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Determination of phosphorylated proteins in tissue specimens requires high‐quality samples collected under stringent conditions
Author(s) -
Wolf Corinna,
Jarutat Tiantom,
Vega Harring Suzana,
Haupt Kristin,
Babitzki Galina,
Bader Sabine,
David Kerstin,
Juhl Hartmut,
Arbogast Susanne
Publication year - 2014
Publication title -
histopathology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.626
H-Index - 124
eISSN - 1365-2559
pISSN - 0309-0167
DOI - 10.1111/his.12268
Subject(s) - phosphorylation , fixation (population genetics) , immunohistochemistry , protein phosphorylation , pathology , biology , in vivo , biomarker , biopsy , cancer research , microbiology and biotechnology , medicine , biochemistry , protein kinase a , gene , genetics
Aims For selection of patients who will benefit from targeted therapies, identification of biomarkers predictive of treatment response is desirable. Activation of the targeted pathway becomes apparent by protein phosphorylation. Determination of this phenomenon is therefore considered a promising biomarker approach. To date, however, it is unclear whether routinely collected tissue specimens allow determination of in‐vivo phosphorylation states. Methods and results To investigate whether routinely collected tissue specimens retain the true phosphorylation states of a tumour's proteins, we compared protein phosphorylation states between matched tumour samples that were subjected to different ischaemic times by immunohistochemistry. The influence of formalin fixation and paraffin‐embedding on phosphorylation states was investigated by comparison of matched fresh frozen and formalin‐fixed paraffin‐embedded surgical specimens as well as small biopsies. We show that ischaemia influences protein phosphorylation in a tumour‐specific, unpredictable manner. Formalin fixation and paraffin‐embedding lead to a decrease in detectable protein phosphorylation in larger surgical specimens, but not in small biopsies. Conclusions Determination of protein phosphorylation using routinely collected surgical specimens results in artefacts which do not reflect a tumour's true states of pathway activation. Valid measurement of phosphorylated biomarkers requires that tissue collection procedures are tightly controlled, avoiding ischaemia and large‐specimen fixation.