Droplet digital PCR-based EGFR mutation detection with an internal quality control index to determine the quality of DNA.

In clinical translational research and molecular in vitro diagnostics, a major challenge in the detection of genetic mutations is overcoming artefactual results caused by the low-quality of formalin-fixed paraffin-embedded tissue (FFPET)-derived DNA (FFPET-DNA). Here, we propose the use of an 'internal quality control (iQC) index' as a criterion for judging the minimum quality of DNA for PCR-based analyses. In a pre-clinical study comparing the results from droplet digital PCR-based EGFR mutation test (ddEGFR test) and qPCR-based EGFR mutation test (cobas EGFR test), iQC index ≥ 0.5 (iQC copies ≥ 500, using 3.3 ng of FFPET-DNA [1,000 genome equivalents]) was established, indicating that more than half of the input DNA was amplifiable. Using this criterion, we conducted a retrospective comparative clinical study of the ddEGFR and cobas EGFR tests for the detection of EGFR mutations in non-small cell lung cancer (NSCLC) FFPET-DNA samples. Compared with the cobas EGFR test, the ddEGFR test exhibited superior analytical performance and equivalent or higher clinical performance. Furthermore, iQC index is a reliable indicator of the quality of FFPET-DNA and could be used to prevent incorrect diagnoses arising from low-quality samples.

Employing Digital Droplet PCR to Detect BRAF V600E Mutations in Formalin-fixed Paraffin-embedded Reference Standard Cell Lines.

ddPCR is a highly sensitive PCR method that utilizes a water-oil emulsion system. Using a droplet generator, an extracted nucleic acid sample is partitioned into ~20,000 nano-sized, water-in-oil droplets, and PCR amplification occurs in individual droplets. The ddPCR approach is in identifying sequence mutations, copy number alterations, and select structural rearrangements involving targeted genes. Here, we demonstrate the use of ddPCR as a powerful technique for precisely quantitating rare BRAF V600E mutations in FFPE reference standard cell lines, which is helpful in identifying individuals with cancer. In conclusion, ddPCR technique offers the potential to precisely profile the specific rare mutations in different genes in various types of FFPE samples.

Development and evaluation of a highly sensitive human papillomavirus genotyping DNA chip.

OBJECTIVES: Test of human papillomavirus (HPV) is a useful adjunctive tool of Pap smear to screen cervical cancer. We have developed a novel HPV genotyping DNA chip arrayed by multiple oligonucleotide probes of both L1 and E6/E7 gene sequence of 42 types of anogenital HPV. METHODS: Consensus PCR products of L1 and E6/E7 gene sequences of HPV are hybridized to arrayed probes on the HPV chip and HPV genotypes are identified by fluorescence scanner. We have comparatively analyzed the value of HPV DNA chip and DNA sequencing in 100 cervical cancer tissues. RESULTS: Overall, 98 cervical cancer tissues were found to harbor DNA sequences of high-risk type HPVs, of which 88 (89.8%) were detected by PCR-sequencing of L1 alone, 98 (100%) by PCR-sequencing of both L1 and E6/E7, and 98 (100%) by HPV DNA chip, respectively. All of the genotypes of HPV detected on sequencing analysis were also found on DNA chip analysis. HPV DNA chip was superior to direct DNA sequencing in detection of mixed infection. CONCLUSIONS: These results suggest that HPV DNA chip analysis in the present study is highly accurate for detection and genotyping of HPV and may have potential value as a robust, high-throughput screening test of uterine cervix cancer.

High prevalence of c-RET expression in papillary thyroid carcinomas from the Korean population.

BACKGROUND: Activation of the RET proto-oncogene, located on the long arms of chromosome 10, contributes to the development of thyroid cancers in two different ways. First, somatic rearrangements of RET with variable activation genes are frequently found in papillary thyroid carcinomas. Second, germ-line point mutations are responsible for the development of medullary thyroid carcinomas and multiple endocrine neoplasia type 2 (MEN 2). There are several conflicting reports on the influences of RET expression and RET/PTC rearrangements on the clinical outcome of thyroid cancers. Therefore, the wild-type RET gene expression and RET/PTC-1, RET/PTC-2, RET/PTC-3 rearrangements were examined in thyroid carcinomas and other thyroid diseases. MATERIALS AND METHODS: Thirty-six papillary thyroid carcinomas (PTCs), 8 follicular thyroid carcinomas (FTCs), 4 anaplastic thyroid carcinomas (ATC), 7 follicular adenomas (FAs), 23 hyperplasias, 6 normal thyroid tissues, and 39 normal portions from each tumor were included in this study. Reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemical analyses were used to identify the RET gene and RET/PTC rearrangements. RESULTS: From the RT-PCR analysis, 68.9% of the PTCs, a single case of FTC, and 22.2% of the hyperplasias expressed the RET gene. No RET gene expression was observed in ATCs, FAs, or normal thyroid tissues. One RET/PTC-1 and one RET/PTC-2 rearrangement were detected in the PTCs. No RET/PTC-3 rearrangement was detected in any specimen. The immunohistochemical results revealed that 66.7% of PTCs, 28.6% of FAs, and 18.2% of hyperplastic thyroid tissue specimens showed high levels of RET protein expression. Neither the normal thyroid tissues nor the FTCs and ATC, showed high levels of RET protein expression. The two methods are agreed in PTC and hyperplastic nodules, but not in FA and FTC. CONCLUSION: PTCs among Koreans rarely showed RET/PTC rearrangements, but commonly showed increased RET gene expression. Compared to earlier reports indicating that the expression of the RET gene was limited to PTCs, the RET gene was also expressed in hyperplasias in this study.

Papillary thyroid carcinoma associated with familial adenomatous polyposis: molecular analysis of pathogenesis in a family and review of the literature.

We found a case of a papillary thyroid carcinoma that was accompanied by a familial adenomatous polyposis (FAP) in a 29-year-old female. She had hundreds of adenomas inside the entire colon and a congenital hypertrophy of the retinal pigmented epithelium (CHRPE). The patient underwent a total thyroidectomy and a central compartment neck node dissection. Gross examination of the thyroid identified two solid and cystic lesions. The pathological finding of thyroid cancer revealed a mixture of a peculiar nuclear clearing, cribriform, morula formation, trabecular and papillary pattern. The patient's brother had undergone a total colectomy due to FAP at the age of 25. Genetic analyses of the patient's family members revealed that she and her brother had the same germline mutation, in which five nucleotides (AAAGA) were deleted from codon 1309 of the adenomatous polyposis coli (APC) gene exon 15. Strong and frequent immunoreactivities of beta-catenin and p53 were evident in the tumor tissue. At the time of writing, a preventive colectomy was still under consideration for the patient. Genetic counseling was given to the other family members, who were not attacked by this disease, in order to allay their fears of cancer.

Frequency analysis and clinical characterization of spinocerebellar ataxia types 1, 2, 3, 6, and 7 in Korean patients.

BACKGROUND: By genetic analysis, the CAG repeat expansion has been established in spinocerebellar ataxia (SCA) types 1, 2, 3, 6, and 7. Despite the genetic differentiation of SCA, the characterization of the phenotypes of various SCAs has been challenging for better clinical diagnosis. OBJECTIVE: To analyze the frequencies and the clinical manifestations of SCA1, SCA2, SCA3, SCA6, and SCA7 in Korean patients. PATIENTS AND METHODS: We performed genetic analysis in 253 unrelated Korean patients with progressive cerebellar ataxia. We compared the frequencies, inheritance patterns, and various clinical manifestations of patients with genetically confirmed SCA. RESULTS: Among the 52 patients with expanded CAG repeat, the most frequent SCA type was SCA2, followed by SCA3, SCA6, SCA1, and SCA7. Nine patients (17%) had a negative family history of ataxia, mostly in SCA6. There were characteristic clinical features such as hypotonia and optic atrophy for SCA1; hyporeflexia for SCA2; nystagmus, bulging eye, and dystonia for SCA3; and macular degeneration for SCA7. Interestingly, 4 patients (1 with SCA2, 1 with SCA3, and 2 with SCA6) were misdiagnosed as having multiple-system atrophy because of the absence of family history and the presence of parkinsonism and urinary incontinence. CONCLUSIONS: This study provides a detailed analysis of the clinical characteristics of the genetically defined CAG-repeat SCAs in Korean patients. Although phenotypes were heterogeneous, some clinical features may be helpful for clinical diagnosis. However, genetic studies for SCA are needed despite uncertain family history or the presence of atypical clinical features causing misdiagnosis as atypical parkinsonism.