Determination of phosphorylated proteins in tissue specimens requires high-quality samples collected under stringent conditions

Histopathology. 2014 Feb;64(3):431-44. doi: 10.1111/his.12268. Epub 2013 Nov 23.


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.

Keywords: assay validity; biomarker; biospecimens quality; pre-analytics; protein phosphorylation; tissue ischaemia.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biomarkers, Tumor / metabolism
  • Biopsy
  • Blotting, Western
  • Breast Neoplasms / metabolism
  • Breast Neoplasms / pathology
  • Carcinoma, Non-Small-Cell Lung / metabolism
  • Carcinoma, Non-Small-Cell Lung / pathology
  • Extracellular Signal-Regulated MAP Kinases / metabolism
  • Female
  • Formaldehyde
  • Frozen Sections
  • Histological Techniques*
  • Humans
  • Immunohistochemistry
  • Ischemia / metabolism
  • Lung Neoplasms / metabolism
  • Lung Neoplasms / pathology
  • Male
  • Neoplasm Proteins / metabolism*
  • Neoplasms / metabolism*
  • Neoplasms / pathology
  • Paraffin Embedding
  • Phosphoproteins / metabolism*
  • Phosphorylation
  • Proto-Oncogene Proteins c-akt / metabolism
  • TOR Serine-Threonine Kinases
  • Tissue Fixation


  • Biomarkers, Tumor
  • Neoplasm Proteins
  • Phosphoproteins
  • Formaldehyde
  • MTOR protein, human
  • TOR Serine-Threonine Kinases
  • Proto-Oncogene Proteins c-akt
  • Extracellular Signal-Regulated MAP Kinases