Multiplex Polymerase Chain Reaction for the Detection of High-risk-Human Papillomavirus Types in Formalin-fixed Paraffin-embedded Cervical Tissues

Context.—Testing for high-risk human papillomavirus (HR-HPV) in head and neck squamous cell carcinomas (HNSCCs) is important for both prognostication and clinical management. Several testing platforms are available for HR-HPV; however, effective alternative automated approaches are needed. Objective.—To assess the performance of the automated Roche cobas 4800 HPV real-time polymerase chain reaction-based system on formalin-fixed, paraffin-embedded HNSCC specimens and compare results with standard methods of in situ hybridization (ISH) and p16 immunohistochemistry. Design.—Formalin-fixed, paraffin-embedded samples of HNSCC were collected from archival specimens in the Department of Pathology, Massachusetts General Hospital (Boston), and prepared using the automated system by deparaffinization and dehydration followed by tissue lysis. Samples were integrated into routine cervical cytology testing runs by cobas. Corresponding formalin-fixed, paraffin-embedded samples were evaluated for HR-HPV by ISH and p16 by immunohistochemistry. Discrepant cases were adjudicated by polymerase chain reaction. Results.—Sixty-two HNSCC samples were analyzed using the automated cobas system, ISH, and immunohistochemistry. Fifty-two percent (n = 32 of 62) of formalin-fixed, paraffin-embedded tumors were positive for HR-HPV by cobas. Eighty-eight percent (n = 28 of 32) of cases were the HPV 16 subtype and 12% (n = 4 of 32) were other HR-HPV subtypes. Corresponding testing with ISH was concordant in 92% (n = 57 of 62) of cases. Compared with the adjudication polymerase chain reaction standard, there were 3 false-positive cases by cobas. Conclusions.—Concordance in HNSCC HR-HPV status between cobas and ISH was more than 90%. The cobas demonstrated a sensitivity of 100% and a specificity of 91% for detection of HR-HPV. Advantages favoring cobas include its automation, cost efficiency, objective results, and ease of performance.

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KRas and BRaf Mutational Status Analysis From Formalin-Fixed, Paraffin-Embedded Tissues Using Multiplex Polymerase Chain Reaction–Based Assay

Archives of Pathology & Laboratory Medicine ◽

10.5858/134.4.620 ◽

2010 ◽

Vol 134 (4) ◽

pp. 620-624

Author(s):

Arturas Jakubauskas ◽

Laimonas Griskevicius

Keyword(s):

Polymerase Chain Reaction ◽

Epidermal Growth Factor Receptor ◽

Multiplex Polymerase Chain Reaction ◽

Chain Reaction ◽

Mutational Status ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Epidermal Growth ◽

Polymerase Chain ◽

Formalin Fixed

Abstract Context.—Monoclonal anti–epidermal growth factor receptor antibodies bind to the epidermal growth factor receptor and inhibit receptor kinase activity. Clinical trials have indicated that evaluation of the mutational status of KRas and BRaf is necessary to exclude patients who are resistant to the clinical benefit of anti–epidermal growth factor receptor therapy. Objective.—To develop a multiplex polymerase chain reaction–based assay for the evaluation of KRas and BRaf mutational status. Design.—A sample-saving and cost-effective, multiplex polymerase chain reaction–based assay to detect somatic mutations in KRAS exon 2 and exon 3 as well as in BRAF exon 15 was developed. The same primer pairs could be successfully used in amplification of a single DNA fragment under the same conditions. Results.—DNA isolated from 20 retrospective formalin-fixed, paraffin-embedded samples of colorectal cancer was screened for mutations using the multiplex polymerase chain reaction assay followed by dideoxy-termination sequencing. Five samples bearing mutations—p.G12D (identified twice), p.G12V, p.G12S, and p.G13D, all encoded in KRAS exon 2—were identified. Three samples were found bearing amino acid substitution p.V600E of BRaf. The detected KRas and BRaf mutations were found to be mutually exclusive. Conclusions.—A multiplex polymerase chain reaction–based amplification followed by dideoxy-termination sequencing may be used advantageously for the evaluation of KRas and BRaf mutational status from formalin-fixed, paraffin-embedded samples.

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Laboratory-Developed L1 Sequencing and Type-Specific, Real-Time Polymerase Chain Reaction for the Detection and Typing of Human Papillomaviruses in Formalin-Fixed, Paraffin-Embedded Tissues

Archives of Pathology & Laboratory Medicine ◽

10.5858/arpa.2011-0392-oa ◽

2013 ◽

Vol 137 (1) ◽

pp. 50-54 ◽

Cited By ~ 6

Author(s):

Anne Mills ◽

Rajeev Balasubramaniam ◽

Teri A. Longacre ◽

Christina S. Kong ◽

Benjamin A. Pinsky

Keyword(s):

Polymerase Chain Reaction ◽

High Risk ◽

Real Time ◽

Clinical Laboratory ◽

Low Risk ◽

Chain Reaction ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Polymerase Chain ◽

Formalin Fixed

Context.—The detection and typing of high-risk and low-risk human papillomavirus (HPV) in archival formalin-fixed, paraffin-embedded tissues by nucleic acid amplification testing is an important adjunct to immunohistochemical staining in evaluation of squamous cell proliferations of the oropharynx, larynx, and anal canal. Objective.—To evaluate semiautomated, xylene-free extraction from formalin-fixed, paraffin-embedded tissues combined with laboratory-developed HPV L1 sequencing and type-specific HPV 6, 11, 16, and 18 real-time polymerase chain reaction for identification and typing of HPV in the clinical laboratory. Design.—We evaluated the adequacy of extraction using β-globin amplification and compared L1 sequencing and real-time polymerase chain reaction methods for typing accuracy using 68 formalin-fixed, paraffin-embedded tissues, including 56 anorectal biopsy or surgical resection specimens and 12 laryngeal papilloma specimens from patients with recurrent respiratory papillomatosis. Results.—Adequate DNA was obtained from 68 of 68 specimens analyzed and all were HPV positive. In 47 cases where L1 sequencing demonstrated that the predominant HPV type was 6, 11, 16, or 18, type-specific, real-time polymerase chain reaction provided concordant results. Sequencing revealed additional low-risk (HPV 40) and high-risk HPV types (HPV 31, 33, 56, and 58) in anorectal specimens, whereas HPV 6 or 11 were the types found in laryngeal papillomas. Conclusion.—Both L1 sequencing and type-specific, real-time polymerase chain reaction are suitable methods for routine HPV testing of formalin-fixed, paraffin-embedded tissues in a clinical laboratory setting.

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