Seo-Jin Park

Application of BRAF, NRAS, KRAS mutations as markers for the detection of papillary thyroid cancer from FNAB specimens by pyrosequencing analysis

Abstract Background: BRAFV600E, the most common BRAF gene mutation, is detected in approximately 50% of sporadic papillary thyroid carcinoma (PTC) and may be associated with triggering tumorigenesis of PTC. The aim of our study was to discover additional mutations to increase the diagnostic performance of molecular tests in screening for thyroid cancer from fine needle aspiration biopsy (FNAB) specimens. Methods: DNA was extracted from 120 freshly obtained FNAB specimens selected according to cytopathology grades of the Bethesda system. A conventional BRAF V600E test was carried out with real-time PCR, and further mutation screening for BRAF mutations in codons 464, 466, 469, NRAS and KRAS codons 12/13 and 61 was done by pyrosequencing. Histopathology reports were reviewed for those who underwent thyroidectomy (n=83). Results: The real-time PCR method detected 45 BRAF V600E- positive cases whereas pyrosequencing detected 30 cases. Additional BRAF (n=4), NRAS (n=11) and KRAS (n=3) mutations were detected in 17 cases (one overlapping BRAF and NRAS mutation). Among 11 NRAS-mutated cases, eight were confirmed as PTC and one as FVPTC on histopathology reports. Five PTC-confirmed cases with BRAF V600E mutation showed additional mutations, all of which were NRAS mutations. Discussion: Despite the higher sensitivity of real-time PCR for detecting BRAFV600E mutations, pyrosequencing easily detected additional point mutations. NRAS mutations were the most prevalently identified additional mutations and were highly associated with malignancy. In conclusion, our findings demonstrate that additional mutations identified by pyrosequencing may help in the pre-operative process in determining the possibility of malignancy and further studies on the occurrence of simultaneous mutations of BRAF, KRAS and NRAS may be warranted.

Interobserver variability and diagnostic performance in US assessment of thyroid nodule according to size.

PURPOSE To evaluate the interobserver variability for US assessments of thyroid nodules and analyze the diagnostic performances of US assessments in thyroid nodules according to nodule size. MATERIALS AND METHODS This was an IRB-approved retrospective study with waiver of informed consent. A total of 400 surgically-confirmed thyroid nodules were included. Nodules were divided into 4 groups by size; group 1 (nodule size < 5 mm), group 2 (5 mm ≤ nodule size < 10 mm), group 3 (10 mm ≤ nodule size < 20 mm), and group 4 (nodule size ≥ 20 mm). Three experienced (7 - 10 years) radiologists retrospectively reviewed the US images. Agreement of each US descriptor and final US assessment, and diagnostic performances were calculated in each group and compared. RESULTS Composition represented substantial or good agreement (k = 0.719 - 0.89). Margin showed the lowest agreement (k = 0.322 - 0.365). Individual kappa values for final assessment according to nodule size were as follows: group 1 (k = 0.674), group 2 (k = 0.596), group 3 (k = 0.674), and group 4 (k = 0.673). Specificity, PPV, and accuracy were significantly different among the groups with different size (p value < 0.05) and lowest in group 1. NPV, specificity, PPV and accuracy except PPV of observer 3 increased with nodule size (p < 0.05). CONCLUSION Interobserver agreements were relatively good (k = 0.637) in final US assessment regardless of nodule size in experienced radiologists. High false-positive rate was observed in US assessment in nodules less than 5 mm in maximum diameter.

Clinical implications of non-A-type NPM1 and FLT3 mutations in patients with normal karyotype acute myeloid leukemia.

Mutations in the nucleophosmin (NPM1) and fms-like tyrosine kinase-3 (FLT3) genes are the most commonly observed mutations in patients with normal-karyotype acute myeloid leukemia (AML-NK). We analyzed the prognostic effects and interactions of these mutations in 201 AML-NK patients. NPM1 and FLT3 mutations were found in 38.3 and 24.9% of AML-NK patients, respectively. NPM1 mutations (NPM1mut), especially in patients without FLT3 mutations (FLT3mut), were associated with a favorable outcome. However, NPM1mut did not affect survival. FLT3mut tended to be associated with a poor survival outcome. FLT3mut showed no prognostic effects in patients with A-type NPM1mut. However, FLT3mut were associated with a significantly worse prognosis in patients with non-A-type NPM1mut. The prognostic interaction between the NPM1 and FLT3 mutations was significant in patients with non-A-type NPM1mut.

Submicroscopic Deletion of FGFR1 Gene Is Recurrently Detected in Myeloid and Lymphoid Neoplasms Associated with ZMYM2-FGFR1 Rearrangements: A Case Study

a Department of Laboratory Medicine, School of Medicine, Kyung Hee University, b Department of Laboratory Medicine, Yonsei University College of Medicine, Seoul , Departments of c Laboratory Medicine and d Pathology, Inje University College of Medicine, Busan , and e School of Life Science and Biotechnology, Kyungpook National University, Daegu , Republic of Korea; f Institute of Pharmaceutical Biology, ZAFES, Diagnostic Center of Acute Leukemia, Goethe University of Frankfurt, Frankfurt/Main , Germany

Detection of bone marrow metastases of neuroblastoma with immunohistochemical staining of CD56, chromogranin A, and synaptophysin.

Neuroblastoma is one of the most common solid tumors occurring in childhood. Bone marrow evaluation is an important part of clinical staging in neuroblastoma patients. In view of the difficulty of detecting neuroblastoma cells with conventional bone marrow aspirate smears and biopsy (BMB) and specimens in cases in which metastasis is not prominent, we propose the use of immunohistochemistry (IHC) as a potential diagnostic tool. We examined 116 BMB and 115 bone marrow clot (BMC) specimens from 60 newly diagnosed neuroblastoma patients for tumor cells. Neuroendocrine IHC markers, such as CD56, chromogranin A, and synaptophysin were applied to diagnose neuroblastoma. Eighteen out of the 60 patients (25.4%) displayed BM metastasis, as observed with conventional hematoxylin and eosin staining. IHC staining of BMC sections was generally more sensitive than that of BMB sections for tumor cell detection. We detected tumors in 5 and 7 additional hematoxylin and eosin-negative BMB and BMC sections, respectively, using CD56. Overall, CD56 or a combination of CD56 and chromogranin A was effective in detecting neuroblastoma cells. IHC analysis of BMB and BMC sections is warranted as a routine component of the diagnostic work-up of neuroblastoma to overcome discrepancies between routine smears and IHC stains.

The First Korean Case of Camurati-Engelmann Disease (Progressive Diaphyseal Dysplasia) Confirmed by TGFB1 Gene Mutation Analysis

Camurati-Engelmann disease (CED) is an autosomal dominant progressive diaphyseal dysplasia caused by mutations in the transforming growth factor-β1 (TGFB1) gene. We report the first Korean family with an affected mother and son who were diagnosed with CED. The proband is a 19-yr-old male with a history of abnormal gait since the age of 2. He also suffered from proximal muscle weakness, pain in the extremities, and easy fatigability. Skeletal radiographs of the long bones revealed cortical, periosteal, and endosteal thickenings, predominantly affecting the diaphyses of the upper and lower extremities. No other bony abnormalities were noted in the skull and spine and no remarkable findings were seen on laboratory tests. The patients mother had a long-standing history of mild limb pain. Under the impression of CED on radiographic studies, we performed mutation analysis. A heterozygous G to A transition at cDNA position +653 in exon 4 of the TGFB1 gene (R218H) was detected in the patient and his mother.

Detection of SET-NUP214 rearrangement using multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) in acute leukemias: a case report and literature review on a Korean case series

Dear Editor, We read an interesting article in a recent issue of the Annals of Hematology by Chae et al. entitled “Phenotypic and genetic characterization of adult T-cell acute lymphoblastic leukemia with del (9) (q34); SET-NUP214 rearrangement” [1]. Here, we would like to point out why the number of recent reports on the above gene rearrangement in Korea is increasing and suggest the most appropriate molecular diagnostic method for the detection of the SET-NUP214 rearrangement, by reporting a new case of SET-NUP214 from a T-cell acute lymphoblastic leukemia (T-ALL) patient and through literature review of the ten reported cases, including our new case, that reports on acute leukemias with SET-NUP214 in Korea between the 2-year period of 2010–2011 from the literature [1–4]. A 43-year-old Korean woman was examined in the outpatient clinic with fever and skin rash lasting from 50 days ago. She received uterine leiomyomectomy a year ago and had her breast mass monitored periodically. A chest X-ray revealed bilateral mediastinal widening, suggesting huge mediastinal mass or lymphadenopathy and abdominal ultrasonography showed splenomegaly. An initial complete blood count showed a hemoglobin level of 8.8 g/dL, a platelet count of 114×10/L, and a white blood cell count of 60.6×10/L with 85% blasts, 2% neutrophils, and 13% lymphocytes (Fig. 1a). Bone marrow study showed a hypercellularity with markedly increased leukemic blasts (91%, Fig. 1a). The karyotype of the bone marrow cells was 46,XX,dup(1)(p22p36.1) in 16 out of 20 metaphase cells analyzed (Fig. 1b). According to immunophenotyping, blasts were positive for CD3 (84%), CD5 (78%), CD7 (99%), CD13 (43%), CD33 (48%), and CD34 (80%). However, these cells did not express CD10, CD19, CD20, cCD22, CD14, HLA-DR, and myeloperoxidase. Multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) analysis using bone marrow specimen was performed with HemaVision kit (HemaVision; DNA technology, Aarhus, Denmark) and revealed the presence of SET-NUP214 fusion transcript measuring 393 bp (Fig. 1c). Eun Young Lee and Tae Sung Park equally contributed to this study as first authors. E. Y. Lee : S.-J. Park : J. R. Choi : J.-H. Yoo (*) Department of Laboratory Medicine, Yonsei University College of Medicine, Seoul, Republic of Korea e-mail: jhyoo92@nhimc.or.kr

CD5-negative Blastoid Variant Mantle Cell Lymphoma with Complex CCND1/IGH and MYC Aberrations

The coexistence of CCND1/IGH and MYC rearrangements in mantle cell lymphoma (MCL) is a rare finding associated with a very poor prognosis. In this study, a patient with blastoid variant (MCL) is reported. The disease was clinically aggressive and refractory to chemotherapy, and the patient only survived for 1 month following diagnosis. Conventional cytogenetic study, FISH, and multicolor FISH (mFISH) demonstrated the involvement of the BCL1/CCND1 locus in a complex translocation, t(3;11)(q25;p15)t(11;14)(q13;q32). In addition, subclonal abnormalities in the 8q24 region, manifested as a t(8;14)(q24;q32)/MYC rearrangement, were identified. To the best of our knowledge, this is the first MCL case in Korea bearing these complex genomic aberrations.

A novel missense MSH2 gene mutation in a patient of a Korean family with hereditary nonpolyposis colorectal cancer.

Hereditary nonpolyposis colorectal cancer (HNPCC) is an autosomal dominant cancer-susceptible syndrome that predisposes to the early development of colorectal cancer. Germline mutations in DNA mismatch repair genes, particularly MLH1 and MSH2, are associated with the clinical phenotype of HNPCC. A previously unreported, novel missense mutation in exon 3 of the MSH2 gene (c.380A>T) was identified in the proband and a different missense mutation in exon 3 of MSH2 gene (c.505A>G) was noted in the mother, with a mutual splice mutation in intron 12 of the MSH2 gene in the proband, mother, and younger brother. Here, we report the clinical implications of a novel mutation in a patient with early-onset colorectal cancer and the significance of a common underlying splice site mutation occurring within a family with HNPCC.

Next-generation sequencing of BRCA1/2 in breast cancer patients: potential effects on clinical decision-making using rapid, high-accuracy genetic results

Purpose We evaluated the clinical role of rapid next-generation sequencing (NGS) for identifying BRCA1/2 mutations compared to traditional Sanger sequencing. Methods Twenty-four paired samples from 12 patients were analyzed in this prospective study to compare the performance of NGS to the Sanger method. Both NGS and Sanger sequencing were performed in 2 different laboratories using blood samples from patients with breast cancer. We then analyzed the accuracy of NGS in terms of variant calling and determining concordance rates of BRCA1/2 mutation detection. Results The overall concordance rate of BRCA1/2 mutation identification was 100%. Variants of unknown significance (VUS) were reported in two cases of BRCA1 and 3 cases of BRCA2 after Sanger sequencing, whereas NGS reported only 1 case of BRCA1 VUS, likely due to differences in reference databases used for mutation identification. The median turnaround time of Sanger sequencing was 22 days (range, 14–26 days), while the median time of NGS was only 6 days (range, 3–21 days). Conclusion NGS yielded comparably accurate results to Sanger sequencing and in a much shorter time with respect to BRCA1/2 mutation identification. The shorter turnaround time and higher accuracy of NGS may help clinicians make more timely and informed decisions regarding surgery or neoadjuvant chemotherapy in patients with breast cancer.