Use of Formalin-Fixed and Paraffin-Embedded Tissues for Diagnosis and Therapy in Routine Clinical Settings

Berg, Daniela; Malinowsky, Katharina; Reischauer, Bilge; Wolff, Claudia; Becker, Karl‐Friedrich

doi:10.1007/978-1-61779-286-1_8

Cited by 40 publications

(27 citation statements)

References 34 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…It has been assumed that the cross-linking of proteins prevents a proteomic analysis. Recently, it could be shown that proteins can be effectively extracted from formalin-fixed and paraffin-embedded samples and that the proteins and phosphorylations are quantitatively preserved compared with fresh frozen tissues (56,61,62).…”

Section: Resultsmentioning

confidence: 99%

Phosphosignature Predicts Dasatinib Response in Non-small Cell Lung Cancer

Klammer

Kaminski

Zedler

et al. 2012

Molecular & Cellular Proteomics

100

View full text Add to dashboard Cite

Targeted drugs are less toxic than traditional chemotherapeutic therapies; however, the proportion of patients that benefit from these drugs is often smaller. A marker that confidently predicts patient response to a specific therapy would allow an individual therapy selection most likely to benefit the patient. Here, we used quantitative mass spectrometry to globally profile the basal phosphoproteome of a panel of non-small cell lung cancer cell lines. The effect of the kinase inhibitor dasatinib on cellular growth was tested against the same panel. From the phosphoproteome profiles, we identified 58 phosphorylation sites, which consistently differ between sensitive and resistant cell lines. Many of the corresponding proteins are involved in cell adhesion and cytoskeleton organization. We showed that a signature of only 12 phosphorylation sites is sufficient to accurately predict dasatinib sensitivity. The introduction of targeted drugs for treating cancer is a major biomedical achievement of the past decade (1, 2). Because these drugs selectively block molecular pathways that are typically overactivated in tumor cells, they are more precise and less toxic than traditional chemotherapeutics. However, although many cancer patients benefit from a specific targeted therapy, many others do not. Therefore, predictive molecular markers are needed to confidently predict patient response to a specific therapy. Such markers would facilitate therapy personalization, where the selected therapy is based on the molecular profile of the patient.Predictive tests currently used in the clinic are frequently based on one particular marker that is often linked to the drug target. A well known example for a predictive test is assessing HER2/neu overexpression using immunohistochemistry or fluorescent in situ hybridization to predict the response to therapy with trastuzumab (Herceptinா; Roche) (3, 4). However, in some cases the expression or mutational status of the target or other singleton markers might not be sufficient to predict a therapeutic response. Recently, several studies tried to identify molecular signatures comprising multiple markers for response predictions, usually based on gene expression profiling (5, 6). To our knowledge, no study successfully identified a signature from global phosphoproteomic profiles so far.Recent advances in mass spectrometry, methods for enriching phosphorylated proteins or peptides, and computer algorithms for analyzing proteomics data have enabled the application of mass spectrometry-based proteomics to monitor phosphorylation events in a global and unbiased manner. These methods have become sufficiently sensitive and robust to localize and quantify the phosphorylation sites within a peptide sequence (7-9). Phosphorylation events are important in signal transduction, where signals caused by external stimuli are transmitted from the cell membrane to the nucleus. Aberrations in these signal transduction pathways are particularly important for understanding the mechanisms of certain diseases,...

show abstract

Section: Resultsmentioning

confidence: 99%

Phosphosignature Predicts Dasatinib Response in Non-small Cell Lung Cancer

Klammer

Kaminski

Zedler

et al. 2012

Molecular & Cellular Proteomics

100

View full text Add to dashboard Cite

show abstract

“…Most DNA for genetic testing from cancer patients is extracted from formalin-fixed paraffin-embedded tumour biopsies, where the primary intent is to preserve tumour cellular structure for histological examination and diagnosis [5]. However, the formalin fixation process is detrimental to downstream genomic applications, causing issues, such as DNA cross-linking to DNA and proteins, that can stall polymerases and DNA-DNA crosslinks that can inhibit denaturation [6].…”

Section: Introductionmentioning

confidence: 99%

Sequence artefacts in a prospective series of formalin-fixed tumours tested for mutations in hotspot regions by massively parallel sequencing

et al. 2014

View full text Add to dashboard Cite

BackgroundClinical specimens undergoing diagnostic molecular pathology testing are fixed in formalin due to the necessity for detailed morphological assessment. However, formalin fixation can cause major issues with molecular testing, as it causes DNA damage such as fragmentation and non-reproducible sequencing artefacts after PCR amplification. In the context of massively parallel sequencing (MPS), distinguishing true low frequency variants from sequencing artefacts remains challenging. The prevalence of formalin-induced DNA damage and its impact on molecular testing and clinical genomics remains poorly understood.MethodsThe Cancer 2015 study is a population-based cancer cohort used to assess the feasibility of mutational screening using MPS in cancer patients from Victoria, Australia. While blocks were formalin-fixed and paraffin-embedded in different anatomical pathology laboratories, they were centrally extracted for DNA utilising the same protocol, and run through the same MPS platform (Illumina TruSeq Amplicon Cancer Panel). The sequencing artefacts in the 1-10% and the 10-25% allele frequency ranges were assessed in 488 formalin-fixed tumours from the pilot phase of the Cancer 2015 cohort. All blocks were less than 2.5 years of age (mean 93 days).ResultsConsistent with the signature of DNA damage due to formalin fixation, many formalin-fixed samples displayed disproportionate levels of C>T/G>A changes in the 1-10% allele frequency range. Artefacts were less apparent in the 10-25% allele frequency range. Significantly, changes were inversely correlated with coverage indicating high levels of sequencing artefacts were associated with samples with low amounts of available amplifiable template due to fragmentation. The degree of fragmentation and sequencing artefacts differed between blocks sourced from different anatomical pathology laboratories. In a limited validation of potentially actionable low frequency mutations, a NRAS G12D mutation in a melanoma was shown to be a false positive.ConclusionsThese findings indicate that DNA damage following formalin fixation remains a major challenge in laboratories working with MPS. Methodologies that assess, minimise or remove formalin-induced DNA damaged templates as part of MPS protocols will aid in the interpretation of genomic results leading to better patient outcomes.

show abstract

“…The dot blot approach, which is dependent on the detection of a single epitope by an affinity reagent, usually an antibody, is particularly applicable to clinical samples, as it is less sensitive to protein quality than is a sandwich antibody-like approach in which two independent epitopes and the intervening region must be intact for quantitative analysis. Indeed, with a number of caveats, RPPA can be applied to at least a subset of targets from formalin-fixed paraffin-embedded patient samples (33)(34)(35).…”

mentioning

confidence: 99%

Realizing the Promise of Reverse Phase Protein Arrays for Clinical, Translational, and Basic Research: A Workshop Report

Akbani

Becker

Carragher

et al. 2014

Molecular & Cellular Proteomics

Self Cite

155

132

View full text Add to dashboard Cite

Reverse phase protein array (RPPA) technology introduced a miniaturized "antigen-down" or "dot-blot" immunoassay suitable for quantifying the relative, semi-quantitative or quantitative (if a well-accepted reference standard exists) abundance of total protein levels and post-translational modifications across a variety of biological samples including cultured cells, tissues, and body fluids. The recent evolution of RPPA combined with more sophisticated sample handling, optical detection, quality control, and better quality affinity reagents provides exquisite sensitivity and high sample throughput at a reasonable cost per sample. This facilitates large-scale multiplex analysis of multiple post-translational markers across samples from in vitro, preclinical, or clinical samples. The technical power of RPPA is stimulating the application and widespread adoption of RPPA methods within academic, clinical, and industrial research laboratories.Advances in RPPA technology now offer scientists the opportunity to quantify protein analytes with high precision, sensitivity, throughput, and robustness. As a result, adopters of RPPA technology have recognized critical success factors for useful and maximum exploitation of RPPA technologies, including the following:• preservation and optimization of pre-analytical sample quality,• application of validated high-affinity and specific antibody (or other protein affinity) detection reagents,• dedicated informatics solutions to ensure accurate and robust quantification of protein analytes, and• quality-assured procedures and data analysis workflows compatible with application within regulated clinical environments.In 2011, 2012, and 2013, the first three Global RPPA workshops were held in the United States, Europe, and Japan, respectively. These workshops provided an opportunity for RPPA laboratories, vendors, and users to share and discuss results, the latest technology platforms, best practices, and future challenges and opportunities. The outcomes of the workshops included a number of key opportunities to advance the RPPA field and provide added benefit to existing and future participants in the RPPA research community. The purpose of this report is to share and disseminate, as a community, current knowledge and future directions of the RPPA technology. Molecular & Cellular Proteomics 13: 10.1074/mcp.O113.034918, 1625-1643, 2014.Reverse phase protein array (RPPA) 1 technology represents a highly efficient and cost-effective descendent of miniaturized immunoassays that use a sandwich format for antigen capture (1, 2). Immunoassay arrays were generally sandwichstyle assays in which a series of antibodies immobilized on the solid phase were used to capture the analyte of interest ("antibody capture"), with a second antibody, directed against a different epitope on the same protein, used as a detection molecule. In contrast, in "reverse phase" the analytes (antigens) are immobilized on the solid phase (usually nitrocellulose) and subsequently probed with an antibody or other affini...

show abstract

Use of Formalin-Fixed and Paraffin-Embedded Tissues for Diagnosis and Therapy in Routine Clinical Settings

Cited by 40 publications

References 34 publications

Phosphosignature Predicts Dasatinib Response in Non-small Cell Lung Cancer

Phosphosignature Predicts Dasatinib Response in Non-small Cell Lung Cancer

Sequence artefacts in a prospective series of formalin-fixed tumours tested for mutations in hotspot regions by massively parallel sequencing

Realizing the Promise of Reverse Phase Protein Arrays for Clinical, Translational, and Basic Research: A Workshop Report

Contact Info

Product

Resources

About