Utility of the Roche Cobas 4800 for detection of high-risk human papillomavirus in formalin-fixed paraffin-embedded oropharyngeal squamous cell carcinoma.

Clinical laboratories are expected to reliably identify human papilloma virus (HPV) associated oropharyngeal squamous cell carcinoma (OPSCC) for prognostic and potential therapeutic applications. In addition to surrogate p16 immunohistochemistry (IHC) testing, DNA-based HPV-specific testing strategies are widely utilized. Recognizing the efficiency of the Roche Cobas 4800 platform for testing gynecological cytology specimens for high-risk HPV, we elected to evaluate the potential utility of this platform for testing formalin-fixed paraffin-embedded (FFPE) OPSCC tissue. Using the Roche Linear Array assay for comparison, we tested twenty-eight samples (16 primary OPSCC, 2 lymph node metastases from primary OPSCC, 1 oral tongue carcinoma, 3 benign squamous papillomas, and 3 non-oropharyngeal carcinoma tissues). Excluding two invalid results, the Roche Cobas 4800 testing resulted in excellent inter-assay concordance (25/26, 96.2%) and 100% concordance for HPV-16/HPV-18 positive samples. This data suggests that the Roche Cobas 4800 platform may be a cost-effective method for testing OPSCC FFPE tissues in a clinical molecular pathology laboratory setting.

Improving Adequacy of Small Biopsy and Fine-Needle Aspiration Specimens for Molecular Testing by Next-Generation Sequencing in Patients With Lung Cancer: A Quality Improvement Study at Dartmouth-Hitchcock Medical Center.

CONTEXT: - At our medical center, cytopathologists perform rapid on-site evaluation for specimen adequacy of fine-needle aspiration and touch imprint of needle core biopsy lung cancer samples. Two years ago the molecular diagnostics laboratory at our institution changed to next-generation sequencing using the Ion Torrent PGM and the 50-gene AmpliSeq Cancer Hotspot Panel v2 for analyzing mutations in a 50-gene cancer hot spot panel. This was associated with a dramatic fall in adequacy rate (68%). OBJECTIVE: - To improve the adequacy rate to at least 90% for molecular testing using next-generation sequencing for all specimens collected by rapid on-site evaluation by the cytology laboratory. DESIGN: - After baseline data on adequacy rate of cytology specimens with rapid on-site evaluation for molecular testing had been collected, 2 changes were implemented. Change 1 concentrated all the material in one block but did not produce desired results; change 2, in addition, faced the block only once with unstained slides cut up front for molecular testing. Data were collected in an Excel spreadsheet and adequacy rate was assessed. RESULTS: - Following process changes 1 and 2 we reached our goal of at least 90% adequacy rate for molecular testing by next-generation sequencing on samples collected by rapid on-site evaluation including computed tomography-guided needle core biopsies (94%; 17 of 18) and fine-needle aspiration samples (94%; 30 of 32). CONCLUSION: - This study focused on factors that are controllable in a pathology department and on maximizing use of scant tissue. Optimizing the adequacy of the specimen available for molecular tests avoids the need for a second procedure to obtain additional tissue.

Small cell neuroendocrine carcinomas of the lung do not harbor high-risk human papillomavirus.

High-risk subtypes of the human papillomavirus (HPV) are known to drive the pathogenesis of cervical, anogenital, and oropharyngeal squamous cell carcinomas. Recent reports have shown that HPV is also associated with small cell neuroendocrine carcinomas of the cervix and oropharynx. Little is known about HPV as a driver of neuroendocrine tumors at other sites, in particular, small cell lung cancer (SCLC). The aim of this study was to evaluate SCLC for the presence of high-risk HPV to further elucidate the role of HPV in SCLC. Archived formalin-fixed, paraffin-embedded surgical resection specimens from 20 primary SCLC from 19 patients were identified from 2004 to 2013. Two cervical small cell carcinomas were included as controls. Small cell neuroendocrine phenotype was confirmed by review of morphology and prior immunohistochemistry staining. Immunohistochemistry for p16 (INK4a) expression was performed in all cases. DNA was extracted from formalin-fixed, paraffin-embedded specimens and run on the Roche Linear Array HPV Genotyping test and a real-time polymerase chain reaction HPV assay. Pathologic tumor stage was collected from surgical pathology reports. High-risk HPV genotypes were not detected in any of the 20 SCLC specimens, whereas p16 was up-regulated in 14 (70%) of 20. p16 up-regulation can be used as an indicator of disruption of the Rb pathway either by integration of the HPV E7 oncoprotein or other mechanisms. In conclusion, our findings indicate that, unlike some other small cell neuroendocrine carcinomas, the pathogenesis of SCLC does not appear to be associated with high-risk HPV infection, a potentially very useful characteristic when determining primary from metastatic tumors.

Rapid fluorescence in situ hybridisation (FISH) for HER2 (ERBB2) assessment in breast and gastro-oesophageal cancer.

Evaluation of HER2 (ERBB2) gene amplification or protein expression is standard of care in breast (BR) and advanced stage gastro-oesophageal cancers to identify patients eligible for anti-HER2 therapies. Here, we evaluate a rapid fluorescence in situ hybridisation (FISH) technology (HER2 instant quality (IQ) FISH pharmDx Kit) for detection of HER2 in patients with BR and gastro-oesophageal cancer using 30 FFPE samples that had been previously evaluated with the PathVysion HER2 DNA Probe Kit. Cases were scored as positive (HER2:CEN-17 ≥2.0), negative (HER2:CEN-17 <2.0) or equivocal according to the ASCO/CAP 2013 BR cancer guidelines. Ten samples were positive for HER2 amplification while 20 were negative; none were equivocal. The IQ FISH was able to detect low level amplification (HER2:CEN-17 ratio 2.4). The HER2 IQ FISH pharmDx Kit is a FDA approved kit that offers a rapid turnaround time (approximately 3.5 h) and in our laboratory was 100% concordant with prior PathVysion results.

Automated processing of fluorescence in-situ hybridization slides for HER2 testing in breast and gastro-esophageal carcinomas.

BACKGROUND: HER2 fluorescence in-situ hybridization (FISH) is used in breast and gastro-esophageal carcinoma for determining HER2 gene amplification and patients' eligibility for HER2 targeted therapeutics. Traditional manual processing of the FISH slides is labor intensive because of multiple steps that require hands on manipulation of the slides and specifically timed intervals between steps. This highly manual processing also introduces inter-run and inter-operator variability that may affect the quality of the FISH result. Therefore, we sought to incorporate an automated processing instrument into our FISH workflow. METHODS: Twenty-six cases including breast (20) and gastro-esophageal (6) cancer comprising 23 biopsies and three excision specimens were tested for HER2 FISH (Pathvysion, Abbott) using the Thermobrite Elite (TBE) system (Leica). Up to 12 slides can be run simultaneously. All cases were previously tested by the Pathvysion HER2 FISH assay with manual preparation. Twenty cells were counted by two observers for each case; five cases were tested on three separate runs by different operators to evaluate the precision and inter-operator variability. RESULTS: There was 100% concordance in the scoring between the manual and TBE methods as well as among the five cases that were tested on three runs. Only one case failed due to poor probe hybridization. In total, seven cases were positive for HER2 amplification (HER2:CEP17 ratio >2.2) and the remaining 19 were negative (HER2:CEP17 ratio <1.8) utilizing the 2007 ASCO/CAP scoring criteria. Due to the automated denaturation and hybridization, for each run, there was a reduction in labor of 3.5h which could then be dedicated to other lab functions. CONCLUSION: The TBE is a walk away pre- and post-hybridization system that automates FISH slide processing, improves work flow and consistency and saves approximately 3.5h of technologist time. The instrument has a small footprint thus occupying minimal counter space. TBE processed slides performed exceptionally well in comparison to the manual technique with no disagreement in HER2 amplification status.

Validation of interleukin 28B genotyping assay for clinical use.

OBJECTIVES: The favorable CC genotype at rs12979860 upstream of the interleukin (IL)-28B gene is correlated with a greater post-treatment sustained virologic response rate in chronic hepatitis C infected patients. We report on our validation of a clinical genotyping assay for rs12979860 polymorphisms in the IL28B locus. DESIGN AND METHODS: The rs12979860 genotype was determined using a TaqMan® Real-Time PCR allelic discrimination assay with primers and probes specific for the C and T alleles on the Applied Biosystems 7500 Fast Real-Time PCR System. RESULTS: The rs12979860 genotype determined by our assay was concordant with the genotypes obtained from a reference laboratory. The allelic frequency was similar to that reported in the HapMap project (rs12979860 C=0.65, T=0.35) and did not deviate from Hardy-Weinberg equilibrium. CONCLUSION: Clinical availability of this assay in conjunction with other factors will allow the prediction of the individual patient's response to therapy.

Validation of a solid-phase electrochemical array for genotyping hepatitis C virus.

Hepatitis C viral infection is a major cause of progressive liver disease. HCV genotype is one of the most significant baseline predictors of response to HCV antiviral therapy. The objective was to evaluate an HCV genotyping method that targets the 5'-untranslated region (UTR) to detect genotypes/subtypes using the GenMark eSensor® XT-8 system. The HCV amplicon of major genotypes/subtypes from the Roche TaqMan® HCV assay served as a template for the nested PCR followed by a direct analysis on the XT-8 detection system. The assay was validated for limit of detection (LOD), specificity, accuracy and precision. The LOD determined was below 175 IU/ml for all the subtypes except 6ab. The genotypes detected using this assay were in concordance with the LiPA assay. The high performance characteristics (LOD, specificity, intra- and inter-assay precision, and accuracy), make this assay particularly well suited for clinical HCV genotyping in order to guide antiviral therapy.

Evaluation of the Cobas 4800 HPV Test for Detecting High-Risk Human Papilloma-Virus in Cervical Cytology Specimens.

As new platforms for high-risk strains of human papillomavirus (HR HPV) testing are introduced into the clinical laboratory, it is important to verify their performance and agreement. In this validation study, post-aliquot cervical cytopathology specimens (n = 226) were used to analyze agreement between the Invader HPV ASR assay (Hologic) and the recently FDA-approved Cobas 4800 high-risk HPV assay (Roche). Residual sample from 92 Invader positive and 134 Invader negative samples were analyzed with the Cobas 4800 test. Discordant results were further analyzed by Linear Array HPV genotype testing (Roche). To assess intra- and inter-run precision, 31 Invader positive samples were run in duplicate on the Cobas 4800 by different operators over multiple days and purchased HR HPV DNA control was run in ten replicates. Cross-contamination during cytology processing was evaluated by spiking 6 Invader negative samples with different volumes of Acrometrix HPV High Risk Positive Control and analyzed on the Cobas with 4 negative samples in between. There was significant discordance between the assays (p < 0.001; exact McNemar X2 test), with overall agreement of 82%. Of the 92 Invader positive samples, 58 (63%) were positive with the Cobas assay, while 34 (37%) were negative. Of the 134 Invader negative samples, 6 (4%) were positive with the Cobas while 128 (96%) were negative. The observed discordance may be attributed to the previously described false positive rate of the Invader ASR assay. The Cobas 4800 high-risk HPV assay is a viable new tool for use in the clinical setting to identify high-risk HPV.