Comprehensive DNA repair gene expression analysis and its prognostic significance in acute myeloid leukemia.

BACKGROUND: Deficiency in DNA damage response (DDR) pathway and accumulation of DNA damage increases mutation rates resulting in genomic instability and eventually increases the risk of cancer. The aim of our study was to investigate expressions of DNA repair genes as new prognostic biomarkers in acute myeloid leukemia (AML). METHODS: We utilized The Cancer Genome Atlas AML project (TCGA-LAML cohort, 15 acute promyelocytic leukemia (APL) and 155 non-APL AML) for the expression data of DNA repair genes. For validation, clinical samples (Ewha study group, 9 APL and 72 non-APL AML patients) were analyzed for the expression of 22 DNA repair genes using a custom RT2 Profiler PCR Array. RESULTS: APL patients presented significantly lower expression of DNA repair genes than non-APL AML patients in both study groups. Among non-APL AML patients, high expression levels of PARP1, XRCC1, and RAD51 were associated with poor overall survival (OS) probability in both study groups. Furthermore, Cox regression analysis showed that increased expression levels of PARP1, XRCC1, RAD51, BRCA1 and MRE11A could be independent risk factors for OS in the Ewha study group. Among non-APL patients of the Ewha study group, the OS probability of DDR-overexpressed group with at least one gene or more showing Z score greater than 1.5 was poorer than that of DDR non-overexpressed group. CONCLUSION: In the current study, the DNA repair gene expression profile of APL patients was different from that of non-APL AML patients. Overexpression of DNA repair genes could be a poor prognostic biomarker in non-APL AML.

Laboratory Diagnosis of Clostridium difficile Infection in Korea: The First National Survey.

In May 2015, we conducted a voluntary online survey on laboratory diagnostic assays for Clostridium difficile infection (CDI) across clinical microbiology laboratories in Korea. Responses were obtained from 66 laboratories, including 61 hospitals and five commercial laboratories. Among them, nine laboratories reported having not conducted CDI assays. The toxin AB enzyme immunoassay (toxin AB EIA), nucleic acid amplification test (NAAT), and C. difficile culture, alone or in combination with other assays, were used in 51 (89.5%), 37 (64.9%), and 37 (64.9%) of the remaining 57 laboratories, respectively, and 23 (40.4%) of the laboratories performed all three assays. Only one laboratory used the glutamate dehydrogenase assay. Nine laboratories used the toxin AB EIA as a stand-alone assay. The median (range) of examined specimens in one month for the toxin AB EIA, NAAT, and C. difficile culture was 160 (50-2,060), 70 (7-720), and 130 (9-750), respectively. These findings serve as valuable basic data regarding the current status of laboratory diagnosis of CDI in Korea, offering guidance for improved implementation.

Antibiotics-Associated Hemorrhagic Colitis Caused by Klebsiella oxytoca: Two Case Reports.

Nowadays, Klebsiella oxytoca is described as a causative organism for antibiotic-associated hemorrhagic colitis (AAHC). Here we report two cases of pediatric AAHC, from which K. oxytoca was cultured after starting amoxicillin-clavulanate or amoxicillin treatment. The patients developed severe abdominal pain and a large amount of bloody diarrhea. K. oxytoca was obtained in intestinal fluid culture of a boy through the colonoscopy. On the other hand, colonic tissue culture and intestinal fluid culture were negative of the other patient. K. oxytoca was detected in stool culture when he was admitted. These cases showed characteristic endoscopic findings of segmental hemorrhagic colitis, and both boys recovered spontaneously within 2-3 days after they stopped taking the antibiotics. Therefore, in children who develop relatively large amount of bloody diarrhea after antibiotic treatment, we should consider AAHC caused by K. oxytoca.

Calreticulin mRNA expression and clinicopathological characteristics in acute myeloid leukemia.

Calreticulin, encoded by CALR, is a multifunctional protein with roles in calcium homeostasis and chaperoning molecular processes. This study aimed to evaluate calreticulin mRNA expression levels in acute myeloid leukemia (AML) compared with other hematologic malignancies, and to investigate the clinicopathological characteristics associated with expression in AML patients. The study group included 43 patients diagnosed with AML, 57 with other hematologic malignancies, and 21 benign hematologic conditions. CALR mRNA quantification using real-time polymerase chain reaction revealed it to be significantly higher in AML compared with other hematologic malignancies (P < 0.0001). There was no difference in CALR mRNA expression between AML subgroups by karyotype (P = 0.3201). No differences were found in age, white blood cell counts, platelet counts, bone marrow blast percentage, calcium, lactate dehydrogenase or CD34 expression rate between the high and low CALR groups (CALR mRNA ≥ 1.2 fold and <1.2 fold, respectively), although hemoglobin and sex differences were observed. Although statistically not significant, there was a trend that Relapse rate was lower (54.5% vs. 84.6%) (P = 0.1063) and disease-free survival was longer (22 months vs. 7 months) (P = 0.0784) in low CALR group, whereas overall survival was similar between the two groups (11 months and 8 months). The clinical relevance of CALR expression in AML remains to be clarified in a larger cohort.

KMT2A (MLL)-MLLT1 rearrangement in blastic plasmacytoid dendritic cell neoplasm.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare hematologic malignancy characterized by CD4 and CD56 coexpression without apparent lineage commitment. The molecular pathogenesis of BPDCN has been studied in only a limited number of cases, and specific chromosomal aberrations are lacking thus far. KMT2A (MLL) rearrangements are observed in various types of pediatric and adult leukemia, but only one adult case report has so far showed KMT2A (MLL)-MLLT1 gene rearrangements in BPDCN. We present the first pediatric case of BPDCN with a KMT2A (MLL)-MLLT1 rearrangement confirmed by molecular study. The karyotype demonstrated a t(11;19)(q23;p13.3), trisomy 8, and trisomy 19 in all 20 metaphase cells analyzed: 48,XX,+8,t(11;19)(q23;p13.3),+19[20]. Fluorescence in situ hybridization analysis showed KMT2A (MLL) gene rearrangement in 83% of interphase cells. The KMT2A (MLL)-MLLT1 gene rearrangement was confirmed by multiplex reverse transcriptase polymerase chain reaction. We suggest that the pathogenesis of BPDCN could be associated with KMT2A (MLL) rearrangement (especially with KMT2A (MLL)-MLLT1) and further study on a larger number of cases is needed.

Comparison of the clinical performances of the AdvanSure HPV Screening Real-Time PCR, the Abbott Real-Time High-Risk HPV Test, and the Hybrid Capture High-Risk HPV DNA Test for Cervical Cancer Screening.

The clinical performance of three human papillomavirus (HPV) DNA commercial assays for cervical cancer screening was evaluated; the AdvanSure HPV Screening Real-Time PCR (AdvanSure PCR; LG Life Sciences) that was developed recently for the detection of both high-risk and low-risk genotypes, the Abbott RealTime High-Risk HPV Test (Abbott PCR; Abbott Molecular) and the Hybrid Capture High-Risk HPV DNA test (HC2; Qiagen). The three different HPV DNA tests were compared using cytology samples obtained from 619 women who underwent routine cervical cancer screening. The gold-standard assay was histopathological confirmation of cervical intraepithelial neoplasia of grade 2 or worse. The clinical sensitivities of the AdvanSure PCR, the Abbott PCR and the HC2 for the detection of cervical intraepithelial neoplasia of grade 2 or worse were 95.5%, 95.5% and 100%, respectively, while the clinical specificities were 61.6%, 86.4% and 83.3%, respectively. There were no significant differences in the clinical sensitivities of the Abbott PCR and the AdvanSure PCR compared to the HC2. The clinical specificities of the Abbott PCR and the AdvanSure PCR for the detection of HPV types 16/18 were 97.8% and 98.5%, respectively. For cervical cancer screening, all three tests showed relatively good clinical sensitivities, but the AdvanSure PCR had lower clinical specificity than the Abbott PCR and the HC2. The AdvanSure PCR and the Abbott PCR assays have the advantage of being automated and the ability to distinguish between HPV types 16/18 and other HPV types. The two real-time PCR assays could be useful tools in HPV testing for cervical cancer screening.

Comparison of the AdvanSure HPV GenoBlot assay with the INNO-LiPA HPV genotyping assay for human papillomavirus genotyping.

BACKGROUND: Persistent infection with oncogenic human papillomavirus (HPV) genotypes has been shown to be necessary for the development of cervical cancer. The recently developed AdvanSure HPV GenoBlot assay (AdvanSure assay) is based on real-time PCR amplification and reverse line-blot hybridization. OBJECTIVE: We first evaluated the performance of the AdvanSure assay compared to the INNO-LiPA HPV Genotyping assay (INNO-LiPA assay) for the molecular genotyping of HPV. STUDY DESIGN: A total of 200 cervical swab specimens were genotyped by the AdvanSure and INNO-LiPA assays. The AdvanSure and INNO-LiPA assays can detect 35 and 28 HPV genotypes, respectively. Samples that showed discrepancy were genotyped using sequencing. RESULTS: The percent agreement between the two assays ranged from 95.4 to 100% according to genotype. Analyzing the kappa values showed almost perfect agreement (>0.9) for high-risk HPV genotypes 16, 18 and 33 and substantial agreement (0.724) for HPV genotype 31. The positive rates of the two assays for frequent HPV genotypes (16, 18, 31, 33, 53, 58, 66, 68, and 70) were not statistically different, but the INNO-LiPA assay showed higher positive rates than the AdvanSure assay for HPV genotypes 51, 52, and 56 (P<0.05). All HPV genotypes that can be detected by only the AdvanSure assay (HPV genotypes 32, 34, 42, 55, 61, 62, and 81) were confirmed by sequencing. CONCLUSION: The AdvanSure assay was comparable with the well-established INNO-LiPA assay. The AdvanSure assay is a new alternative test for HPV genotyping in clinical laboratories.

Novel and recurrent mutations of ITGA2B and ITGB3 genes in Korean patients with Glanzmann thrombasthenia.

Glanzmann thrombasthenia (GT) is an autosomal recessive bleeding disorder caused by defective glycoprotein, αIIb and ß3, encoded by ITGA2B and ITGB3 genes, respectively. We herein describe four unrelated Korean patients with genetically confirmed GT. Two patients were homozygous for c.1913+5G>T (IVS11+5G>T) mutation of ITGB3 with a signature of founder effect. The other two patients were compound heterozygous for two mutations of ITGA2B: c.[2333A>C];[2975delA] (p.[Q778P];[E992Gfs*30]) and c.[1750C>T];[2333A>C] (p.[R584X];[Q778P]). The c.2975delA mutation was a novel frameshift mutation of ITGA2B. Although from a limited number of patients, these results suggests c.1913+5G>T of ITGB3 is a recurrent mutation in Korean patients with GT.

A novel nonsense mutation in the MPL gene in congenital amegakaryocytic thrombocytopenia.

Congenital amegakaryocytic thrombocytopenia (CAMT) is a rare autosomal recessive disorder characterized by thrombocytopenia from failure of megakaryopoiesis. CAMT is one of the bone marrow failure syndromes, and the disease progression may involve other lineages leading to pancytopenia. The genetic background of CAMT is mutations in the MPL gene encoding the thrombopoietin receptor. Here, we describe a Korean male with CAMT. Molecular genetic analyses by direct sequencing revealed that he was compound heterozygous for two nonsense mutations in MPL, Tyr63X (c.189C>A), and Arg357X (c.1069C>T), the latter being a novel mutation.

Association of ATP7B mutation detection rate with biochemical characteristics in Korean patients with Wilson disease.

Wilson disease (WD) is an autosomal recessive disorder caused by mutations in the ATP7B gene, yet many patients have either one mutation, or no mutation. We investigated whether the mutation detection rate is associated with any biochemical characteristics of WD. In a study of 71 patients, we used PCR-sequencing to screen for ATP7B mutations in 7 exons (exons 8, 10, 11, 14, 15, 16, and 18) covering 95% of known mutations in Korean patients with WD. We also investigated serum concentrations of various biochemical analytes. Data were analyzed by linear association test and one-way ANOVA. Based on the number of detected ATP7B mutations, a significant difference in serum ceruloplasmin concentration was found among the 3 groups (p <0.001). Serum ceruloplasmin concentration averaged 3.32 +/- 1.74, 10.8 +/- 5.50, and 14.9 +/- 3.88 mg/dl (mean +/- SD) in the 25, 20, and 26 patients with two, one, and no ATP7B mutations, respectively. We observed 82.9% and 16.7% of mutant allele frequency in WD patients with ceruloplasmin concentration <10 mg/dl and 10-20 mg/dl, respectively (p <0.001). Thus serum ceruloplasmin concentrations among WD patients differed according to the number of ATP7B mutations detected.

UNC13D is the predominant causative gene with recurrent splicing mutations in Korean patients with familial hemophagocytic lymphohistiocytosis.

BACKGROUND: Familial hemophagocytic lymphohistiocytosis is a fatal disease characterized by immune dysregulation from defective function of cytotoxic lymphocytes. Three causative genes have been identified for this autosomal recessive disorder (PRF1, UNC13D, and STX11). We investigated the molecular genetics of familial hemophagocytic lymphohistiocytosis in Korea. DESIGN AND METHODS: Pediatric patients who fulfilled the HLH-2004 criteria were recruited from the Korean Registry for Histiocytosis. Molecular genetic studies were performed on the patients' DNA samples by direct sequencing of all coding exons and flanking sequences of PRF1, UNC13D, and STX11. RESULTS: Forty patients were studied and familial hemophagocytic lymphohistiocytosis mutations were identified in nine; eight patients had UNC13D mutations (89%) and one had a mutation in PRF1. No patient had a STX11 mutation. Notably, four patients had only one UNC13D mutant allele, suggesting that the other mutation was missed by conventional direct sequencing. All UNC13D mutations were deleterious in nature. One known splicing mutation, c.754-1G>C, was recurrent, accounting for 58% of all the mutant alleles (7/12). Five UNC13D mutations were novel (p.Gln98X, p.Glu565SerfsX7, c.1993-2A>G, c.2367+1G>A, and c.2954+5G>A). The one patient with PRF1 mutation was homozygous for a frameshift mutation (p.Leu364GlufsX93), which was previously reported to be the most frequent PRF1 mutation in Japan. CONCLUSIONS: This is the first investigation on the molecular genetics of familial hemophagocytic lymphohistiocytosis in Korea. The data showed that UNC13D is the predominant causative gene in the Korean population. The identification of mutations missed by conventional sequencing would better delineate the mutation spectrum and help to establish the optimal molecular diagnostic strategy for familial hemophagocytic lymphohistiocytosis in Korea, which might need an RNA-based screening strategy.

[Evaluation of Viva-E drug testing system].

BACKGROUND: The importance and usefulness of therapeutic drug monitoring (TDM) have been emphasized, and analysis of drugs has been increased in clinical laboratories. We evaluated the analytical performance and clinical usefulness of a recently introduced enzyme multiplied immunoassay instrument, Viva-E Drug Testing System (Dade Behring Inc., USA). METHODS: Using patients' samples and quality control material, we evaluated the analytical performance of Viva-E for a total of 11 drugs (cyclosporine, tacrolimus, mycophenolic acid, valproic acid, digoxin, theophylline, carbamazepine, phenytoin, phenobarbital, vancomycin, and gentamicin) with respect to linearity, precision, and correlations with other methods according to CLSI guidelines. Cobas Integra 800 (Roche Diagnostics, Switzerland) and API 4000 LC-MS/MS System (Applied Biosystems, USA) were used to make a comparison. In addition, we analyzed analysis time. RESULTS: Viva-E showed a good linearity (r2 >or= 0.97) for all items. Within-run CVs were within 5% and total CVs were within 10% for all drugs except for tacrolimus and digoxin at low concentrations. The system correlated well with the other methods (r=0.9283-0.9778). The time required for reporting the first sample was 11 min and the analysis time was 1.1 min. CONCLUSIONS: Since Viva-E showed a good analytical performance required for TDM in its linearity, precision, and accuracy with its wide drug menus including cyclosporine, tacrolimus, and mycophenolic acid, stat and random accessing functions, and the consolidation to a single workstation, it could be very useful in the clinical laboratory for various needs.