Consensus Definition of Blood Samples from the Subcategorized Normal Controls in the Korea Biobank Network.

A control group is defined as a group of people used for comparison. Depending on the type of study, it can be a group of healthy people or a group not exposed to risk factors. It is important to allow researchers to select the appropriate control participants. The Korea Biobank Project-sponsored biobanks are affiliated with the Korea Biobank Network (KBN), for which the National Biobank of Korea plays a central coordinating role among KBN biobanks. KBN organized several working groups to address new challenges and needs in biobanking. The "Normal Healthy Control Working Group" developed standardized criteria for three defined control groups, namely, normal, normal-plus, and disease-specific controls. Based on the consensus on the definition of a normal control, we applied the criteria for normal control participants to retrospective data. The main reason for exclusion from the "Normal-plus" group was blood test results beyond 5% of the reference range, including hypercholesterolemia. Subclassification of samples of normal controls by detailed criteria will help researchers select optimal normal controls for their studies.

BRCA1/2 somatic mutation detection in formalin-fixed paraffin embedded tissue by next-generation sequencing in Korean ovarian cancer patients.

INTRODUCTION: The detection of BRCA1/2 mutations is important because PARP1 inhibitors are approved for germline and/or somatic BRCA-mutated advanced ovarian cancer. Next-generation sequencing (NGS) is increasingly used in clinical practice for BRCA1/2 mutations. The purpose of this study was to consider several conditions of NGS BRCA1/2 assay applicable to clinical laboratory tests, in particular for using formalin fixed paraffin embedded (FFPE) ovarian tissues. MATERIALS AND METHODS: We selected 64 ovarian cancer patients and performed Oncomine™ BRCA assay using FFPE tissue. Effect of FFPE sample quality was analyzed by NGS quality parameters including deamination metric. Somatic variants were selected by removing germline variants of peripheral blood and interpreted as pathogenic, variants of unknown significance, and false positive. RESULTS: We found a positive relationship between the number of variants over the deamination metric and FFPE age (P < 0.001) with a cutoff values of approximately 0.7 and 60 months, respectively. When comparing NGS results with Sanger sequencing, NGS misreported 3 of 15 variants using default parameters which were corrected after changing parameters. We detected somatic variants in eight patients and classified them into pathogenic (n = 3), VUS (n = 3) and false positive (n = 2). CONCLUSIONS: This study is important for improving BRCA1/2 mutation detection capabilities of NGS analytical pipelines and strategy to overcome their limitations using FFPE tissue in ovarian cancer patients.

Quantification of BRAF V600E alleles predicts papillary thyroid cancer progression.

The BRAF V600E mutation is the most common genetic alteration in thyroid cancer. However, its clinicopathological significance and clonal mutation frequency remain unclear. To clarify the inconsistent results, we investigated the association between the allelic frequency of BRAF V600E and the clinicopathological features of classic papillary thyroid carcinoma (PTC). Tumour tissues from two independent sets of patients with classic PTC were manually microdissected and analysed for the presence or absence of the BRAF mutation and the mutant allelic frequency using quantitative pyrosequencing. For external validation, the Cancer Genome Atlas (TCGA) data were analysed. The BRAF V600E mutation was found in 264 (82.2%) out of 321 classic PTCs in the training set. The presence of BRAF V600E was only associated with extrathyroidal extension and the absence of thyroiditis. In BRAF V600E-positive tumours, the mutant allelic frequency varied from 8 to 41% of the total BRAF alleles (median, 20%) and directly correlated with tumour size and the number of metastatic lymph nodes. Lymph node metastases were more frequent in PTCs with a high (≥20%) abundance of mutant alleles than in those with a low abundance of mutant alleles (P=0.010). These results were reinforced by validation dataset (n=348) analysis but were not reproduced in the TCGA dataset. In a population with prevalent BRAF mutations, quantitative analysis of the BRAF mutation could provide additional information regarding tumour behaviour, which is not reflected by qualitative analysis. Nonetheless, prospective studies are needed before the mutated allele percentage can be considered as a prognostic factor.

Asymptomatic hematuria associated with urinary polyomavirus infection in immunocompetent patients.

Unlike the case for immunodeficient patients, little is known about polyomavirus (PV) infection in immunocompetent patients. PV infection in immunocompetent individuals has been reported sporadically, but little is known about asymptomatic hematuria. To determine the clinical significance and prevalence of urinary PV infection in immunocompetent patients, a total of 95 individuals admitted to Seoul St. Mary's hospital were investigated. Sixty-four patients were enrolled for evaluation of asymptomatic hematuria, and 31 healthy individuals served as controls. Clinical screening for PV infection was performed by urine cytology analysis by liquid-based preparation and urine RT-PCR for BK virus (BKV) and JC virus (JCV), respectively. The average age of the patients in the PV(+) - and PV(-) -groups with asymptomatic hematuria were 60 years and 46 years, respectively. Urine cytology analysis revealed decoy cells in 37/64 hematuria patients (38.9%), but not in healthy controls. They were more prevalent in male patients. Eighty-two patients (86.3%) had PV viruria, viz., 54/64 patients in the hematuria group and 28/31 in the control group. Interestingly, 28/31 (90.3%) cases in the healthy control group were positive for PV viruria, which exceeded the number in the hematuria group (84.4%). PV viruria was associated primarily with JCV, rather than BKV. PV viruria, including JCV viruria, correlated with urine decoy cells and increased age. In conclusion, urinary PV infection is common in immunocompetent patients with asymptomatic hematuria and is age-related. These data may provide an insight into the pathogenesis and future treatment of asymptomatic hematuria associated with urinary PV infection in immunocompetent patients.

Mutational patterns and novel mutations of the BRAF gene in a large cohort of Korean patients with papillary thyroid carcinoma.

BACKGROUND: BRAF mutation is the most common genetic event in papillary thyroid carcinoma (PTC); however, the prevalence and patterns of the mutation vary worldwide. We investigated the frequency and type of BRAF mutations based on the histologic subtypes in a large cohort of Korean patients with PTC. METHODS: A total of 1041 consecutive PTCs were classified according to histologic subtypes. BRAF mutations were examined by denaturing high-performance liquid chromatography and direct sequencing. Rare complex mutations were confirmed by molecular cloning of polymerase chain reaction amplicons and sequencing of the products. RESULTS: BRAF mutations were found in 839 (80.6%) of 1041 patients with PTC. The histologic subtype-specific prevalence of BRAF mutation was as follows: 85.3% (249/292) were classic, 45.8% (11/24) were follicular, 79.9% (576/721) were microcarcinoma, and 75.0% (3/4) were other variants. In addition to the usual c.1799T>A mutation, we identified other four mutation types: c.[1795_1796insA;1770_1795dup26], c.[1742-10T>C;1799T>A] and c.[1796C>G;1799T>A], and c.1799_1800TG>AA, respectively. The former three were novel mutations in thyroid tumors. Within the series of microcarcinoma variants, the BRAF mutation rate was lower in tumors with follicular morphology than those with nonfollicular types (66.7% vs. 80.9%, p=0.0145). CONCLUSION: Out of 1041 Korean patients with PTC, 0.4% had rare types of BRAF mutation and three new somatic mutations were identified. The BRAF mutation rate was quite low in PTC with follicular morphology regardless of tumor size. However, the prevalence of BRAF mutation in microcarcinoma and follicular variants of PTC is relatively high in Korea and its analysis may be clinically useful for managing the patients.

A prospective, multicenter, phase 2 study of imatinib mesylate in korean patients with metastatic or unresectable gastrointestinal stromal tumor.

OBJECTIVES: This prospective, multicenter, phase 2 study evaluated the efficacy and safety of imatinib mesylate and assessed KIT and PDGFRA gene mutation status in Korean patients with gastrointestinal stromal tumors (GISTs). METHODS: Forty-seven patients with pathologically proven KIT-positive metastatic or unresectable GISTs were accrued from eight institutions in Korea. Imatinib was administered orally at 400 mg once daily. In case of disease progression, the dose was escalated to 600 mg once daily, then 400 mg twice daily. KIT and PDGFRA mutations were analyzed in 29 of the 47 patients. RESULTS: Imatinib produced partial responses in 30 patients (63.8%; 95% confidence interval, 50.1-77.6%) and stable disease in 13 patients (27.7%). The median time to response was 2.6 months (range, 1.0-6.2 months). With a median follow-up of 62 months (range, 32-67 months), 4-year progression-free survival and overall survival rates were 50 and 65%, respectively. The most common adverse events were anemia, neutropenia, edema, and skin rash (predominantly of grade 1-2). There were no treatment-related deaths. In the subset evaluated for mutational status, 24 patients (82.8%) had KIT exon 11 mutations and 1 (3.4%) had a KIT exon 9 mutation. CONCLUSIONS: Imatinib is effective and safe in Korean patients with metastatic or unresectable GIST.

Dose escalation of imatinib after failure of standard dose in Korean patients with metastatic or unresectable gastrointestinal stromal tumor.

OBJECTIVE: We evaluated the results of imatinib dose escalation in patients with advanced gastrointestinal stromal tumors (GISTs) after disease progression on standard-dose imatinib. METHODS: Clinical data from patients with metastatic or unresectable GISTs whose dose of imatinib was increased after disease progression on imatinib 400 mg/day were retrospectively reviewed. RESULTS: The 24 patients studied had a median age of 52 years. Imatinib dosing was escalated to 600 mg/day in 12 patients, then to 800 mg/day in four patients. The other 12 patients had dose escalation directly to 800 mg/day. Two patients (8.3%) achieved a partial response, and seven (29.2%) had stable disease. Six-month progression-free and overall survival rates were 33.3 and 70.7%, respectively. Dose escalation to 600 or 800 mg/day was generally well tolerated. CONCLUSION: Imatinib dose escalation is feasible and well tolerated in patients with advanced GIST who progress on standard-dose therapy, producing clinical benefit in approximately 37% of patients.

PDGFRalpha gene mutation and protein expression in gastrointestinal stromal tumors.

OBJECTIVE: Mutation of the PDGFRalpha is a potential candidate in the pathogenesis of KIT wild-type gastrointestinal tumors (GISTs). In this study, we evaluated the prevalence of PDGFRalpha mutations and corresponding protein expression in GISTs, to determine their usefulness in obtaining a prognosis. METHODS: Genomic DNA was extracted from paraffin-embedded tumor tissues from 194 GISTs. Exons 12, 14 and 18 of the PDGFRalpha were amplified and sequenced. Immunohistochemical staining was performed in 179 patients. RESULTS: Mutations in the PDGFRalpha were detected in 6 (3.1%) patients, and were observed solely in KIT wild-type GISTs. Among the 6 patients with PDGFRalpha gene mutations, 5 patients with localized GISTs showed no relapse after resection during the 19- to 80-month follow-up period. Intensity of PDGFRalpha expression was classified as 0 in 26 (14.5%), 1+ in 69 (38.5%), 2+ in 71 (39.7%) and 3+ in 13 (7.3%) patients. Levels of PDGFRalpha expression showed no correlation with relapse-free survival. CONCLUSION: PDGFRalpha mutations in GISTs were found to be rare in this Korean population. Although localized GISTs with PDGFRalpha mutations showed relatively good prognosis after resection, the difference was not statistically significant.

Chlamydia psittaci infection and clinicopathologic analysis of ocular adnexal lymphomas in Korea.

Ocular adnexal lymphoma (OAL) is a mostly extranodal marginal zone lymphoma (EMZL). Recent findings have suggested an association between Chlamydia psittaci (Cp) infection and OAL. We sought to confirm this issue and to analyze the clinicopathologic characteristics of OAL in Korea. Between 1993 and 2004, 33 OAL cases were identified at the Asan Medical Center, Seoul, Korea. DNA was extracted from paraffin-embedded tissues, and touchdown enzyme time release polymerase chain reaction was performed to identify three Chlamydia species (Cp, C. tracomatis, and C. pneumoniae). The same procedures were also performed in 21 samples from patients with non-neoplastic ocular adnexal disease (NNOAD). All OAL cases were EMZL. Cp DNA was detected in 26/33 (79%) OAL samples compared with 5/21 (23%) NNOAD samples (P < 0.001). With a median follow-up of 38.5 months (range: 1-105 months), the 5-year progression-free survival (PFS) and overall survival (OS) rates of OAL patients were 72% and 93%, respectively. Clinicopathologic characteristics, recurrence rate, PFS, and OS were not associated with Cp infection. Our study demonstrates an association between OAL and Cp infection in Korea, suggesting that Cp plays a role as a causative antigen in Korean OAL patients.

Prognostic significance of c-kit mutation in localized gastrointestinal stromal tumors.

PURPOSE: Constitutive mutational activation of c-kit has been found to be associated with the pathogenesis of gastrointestinal stromal tumors (GISTs). The prognostic significance of c-kit mutations, however, is still controversial. EXPERIMENTAL DESIGN: We examined 86 patients curatively resected for localized GIST. Genomic DNA was extracted from paraffin-embedded tumor tissues. Exons 9, 11, 13, and 17 of the c-kit gene were amplified by PCR and sequenced. RESULTS: Mutations in exon 11 were detected in 61 tumors, and mutations in exon 9 were observed in three tumors, whereas no mutations were detected in exons 13 or 17. The overall c-kit mutation frequency was 74%. Amino acid alterations in the 61 tumors with exon 11 mutations were deletion in 33 tumors, substitution in 20, both deletion and substitution in 4, insertion in 1, and duplication in 3. Histologically, tumors with c-kit mutations showed higher mitotic counts and higher cellularity. The 5-year relapse-free survival (RFS) in patients having GISTs with c-kit mutations was 21%, compared with 60% in those without c-kit mutations. Significantly higher RFS rates were observed in patients with tumors having mitotic counts < 5 mitoses/50 high power field, spindle-cell histology, tumor size < 5 cm, or gastric GISTs. Multivariate analyses indicated association of poorer RFS with a higher mitotic count > or = 5 of 50 high power fields; odds ratio (OR) = 3.0], presence of c-kit mutations (OR = 5.6), and a larger tumor size (> or = 5 cm; OR = 4.2). CONCLUSIONS: The presence of c-kit mutation, along with high mitotic count and larger tumor size, was an independent factor for poor prognosis in patients with localized GISTs.