Seo Young Oh

Preoperative RAS Mutational Analysis Is of Great Value in Predicting Follicular Variant of Papillary Thyroid Carcinoma

Follicular variant of papillary thyroid carcinoma (FVPTC), particularly the encapsulated subtype, often causes a diagnostic dilemma. We reconfirmed the molecular profiles in a large number of FVPTCs and investigated the efficacy of the preoperative mutational analysis in indeterminate thyroid nodules. BRAF V600E/K601E and RAS mutational analysis was performed on 187 FVPTCs. Of these, 132 (70.6%) had a point mutation in one of the BRAF V600E (n = 57), BRAF K601E (n = 11), or RAS (n = 64) genes. All mutations were mutually exclusive. The most common RAS mutations were at NRAS codon 61. FNA aspirates from 564 indeterminate nodules were prospectively tested for BRAF and RAS mutation and the surgical outcome was correlated with the mutational status. Fifty-seven and 47 cases were positive for BRAF and RAS mutation, respectively. Twenty-seven RAS-positive patients underwent surgery and all except one patient had FVPTC. The PPV and accuracy of RAS mutational analysis for predicting FVPTC were 96% and 84%, respectively. BRAF or RAS mutations were present in more than two-thirds of FVPTCs and these were mutually exclusive. BRAF mutational analysis followed by N, H, and KRAS codon 61 mutational analysis in indeterminate thyroid nodules would streamline the management of patients with malignancies, mostly FVPTC.

Primary pure squamous cell carcinoma of the thyroid: Report and histogenic consideration of a case involving a BRAF mutation

Primary squamous cell carcinoma of the thyroid (SCC‐T) is extremely rare. Its clinical presentation is similar to that of anaplastic carcinoma. Metastasis or extension from the head and neck area should be ruled out, as patients with SCC‐T have a poorer prognosis than patients who have a thyroid extension from an adjacent tumor. An 87‐year‐old man presented with a longstanding painless mass in the right thyroid and had experienced 2 months of pain upon swallowing. A right lobectomy was performed with resection of thyroid cartilage, cricoid cartilage, a portion of the first to third tracheal ring and the right neck lymph node. A histological examination revealed pure SCC. The tumor cells showed diffuse immunoreactivity to CK5/6, CK19 and p63. Immunoreactivity to EMA and p53 was focally positive. TTF‐1, galectin 3 and thyroglobulin immunoreactivity was restricted to the non‐neoplastic thyroid tissue. Both tumor cells and non‐neoplastic follicular cells were negative for CD5. The MIB‐1 index was 36%. DNA extracted from the tumor identified a BRAF V600E mutation in exon 15 and a BRAF G468A mutation in exon 11, whereas DNA from non‐tumorous cells did not contain a mutation. These molecular findings may suggest a direct transformation from papillary carcinoma to SCC‐T.

DNA degradation in liquid‐based cytology and its comparison with conventional smear

In fine needle aspiration biopsy (FNAB) of thyroid nodules, LBC is adopted in most of the hospitals and clinics in Korea for its convenience. BRAF mutation test has been introduced as an important ancillary test, but its applicability has not been completely proven with LBC samples.

Development of an Ammonium Sulfate DNA Extraction Method for Obtaining Amplifiable DNA in a Small Number of Cells and Its Application to Clinical Specimens

DNA extraction from microdissected cells has become essential for handling clinical specimens with advances in molecular pathology. Conventional methods have limitations for extracting amplifiable DNA from specimens containing a small number of cells. We developed an ammonium sulfate DNA extraction method (A) and compared it with two other methods (B and C). DNA quality and quantity, β-globin amplification, and detectability of two cancer associated gene mutations were evaluated. Method A showed the best DNA yield, particularly when the cell number was very low. Amplification of the β-globin gene using DNA from the SNU 790 cell line and papillary thyroid carcinoma (PTC) cells extracted with Method A demonstrated the strongest band. BRAF V600E mutation analysis using ethanol-fixed PTC cells from a patient demonstrated both a “T” peak increase and an adjacent “A” peak decrease when 25 and 50 cells were extracted, whereas mutant peaks were too low to be analyzed using the other two methods. EGFR mutation analysis using formalin-fixed paraffin-embedded lung cancer tissues demonstrated a mutant peak with Method A, whereas the mutant peak was undetectable with Methods B or C. Method A yielded the best DNA quantity and quality with outstanding efficiency, particularly when paucicellular specimens were used.

Detection of EGFR and KRAS Mutation by Pyrosequencing Analysis in Cytologic Samples of Non-Small Cell Lung Cancer

EGFR and KRAS mutations are two of the most common mutations that are present in lung cancer. Screening and detecting these mutations are of issue these days, and many different methods and tissue samples are currently used to effectively detect these two mutations. In this study, we aimed to evaluate the testing for EGFR and KRAS mutations by pyrosequencing method, and compared the yield of cytology versus histology specimens in a consecutive series of patients with lung cancer. We retrospectively reviewed EGFR and KRAS mutation results of 399 (patients with EGFR mutation test) and 323 patients (patients with KRAS mutation test) diagnosed with lung cancer in Konkuk University Medical Center from 2008 to 2014. Among them, 60 patients had received both EGFR and KRAS mutation studies. We compared the detection rate of EGFR and KRAS tests in cytology, biopsy, and resection specimens. EGFR and KRAS mutations were detected in 29.8% and 8.7% of total patients, and the positive mutation results of EGFR and KRAS were mutually exclusive. The detection rate of EGFR mutation in cytology was higher than non-cytology (biopsy or resection) materials (cytology: 48.5%, non-cytology: 26.1%), and the detection rate of KRAS mutation in cytology specimens was comparable to non-cytology specimens (cytology: 8.3%, non-cytology: 8.7%). We suggest that cytology specimens are good alternatives that can readily substitute tissue samples for testing both EGFR and KRAS mutations. Moreover, pyrosequencing method is highly sensitive in detecting EGFR and KRAS mutations in lung cancer patients.

A Case of Multifocal Papillary Thyroid Carcinoma Consisting of One Encapsulated Follicular Variant with BRAF K601E Mutation and Three Conventional Types with BRAF V600E Mutation

Multifocal papillary thyroid carcinoma (mPTC) comprises about 20-30% of PTC. In mPTC, individual tumor foci can be identical or frequently composed of different histological types including follicular, solid, tall-cell or conventional patterns. We report a case of mPTC consisting of one encapsulated follicular variant of papillary thyroid carcinoma (FVPTC) and three conventional PTCs in a 44-year-old woman. This case genetically demonstrates unique features including the simultaneous presence of the BRAF V600E (T1799A) mutation and the BRAF K601E (A1801G) mutation in conventional PTC and FVPTC, respectively.

Comparison Between Real-Time PCR and Pyrosequencing for Detection of BRAF V600E Mutation in Thyroid Fine-Needle Aspirates.

The BRAF V600E mutation test has proven diagnostic value in thyroid fine-needle aspiration. The real-time polymerase chain reaction (RT-PCR) has recently been introduced as a new method for BRAF mutation detection. We performed BRAF V600E detection in 126 cases of thyroid fine-needle aspiration, using RT-PCR and pyrosequencing. BRAF V600E mutation was detected in 78 (61.9%) of 126 cases by RT-PCR and in 74 (57.8%) by pyrosequencing. Of the 98 papillary thyroid carcinoma samples, the BRAF V600E mutation was identified in 72 by RT-PCR and in 70 by pyrosequencing (sensitivities of 71.6% and 71.4%, respectively). Among 28 benign nodules, 6 false-positive cases were detected by RT-PCR, whereas 4 false-positive cases were detected by pyrosequencing (specificities of 78.6% and 85.7%, respectively). When analyzing 104 cases after excluding equivocal samples, pyrosequencing had a marginally higher specificity than RT-PCR (100% vs. 78.3%, P=0.074). After modifying the cut-off criteria, the low RT-PCR specificity improved to a similar or a slightly lower specificity compared with that of pyrosequencing. In the titration assay mixing the mutant DNA with the wild-type DNA in varying proportions, RT-PCR was sensitive enough to detect the mutation in a mixture containing 0.001% mutant DNA, whereas the limit of detection was 10% for pyrosequencing. In conclusion, compared with pyrosequencing, RT-PCR was more sensitive, faster, and more convenient, but less specific, for detecting the BRAF V600E mutation. A readjustment or modification of the interpretation criteria may be necessary to reduce false-positive RT-PCR results and improve the specificity while maintaining the sensitivity.

Comparison of Analytical and Clinical Performance of HPV 9G DNA Chip, PANArray HPV Genotyping Chip, and Hybrid-Capture II Assay in Cervicovaginal Swabs

Background: Human papillomavirus (HPV) infection can be detected by using several molecular methods, including Hybrid-Capture II (HC2) assay and variable HPV DNA chip tests, although each method has different sensitivities and specificities. Methods: We performed HPV 9G DNA Chip (9G) and PANArray HPV Genotyping Chip (PANArray) tests on 118 cervicovaginal swabs and compared the results with HC2, cytology, histology, and direct sequencing results. Results The overall and high-risk HPV (HR-HPV) positivity rates were 62.7% and 44.9% using 9G, and 61.0% and 30.5% using PANArray, respectively. The positivity rates for HR-HPV with these two chips were significantly lower than 55.1% when HC2 was used. The sensitivity of overall HPV positivity in detecting histologically confirmed low-grade cervical squamous intraepithelial lesions or higher was 88.7% for all three tests. The specificity was 58.5% for 9G and 61.5% for PANArray, which was significantly lower than the 72.3% for HC2. With the HR-HPV+ genotype threshold, the sensitivity decreased to 75.5% for 9G and 52.8% for PANArray, which was significantly lower than the 88.7% for HC2. Comparison of the two chips showed concordant results in 55.1% of the samples, compatible results in 16.9%, and discordant results in 28.0%, exhibiting poor agreement in detecting certain HPV genotypes. Compared with direct sequencing, 9G yielded no discordant results, whereas PANArray yielded 31 discordant results (26.7%). Conclusions Compared with HC2, the HPV genotyping tests showed lower sensitivity in histologic correlation. When the two chips were compared, the 9G was more sensitive and accurate for detecting HR-HPV than the PANArray.

Efficiency of EGFR mutation analysis for small microdissected cytological specimens using multitech DNA extraction solution

The microdissection method has greatly facilitated the isolation of pure cell populations for accurate analysis of mutations. However, the absence of coverslips in these preparations leads to poor resolution of cellular morphological features. In the current study, the authors developed the MultiTech DNA extraction solution to improve the visualization of cell morphology for microdissection and tested it for the preservation of morphological properties of cells, quality of DNA, and ability to detect mutations.

Abstract A23: Detection of K ras mutations in Korean non small cell lung cancer patients

Background: Point mutation of the K ras gene is known to be a poor prognostic marker of an adenocarcinoma of the lung. This study aims to define frequency of K ras mutations and evaluate clinical features of patients with Non small cell lung cancer (NSCLC) who harbor K ras mutation. Methods: NSCLC patient who were diagnosed from Sep. 2005 to Aug. 2011 at Konkuk University Hospital were included in this retrospective study. K ras mutation was assessed by pyrosequencing on biopsy or cytology specimen. Medial chart review was performed and clinical characteristics were compared according to K ras mutation status of their tumors. Results: K ras mutation was found in 25 (9.0%) of 277 patients and which was similar to those in East Asian countries, but lower than those in western countries. Fifteen mutations (60.0%) were point mutation at codon 12 (GGT), 7 mutations (28.0%) were point mutation at codon 61 (CAA), and 3 mutations (12.0%) were found at codon 13 (GGC) in exon 2. Median age of 25 patients with KRAS mutation was 6 years (range 43–85 years). There were 20 males (80.0%) and 3 never-smokers (12.0%). The majority of histological diagnoses were adenocarcinomas (21 cases; 84.0%) and squamous cell carcinomas were followed (3 cases; 12.0%). Interestingly, 2 patients had both mutations of EGFR and KRAS unlike previous studies, they showed favorable response to EGFRTKIs. Conclusions: The data indicate that KRAS mutation is less frequent in Korean than Caucasian patients. Although it is suggested that EGFR and KRAS mutations occur in mutually exclusive, very few of the patients with KRAS mutation showed also EGFR mutation and relatively favorable response to EGFR-TKIs. Additional large scale prospective studies are needed in order to validate the clinical significance of Kras mutation in Asian patients.