Comparison of two FFPE preparation methods using label-free shotgun proteomics: Application to tissues of diverticulitis patients.

Formalin-fixed paraffin-embedded (FFPE) specimens of patients are useful sources of materials for clinical research and have recently gained interest for use in the discovery of clinical proteomic biomarkers. However, the critical step in this field is the ability to obtain an efficient and repeatable extraction using the limited quantities of material available for research in hospital biobanks. This work describes the evaluation of the peptide/protein extraction using FFPE sections treated by the following two methods before shotgun proteomic analysis: a commercial solution (FFPE-FASP) (filter aided sample preparation) and an antigen retrieval-derived protocol (On Slice AR). Their efficiencies and repeatabilities are compared using data-independent differential quantitative label-free analysis. FFPE-FASP was shown to be globally better both qualitatively and quantitatively than On Slice AR. FFPE-FASP was tested on several samples, and differential analysis was used to compare the tissues of diverticulitis patients (healthy and inflammatory tissues). In this differential proteomic analysis using retrospective clinical FFPE material, FFPE-FASP was reproducible and provided a high number of confident protein identifications, highlighting potential protein biomarkers. BIOLOGICAL SIGNIFICANCE: In clinical proteomics, FFPE is an important resource for retrospective analysis and for the discovery of biomarkers. The challenge for FFPE shotgun proteomic analysis is preparation by an efficient and reproducible protocol, which includes protein extraction and digestion. In this study, we analyzed two different methods and evaluated their repeatabilities and efficiencies. We illustrated the reproducibility of the most efficient method, FFPE-FASP, by a pilot study on diverticulitis tissue and on FFPE samples amount accessible in hospital biobanks. These data showed that FFPE is suitable for use in clinical proteomics, especially when the FFPE-FASP method is combined with label-free shotgun proteomics as described in the workflow presented in this work.

Evaluation of CellSolutions BestPrep® automated thin-layer liquid-based cytology Papanicolaou slide preparation and BestCyte® cell sorter imaging system.

OBJECTIVE: A double-blind study was conducted to compare the performance of the new BestPrep® (CellSolutions) liquid-based thin-layer Papanicolaou (Pap) test with ThinPrep® (Hologic). STUDY DESIGN: Samples from the study patients (n = 105) were collected twice in the same encounter with the ThinPrep sample always taken first and the BestPrep sample collected second. Slides were prepared according to both manufacturers' protocols and evaluated using manual microscopic review and the BestCyte® cell sorter imaging system (CellSolutions). Diagnostic truth for each case was determined by independent manual review of both slides by multiple pathologists and histology when available. The presence of atypical squamous cells of undetermined significance was the threshold for positive for sensitivity and specificity calculations. RESULTS: BestPrep and ThinPrep, by manual review, had sensitivities for high-grade squamous intraepithelial lesion (HSIL) cases of 100 and 95.6%, respectively. Using the BestCyte cell sorter, both had 100% sensitivity. For the same HSIL cases, the digene HC2 high-risk human papillomavirus DNA test had sensitivities of 100% (BestPrep) and 95.6% (ThinPrep). Specificities were 71.4% (BestPrep) and 54.8% (ThinPrep). CONCLUSIONS: BestPrep was equivalent to ThinPrep for manual review even though BestPrep was always the second sample collected. The BestCyte cell sorter provides a practical alternative to manual review for both BestPrep and ThinPrep slides.

Clinical utility of an epigenetic assay to detect occult prostate cancer in histopathologically negative biopsies: results of the MATLOC study.

PURPOSE: Concern about possible false-negative prostate biopsy histopathology findings often leads to rebiopsy. A quantitative methylation specific polymerase chain reaction assay panel, including GSTP1, APC and RASSF1, could increase the sensitivity of detecting cancer over that of pathological review alone, leading to a high negative predictive value and a decrease in unnecessary repeat biopsies. MATERIALS AND METHODS: The MATLOC study blindly tested archived prostate biopsy needle core tissue samples of 498 subjects from the United Kingdom and Belgium with histopathologically negative prostate biopsies, followed by positive (cases) or negative (controls) repeat biopsy within 30 months. Clinical performance of the epigenetic marker panel, emphasizing negative predictive value, was assessed and cross-validated. Multivariate logistic regression was used to evaluate all risk factors. RESULTS: The epigenetic assay performed on the first negative biopsies of this retrospective review cohort resulted in a negative predictive value of 90% (95% CI 87-93). In a multivariate model correcting for patient age, prostate specific antigen, digital rectal examination and first biopsy histopathological characteristics the epigenetic assay was a significant independent predictor of patient outcome (OR 3.17, 95% CI 1.81-5.53). CONCLUSIONS: A multiplex quantitative methylation specific polymerase chain reaction assay determining the methylation status of GSTP1, APC and RASSF1 was strongly associated with repeat biopsy outcome up to 30 months after initial negative biopsy in men with suspicion of prostate cancer. Adding this epigenetic assay could improve the prostate cancer diagnostic process and decrease unnecessary repeat biopsies.