Highly Sensitive and Specific Molecular Test for Mutations in the Diagnosis of Thyroid Nodules: A Prospective Study of BRAF-Prevalent Population.

Molecular testing offers more objective information in the diagnosis and personalized decision making for thyroid nodules. In Korea, as the BRAF V600E mutation is detected in 70-80% of thyroid cancer specimens, its testing in fine-needle aspiration (FNA) cytology specimens alone has been used for the differential diagnosis of thyroid nodules until now. Thus, we aimed to develop a mutation panel to detect not only BRAF V600E, but also other common genetic alterations in thyroid cancer and to evaluate the diagnostic accuracy of the mutation panel for thyroid nodules in Korea. For this prospective study, FNA specimens of 430 nodules were obtained from patients who underwent thyroid surgery for thyroid nodules. A molecular test was devised using real-time PCR to detect common genetic alterations in thyroid cancer, including BRAF, N-, H-, and K-RAS mutations and rearrangements of RET/PTC and PAX8/PPARr. Positive results for the mutation panel were confirmed by sequencing. Among the 430 FNA specimens, genetic alterations were detected in 293 cases (68%). BRAF V600E (240 of 347 cases, 69%) was the most prevalent mutation in thyroid cancer. The RAS mutation was most prevalently detected for indeterminate cytology. Among the 293 mutation-positive cases, 287 (98%) were diagnosed as cancer. The combination of molecular testing and cytology improved sensitivity from 72% (cytology alone) to 89% (combination), with a specificity of 93%. We verified the excellent diagnostic performance of the mutation panel applicable for clinical practice in Korea. A plan has been devised to validate its performance using independent FNA specimens.

Molecular genotyping of the non-invasive encapsulated follicular variant of papillary thyroid carcinoma.

AIMS: The non-invasive encapsulated follicular variant of papillary thyroid carcinoma (FVPTC) has been managed as a low-risk malignancy. Recently, a proposal was made to reclassify this tumour type as a premalignant lesion and rename it non-invasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP). This study aims to provide the first comprehensive study on molecular genotype-phenotype correlations of encapsulated FVPTC. METHODS AND RESULTS: This study was performed on 177 consecutive FVPTCs from January 2014 to April 2016. These were classified as non-invasive encapsulated FVPTC (n = 74) invasive encapsulated FVPTC (n = 51), and infiltrative FVPTC (n = 52), according to standard criteria, by two independent pathologists. Genetic alterations and other clinicopathological information were compared. BRAFV600E was found in 12.2% (non-invasive) and 11.8% (invasive) of encapsulated FVPTCs, and in 34.6% of infiltrative FVPTCs (P = 0.001). Mutation in encapsulated FVPTCs was limited to cases with rare or abortive papillae. RET-PTC1 and RET-PTC3 rearrangements were present (11.5%) only in infiltrative FVPTCs. In contrast, NRAS, HRAS and KRAS mutations were observed more often in encapsulated FVPTCs (48.6% in non-invasive and 66.7% in invasive) than in infiltrative FVPTCs (15.4%) (P < 0.001). Preoperative cytological examination did not distinguish between non-invasive and invasive encapsulated FVPTCs, whereas infiltrative FVPTC was more likely to be Bethesda class V/VI than the encapsulated type (60.4% versus 38.1%; P = 0.01). CONCLUSIONS: There were no differences in clinicopathological or molecular profiles between non-invasive and invasive encapsulated FVPTCs, except in vascular and capsular invasion. Therefore, the diagnosis of NIFTP, like that of follicular adenoma, may require surgical resection and exclusion of those tumours with any papillae.

Diffuse sclerosing variant of papillary thyroid carcinoma: major genetic alterations and prognostic implications.

AIM: Diffuse sclerosing variant of papillary thyroid carcinoma (DSV-PTC) is an uncommon variant of PTC, and its prognostic significance remains controversial. The aim of this study was to investigate the major genetic alterations of DSV-PTC and their prognostic implications. METHODS AND RESULTS: We included 37 patients with DSV-PTC who underwent thyroid surgery and had formalin-fixed paraffin-embedded samples. We tested for a panel of genetic alterations, including BRAF(V) (600E) , NRAS codon 61, HRAS codon 12/13/61 and KRAS codon 12/13 point mutations as well as RET/PTC1, RET/PTC3 and PAX8/PPARγ rearrangements using reverse transcription real-time polymerase chain reaction (PCR). All genetic alterations found on PCR were confirmed by Sanger sequencing. Associations between the identified genetic alterations and clinicopathological characteristics were evaluated. Among 37 cases of DSV-PTC, 17 were positive for RET/PTC1 (46%), six for RET/PTC3 (16%) and nine for BRAF(V) (600E) (24%). All mutations/rearrangements were mutually exclusive. The remaining five cases had none of the above genetic alterations. DSV-PTC with RET/PTC3 rearrangement was associated with advanced-stage disease, including T4 and distant metastasis (P < 0.05). Patients with RET/PTC3 showed a higher frequency of persistent disease (P < 0.01). In contrast, DSV-PTC with RET/PTC1 was associated with a higher prevalence of disease remission (P < 0.05) and coexistent Hashimoto's thyroiditis (P < 0.01). CONCLUSION: Taken together, RET/PTC rearrangement was the major genetic alteration seen in patients with DSV-PTC, and the RET/PTC3 rearrangement was associated with advanced stage at diagnosis and poor clinical outcome.

BRAFV600E mutation testing in fine needle aspirates of thyroid nodules: potential value of real-time PCR.

The BRAF(V600E) mutation is a valuable adjunctive diagnostic tool to ultrasound (US)-guided fine-needle aspiration (US-FNA). The objective of this study was to investigate the potential value of realtime PCR to detect BRAF(V600E) mutation. This study included 447 thyroid nodules in 420 patients who underwent US-FNA and BRAF(V600E) mutation analysis using dual priming oligonucleotide-based multiplex polymerase chain reaction (DPO-PCR) and real-time PCR. We calculated and compared the diagnostic performances of DPO-PCR and real-time PCR to detect BRAF(V600E) mutation in the thyroid nodules. Receiver operating characteristic (ROC) analysis was used to quantify the cut-off value of the Ct values of BRAF(V600E) mutation on real-time PCR. Optimal thresholds were determined (Youden index). We also compared the diagnostic performances between DPO-PCR and real-time PCR after applying the cut-off value on real-time PCR. Sensitivity, accuracy, and NPV were significantly higher in real-time PCR than DPO-PCR. When the optimal cut-off value of 32.4 at Ct values of BRAF(V600E) mutation was adjusted on real-time PCR, sensitivity was 66.2% and specificity was 100%. Sensitivity, accuracy, and NPV of real-time PCR were also significantly higher than DPO-PCR. In contrast, specificity and PPV were not significantly different between DPO-PCR and real-time PCR. Real time PCR can be a promising diagnostic method in detecting BRAF(V600E) mutation using optimal cut-off value.

[Evaluation of Biosewoom Real-Q Cytomegalovirus Quantification kit for Cytomegalovirus viral load measure].

BACKGROUND: Rapid and accurate laboratory tests are essential to detect cytomegalovirus (CMV) infections in solid organs and haematopoietic stem cell transplant recipients. We assessed the realtime quantitative PCR (RQ-PCR) technology for its usefulness in detecting CMV DNA. METHODS: We evaluated the analytical performance of CMV RQ-PCR using Real-Q Cytomegalovirus Quantification kit (BioSewoom Inc., Korea). To evaluate its clinical utility, we also compared it to pp65 antigenemia test, an immunostaining method, on 343 samples of total 84 patients, including 63 transplant recipients. RESULTS: The detection limit of RQ-PCR was 63 copies/mL and none of hepatitis B virus, hepatitis C virus, or human immunodeficiency virus showed a cross-reactivity with CMV. Total coefficient of variation (CV) was 10.4-19.5%. It detected CMV DNA in a linear range from 1 x 10(2) to 5 x 10(11) copies/mL (P<10(-13), R(2)=0.9994). The qualitative positive rates of pp65 antigenemia test and RQ-PCR were 4.7%, 16.3%, respectively and concordance rate between the two tests was 84.8% (Kappa=0.221, P<10(-6)). In comparison of quantitative results, the correlation between two tests was significant (r=0.45, P<10(-17)). In comparison among three groups by pp65 antigen level, CMV DNA level obtained with RQ-PCR increased significantly (P<10(-3) and P<10(-7), respectively). CONCLUSIONS: The RQ-PCR is easier to perform than the immunostaining method, has good analytical performance and reflects the blood level of viral DNA well. It may be a new method substituting the pp65 antigenemia test. Further studies determining RQ-PCR value starting pre-emptive therapy will be required.

Identification of leukemia-specific fusion gene transcripts with a novel oligonucleotide array.

BACKGROUND: Identification of specific chromosomal translocations is essential for the diagnosis and prognosis of leukemia. In this study, we employ DNA microarray technology to detect chromosomal aberrations in patients with chronic myeloid leukemia (CML) and acute myeloid leukemia (AML), as well as in leukemic cell lines. METHODS: Reverse transcription using a random 9-mer primer was performed with total RNA from patients and leukemic cells lines. Multiplex PCR reactions using four groups of primer sets were then performed for amplification of cDNA from reverse-transcribed total RNA samples. Normal and fusion sequences were distinguished by hybridization of the amplified cDNA to a selective oligonucleotide array (SOA) containing 20-30mer synthetic probes. A total of 23 sets of oligomers were fabricated on glass slides for the detection of normal and fusion genes, as follows: BCR/ABL, AML/EAP, AML/ETO, AML/MDS, PML/RARA, NUMA1/RARA, PLZF/RARA, and CBFB/MYH. RESULTS: Gene translocation in leukemia was effectively identified with the SOA containing various leukemia-specific fusion and normal control sequences. Leukemic fusion sequences from patients and cell lines hybridized specifically to their complementary probes. The probe sets differing by approximately 50% at their 5' or 3' ends could distinguish between normal and fusion sequences. The entire process of detection was completed within 8 hours using the SOA method. CONCLUSIONS: Probe sets on SOA can effectively discriminate between leukemia-specific fusion and normal sequences with a chip hybridization procedure. The oligonucleotide array presents several advantages in identifying leukemic gene translocations, such as multiplex screening, relatively low cost, and speed.

Comprehensive analysis of BCR-ABL transcript types in Korean CML patients using a newly developed multiplex RT-PCR.

Diagnosis of chronic myeloid leukemia (CML) is based on the detection of BCR-ABL gene or Philadelphia chromosome (Ph chromosome), and fusion proteins with different sizes are encoded depending on the breakpoint in the BCR gene. In general, 3 breakpoint cluster regions in the BCR gene have been described: major (M-bcr), minor (m-bcr), and micro (mu-bcr). This study was designed to determine the frequency of BCR-ABL transcripts using one-step multiplex reverse transcription polymerase chain reaction (RT-PCR). Bone marrow (BM) or peripheral blood (PB) samples at diagnosis from 548 patients were obtained with a referring diagnosis of Ph-positive (Ph+) CML, and multistep RT-PCR and newly developed one-step multiplex RT-PCR were applied on each sample. Compared with the previous multistep RT-PCR, one-step multiplex RT-PCR with the primers is the more rapid and accurate method to identify the BCR-ABL breakpoints. Most patients (538/548, 98.18%) were found to have b3a2 or b2a2, and total frequency of occurrence of c3a2, e1a2, b2a3, b1a1, and e1a3 or coexpression of b2a2 and b3a2 was less than 2.00%. No differences were observed between women and men. As the multiplex RT-PCR technique distinguishes BCR-ABL transcripts in all samples with high sensitivity and specificity, it easily could be applied at early stages of diagnosis. The incidence of one or the other rearrangement in CML patients varies in different reported series, and the frequency in each type of BCR-ABL transcript in Korean CML patients seems to be different from those of Western countries.

[Performance evaluation of real-q HBV quantification kit for HBV DNA by real-time PCR.].

BACKGROUND: Hepatitis B virus (HBV) DNA quantification is important for the management of HBV infection and identification of the development of resistance. The susceptibility to contamination and more variable reproducibility of results with the conventional HBV DNA quantification method have raised the need of a more simple and accurate method for HBV DNA quantification. Real-time quantitative PCR assays recently introduced in the laboratory can meet these needs. In this study, we evaluated the performance of the Real-Q HBV Quantification kit developed in Korea. METHODS: We evaluated the recovery of DNA extraction, the interference of internal control, an analytical sensitivity, specificity, and reproducibility, a clinical specificity, and a reportable range of the Real-Q HBV Quantification kit. The quantification result was also compared to that obtained by the Digene Hybrid-Capture II. RESULTS: The mean percent recovery was 108.6% and there was no interference with the internal control on DNA extraction. None of HIV, hepatitis C virus, or cytomegalovirus showed a cross-reactivity with HBV. This assay detected HBV DNA in a linear range from 10(2) to 10(10) copies/mL, with the detection limit of 56 copies/mL. The assay exhibited a low within-run CV (coefficient of variation) (8.7-11.9%), between-run CV (10.5-14.7%), and between-day CV (13.2-21.4%). No HBV DNA was detected in any of 100 samples without HBV, resulting in a clinical specificity of 100%. The levels of HBV DNA showed a good correlation with those determined with Digene Hybrid-Capture II (R(2)=0.9827). CONCLUSIONS: The Real-Q HBV Quantification kit showed a good analytical sensitivity, specificity, and high reliability with a broad reportable range. This assay should be clinically useful in managing patients with HBV infection.

Early prediction of molecular remission by monitoring BCR-ABL transcript levels in patients achieving a complete cytogenetic response after imatinib therapy for posttransplantation chronic myelogenous leukemia relapse.

Imatinib induces a high complete cytogenetic response (CCR) rate in relapsed chronic myelogenous leukemia. By analyzing minimal residual disease (MRD) under the levels of CCR, we tried to assess the molecular response after imatinib therapy. By using real-time quantitative reverse transcriptase-polymerase chain reaction (Q-RT-PCR), MRD was evaluated in 23 patients (3 in cytogenetic relapse, 6 in chronic phase, 9 in accelerated phase, and 5 in blast crisis) who were treated with standard-dose imatinib for relapsed chronic myelogenous leukemia after allogeneic stem cell transplantation. With a median therapy time of 399 days (range, 35-817 days), 19 (83%) patients achieved a CCR. Meanwhile, 11 (58%) of them achieved a molecular remission (MR), which was associated with improved survival. The Q-RT-PCR data were compared according to the best response (MR, n = 11; CCR, n = 8) in the patients achieving a CCR. The BCR-ABL/ABL ratios were similar in 2 groups at 3 months but were significantly different at 6 months (median, 0.0000012 for MR and 0.00022 for CCR; P =.003). The probability of a subsequent MR was significantly higher in patients with a lower BCR-ABL/ABL ratio at 6 months (100% for <0.0001 versus 33% for >/=0.0001; P =.006) or a greater reduction in the level between 3 and 6 months (log-reduction >/=1.0;, 100%; <1.0, 17%; P =.003). Q-RT-PCR is a reliable method for monitoring MRD: the early trends in the BCR-ABL/ABL ratio may be clinically useful in discriminating patients who will achieve an MR from those who will remain in CCR.

Cotransplantation of third-party mesenchymal stromal cells can alleviate single-donor predominance and increase engraftment from double cord transplantation.

Although the infusion of umbilical cord blood (UCB) from multiple donors can be a strategy to overcome the cell dose limitation frequently encountered in UCB transplantation, clinical trials have revealed that cells from one donor dominate engraftment. To investigate the origin of and the factors influencing this inequality, we performed mixed transplantation of 2 UCB units with varying degrees of HLA disparities into NOD/SCID mice and determined donor origins by polymerase chain reaction-sequence-specific oligonucleotide probe (PCR-SSOP) or real-time quantitative (RQ)-PCR for human short tandem repeats (STRs). When total mononuclear cells from 2 units were transplanted as a mixture, cells from one donor predominated (ratio, 81:19), despite comparable overall engraftment when infused as single units, and no augmentation in overall engraftment was observed when compared with the single-unit controls. However, lineage depletion or cotransplantation of mesenchymal stromal cells (MSCs) expanded from third-party bone marrow resulted in more balanced coengraftment. Direct comparison of double UCB transplantation in the presence or absence of MSCs showed that the reduced deviation in the donor ratio (1.8:1 vs. 2.8:1) correlated with a higher overall level of engraftment with MSC cotransplantation. These results indicate that third-party MSCs can be used to alleviate donor deviation and to facilitate engraftment of multidonor UCB.

Allele frequencies of 15 STR loci using AmpF/STR Identifiler kit in a Korean population.

The genetic variations for 15 short tandem repeat (STR) loci D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818 and FGA were performed on 231 unrelated Korean population using commercially available AmpF/STR Identifiler kit.

Minimal residual disease-based role of imatinib as a first-line interim therapy prior to allogeneic stem cell transplantation in Philadelphia chromosome-positive acute lymphoblastic leukemia.

Fourteen adults with Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) were studied to evaluate the role of imatinib prior to allogeneic stem cell transplantation (SCT). Of these, 12 patients were in complete hematologic response (CHR), and 2 were refractory. Imatinib was administered as an interim schedule after each chemotherapy course. After the first imatinib cycle, 11 patients remained in sustained CHR with a decrease in the BCR-ABL/ABL ratios (0.89 logs), and one refractory patient achieved CHR. Meanwhile, 2 patients were resistant to imatinib. Ten patients receiving a second imatinib cycle following consolidation showed sustained CHR, including 2 molecular CR, with a further decrease in the BCR-ABL/ABL ratios (0.19 logs). Twelve patients underwent SCT in a favorable status, and of these, 11 are still alive in a leukemia-free status at 9 to 28+ months after SCT. First-line imatinib interim therapy appears to be a useful strategy to bridge the time to SCT for patients with Ph+ ALL.

Risk factors for adults with Philadelphia-chromosome-positive acute lymphoblastic leukaemia in remission treated with allogeneic bone marrow transplantation: the potential of real-time quantitative reverse-transcription polymerase chain reaction.

The aim of this study was to evaluate the outcomes for Philadelphia-chromosome-positive acute lymphoblastic leukaemia (Ph+ ALL) patients in remission treated with allogeneic bone marrow transplantation (BMT). Twenty-three adults were entered onto this study. The 2-year probabilities of relapse and disease-free survival (DFS) were 39.4 +/- 11.6% and 43.5 +/- 10.3% respectively. The presence of chronic graft-versus-host disease (GVHD) was found to be an independent predictive factor affecting lower relapse and DFS. To monitor the BCR-ABL transcript, we also analysed 48 bone marrow samples of eight patients using real-time quantitative reverse-transcription polymerase chain reaction (RT-PCR). The kinetics of the BCR-ABL transcript correlated well with the patients' clinical course. In six patients who were in continuous remission after BMT, a rapid decrease in BCR-ABL copy number to the PCR-negative status was observed after the development of chronic GVHD. Meanwhile, routine bone marrow examination of two patients showed PCR positivity with a 3 or 4-log increase of BCR-ABL copy number and subsequent haematological relapse, which occurred 2 and 4 months later respectively. Although our data should be interpreted cautiously, the presence of chronic GVHD may reduce the risk of relapse in Ph+ ALL. Real-time quantitative RT-PCR appears to be a useful test for BCR-ABL transcript monitoring.

Detection of the BCR-ABL gene by interphase fluorescence in situ hybridization (iFISH) in chronic myelogenous leukemia patients after hemopoietic stem cell transplantation: the feasibility of iFISH monitoring of therapeutic response in peripheral blood.

The detection of the Philadelphia (Ph) translocation has been accomplished primarily by cytogenetic analysis and reverse transcriptase polymerase chain reaction (RT-PCR). RT-PCR is highly sensitive (1/10(4)-10(6)) but not quantitatively reliable and is thus unsuitable for the monitoring of Ph-positive cells during therapy. Interphase fluorescence in situ hybridization (iFISH) allows analysis of a large number of cells (> 500) in a timely and efficiently quantitative manner. We obtained 118 peripheral blood (PB) and 127 bone marrow (BM) samples from 75 adult chronic myelogenous leukemia (CML) patients undergoing stem cell transplantation. We simultaneously performed nested RT-PCR and iFISH for all samples. False-positive cells were detected in 2.48% +/- 0.93% (mean +/- SD) of PB samples and 2.75% +/- 0.83% of BM samples. The iFISH results for PB and BM ranged from 1.4% to 92.8% and 1.0% to 93.8%, respectively. Correlation analysis of iFISH results for PB versus BM samples showed a strong relation (r = .993). A significant correlation (P < .05) was also found between iFISH and first-round RT-PCR. The sensitivity of BCR-ABL iFISH was similar to that of first-round RT-PCR, and iFISH results for PB and BM were also well correlated. Thus, iFISH analysis of PB and/or BM samples may be more clinically reliable than RT-PCR in the quantitative monitoring of BCR-ABL fusion in CML after transplantation.

Comprehensive comparison of FISH, RT-PCR, and RQ-PCR for monitoring the BCR-ABL gene after hematopoietic stem cell transplantation in CML.

The reverse transcriptase-polymerase chain reaction (RT-PCR) was compared with fluorescence in situ hybridization (FISH) and real-time quantitative RT-PCR (RQ-PCR) for minimal residual disease (MRD) monitoring in 266 post-transplant bone marrow samples from 78 patients with chronic myelogenous leukemia (CML). The sensitivities of FISH to BCR-ABL positive samples determined by first-round (1st) RT-PCR, second-round (2nd) RT-PCR, and RQ-PCR were 64.2%, 25.8%, and 20.7%, respectively. The BCR-ABL/ABL ratio by RQ-PCR had a mean of 0.000 13 in the 1st RT-PCR-negative samples and 1.42 in the 1st RT-PCR-positive samples (P<0.001), and means of 0.000 39 and 0.51 in the 2nd RT-PCR-negative and -positive samples (P< 0.001). The mean ratios of BCR-ABL/ABL by RQ-PCR were significantly different in N/N (1st/2nd RT-PCR) or N/P and P/P (P<0.001), but not in N/N and N/P, which showed that the discriminative power of RQ-PCR is confined to the 1st RT-PCR level. In this respect, monitoring of the 1st RT-PCR might be useful for estimating normalized BCR-ABL levels after transplantation. Nested RT-PCR was of limited use, as RQ-PCR quantified the BCR-ABL transcripts in 60 (91%) of 66 samples determined to be negative by 2nd RT-PCR. FISH was significantly correlated with RQ-PCR in FISH-positive samples (n=24, r=0.79, P=0.001). An increase of FISH preceded that of RQ-PCR in a few cases with molecular relapse. By analyzing a large number of samples post-transplant, we found that RQ-PCR might be the most useful assay for MRD monitoring; however, FISH and RT-PCR were found to be useful complementary tools.