Allele-specific PCR with competitive probe blocking for sensitive and specific detection of BRAF V600E in thyroid fine-needle aspiration specimens.

OBJECTIVE: To detect BRAF V600E mutation in thyroid fine-needle aspiration (FNA) slides and needle rinses (NR). STUDY DESIGN: Tumor-enriched DNA was extracted from FNA smears, formalin-fixed paraffin-embedded (FFPE) sections, or NR specimens from 37 patients with confirmed papillary thyroid carcinoma or benign findings. An allele-specific primer selectively amplified the 1799 T>A BRAF mutation while simultaneously blocking amplification of wild-type (WT) BRAF with an unlabeled probe during PCR. Mutation detection was accomplished by melting analysis of the probe. RESULTS: Allele-specific/blocking probe PCR confirmed the BRAF mutation status for 20 of 24 paired FNA/FFPE samples previously tested by fluorescent probe real-time PCR. For the other 4 cases, the sensitive PCR method detected the BRAF mutation in all paired FNA/FFPE samples. Previously, the mutation had been detected in only the FFPE samples. The BRAF mutation was also detected in some NR specimens. CONCLUSION: Treatment of patients with thyroid nodules is guided by FNA biopsy, which can be scantly cellular, necessitating a sensitive test that can detect low levels of BRAF V600E mutation in a WT background. We report increased detection of BRAF V600E in FNA specimens using allele-specific/blocking probe PCR, which has an analytical sensitivity of 0.01%.

Comparison of ThinPrep UroCyte and cytospin slide preparations for gastrointestinal specimens: evaluation and retrospective performance review.

Gastrointestinal (GI) tract cytology has high specificity but poor sensitivity for detecting GI tract cancer. Newer methods of slide preparation may improve cytology performance and additionally permit molecular slide-based assays that could improve diagnostic accuracy. A split-sample validation study compared slides prepared using ThinPrep UroCyte filters or a cytocentrifuge method with respect to cellularity, stain quality, and interpretation. In this 15-slide split-sample study, UroCyte slide preparations were judged to be superior to cytocentrifuge preparations, and the method was implemented for GI cytology in December 2006. To assess diagnostic performance for GI cytology, we retrospectively reviewed outcomes for one year before and after implementation of UroCyte filter slide preparation. Sensitivity, specificity, positive predictive value, and negative predictive value for both slide preparations were largely equivalent to one another and compared favorably with values in the literature, but varied greatly depending on how atypical and suspicious-atypical cases were defined for calculations. For biopsied biliary samples, the highest sensitivities were observed when all atypical and suspicious-atypical cases were considered positive for malignancy, but were lower when suspicious-atypical cases were considered positive and atypical cases were considered negative for malignancy. This highlights the difficulty with comparing studies that define atypical classes differently, and points to the need for a well-defined approach to performance evaluation that relates directly to how diagnostic information is used clinically. We conclude that the UroCyte filter slide preparation is valid for evaluation of GI cytology specimens and may simplify adjunct molecular testing such as FISH. This is the first reported use of UroCyte filters for preparation of GI specimens.

Integrating a FISH imaging system into the cytology laboratory.

We have implemented an interactive imaging system for the interpretation of UroVysion fluorescence in situ hybridization (FISH) to improve throughput, productivity, quality control and diagnostic accuracy. We describe the Duet imaging system, our experiences with implementation, and outline the financial investment, space requirements, information technology needs, validation, and training of cytotechnologists needed to integrate such a system into a cytology laboratory. Before purchasing the imaging system, we evaluated and validated the instrument at our facility. Implementation required slide preparation changes, IT modifications, development of training programs, and revision of job descriptions for cytotechnologists. A darkened room was built to house the automated scanning station and microscope, as well as two imaging stations. IT changes included generation of storage for archival images on the LAN, addition of external hard drives for back-up, and changes to cable connections for communication between remote locations. Training programs for cytotechnologists, and pathologists/fellows/residents were developed, and cytotechnologists were integrated into multiple steps of the process. The imaging system has resulted in increased productivity for pathologists, concomitant with an expanded role of cytotechnologists in multiple critical steps, including FISH, scan setup, reclassification, and initial interpretation.

Enrichment and detection of rare alleles by means of snapback primers and rapid-cycle PCR.

BACKGROUND: Selective amplification of minority alleles is often necessary to detect cancer mutations in clinical samples. METHODS: Minor-allele enrichment and detection were performed with snapback primers in the presence of a saturating DNA dye within a closed tube. A 5' tail of nucleotides on 1 PCR primer hybridizes to the variable locus of its extension product to produce a hairpin that selectively enriches mismatched alleles. Genotyping performed after rapid-cycle PCR by melting of the secondary structure identifies different variants by the hairpin melting temperature (T(m)). Needle aspirates of thyroid tissue (n = 47) and paraffin-embedded biopsy samples (n = 44) were analyzed for BRAF (v-raf murine sarcoma viral oncogene homolog B1) variant p.V600E, and the results were compared with those for dual hybridization probe analysis. Needle aspirates of lung tumors (n = 8) were analyzed for EGFR [epidermal growth factor receptor (erythroblastic leukemia viral (v-erb-b) oncogene homolog, avian)] exon 19 in-frame deletions. RESULTS: Use of 18-s cycles and momentary extension times of "0 s" with rapid-cycle PCR increased the selective amplification of mismatched alleles. A low Mg(2+) concentration and a higher hairpin T(m) relative to the extension temperature also improved the detection limit of mismatched alleles. The detection limit was 0.1% for BRAF p.V600E and 0.02% for EGFR exon 19 in-frame deletions. Snapback and dual hybridization probe methods for allele quantification of the thyroid samples correlated well (R(2) = 0.93) with 2 more BRAF mutations (45 and 43, respectively, of 91 samples) detected after snapback enrichment. Different EGFR in-frame deletions in the lung samples produced different hairpin T(m)s. CONCLUSIONS: Use of snapback primers for enrichment and detection of minority alleles is simple, is inexpensive to perform, and can be completed in a closed tube in <25 min.

"FISHing" to detect urinary and other cancers: validation of an imaging system to aid in interpretation.

BACKGROUND: The UroVysion Bladder Cancer Kit detects amplifications of chromosomes 3, 7, and 17, and the deletion of 9p21, by fluorescence in situ hybridization (FISH). Because manual interpretation of UroVysion FISH is time consuming and can be challenged by variable probe signal strengths and background labeling, the authors investigated an automated image analysis system to improve throughput, productivity, quality control, and accuracy. METHODS: The authors evaluated the interactive BioView Duet imaging system as an aid to UroVysion FISH interpretation in a 2-armed, blinded comparison with manual screens of the same 135 consecutive cases. Manual and Duet-assisted interpretations were compared with respect to concordance, reproducibility, and timing. RESULTS: Eighty-one cases were interpreted as positive or negative with 94% concordance and a kappa value of 0.84 between manual and Duet-aided interpretations. Three cases that ultimately were judged positive were detected with the aid of Duet but were missed with a manual screen. A final interpretation could not be given for approximately 25% of Duet-scanned cases. Duet-aided interpretation was highly reproducible for patient and control slides. Pathologist evaluation time per case was 4 minutes compared with 30 minutes for manual interpretation. Cytotechnologist involvement added 18 minutes for a total of 22 minutes, a savings of 8 minutes per case. CONCLUSIONS: Duet-aided interpretations were at least equivalent to manual interpretations. The system permitted interactive review of abnormal cells and had the ability to evaluate the same cells for brightfield cytology followed by FISH. The image processing and analysis tools of the Duet system enhanced the morphology skills of cytology professionals in providing accurate interpretations.

A comparison of the clinical utility of p16(INK4a) immunolocalization with the presence of human papillomavirus by hybrid capture 2 for the detection of cervical dysplasia/neoplasia.

BACKGROUND: Evidence suggests that overexpression of p16(INK4a) protein indicates infection and genomic integration of high-risk human papillomavirus (HR HPV) and predicts progression to cervical high-grade squamous intraepithelial lesions (HSILs) and carcinoma. The authors compared the ability of p16(INK4a) and HR HPV detection by Hybrid Capture 2 (HC2) to detect the presence of significant cervical disease. METHODS.: Four hundred ThinPrep specimens (100 each in 4 categories: 100 specimens that were negative for intraepithelial lesions, 100 specimens of atypical squamous cells of undetermined significance [ASC-US], 100 specimens of low-grade squamous intraepithelial lesions [LSILs], and 100 specimens of HSILs) were analyzed. p16(INK4a) protein was immunolocalized using a specific monoclonal antibody, and the detection of HR HPV in all 400 specimens was determined using HC2. RESULTS: p16(INK4a) was found to be positive in 78% of HSIL specimens, 42% of LSIL specimens, and 36% of ASC-US specimens; whereas HC2 was positive in 92% of HSIL specimens, 81% of LSIL specimens, and 45% of ASC-US specimens. In the HSIL category, the sensitivity, which was calculated using Grade 2 or greater cervical intraepithelial neoplasia as the endpoint, was 78% (50 of 66 specimens) for p16(INK4a) and 91% (60 of 66 specimens) for HC2. For LSIL, the sensitivity was 75% (3 of 4 specimens) for p16(INK4a) and 100% (4 of 4 specimens) for HC2. In the ASC-US category, the sensitivity was 89% (8 of 9 specimens) for p16(INK4a) and 100% (9 of 9 specimens) for HC2. Overall, the sensitivity for HSIL was 92% for HC2 and 78% for p16(INK4a). The specificity for HC2 was 8.3% for HSIL, 16.9% for LSIL, and 48.7% for ASC-US; whereas the specificity for p16(INK4a) was 25% in HSIL, 59.1% in LSIL, and 68.4% in ASC-US. The overall specificity was 25% for HC2 and 56% for p16(INK4a). CONCLUSIONS: Although both p16(INK4a) and HC2 may aid in the clinical management of patients with clinically significant lesions, HC2 was found to have greater sensitivity, and p16(INK4a) greater specificity. The labeling of normal cells and bacteria may preclude the use of p16(INK4a) in automated screening or nonmorphologic assays.

The utilization of lipovitellin during blue crab (Callinectes sapidus) embryogenesis.

Embryos of the blue crab Callinectes sapidus develop in egg sacs carried on the abdomen of the female. They develop over a period of 10-13 days at 28 degrees C and are nutritionally dependent on yolk until they emerge from the egg sacs as free-swimming zoeae. The principal component of blue crab yolk is lipovitellin (LpII), a water-soluble lipoprotein composed of approximately equal amounts of lipid and protein. We followed changes in the concentration of apoproteins of LpII during embryogenesis by ELISA and Western blots, using monoclonal antibodies against two LpII apoprotein associated peptides identified as Protein A (107 kDa) and Protein B (75 kDa). During embryogenesis there was a decrease in Protein B but an increase in two smaller peptides (52 and 35 kDa) that reacted with the Protein B antibody. Utilization of LpII during embryogenesis was also followed morphologically by immunohistochemistry. Utilization of LpII was slow in early embryonic stages, followed by rapid utilization in late embryonic stages, such that only traces of LpII were present at the end of embryogenesis. The cells of the developing hepatopancreas appear to play an important role in the utilization of LpII.