Instability of Plasma and Serum Progastrin-Releasing Peptide During Repeated Freezing and Thawing.

OBJECTIVES: Progastrin-releasing peptide (proGRP) is a promising biomarker for small cell lung cancer. However, not much is known about how sample processing and storage conditions affect the stability of proGRP. Here, we examined the effects of repeated freeze-thaw cycles on the stability of proGRP in plasma and serum. METHODS: Concentrations of proGRP were measured in plasma and serum samples exposed to two, three, or four freeze-thaw cycles and these were compared with values of corresponding samples exposed to one cycle (baseline). We also performed the area under the receiver-operating-characteristic curve (AUC) analysis to determine whether the differences of proGRP concentrations between each paired plasma and serum sample (ΔproGRP) can be used for identifying the samples that have been exposed to multiple freeze-thaw cycles. RESULTS: Concentrations of proGRP gradually decreased in both plasma and serum samples with increasing numbers of freeze-thaw cycles. Reduction rates of proGRP concentrations were greater in serum than in plasma samples and serum proGRP levels declined with statistical significance (p < 0.001) up to 10.1% after four freeze-thaw cycles. The ΔproGRP measurement showed fair accuracy (AUC = 0.741) for identifying samples that had been through four freeze-thaw cycles. The sensitivity was 82.8% and specificity was 62.1% at an optimal cut-off point of > 4.9. CONCLUSION: Our study shows that the stability of circulating proGRP is affected in both plasma and serum samples by repeated freezing and thawing. We also show that ΔproGRP could be used for identifying paired plasma and serum samples subjected to multiple freeze-thaw cycles.

Incidence, clinical features, and prognostic impact of CALR exon 9 mutations in essential thrombocythemia and primary myelofibrosis: an experience of a single tertiary hospital in Korea.

We evaluated the incidence, clinical characteristics, and prognostic impact of calreticulin (CALR) mutations in essential thrombocythemia (ET) and primary myelofibrosis (PMF) patients. In all, 48 ET and 14 PMF patients were enrolled, and the presence of CALR mutations was analyzed by direct sequencing. Patients were classified into three subgroups according to Janus kinase 2 (JAK2) V617F and CALR mutation status, and their clinical features and prognosis were compared. CALR mutations were detected in 15 (24.2%) patients, and the incidence increased to 50.0% in 30 JAK2 V617F mutation-negative cases. These included 11 patients with three known mutations (c.1092_1143del [seven cases], c.1154_1155insTTGTC [three cases], and c.1102_1135del [one case]) and 4 patients with novel mutations. ET patients carrying CALR mutation were younger, had lower white blood cell counts, and experienced less thrombosis during follow-up than those carrying JAK2 V617F mutation, while both patient groups showed similar clinical features and prognosis. In ET patients without JAK2 V617F mutation, CALR mutation did not significantly affect clinical manifestation and prognosis. In conclusion, CALR mutation analysis could be a useful diagnostic tool for ET and PMF in 50% of the cases without JAK2 V617F mutations. The prognostic impact of CALR mutations needs further investigation.

Incidence and prognostic impact of DNMT3A mutations in Korean normal karyotype acute myeloid leukemia patients.

BACKGROUND: DNA methyltransferase 3A (DNMT3A) mutation was recently introduced as a prognostic indicator in normal karyotype (NK) AML and we evaluated the incidence and prognostic impact of DNMT3A mutations in Korean NK AML patients. METHODS: Total 67 NK AML patients diagnosed during the recent 10 years were enrolled. DNMT3A mutations were analyzed by direct sequencing and categorized into nonsynonymous variations (NSV), deleterious mutations (DM), and R882 mutation based on in silico analysis results. Clinical features and prognosis were compared with respect to DNMT3A mutation status. RESULTS: Three novel (I158M, K219V, and E177V) and two known (R736H and R882H) NSVs were identified and the latter three were predicted as DMs. DNMT3A NSVs, DMs, and R882 mutation were identified in 14.9%-17.9%, 10.3%-10.4%, and 7.5% of patients, respectively. DNMT3A mutations were frequently detected in FLT3 ITD mutated patients (P=0.054, 0.071, and 0.071 in NSV, DMs, and R882 mutation, resp.) but did not affect clinical features and prognosis significantly. CONCLUSIONS: Incidences of DNMT3A NSVs, DMs, and R882 mutation are 14.9%-17.9%, 10.3%-10.4%, and 7.5%, respectively, in Korean NK AML patients. DNMT3A mutations are associated with FLT3 ITD mutations but do not affect clinical outcome significantly in Korean NK AML patients.

Clinical relevance of high-resolution single nucleotide polymorphism array in patients with relapsed acute lymphoblastic leukemia with normal karyotype: a report of three cases.

We report three patients with normal karyotype (NK) ALL, who showed genetic aberrations as determined by high-resolution single nucleotide polymorphism array (SNP-A) analysis at both diagnosis and relapse. We evaluated the clinical relevance of the SNP-A assay for the detection of subtle changes in the size of affected genetic lesions at relapse as well as the prognostic value of the assay. In our patients, application of the SNP-A assay enabled sensitive detection of cryptic changes affecting clinically important genes in NK ALL. Therefore, this assay seems to be more advantageous compared to other conventional methods such as FISH assay, HemaVision (DNA Technology, Denmark), and conventional karyotyping for the detection of an "unstable genotype" at relapse, which may be associated with microscopic clonal evolution and poor prognosis. Further comprehensive studies are required to confirm the issues presented by our case patients in this report.

Effect of Repeated Freezing and Thawing on Biomarker Stability in Plasma and Serum Samples.

OBJECTIVES: The stability of circulating proteins can be affected by repeated freezing and thawing. The aim of our study was to identify the effect of repeated freezing and thawing on the plasma and serum concentrations of eight proteins [interferon-γ, interleukin (IL)-8, IL-15, IL-17A, matrix metalloproteinase (MMP)-7, tumor necrosis factor-α, vascular endothelial growth factor (VEGF), and VEGF receptor 2 (VEGF-R2)]. METHODS: We assessed the concentration changes of these proteins in 30 plasma and serum samples subjected to three, four, or five freeze-thaw cycles, and compared these with the concentration changes in the samples that were subjected to two freeze-thaw cycles before analysis. RESULTS: Repeated freezing and thawing by up to five cycles did not modify the plasma and serum concentrations of interferon-γ, IL-8, and VEGF-R2, while levels of MMP-7, tumor necrosis factor-α, and VEGF were significantly changed in both plasma and serum samples. Moreover, MMP-7 and VEGF concentrations tended to increase with freeze-thaw cycles. They were more elevated in plasma samples (up to about 15%) than in serum samples (up to about 7%), suggesting that serum is the preferred sample type for the analysis of circulating proteins. CONCLUSION: This is the first report on the effect of repeated freezing and thawing on plasma concentrations of MMP-7 and VEGF-R2. Our findings propose that researchers should consider the number of freeze-thaw cycles to select plasma or serum samples, depending on the type of analyte.

Differential expression levels of plasma-derived miR-146b and miR-155 in papillary thyroid cancer.

BACKGROUND: Specific circulating microRNAs (miRNAs) in each organ may contribute to the diagnosis and prognosis in some cancers. miRNA from papillary thyroid cancer (PTC) may be released into the bloodstream. This study was performed to detect miRNAs in the plasma and estimate their diagnostic usefulness for discriminating between benign and malignant lesions. METHODS: Patients who underwent thyroidectomy for benign thyroid nodules or PTC were enrolled in this study. The patients were divided into three groups: benign, PTC without lymph node metastasis (LNM), and PTC with LNM. The levels of miR-146b, miR-221, miR-222, and miR-155miRNA expression in blood samples before surgery were evaluated. RESULTS: Of 89 patients enrolled in this study, 19 and 70 had benign lesions (21.3%) and PTC (78.7%), respectively. The mean levels of miR-146b and miR-155 expression were higher in the PTC group than the benign group. For discrimination between benign and PTC lesions, the area under the ROC curve (AUC) for miR-146b was 0.649 with 61.4% sensitivity and 57.9% specificity. The AUC for miR-155 was 0.695 with 74.3% sensitivity and 63.2% specificity (P<0.05). The levels of miR-146b, miR-221, and miR-222 were slightly higher in the N1 group than the N0 group. The levels of miR-146b, miR-155, and miR-222 increased in proportion to tumor size. CONCLUSIONS: miR-146b and miR-155 helped to discriminate between benign and malignant lesions. Circulating miRNA is likely a useful alternate serological marker for PTC. This preliminary study suggested that circulating miRNAs may be useful as follow-up tools as well as diagnostic tools.

All-in-one centrifugal microfluidic device for size-selective circulating tumor cell isolation with high purity.

Circulating tumor cells (CTCs) have gained increasing attention owing to their roles in cancer recurrence and progression. Due to the rarity of CTCs in the bloodstream, an enrichment process is essential for effective target cell characterization. However, in a typical pressure-driven microfluidic system, the enrichment process generally requires complicated equipment and long processing times. Furthermore, the commonly used immunoaffinity-based positive selection method is limited, as its recovery rate relies on EpCAM expression of target CTCs, which shows heterogeneity among cell types. Here, we propose a centrifugal-force-based size-selective CTC isolation platform that can isolate and enumerate CTCs from whole blood within 30 s with high purity. The device was validated using the MCF-7 breast cancer cell line spiked in phosphate-buffered saline and whole blood, and an average capture efficiency of 61% was achieved, which is typical for size-based filtration. The capture efficiency for whole blood samples varied from 44% to 84% under various flow conditions and dilution factors. Under the optimized operating conditions, a few hundred white blood cells per 1 mL of whole blood were captured, representing a 20-fold decrease compared to those obtained using a commercialized size-based CTC isolation device. In clinical validation, normalized CTC counts varied from 10 to 60 per 7.5 mL of blood from gastric and lung cancer patients, yielding a detection rate of 50% and 38%, respectively. Overall, our CTC isolation device enables rapid and label-free isolation of CTCs with high purity, which should greatly improve downstream molecular analyses of captured CTCs.

The first Korean case of Sphingobacterium spiritivorum bacteremia in a patient with acute myeloid leukemia.

Sphingobacterium spiritivorum has been rarely isolated from clinical specimens of immunocompromised patients, and there have been no case reports of S. spiritivorum infection in Korea to our knowledge. We report a case of S. spiritivorum bacteremia in a 68-yr-old woman, who was diagnosed with acute myeloid leukemia and subsequently received chemotherapy. One day after chemotherapy ended, her body temperature increased to 38.3℃. A gram-negative bacillus was isolated in aerobic blood cultures and identified as S. spiritivorum by an automated biochemical system. A 16S rRNA sequencing analysis confirmed that the isolate was S. spiritivorum. The patient received antibiotic therapy for 11 days but died of septic shock. This is the first reported case of human S. spiritivorum infection in Korea. Although human infection is rare, S. spiritivorum can be a fatal opportunistic pathogen in immunocompromised patients.

Validation of circulating miRNA biomarkers for predicting lymph node metastasis in gastric cancer.

We validated candidate biomarkers using circulating miRNAs by analyzing serum miRNA concentrations from patients with gastric cancer (GC) to predict lymph node (LN) metastasis. In a pilot study, serum levels of miR-21, miR-27a, miR-106b, miR-146a, miR-148a, miR-223, and miR-433 were compared in 10 healthy donors, 16 LN-positive patients with GC, and 15 LN-negative patients with GC. Then, we compared the level of three miRNAs (miR-21, miR-146a, and miR-148a) with the total of 79 GC patients with or without LN metastasis. In the pilot study, miR-21, miR-27a, miR-106b, miR-146a, miR-148a, and miR-223 concentrations from LN-positive patients with GC were significantly different from those of LN-negative patients with GC (P < 0.001, P = 0.003, P = 0.033, P < 0.001, P <0.001, and P = 0.017, respectively). In the validation study, levels of miR-21, miR-146a, and miR-148a increased as pN stage increased (P < 0.001, P = 0.001, and P < 0.001, respectively). Levels of the miRNAs were significantly different between pN0 and pN0 in the pT1 group (P = 0.013, P = 0.004, and P = 0.035, respectively) and among clinical stages (P = 0.001, P = 0.002, and P < 0.001, respectively). No differences in miRNA levels were observed by pT stage, Lauren's classification, sex, or age. Serum concentrations of miR-21, miR-146a, and miR-148a were closely associated with GC pN stage. These serum miRNA levels could be biomarker candidates to predict the presence of LN metastasis.

Bacteroides fragilis vertebral osteomyelitis complicated by percutaneous epidural adhesiolysis.

STUDY DESIGN: A case report of anaerobic vertebral osteomyelitis after percutaneous epidural adhesiolysis. OBJECTIVE: To present a case of Bacteroides fragilis spondylodiscitis (BFS) secondary to percutaneous epidural adhesiolysis in a 38-year-old woman without predisposing factors. SUMMARY OF BACKGROUND DATA: Most cases of BFS result from hematogenous spread from a perianal abscess or sigmoidoscopy or local spread from an adjacent infection. However, BFS due to direct inoculation after percutaneous epidural adhesiolysis has not been previously reported. METHODS: A 38-year-old woman presented with spondylodiscitis at the L4-L5 level 2 weeks after percutaneous epidural adhesiolysis. Despite empirical antibiotherapy, the spondylodiscitis and an epidural abscess became much aggravated. Open biopsy and curettage was performed, and metronidazole sensitive Bacteroides fragilis was identified by tissue culture. RESULTS: Metronidazole was administrated for 5 weeks and symptoms were completely resolved. Follow-up magnetic resonance imaging showed that the spondylodiscitis was completely cured. CONCLUSION: This is the first report to be issued regarding BFS secondary to percutaneous epidural adhesiolysis. In our case, the pathogenesis may have been direct inoculation of Bacteroides fragilis into the epidural space and disc during percutaneous epidural adhesiolysis because the procedural approach used was adjacent to the anus.

Clinical relevance of elevated levels of serum soluble interleukin-2 receptor alpha (sIL-2Rα) in patients with non-Hodgkin's lymphoma.

Levels of soluble interleukin-2 receptor alpha (sIL-2Rα) are known to increase in the sera of patients with certain malignancies, including malignant lymphoma. This study aimed to assess the clinical significance of the sIL-2Rα level in non-Hodgkin's lymphoma (NHL). We used ELISA to measure the sIL-2Rα levels in 48 newly diagnosed and untreated patients with NHL and evaluated the correlation between the sIL-2Rα levels and clinical characteristics and the International Prognostic Index (IPI). We monitored serum sIL-2Rα in 7 patients to compare the changes in their clinical progress with these levels. High levels of serum sIL-2Rα (≥ 2,000 U/mL) correlated well with parameters defining the high risk group according to the IPI, i.e., high tumor burden at diagnosis (stage III+IV) and lactate dehydrogenase ≥ 472 U/L. The levels were also associated with B symptoms, bone marrow involvement, and poor response to therapy. The sIL-2Rα level decreased during complete remission and was elevated during disease progression or relapse. A high level of sIL-2Rα was significantly associated with a low survival rate. These results suggest that serum sIL-2Rα might be useful as a biomarker for evaluating the prognosis of patients with NHL at the time of diagnosis and during therapy. A well-controlled, large-scale study is needed to clarify the clinical significance of sIL-2Rα in specific groups of NHL.

[Evaluation of ARCHITECT HCV core antigen assay].

BACKGROUND: Hepatitis C virus (HCV) core antigen (Ag) levels are known to be well correlating with HCV RNA levels, and may be used as an alternative marker of HCV replication for monitoring the response to HCV treatment. However, the low sensitivity of HCV core Ag assay has been an obstacle for clinical use. In this study, recently developed ARCHITECT HCV Ag assay (Abbott Laboratories, USA) was evaluated for analytical performance and clinical usefulness. METHODS: A total of 109 sera from HCV infected patients including various genotypes of HCV (1b, 2, 2a/2c, 2b, and 3a) and 20 sera from healthy donors were used for evaluating the sensitivity, precision, and linearity of the HCV core Ag assay. The cross reactivity with HIV, hepatitis B virus and myeloma proteins (N=5, each) and correlation with HCV RNA PCR assay were also evaluated. RESULTS: The sensitivity of the HCV core Ag assay was 97.2% (106/109) and there were no false positive results and cross reactivity. The within-run, between-run and between-day CVs were 3.0%, 2.5% and 3.0%, respectively. The levels of HCV core antigen showed a good correlation with those of HCV RNA quantification (r=0.940). The HCV Ag assay showed an excellent linearity in the range from 0.63 to 17,114 fmol/L (r=0.999). CONCLUSIONS: The ARCHITECT HCV Ag assay was good in sensitivity, precision, and linearity and its results well correlated with HCV RNA levels. This assay could be used as a good marker of viral replication for monitoring the therapy response in chronically HCV infected patients.

Level of HOXA5 hypermethylation in acute myeloid leukemia is associated with short-term outcome.

Hypermethylation of the homeobox (HOX) gene promoter leads to decreased expression of the gene during tumor development and is thought to be correlated with the clinical outcome in leukemia. In this study, we performed pyrosequencing to quantify the methylation level of HOXA5 genes in the bone marrow samples obtained from 50 patients with AML and 19 normal controls. The methylation percentage of HOXA5 in AML patients (median=65.4%, interquartile range=35.9-72.3%) was higher than that of HOXA5 in control patients (median=43.1%, interquartile range=36.7-49.6%, Mann-Whitney U test, P=0.012). The patients of the AML group who had a high methylation percentage (>70%) had a good prognosis with a 3-yr overall survival (OS) of 82.5%, whereas the patients with a low methylation percentage (≤70%) showed a 3-yr OS of 40.5% (P=0.048). Cox proportional hazards regression showed that the methylation percentages of HOXA5 were independently associated with the 3-yr OS of AML patients, regardless of their karyotypes. We propose that the quantification of HOXA5 methylation by pyrosequencing may be useful for predicting short-term prognosis in AML. However, the limitations of our study are the small sample size and its preliminary nature. Thus, a larger study should be performed to clearly determine the relationships among HOXA5 methylation levels, cytogenetics, and prognosis in AML patients.

A case of congenital central hypoventilation syndrome with PHOX2B gene mutation in a Korean neonate.

Congenital central hypoventilation syndrome (CCHS) is a life-threatening disorder with apnea and cyanosis during sleep requiring immediate endotracheal intubation during the first day of life. The PHOX2B gene has been identified as the major gene involved in CCHS. This is the first report of a Korean neonate with CCHS confirmed to have a PHOX2B mutation with expanded alleles containing 20 polyalanine repeats that is a relatively small number compared to previous cases. The patient required intermittent ventilator support during sleep only and did not suffer from any other disorders of the autonomic nerve system. He consistently needs ventilator support during sleep and remains alive. Analysis of PHOX2B gene is useful for diagnosis and appropriate therapeutic intervention of CCHS patients.