subtypes. In 7/16 models (44%), minor clones detected in patients at diagnosis dominantly expanded to be major clones in their PDX models, whereas major clones persisted in similar VAF between primary leukemia and their PDXs in 9/16 models (56%). These subtypes of clonal diversity between primary leukemia at diagnosis and their PDXs were concordant with clonal evolution between patients at diagnosis and relapse in 12/16 models (75%). Furthermore, in 4/16 models (25%), clones selectively expanded in PDXs were detected as major clones in their patients' first relapsed samples predictively.Conclusions: Our findings indicate the existence of diverse clonal evolution in AML PDX models. Primary AML samples harboring treatment-resistant clones have higher potential of engraftment and growth in NOG mice. Furthermore, PDX models recapitulate the clonal evolution from diagnosis through relapse in treatment-resistant patients. Further understanding of expanded clones in PDX models could reveal the pathogenesis of clonal selection in AML patients. [Display omitted] DisclosuresKiyoi:Bristol-Myers Squibb: Honoraria; FUJIFILM Corporation: Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; Sanofi K.K.: Research Funding; Celgene Corporation: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Phizer Japan Inc.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding; Astellas Pharma Inc.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Novartis Pharma K.K.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding.

Genetic Heterogeneity and Evolution in Lymphoid Malignancies

Patterns of mutation and clonal evolution in relapsed acute lymphoblastic leukemiaRelapsed acute lymphoblastic leukemia remains a major cause of childhood cancer death, and this remains true despite the advent of new targeted and immunotherapeutic approaches. Recent years have witnessed the use of broad and deep serial genomic profiling approaches to dissect the relationship of genetic variegation to clonal evolution and relapse. Studies of over 90 children treated on St Jude Total Therapy protocols, incorporating genome, exome and transcriptome sequencing, coupled with limiting dilution xenografting to formally elucidate clonal structure have provided multiple key insights. In the majority of cases, the relapse-fated clone is a minor clone at diagnosis, that harbors resistance-enriched (and thus relapse-promoting) mutations at diagnosis, and/or acquires additional mutations the confer resistance after initial therapy. Approximately one third of cases relapse from a major clone, or show polyclonal evolution, and such cases typically have a shorter time to disease recurrence and relapse. A subset of cases exhibit complete discordance for somatic non-silent mutations, DNA copy number alterations and antigen receptor rearrangements between diagnosis and relapse, suggesting relapse represents a second leukemia; however such cases typically preserve the founding chromosomal rearrangement and a subset of non-coding mutations, indicating that relapse arises from an ancestral clone that has undergone divergent evolution early in leukemogenesis. Conversely, a subset of cases relapse with myeloid or lineage ambiguous leukemia but preserve genomic alterations indicating a common clonal origin but lineage plasticity: thus, careful genomic analysis is required to interpret the nature of disease recurrence/relapse. Approximately 15% of cases exhibit hypermutation, particularly in aneuploid leukemia and second or later relapse, associated with distinct mutational signatures and kinetics of hypermutation, thus identifying this process as a driver of treatment failure in a subset of ALL cases. Integrated analysis has identified over 80 recurrent targets of alteration at relapse that show variable patterns of enrichment in rising and falling clones. Importantly, several targets (e.g. NT5C2) are never identified at diagnosis despite deep sequencing approaches, suggesting adverse effects on leukemic fitness, and/or an absolute requirement of prior drug exposure to initiate mutagenesis. Integration of limiting dilution xenografting, coupled with genomic analysis of xenografts and drug exposure has not only formally confirmed and extended inferential clonal structures, but shown that in a subset of cases resistance is present at initial diagnosis, rather than being acquired after drug exposure. Finally, several groups have shown that the relationship of relapse-enriched mutations and relapse by be drug agnostic (e.g. IKZF1) or drug specific (e.g. NT5C2 and thiopurine resistance, and CREBBP and glucocorticoid resistance). As such mutations may now be detected at levels suitable for tracking of minimal residual disease, these insights offer the opportunity to identify the relapse-fated clone early in disease evolution, and modulate therapy accordingly to circumvent relapse. DisclosuresMullighan:Pfizer: Honoraria, Research Funding, Speakers Bureau; Cancer Prevention and Research Institute of Texas: Consultancy; Loxo Oncology: Research Funding; Abbvie: Research Funding; Amgen: Honoraria, Speakers Bureau.

The percentage of cells with 17p deletion and the size of 17p deletion subclones show prognostic significance in chronic lymphocytic leukemia.

TP53 disruption is considered to be the most important prognostic factor in chronic lymphocytic leukemia (CLL), but not all patients with TP53 disruption have similar dismal outcomes. We evaluated the prognostic value of TP53 disruption in CLL patients without treatment indications. Data of 305 CLL patients were analyzed. 41 of them (13%) had TP53 disruption. Patients with lower percentage of cells with del(17p) had significantly better survival. Patients with mutated IGHV, β2-microglobulin ≤3.5 mg/L, wild-type TP53, age ≤65 years or without complex karyotype (CK) had relatively favorable outcomes in the del(17p) group. Furthermore, patients with del(17p) as a minor clone showed survival advantage compared with those with del(17p) as a major clone. These data suggest that the percentage of cells with del(17p), the size of the del(17p) subclone, CLL International Prognostic Index, and CK should be considered to build refined prognostication models for patients with TP53 disruption.

Longitudinal Multilevel Omic Analysis of Pediatric T-ALL Reveals Distinct Mechanisms for Disease Progression in Type 1 and in Type 2 Relapses

We aimed at understanding the relapse-driving processes in pediatric T-ALL and performed an integrated longitudinal multi-level omics analysis of 13 T-ALL patients at initial diagnosis (INI) and relapse (REL). We compared the mutation (SNV/InDels) and copy number alteration (CNA) patterns as well as gene expression, methylation levels and chromatin accessibility by ATAC-Seq.Aberrant expression of T-ALL transcription factors (TAL1, TAL2,LMO2, TLX1, TLX3, NKX2.4 and NKX2.5) was preserved from initial presentation to relapse in all patients. These leukemia-driving events defined the expression patterns, methylation profiles and the chromatin accessibility landscapes.A global differential analysis of the RNA-Seq data (DESeq2, padj<.05) revealed only 0.3% of the genes to be either up- or down-regulated at relapse when compared to the matched initial sample. Likewise, we detected methylation changes in only 3% of the promoters and differential chromatin accessibility in only 0.26% of the analyzed ATAC-peaks (DESeq2, padj<.05).We then focused our analysis on the 2 types of relapse in pediatric T-ALL, which we have previously defined on the basis of subclonal mutation profiles (Kunz et al., 2015). These types of relapse are characterized by either clonal evolution of cells derived from the major clone at initial presentation (type 1) or emergence and evolution of a minor initial clone showing a molecular profile that is distinct from the predominant initial clone (type 2). When considering type 1 and type 2 relapses separately we identified a strong trend for type 2 relapses to acquire more mutations (p=0.0879, ttest) than type 1 relapses. Further to the known activating mutations in NT5C2 acquired at relapse by 8/13 patients no other mutations or CNAs were recurrently acquired in the relapses of this group of patients. However, mutations in proto-oncogenes or genes involved in DNA surveillance were acquired by 7/8 type 2 relapse patients in our series. Changes of CNAs also occurred more frequently in type 2 than in type 1 relapses (pval= 0.0267, ttest). This increased complexity on the genetic level was also apparent on the epigenetic level, with an increase of changes in the methylation pattern (mean difference in β value between INI and REL: type 1 - 0.00034; type2 - 0.002 (pval< 0.0001, chi2)), chromatin accessibility (number of differentially accessible ATAC-peaks: type 1 - 4 (0.006%) ; type 2 - 1018 (1.3%); (pval< 0.0001, chi2)) and on the expression level (number of differentially expressed genes: type 1 - 11; type 2 - 111, pval<0.0001, chi2).When considering differences between leukemias at the time of initial diagnosis, which later develop either type 1 or type 2 relapses we found 1.016 genes to be differentially expressed (524: up; 492: down in type 1; DE-Seq2: padj<0.05). Differential expression analysis revealed that genes involved in early T-cell differentiation were upregulated at initial diagnosis of type 1 in comparison to initial diagnosis of type 2, which was also reflected by more accessible chromatin surrounding their cis-regulatory regions as analyzed by ATAC-seq suggesting an early arrest of these samples during differentiation process. Altogether 1.4% of all ATAC-peaks were more accessible in type 1, whereas 0.7% of peaks were more accessible in type 2 leukemia (DE-Seq2: p<0.05). Remarkably, the chromatin surrounding DNA-repair genes was more accessible in initial leukemias of type 1, which was reflected by up-regulated mRNA expression level of such genes. These data suggest that an increased propensity of DNA repair may represent an important mechanism of T-ALL to develop a type 1 relapse, an interpretation that is consistent with previous reports linking the epigenetic silencing of the methylguanine-DNA methyltransferase promoter with compromised DNA repair and longer survival in patients with glioblastoma receiving alkylating agents (Esteller et. al., 2000).In sum, the multilevel omic comparison of pediatric T-ALL that develop either a type 1 or a type 2 relapse show remarkably more complex changes of the genetic and epigenetic profiles during the transition from initial to relapsing disease. Notably, pediatric T-ALLs, who later develop a type 1 relapse display an epigenetic and transcriptomic landscape predicting an upregulation of DNA repair functions, which we suggest to potentially play a role in developing resistance to DNA damaging agents in this type of relapse. DisclosuresMuckenthaler:Novartis: Research Funding. Bourquin:Amgen: Other: Travel Support. Kulozik:bluebird bio: Consultancy, Honoraria.

Polyclonal Immune Response in T-LGL Leads to Clonal Expansions Preceding Occurrence of STAT3 Mutations Further Solidifying Clonal Dominance

T cell large granular lymphocyte leukemia (T-LGLL) characterized by excessive clonal cytotoxic T cell proliferation, is often accompanied by cytopenias and is thought to result from immune-mediated suppression, either by misguided immune antigenic recognition or mimicry triggered by auto-, tumor or viral antigens. Irrespective of the mode of evolution, T-LGLL progresses via: i) purely reactive clonal outgrowth in the context of a polyclonal immune response or: ii) a transforming event such as a STAT3 mutation (STAT3MT) within the immunodominant clone.Studies of TCR rearrangement using DNA based NGS of the TCR Vβ complementarity-determining region 3 (TCR VB CDR3) facilitate analyses of T cell clonality, as the CDR3-based “biological barcodes” allow for clonal quantification. We used serial CDR3 clonotyping accompanied by clonal STAT3 mutant burden measurements to recapitulate the pathological cascade in the course of T-LGLL. We asked if a polyclonal/oligoclonal immune response is the primary process that is clonally “imprinted” by STAT3MT, which would likely arise with the most proliferative clone. We were interested in the dynamics of underlying clonal behavior during treatment.Our initial cohort included 207 well characterized LGL patients. Most presented with anemia (46%), and/or neutropenia (46%). VB expansion was present in 94% of cases, with an average LGL count of 2317k/uL .STAT3MT were found by targeted deep sequencing in 38% of patients in 4 common hotspots: 42% Y640F, 34% D661Y, 11% D661V, and 8% N647I, with an average clonal burden of 28%. Multiple STAT3MT variants were found in some patients. From this cohort, serial samples obtained at presentation and throughout clinical course were obtained from 20 representative cases (10 STAT3MT and 10 STAT3WT), and subjected to analysis of clonal dynamics, including simultaneous deep TCR VB and STAT3 NGS. Patients were sequenced at an average of 4 time points (range 2-8). At least one major clonotype was identified in all patients, and multiple major clonotypes were identified in half.Analyses of clonal architecture revealed that STAT3 clones arose with VB expanded clones. In all cases, the TCR clonal burden was greater than that of STAT3MT demonstrating for the first time that STAT3MT is not the ancestral event for clonal expansion; rather, it evolves within the pre-expanded immunodominant clone.More than half of the patients were treated with immunosuppressive therapy (IST) achieving an approximate 40% response rate. Distinct patterns of clonal dynamics were seen following treatment (see Figure). In some patients, both the STAT3 (if present) and the major TCR clone decreased upon successful therapy. A correlation between a specific IST treatment and a clonal burden decrease was not found. In a subset of patients, the clones persisted despite a hematologic response, suggesting the major clonotype was functionally silenced. We also observed a common phenomenon of TCR “clonotype switching”, whereby therapy contracts one major clonotype, while another previously “minor” clonotype emerges. These newly expanded clones did not harbor STAT3MT, and most patients with “switching” were resistant to IST therapy. Multiple clonotypes were present at initial sampling in a few patients without STAT3MT and persisted at the same rate in subsequent samplings, precluding identification of a clear immunodominant clonotype. A stable or increasing clonal burden of both STAT3MT and VB CDR3 was seen in IST non-responders.In sum, STAT3MT were found to be a secondary event to the clonal expansion of the TCR VB clonotype, as a response within an already expanded clonotype and not the initiator of clonal expansion. The dynamics of both the STAT3MT and the TCR VB clonotype can be assessed over disease course and treatment regimens and demonstrate additional clinical ultility when applied to larger prospective clinical trials. The difficulty in finding a direct correlation between response to specific IST and a decrease in TCR VB clonal burden may be due to variable time frames between samplings, heterogeneity of IST regimens and response assessment, or large asymptomatic clonotypes. Prior to this study, the association of remission and elimination of immunodominant clonotypes was unclear. Our results suggest that clonal elimination is not necessary for complete clinical response; rather, the clone can be contracted to a manageable clonal burden or silenced. DisclosuresMustjoki:Novartis: Honoraria, Research Funding; Celgene: Honoraria; Bristol-Myers Squibb: Honoraria, Research Funding; Ariad: Research Funding; Pfizer: Honoraria, Research Funding. Sekeres:Opsona: Membership on an entity's Board of Directors or advisory committees; Opsona: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Ra Pharmaceuticals, Inc: Consultancy; Apellis Pharmaceuticals: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Apellis Pharmaceuticals: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Ra Pharmaceuticals, Inc: Consultancy.

Somatic Mutations in T Cells As Possible Regulators of Immunodeficiency

▪The pathogenesis of common variable immunodeficiency (CVID) and many other immunodeficiencies is elusive, and no cure for these diseases exists. The characteristic features of CVID are immunoglobulin deficiency and recurrent infections, but autoimmunity co-manifests in 30% and lymphoproliferation in 50% of CVID cases. The failure to produce sufficient quantities of immunoglobulins is attributed to B cells, but as T cells are critical players in adaptive immune response and autoimmune disease, they have also suggested to play a role in CVID. As monogenic germline mutations account only for 2-10% of CVID cases, the etiology of CVID remains unknown in most cases.To study if somatic mutations in T cells contribute to immunodeficiencies, we recruited patients with broad immune dysregulation: 8 patients with late-onset CVID, and 9 patients with other type of immunodeficiency and/or severe autoimmunity. All patients signed informed consent and the declaration of Helsinki principles were followed. Study design and key patient characteristics are depicted in the Figure. To discover somatic mutations in T cells, we sequenced both CD4+ and CD8+ cells with a custom gene panel comprising of 2500 immune-related genes with an average coverage of 500x. Somatic variants were identified using the GATK MuTect2 toolset by using a panel of 21 healthy controls' CD4+ and CD8+ cells as a background. Variants were called using paired samples (CD4+ vs CD8+ and vice versa). This approach identifies variants that have occurred in mature T cells. To complement this approach, we performed variant calling also in single-sample mode to identify variants originating from hematopoietic progenitors.Paired-sample analyses revealed 44 somatic mutations in mature T cells in 10/17 (59%) patients: 30 (68%) in CD8+ and 14 (32%) in CD4+ cells. The mutations included 2 frameshift-, 37 missense-, and 5 nonsense variants. In silico tools (both Polyphen-2 and SIFT) predicted 19 (51%) of the missense mutations to be damaging. The Catalogue Of Somatic mutations In Cancer (COSMIC) database included 9/44 (20%) of all mutations. Also, pathway analysis annotated 20% of the mutated genes to be involved in inflammatory response regulation, 27.5% in protein phosphorylation regulation, and 22.5% in positive regulation of cell proliferation. Interesting mutation findings included two patients with STAT5B mutations (N418K and T628S) with a low variant-allele (VAF) frequencies (3.2-3.6% in CD4+ cells), one patient with a KRAS T58I mutation (VAF 7.9% in CD8+), and two patients with distinct C5AR1 mutations (R197W and T62M). Complementing the paired-sample variant calling strategy, single-sample analyses revealed mutations associated with clonal hematopoiesis with low VAFs (2.2-5.5%) in T cells in 4 (23.5%) patients. Three patients had DNMT3A mutations and one patient 4 distinct TET2 mutations (2 nonsense-, 1 frameshift-, and 1 missense mutations).CD4+ and CD8+ T-cell receptor (TCR) repertoires were profiled with deep TCR beta chain (TCRB) sequencing. Healthy controls' CD4+ and CD8+ cells (n=27) were used as comparators. Although some CVID patients harbor major (17-20% of CD8+) T-cell clones, overall CD4+ or CD8+ clonality did not significantly differ from age-matched healthy controls. As a marker of selective pressure, CVID patients' CD4+ and CD8+ cells harbored more clones that shared a CDR3 amino-acid but had distinct nucleotide CDR3 sequences than healthy controls (7.0% vs 3.3% of all amino-acid rearrangements, p=0.021 for CD8+; 4.3% vs 3.5% p=0.013 for CD4+). These TCRs were not enriched with pathogen-specific (such as cytomegalovirus or Epstein-Barr virus) TCRs. Moreover, high CD4+ clonality was associated with a lower frequency of switched-memory-B cells.In conclusion, somatic mutations in T cells occurred in 59% of patients with immunodeficiency in this study, and some of the mutated genes (such as STAT5B) have previously been implicated for the pathogenesis of autoimmunity and lymphoproliferation. Also, clonal hematopoiesis in T cells was discovered in 23.5% of patients. The overall T-cell clonality was not increased, but CVID patients showed slight selective pressure in the TCR repertoire. Our results demonstrate that further research on somatic mutations in immunodeficiencies is needed, as they may contribute to disease pathogenesis in a subset of immunodeficiency patients. DisclosuresMartelius:Gilead: Other: lecture fee; Octapharma: Other: travel grant; CSL Behring: Other: lecture fee and travel grant; MSD: Other: lecture fee and travel grant; Sanguin: Other: travel grant and grants. Kankainen:Medix Biochemica: Consultancy. Seppänen:CSL Behring: Other: Chairing and speaker fees. Mustjoki:Ariad: Research Funding; Pfizer: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Celgene: Honoraria; Novartis: Honoraria, Research Funding.

Pediatric T-ALLs Developing into a Type 2 Relapse Originate from Cells That Carry the Potential of Variable Maturation into Subclones with Distinct Chromatin Landscapes

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy that is classified according to surface marker expression. In order to reveal the cells of origin in pediatric T-ALL and to understand mechanisms of relapse we used ATAC-Seq (Assay for Transposase-Accessible Chromatin Sequencing) to compare chromatin accessibility landscapes of healthy T-cell precursors to those of T-ALL cells obtained at initial diagnosis (INI) and relapse (REL).We have FACS sorted 7 differentiation stages of normal T-cell precursors contained in the thymus of infants undergoing cardiac surgery (DN2, DN3, ISP, DPCD3-, DPCD3+, CD4+ and CD8+) and subjected these to ATAC-Seq. Unsupervised learning by principal component analysis (PCA) clustered sorted populations according to the maturation stage, demonstrating that regulatory chromatin signatures of thymocytes are highly stage-specific and re-shaped during T-cell differentiation.We next compared normal T-cell precursors at different stages of maturation to pediatric T-ALLs and found fundamental differences with 30% of open chromatin regions to be more and 28% being less accessible in T-ALL (DESeq2, padj<.05), respectively. We next identified transcription factor (TF) binding sites in open chromatin regions of normal T-cell precursors and T-ALL cells by HOMER analysis. We found 76% of the accessible TF binding sites in double negative (DN2 and DN3) normal precursors to be also accessible in most (20/24) leukemias comparing to 17% of the sites accessible in the more mature (CD4+ and CD8+) stages. These data indicate that T-ALLs originate from cells with an epigenetic profile of early thymic progenitors.We then subjected the ATAC-seq data of all matched leukemia samples obtained at initial disease and at relapse to PCA. INI and REL samples derived from the same patient always clustered in close proximity and were separated according to the T-ALL driving fusion genes. A global analysis of differential accessibility revealed only 0.26% of ATAC-regions to be less- or more-accessible at relapse when compared to the matched initial samples (DESeq2, padj<.05). These data indicate chromatin accessibility to be largely determined by the cell of origin and to generally remain stable during progression from initial diagnosis to relapse. We then considered the 2 types of relapse separately, which we have previously characterized on the basis of subclonal mutation profiles (Kunz et al. Haematologica, 2015). These relapse types are defined by either clonal evolution of cells derived from the predominant clone at primary disease (type 1) or emergence and evolution of a minor initial clone showing a molecular profile that differs from the major initial clone (type 2). We found that the proportion of differentially accessible ATAC-regions between INI and REL is significantly higher in type 2 (1.3%) than in type 1 (0.006%) (DESeq2, padj<.05).Moreover, we trained the deconvolution algorithm CIBERSORT to recognize particular T-cell differentiation stages using ATAC-profiles of the 7 FACS-sorted healthy T-cell populations. We used regulatory chromatin landscape of non-sorted (total) thymus to assess the accuracy of deconvolution. Comparison of predicted fractions in total thymus to FACS measurements revealed highly accurate identification of the maturation stages (r2 = 0.95). CIBERSORT analysis confirmed that the profiles were largely preserved between INI and REL of each sample pair. Notably, however, while in T-ALLs that later developed into a type 1 relapse only one type of early T-cell progenitor dominated the deconvolution profile, T-ALLs that developed into a type 2 relapse showed heterogeneous profiles with contributions of progenitors at different maturation stages.In sum, these epigenomic analyses revealed that the chromatin landscape of normal T-cell precursors evolves in the course of thymic maturation and that the early maturation stages are the likely origin of T-ALL cells. Remarkably, pediatric T-ALLs that later develop a type 2 relapse consist of subclones with a variable profile of chromatin accessibility that define different stages of maturation. These data indicate that T-ALLs with the propensity to develop a type 2 relapse differ from type 1 in that they originate from early precursors that carry the potential of further development into different stages of maturation before the leukemia becomes apparent with a highly subclonal pattern. DisclosuresMuckenthaler:Novartis: Research Funding. Bourquin:Amgen: Other: Travel Support. Kulozik:bluebird bio: Consultancy, Honoraria.

Longitudinal High-Throughput T Cell Repertoire Profiling of Chronic Lymphocytic Leukemia Patients Under Different Types of Treatment: Implications for Combination Strategies

Using next-generation sequencing (NGS), we recently documented the clonal architecture of the T cell repertoire in treatment-naive chronic lymphocytic leukemia (CLL), with ample immunogenetic evidence indicating selection by restricted antigens. Our preliminary NGS study in 16 patients pre- and 3-month post-treatment indicated a differential impact of standard chemoimmunotherapy (FCR) versus B cell receptor signaling inhibitors (BcRi) on CLL T cells. Prompted by these observations, here we sought to comprehensively assess CLL T cell repertoire changes over treatment in relation to both treatment type and clinical response by combining NGS immunoprofiling, flow cytometry and functional assays.NGS profiling of the T cell receptor (TR) gene repertoire was performed in 28 CLL patients who received FCR (n=9), ibrutinib (IB, n=15) and/or rituximab-idelalisib (R-ID, n=10) at successive timepoints (pre, +3mo, +9mo and at deepest clinical response, total samples: n=113). TRBV-TRBD-TRBJ gene rearrangements were RT-PCR amplified and subjected to paired-end NGS. Raw reads were processed through a purpose-built, validated bioinformatics pipeline, culminating to 20,347,768 productive, filtered-in TRB sequences (median 155,479/sample). For repertoire analysis, clonotypes (i.e. rearrangements with identical TRBV gene usage and amino acid complementarity-determining region 3 sequence) were considered (median 11,420 distinct clonotypes/sample).All cases displayed significant clonal T cell expansions both pre- and post-treatment [median clonality, measured as the cumulative frequency of the 10 most expanded (major) clonotypes/sample: 30.3% and 39.6%, respectively]. Median clonality significantly increased at +3mo in the FCR (29.0% to 46.9%, p<.001) and R-ID group (33.0% to 39.1%, p<.001), but not in the IB group (33.3% to 31.2%, p>.05). Overtime analysis revealed a gradual increase of clonality over deepening clinical response (pre-, +3mo, +9mo, deepest response) in the R-ID group (33.0% to 39.1% to 46.0% to 46.1%, respectively; p<.001), but only a trend in this respect for IB (33.3% to 31.2% to 33.8% to 42.0%; p>.05). Considering that FCR resulted in T cell repertoire reconstitution whereas BcRis retained pre-treatment clones, we then focused on major clones persisting over treatment and found that they significantly expanded in the R-ID group, peaking at +3mo (p<.01). Cross-comparison across all CLL patients and against 767,438 unique TRB sequences retrieved from multiple public databases (HSV infections, T-cell lymphoproliferations, autoimmune disorders, healthy individuals), revealed 23/563 major clonotypes shared exclusively among CLL patients, alluding to selection by conserved CLL-related antigens. We then sought to test the functional effect of treatments on T cells. To this end, we evaluated activation markers on CLL T cell subpopulations for 8 CLL patients (R-ID, n=4; IB, n=4) pre- and +3mo post-treatment by flow cytometry and found statistically significant upregulation of T cell activation markers for R-ID compared to IB, particularly for: (i) CD69 in CD4+ effector memory T cells (p<.01); (ii) CD25 in CD8+ TEMRA T cells (p=0.006); and, (iii) CD38 in CD8+ effector memory T cells (p<.05) and CD8+ TEMRA T cells (p<.05). We also investigated the ability of CD3+ T cells, purified from 13 patients pre- and +3mo post-treatment (FCR, n=3; R-ID, n=5; IB, n=5), to form immune synapses with autologous pre-treatment CD19+ tumor cells. Quantitative relative recruitment index (RRI) analysis for F-actin showed that both R-ID (p<.01) and IB (p<.05) treated T cells form polarized immune synapses in contrast to FCR (p>.05).Taken together, NGS immunoprofiling suggests that BcRis retain T cell clones that may have developed in response to CLL-related antigens, which in the case of R-ID expand and peak at +3mo. Phenotypic and immune synapse bioassays support a concurrent restoration of functionality, mostly evident for R-ID, arguably contributing to clinical response. Overall, this data provides rationale for designing combination strategies, e.g. of R-ID with immunomodulating drugs, aiming to boost cytotoxic anti-tumor responses. Moreover, identifying the relevant neoepitopes may eventually pave the way for stratified treatments by means of engineered T cells or peptide vaccines, especially if these epitopes are conserved among CLL. DisclosuresVardi:Janssen: Honoraria; Gilead: Research Funding. Gemenetzi:Gilead: Research Funding. Ramsay:MedImmune: Research Funding; Roche Glycart AG: Research Funding; Celgene Corporation: Research Funding. Stamatopoulos:Janssen: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding. Hadzidimitriou:Abbvie: Research Funding; Gilead: Research Funding; Janssen: Honoraria, Research Funding.

Prevalence and Characterization of Staphylococcus aureus Isolated From Women and Children in Guangzhou, China.

The prevalent Staphylococcus aureus clones and antibiotic susceptibility profiles are known to change dynamically and geographically; however, recent S. aureus strains causing infections in women and children in China have not been characterized. In this study, we analyzed the molecular epidemiology and antimicrobial resistance of S. aureus isolated from patients in four centers for women and children in Guangzhou, China. In total, 131 S. aureus isolates (100 from children and 31 from women) were analyzed by spa typing, multi-locus sequence typing, virulence gene and antimicrobial resistance profiling, staphylococcal chromosomal cassette mec typing, and mutation analyses of rpoB. A total of 58 spa types, 27 sequence types (STs), and 10 clonal complexes (CCs) were identified. While CC59 (ST59-IV, 48.8%; ST338-III, 35.7%) and CC45 (ST45-IV, 100%) were the major clones (84.4%) among MRSA isolates, CC5 (ST188, 24.3%; ST1, 21.6%) and CC398 (ST398, 70%) were the major ones (70.1%) among MSSA isolates. ST338-MRSA-III mostly found in pus but hardly in respiratory tract samples while ST45-MRSA-IV was on the opposite, even though they both found in blood and cerebrospinal fluid sample frequently. Staphylococcal enterotoxin genes seb-seq-sek were strongly associated with ST59 and ST338, while sec was associated with ST45, ST121, ST22, and ST30. All ST338, ST1232, and SCCmec III isolates carried lukF/S-PV genes. A total of 80% of ST338 isolates were resistant to erythromycin, clindamycin, and tetracycline. All ST45 isolates exhibited intermediate or complete resistance to rifampicin. In total, 481 HIS/ASN mutations in rpoB were found in rifampicin-resistant or intermediate-resistant isolates. ST338-III and ST45-IV emerged as two of three major clones in MRSA isolates from women and children in Guangzhou, China, though ST59-MRSA-IV remained the most prevalent MRSA clone. Clonal distribution of S. aureus varied, depending on the specimen source. Virulence genes and antibiograms were closely associated with the clonal lineage. These results clarified the molecular epidemiology of S. aureus from women and children in Guangzhou, China, and provide critical information for the control and treatment of S. aureus infections.

Staphylococcus aureus Isolated From Retail Meat and Meat Products in China: Incidence, Antibiotic Resistance and Genetic Diversity.

This study was to estimate the prevalence and characteristics of Staphylococcus aureus from 1,850 retail meat and meat products in China during July 2011 to June 2016. The samples were collected covering most provincial capitals in China, including 604 raw meat, 601 quick-frozen meat, and 645 ready-to-eat meat. Using the qualitative and quantitative methods, all 39 cities had S. aureus-positive samples, and S. aureus was detected in 35.0% (647/1,850) of the samples. The levels of S. aureus in retail meat showed that the MPN value of the majority of the positive samples ranged from 0.3 to 100 MPN/g. Twenty-four antibiotics were used to test all 868 S. aureus isolates for antibiotic susceptibility. Only 11 isolates (1.26%) were susceptible to all antibiotics, whereas most isolates (821/868, 94.6%) showed resistance or intermediary resistance to more than three or more antibiotics. Of these strains, 104 (12.0%) were resistant to more than 10 antibiotics. However, the most frequent resistance was observed to ampicillin (85.4%), followed by penicillin (84.6%), erythromycin (52.7%), tetracycline (49.3%), kanamycin (45.3%), telithromycin (30.1%), clindamycin (29.6%), streptomycin (21.1%), norfloxacin (20.4%), gentamicin (19.4%), fusidic acid (18.4%), ciprofloxacin (16.9%), chloramphenicol (13.1%), amoxycillin/clavulanic acid (11.0%), and others (<10%). 7.4% of isolates (62/868) were confirmed as methicillin-resistance S. aureus (MRSA). By molecular typing analysis, there were 164 spa types and 111 STs were identified, including 15 novel spa types and 65 newly STs by multilocus sequence typing (MLST) and spa typing. Despite the wide genetic diversity observed among the 868 isolates, a great proportion of the population belonged to finite number of major clones: ST1-t127 (93/868, 10.7%) and ST7-t091 (92/868, 10.6%), ST5-t002 (42/868, 4.8%), ST398-t034 (40/868, 4.6%), ST188-t034 (38/868, 4.4%), ST59-t437 (30/868, 3.5%), ST6-t701 (29/868, 3.3%), and ST9-t899 (27/868, 3.1%) in China. This study reflects S. aureus was readily detected in Chinese retail meat and meat products but the level were not very excessive. In this study, the high antibiotic resistance is alarming and raising public health concern. In additions, most of molecular types of isolates have been linked to human infections around the world, indicating that these types of S. aureus in China have a theoretical pathogenic potential.

Next-generation antigen receptor sequencing of paired diagnosis and relapse samples of B-cell acute lymphoblastic leukemia: Clonal evolution and implications for minimal residual disease target selection

Antigen receptor gene rearrangements are frequently applied as molecular targets for detection of minimal residual disease (MRD) in B-cell precursor acute lymphoblastic leukemia patients. Since such targets may be lost at relapse, appropriate selection of antigen receptor genes as MRD-PCR target is critical. Recently, next-generation sequencing (NGS) – much more sensitive and quantitative than classical PCR-heteroduplex approaches – has been introduced for identification of MRD-PCR targets. We evaluated 42 paired diagnosis-relapse samples by NGS (IGH, IGK, TRG, TRD, and TRB) to evaluate clonal evolution patterns and to design an algorithm for selection of antigen receptor gene rearrangements most likely to remain stable at relapse. Overall, only 393 out of 1446 (27%) clonal rearrangements were stable between diagnosis and relapse. If only index clones with a frequency >5% at diagnosis were taken into account, this number increased to 65%; including only index clones with an absolute read count >10,000, indicating truly major clones, further increased the stability to 84%. Over 90% of index clones at relapse were also present as index clone at diagnosis. Our data provide detailed information about the stability of antigen receptor gene rearrangements, based on which we propose an algorithm for selecting stable MRD-PCR targets, successful in >97% of patients.