ALBERT

Description: This study examined changes in the clinical characteristics of community-acquired acute pyelonephritis (CA-APN) in South Korea between the period 2010–2011 and 2017–2018. We recruited all CA-APN patients aged ≥19 years who visited eight hospitals in South Korea from September 2017 to August 2018, prospectively. Data collected were compared with those from the previous study in 2010–2012, with the same design and participation from 11 hospitals. A total of 617 patients were enrolled and compared to 818 patients’ data collected in 2010–2011. Escherichia coli was the most common causative pathogen of CA-APN in both periods (87.3% vs. 86.5%, p = 0.680). E. coli isolates showed significantly higher antimicrobial resistance against fluoroquinolone (32.0% vs. 21.6%, p 〈 0.001), cefotaxime (33.6% vs. 8.3%, p 〈 0.001), and trimethoprim/sulfamethoxazole (37.5% vs. 29.2%, p = 0.013) in 2017–2018 than in 2010–2011. Total duration of antibiotic treatment increased from 16.55 ± 9.68 days in 2010–2011 to 19.12 ± 9.90 days in 2017–2018 (p 〈 0.001); the duration of carbapenem usage increased from 0.59 ± 2.87 days in 2010–2011 to 1.79 ± 4.89 days in 2010–2011 (p 〈 0.001). The median hospitalization was higher for patients in 2017–2018 than in 2010–2011 (9 vs. 7 days, p 〈 0.001). In conclusion, antimicrobial resistance of E. coli to almost all antibiotic classes, especially third generation cephalosporin, increased significantly in CA-APN in South Korea. Consequently, total duration of antibiotic treatment, including carbapenem usage, increased.

Description: Background Chromosomal translocations in acute leukemia frequently result in gene fusions that are associated with leukemogenesis. Next-generation sequencing technology has opened up a systematic characterization of transcriptomes including gene expression, novel transcript, and fusion transcripts. We used next-generation RNA sequencing to identify fusion genes responsible for novel chromosomal translocations in acute leukemia and to find their differentially expressed genes. Methods We selected 10 acute leukemia (AML, 6; B-ALL, 3; and T-ALL, 1) patients with novel translocations by G-banding. Total RNA was extracted from leukemia cells and cDNA libraries were constructed with TruSeq RNA kit. Paired-end sequencing was performed on HiSeq2500. Reads were aligned with TopHat/BowTie, and deFuse was used to detect fusion transcripts. Transcript assembly and abundance estimation were done using Cufflinks, and expression levels were quantified by fragments per kilobase of transcript per million mapped reads (FPKM). The candidate fusion transcripts were validated with fluorescence in situ hybridization (FISH), and reverse-transcription PCR followed by Sanger-sequencing. Results We found 5 in-frame fusion genes exactly matched on translocation breakpoints from 3 AML patients and 1 B-ALL patient: USP34-ASAP3/t(1;2)(p36.1;p11.2), MAZ-MKL1/t(16;22)(p11.2;q13), MLL-SEPT6 and SEPT6-CDCA5/t(X;11)(q24;q13), and RCSD1-ABL1/t(1;9)(q24;q34). The USP34-ASAP3 fusion produced a novel transcript between USP34 exon 2 and ASAP3 exon 18. The protein encoded by the ASAP3 gene promotes cell differentiation and migration and has been implicated in cancer cell invasion. Comparing gene expression in this sample to nine other samples, we found six overexpressed genes; CLEC3B, SNAR-A14, H19, HOTS, SNORD35A, and S100A1. CLEC3B is associated with human disorders affecting bone and connective tissue. H19 is located in an imprinted region of chromosome 11 and is associated with Wilms tumorigenesis. S100A1 is involved in the regulation of a number of cellular processes such as cell cycle progression and differentiation. The MAZ-MKL1 fusion transcript was composed of MAZ exon 4 and MKL1 exon 4. MAZ was a novel partner gene of MKL1 which had been reported in acute megakaryoblastic leukemia carrying RBM15-MKL1/t(1;22)(p13;q13). MS4A2, RPLP0, and ARP5J2 genes were overexpressed in this rearrangement. MS4A2 is related PI3K cascade pathway and immune response pathway. RPLP0 is responsible for RNA binding and structural constituent of ribosome. AML patient with t(X;11)(q24;q13) had two fusion transcripts, MLL-SEPT6 and SEPT6-CDCA5 resulting from complex MLL rearrangement. While the MLL-SEPT6 fusion has been known in AML cases, the SEPT6-CDCA5 was a novel fusion. SNORD88B, MYL6, PTMA, MKX, NDUFAF3, and CNTN1 gene were more highly expressed than other samples. Among them, MKX and CNTN1 genes are related with cell adhesion function. The RCSD1-ABL1/t(1;9)(q24;q34) in B-ALL was previously reported to encode an aberrant tyrosine kinase. This translocation had also reciprocal ABL1-RCSD1 fusion transcript which could result in an alteration of cellular function. Six genes were specifically overexpressed in this sample RCBTB2, SERHL2, MIR941-2, FAM150B, GPR110, and SNORA27. RCBTB2 encodes a protein that is related to regulator of chromosome condensation. We also investigated leukemia subtype-specific expression profiles. The five significant genes were higher expressed in AML as compared with ALL (MIR4461, SET, RNU6ATAC, NINJ2, and ATP6V0C). Especially, MIR4461 was over 6000 FPKM in 5 of 6 AML samples, but was never expressed in ALL samples. B-ALL specific overexpressed genes were C17orf62, and MIR941-1, whereas T-ALL specific overexpressed gene was SNORD33. Conclusions Using next-generation RNA sequencing, we have discovered 5 candidate fusion genes in 10 acute leukemia patients with novel translocations, and identified 3 novel fusion genes to be predicted as oncogenic potential. Through the comparison of expression profiling, we were able to define differentially expressed genes in acute leukemia with novel fusion genes and leukemia subtype-specific gene expression. RNA-sequencing is a powerful tool for the discovery of leukemia-associated fusion genes and their related genes as well as molecular pathways. Disclosures No relevant conflicts of interest to declare.

Description: Background: Somatic mutations in RUNX1 gene have been identified in a substantial proportion of patients with de novo acute myeloid leukemia (AML). It is suggested as a new candidate molecular marker and, therefore, is suggested to be routinely performed at the diagnostic stage of AML. Despite its clinical importance, however, previous cohorts have been heterogeneous in terms of cytogenetic and molecular subtypes of AML. Here, the aim of this study was to evaluate the frequency, biologic characteristics, and prognostic significance of RUNX1 mutations focusing on patients with AML, not otherwise specified (NOS). Methods: Diagnostic samples from 202 patients with AML were analyzed for RUNX1 mutations. We excluded AML with recurrent genetic abnormalities, AML with myelodysplasia-related changes, and therapy-related AML because these entities have prognostic relevances of their own. RUNX1 mutations were detected using standard PCR techniques and direct sequencing. Results: RUNX1 mutations were found in 27 (13.4%) patients. The mutations were clustered in Runt homology domain (13, 48.1%) and transactivation domain (9, 33.3%). Frameshift mutations were most common (52.9%), followed by missense mutations (35.3%) and nonsense mutations (11.8%). As shown in Table 1, patients with RUNX1 mutations had a lower platelet count (P = 0.03), a higher rate of trisomy 8 (P = 0.02) and trisomy 13 (P = 0.039), and a trend toward older age (P = 0.063) than patients without mutations. Presence of RUNX1 mutations and NPM1 or CEBPA mutations were mutually exclusive. At the median follow-up of 12.1 months, RUNX1 mutations predicted for shorter overall survival (OS; P = 0.007) and relapse-free survival (RFS; P = 0.003). In the multivariate analysis, RUNX1 mutation was a significant marker for inferior OS (hazard ratio, 3.037; P = 0.014) and RFS (hazard ratio, 5.699; P = 0.001). Conclusion: The findings of our study further strengthen the previous data about RUNX1 mutations in AML. Furthermore, AML NOS with RUNX1 mutations is characterized by distinct biology and is associated with adverse clinical outcome. Our study supports the notion that RUNX1 mutational status would be integrated into diagnostic workup of AML, particularly for AML, NOS subgroup. Table 1. Clinical and biologic features of the cohort by RUNX1 mutations RUNX1 mutations P -value Mutated, n (%) Wild type, n (%) Number 27 (13.4) 175 (86.6) Male sex 17 (63.0) 94 (53.7) 0.489 Median age, years (range) 63 (14 - 80) 55 (1 - 83) 0.063 WBC count, ¡¿109/L (median, range) 7.9 (1.1 - 133.3) 14.0 (0.8 - 231.3) 0.636 Hemoglobin, g/dL (median, range) 8.6 (5.0 - 10.6) 8.8 (4.1 - 17.3) 0.376 Platelet count, ¡¿109/L (median, range) 35 (14 - 230) 59 (9 - 900) 0.03 Blood blasts, % (median, range) 29 (0 - 94) 38.5 (0 - 93) 0.312 FAB subtypes  M0 3 (11.1) 11 (6.3) 0.609  M1, M2 21 (77.8) 129 (73.7) 0.831  M4, M5 3 (11.1) 27 (15.4) 0.767  M6, M7 0 8 (4.6) 0.546 Cytogenetic abnormalities  Normal karyotype (%) 11 (40.7) 104 (59.4) 0.106  Trisomy 8 (%) 5 (18.5) 8 (4.6) 0.02  Trisomy 11 (%) 1 (3.7) 4 (2.3) 0.823  Trisomy 13 (%) 3 (11.1) 3 (1.7) 0.039  Trisomy 21 (%) 1 (3.7) 2 (1.1) 0.866 Distribution of other mutations  FLT3 -ITD 5 (18.5) 50 (28.6) 0.39  FLT3 -TKD 1 (3.7) 4 (2.3) 0.823  NPM1 0 55 (31.4) 0.002  CEBPA 0 17 (9.7) 0.187  MLL -PTD 1 (3.7) 14 (8.0) 0.691 Disclosures No relevant conflicts of interest to declare.

Description: Backgrounds Flow cytometry can rapidly determine immunophenotypes of neoplastic plasma cells (PCs) and quantify PCs in patients with plasma cell myeloma. Flow cytometric immunophenotyping and quantification of neoplastic plasma cells is sensitive and reliable tool for diagnosis and disease monitoring in patients with monoclonal gammopathy. Circulating PCs (cPCs) in peripheral blood (PB) after autologous hematopoietic stem cell transplantation is a marker of high-risk disease in patients with plasma cell myeloma. We assessed the utility of quantification of cPCs using flow cytometry for risk stratification in newly diagnosed plasma cell myeloma patients in the era of novel agents. Methods PB and bone marrow (BM) aspirates of 85 newly diagnosed patients with symptomatic plasma cell myeloma from August 2013 to July 2014 were analyzed by five-color flow cytometry using monoclonal antibodies against CD45, CD19, CD56, CD38, and CD138. The gating strategy employed first used the expression of CD38 and CD138 to identify plasma cells among 100,000 to 200,000 events. cPCs in PB was determined according to the patient's specific immunophenotype of neoplastic PCs in BM. Results The median age of the patient population was 68 years (45~87) and 58% were female. Median follow-up duration was 19.2 months. Six out of 85 patients (7%) did not show cPCs. Among 79 patient (93%) who had detectable cPCs, the median cPCs was 0.09% (0.006~3.612%). Patients without cPCs or cPCs under 0.05% were assigned to low cPCs group (n=32, 38%) and others to high cPCs group (n=53, 62%) according to receiver operating characteristics analysis. High cPCs group showed higher level of BM neoplastic PCs detected by both methodologys of morphology and flow cytometry (P=0.002, 0.033, respectively), higher BM cellularity (P=0.011), higher serum M protein level (P=0.013), lower hemoglobin (P=0.008), and lower platelet level (P=0.034) than low cPCs group. High cPCs group was associated with adverse cytogenetics such as t(4;14) and monosomy 13 (P=0.008), and CD45 negative immunophenotype (P=0.007). In survival analysis, high cPCs presented shorter overall survival (OS) than low cPCs group (P=0.013) (Fig. 1). It was independent with patient age and cytogenetic risks (P =0.011). Conclusion By flow cytometry cPCs was detected in most symptomatic plasma cell myeloma patients. Increased cPCs ≥0.05% among PB leukocytes could be an independent prognostic factor showing adverse effect in overall survival in symptomatic plasma cell myeloma patients. Figure 1. Kaplan-Meier survival curve of patients with plasma cell myeloma who showed 0.05% or more circulating plasma cells in peripheral blood and patients with circulating plasma cells less than 0.05%. Figure 1. Kaplan-Meier survival curve of patients with plasma cell myeloma who showed 0.05% or more circulating plasma cells in peripheral blood and patients with circulating plasma cells less than 0.05%. Disclosures No relevant conflicts of interest to declare.

Description: Background: New-onset pancytopenia or bicytopenia presents a diagnostic challenge and often leads to an invasive bone marrow (BM) aspiration and biopsy. There have been several studies that determined the distribution of BM diagnoses in patients with new-onset cytopenia. However, most of these studies were performed in nations, where the nutritional and environmental status is significantly different than that of Korea. Thus, the objective of this study was to evaluate the distribution of BM diagnoses among patients at a single tertiary care center in Korea and also to provide a recommendation for a practical laboratory approach based on the distribution. Methods: We performed a search of BM data in our laboratory information system regarding new-onset pancytopenia or bicytopenia from January 2010 to December 2014. The BM diagnoses, hematological parameters, and associated clinical findings at presentation were recorded. Results: A total of 2,632 patients were referred for BM examination for bicytopenia or pancytopenia (n = 901) during the study period. Of the BM examinations for bicytopenia (n = 1,731), 1,580 were performed on adults and 151 were performed on pediatric patients. In adults, the most common BM diagnosis was a malignancy (65.8%), including acute myeloid leukemia (AML; 25.9%), BM involvement of lymphoma (12.7%), plasma cell myeloma (PCM; 8.2%), acute lymphoblastic leukemia (ALL; 6.3%), myelodysplastic syndrome (MDS; 4.4%), metastatic carcinoma (3.2%), and other malignancies (5.0%). Benign diagnoses included idiopathic thrombocytopenic purpura (ITP; 1.9%) and aplastic anemia (AA; 1.3%). Non-specific findings were present in the remaining 31.0% of patients. In children, the most common BM diagnosis was a malignancy, including ALL (45.8%), hemophagocytic lymphohistiocytosis (HLH; 18.8%), AML (12.5%), metastatic neuroblastoma (6.3%), MDS (3.3%), and others (1.0%). AA was found in 6.3%, and non-specific findings were present in 6.3% of patients. Of the BM examinations for pancytopenia (n = 901), 791 were performed on adults and 110 were performed on pediatric patients. In adults, the most common BM diagnosis was also a malignancy (50.6%), including MDS (16.5%), AML (15.2%), BM involvement of lymphoma (8.9%), ALL (6.3%), metastatic carcinoma (1.3%), and others (2.5%). Benign diagnoses included AA (10.1%) and ITP (1.3%). Non-specific findings were present in the remaining 38.0% of patients. In children, the most common BM diagnosis was also a malignancy, including ALL (36.4%), HLH (9.1%), and AML (9.1%). AA was found in 27.3%, and non-specific findings were present in 18.2% of patients. Of note, a small number of patients exhibited unique circumstances, including a lymphoma or even metastatic cancer diagnosed in the BM without an available histological diagnosis (1.8%), acute leukemia with no apparent circulating blasts (1.1%), or therapy-related myeloid neoplasms (1.1%) Conclusions: The results of this study revealed the prevalent causes of new-onset cytopenia in the Korean population and can hopefully provide diagnostic insights to both physicians and hematopathologists. Furthermore, our results could justify performing BM examination on patients with new-onset cytopenia as well as refining the diagnostic approaches such as protein electrophoresis, immunohistochemistry for lymphoma or even carcinoma, and recommendations for further imaging studies on these patients. Disclosures No relevant conflicts of interest to declare.

Description: Objective was to analyse bacterial composition and abundance of Clostridioides difficile in gut microbiome of patients with C. difficile infection (CDI) in association with clinical characteristics. Whole metagenome sequencing of gut microbiome of 26 CDI patients was performed, and the relative abundance of C. difficile and its toxin genes was measured. Clinical characteristics of the patients were obtained through medical records. A strong correlation between the abundance of C. difficile and tcdB genes in CDI patients was found. The relative abundance of C. difficile in the gut microbiome ranged from undetectable to 2.8% (median 0.089). Patients with fever exhibited low abundance of C. difficile in their gut, and patients with fewer C. difficile organisms required long-term anti-CDI treatment. Abundance of Bifidobacterium and Bacteroides negatively correlated with that of C. difficile at the genus level. CDI patients were clustered using the bacterial composition of the gut: one with high population of Enterococcus (cluster 1, n = 12) and another of Bacteroides or Lactobacillus (cluster 2, n = 14). Cluster1 showed significantly lower bacterial diversity and clinical cure at the end of treatment. Additionally, patients with CDI exhibited increased ARGs; notably, blaTEM, blaSHV and blaCTX-M were enriched. C. difficile existed in variable proportion of the gut microbiome in CDI patients. CDI patients with Enterococcus-rich microbiome in the gut had lower bacterial diversity and poorer clinical cure.

Description: The objective of this study was to analyse the genetic relatedness of Clostridioides difficile polymerase chain reaction ribotype 017 (RT017) strains from patients with hospital-acquired C. difficile infection (HA-CDI) in a hospital with a high RT017 prevalence. From 2009 to 2013, 200 RT017 strains (26.8%) were collected from 745 HA-CDI patient isolates. They comprised 64 MLVA types, and 197 (98.5%) strains were genetically related to 5 clonal complexes (CCs). The largest cluster, CC-A, included 163 isolates of 40 MLVA types. CC-A accounted for 20% of RT017 strains in 2009 and sharply increased to 94.9% in 2010, 94% in 2011, 86.2% in 2012, and 73.5% in 2013. The other 4 CCs included 20 isolates with 7 MLVA types. The resistance rates of antimicrobials were as follows: clindamycin 100%, moxifloxacin 99%, rifaximin 88.5%, and vancomycin 1%. All isolates were susceptible to metronidazole and piperacillin/tazobactam. Comparing antibiotic resistance among CCs, the geometric mean of the minimum inhibitory concentrations of moxifloxacin, vancomycin, and piperacillin/tazobactam were significantly higher for CC-A isolates than for the other CCs. RT017 clones constantly evolved over the 5 years studied with regard to genetic relatedness. The levels of antibiotic resistance may contribute to the persistence of organisms in the institution.