Background: Molecular methods for histopathology are today increasingly considered to support diagnosis together with classical methods. IMS is one of the most striking technique providing an overall insight of the molecular context of a given physiological or pathological status that cannot not be obtained with classical histopathological examinations. However a systematic optimization and standardization of routine IMS protocols in clinical application has not yet been realized. We performed a method development study for IMS of peptides from FFPE breast and ovarian tumor tissues with the goal of increasing the quality and reproducibility of the IMS data for these specific tumor entities. Methods: Breast and ovarian tumors were collected from invasive breast adenocarcinoma (highly differentiated, n = 3) and endometrioid ovarian adenocarcinoma (moderately differentiated, n = 3). Six sample preparation parameters with two conditions each were selected, including deparaffination, antigen-retrieval buffer/time, digestion time/temperature, and use of reduction/alkylation agents. Using a fractional factorial design of experiments, eight different protocols were defined. Protocols were run on all patients and at two different labs using equivalent instrumentations. Data of all 96 experiments was evaluated using SCiLS Lab software (SCiLS, Bremen) and statistical analysis performed by MATLAB with respect to six quantitative-quality metrics, including mean intensity, spatial measure of chaos, mean total variation, number of observed peptides/proteins, and correlation between peptides of the same protein. Results: Visual inspection was performed comparing peptide profiles of all experiments for total ion current, noise background, intensity and number of observed peaks, yielding 2/8 protocols with significantly higher signal-to-noise ratios and enhanced structures in the ion-images. A multivariate statistical analysis of variance (ANOVA) estimated the individual effects of all process parameters to signal quality. As a result, a strong recommendation for three of the parameters was derived that was identical to the one obtained from visual inspection. Interestingly, the comparison of data from the two labs showed a high inter-lab variability, which we attribute to different characteristics of the IMS devices. Conclusion: An optimized protocol for FFPE breast and ovarian tissues is obtained by a statistical evaluation of a method development and repro- ducibility study, aimed to be used in clinical applications.
Optimized Imaging Mass Spectrometry (IMS) protocol for breast and ovarian cancer tissue obtained by a two-center method development and reproducibility study
Casadonte R;
2018-01-01
Abstract
Background: Molecular methods for histopathology are today increasingly considered to support diagnosis together with classical methods. IMS is one of the most striking technique providing an overall insight of the molecular context of a given physiological or pathological status that cannot not be obtained with classical histopathological examinations. However a systematic optimization and standardization of routine IMS protocols in clinical application has not yet been realized. We performed a method development study for IMS of peptides from FFPE breast and ovarian tumor tissues with the goal of increasing the quality and reproducibility of the IMS data for these specific tumor entities. Methods: Breast and ovarian tumors were collected from invasive breast adenocarcinoma (highly differentiated, n = 3) and endometrioid ovarian adenocarcinoma (moderately differentiated, n = 3). Six sample preparation parameters with two conditions each were selected, including deparaffination, antigen-retrieval buffer/time, digestion time/temperature, and use of reduction/alkylation agents. Using a fractional factorial design of experiments, eight different protocols were defined. Protocols were run on all patients and at two different labs using equivalent instrumentations. Data of all 96 experiments was evaluated using SCiLS Lab software (SCiLS, Bremen) and statistical analysis performed by MATLAB with respect to six quantitative-quality metrics, including mean intensity, spatial measure of chaos, mean total variation, number of observed peptides/proteins, and correlation between peptides of the same protein. Results: Visual inspection was performed comparing peptide profiles of all experiments for total ion current, noise background, intensity and number of observed peaks, yielding 2/8 protocols with significantly higher signal-to-noise ratios and enhanced structures in the ion-images. A multivariate statistical analysis of variance (ANOVA) estimated the individual effects of all process parameters to signal quality. As a result, a strong recommendation for three of the parameters was derived that was identical to the one obtained from visual inspection. Interestingly, the comparison of data from the two labs showed a high inter-lab variability, which we attribute to different characteristics of the IMS devices. Conclusion: An optimized protocol for FFPE breast and ovarian tissues is obtained by a statistical evaluation of a method development and repro- ducibility study, aimed to be used in clinical applications.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
