Pathology Diagnostics Ltd Publish First White Paper in Series - The Use of Immunohistochemistry in Biomarker Research and Clinical Trials

Cambridge, United Kingdom, November 04, 2014 --(PR.com)-- Biomarkers are characteristics that are objectively measured and evaluated as indicators of normal biological or pathological processes or of pharmacological responses to therapeutic intervention (REF 1). Biomarkers may be evaluated at a variety of different stages in the drug development cycle: pharmacodynamic biomarkers to evaluate drug-target interaction in proof of mechanism studies and in proof of concept studies in early clinical development; predictive biomarkers to evaluate drug effect in clinical trials and patient stratification in the clinic; as well as the diagnostic and prognostic biomarker tests in the clinic. Of the recent biomarker tests approved by the FDA (as of October 2013), 5 out of 18 are tissue based and all involve immunohistochemistry (ER, PR, Her2, p63, c-kit) (REF2).

Immunohistochemistry (IHC) remains the most widely used method to detect and characterise proteins in tissues in biomarker research and clinical trials. This is because it is the only method in which morphological information on the cell and tissue is maintained, and it is the most widely used protein detection method in the clinic, being suitable for formalin fixed paraffin embedded tissues (FFPE) as well as frozen tissue material, as well as relatively cheap and quick to perform.

It has been stated that the identification of fit for purpose i.e. clinically validated biomarkers is one of the main barriers to drug development (REF3). For full analytical validation of a biomarker to be completed for use in Phase II or later, testing needs to occur from preclinical/Phase I exploratory stages. The regulatory authorities have published recommendations on the requirements of biomarker assay validation (REF4) and for histopathology testing (REF5). In order to use a biomarker as a surrogate end point in a regulatory submission, researchers must evaluate the method for sensitivity, specificity, reproducibility, interobserver reliability, variability and bias (REF6).

This review discusses some of the technical challenges to performing immunohistochemistry and its analysis in clinical trials.

There are a variety of different immunohistochemistry methods but the commonest used in clinical practice is the indirect immunohistochemistry method. In this method the antigen is bound by a specific primary antibody directed to the antigen followed by amplification with a labelled conjugated to a secondary antibody and visualised with a chromagen. The commonest label and chromagen used in the clinical setting is Horse Radish Peroxidase (HRP) and diaminobenzidine hydrochloride (DAB). Other immunohistochemistry techniques include Avidin-Biotin Complex (ABC), Polymer based detection, tyramide or catalysed signal amplification, and rolling circle amplification (REF7).

A number of different labels and chromagens, such as fluorescein isothiocyanate can be used, and therefore, it is possible to multiplex biomarker analysis in the same tissue section via double or even triple immunohistochemistry and immunofluorescence.

Manual immunohistochemistry is possible but automated IHC platforms are preferred for high throughput staining and for the avoidance of inter-sample variability in staining due to operator variation in technique. These can vary in design from capillary bed platforms to those that incorporate overhead dispensing of reagents and liquid coverslips. Platforms also vary from those where antigen retrieval is performed in a separate platform or by microwave or fully enclosed systems where all steps are performed on the platform with little scope for operator intervention. The latter systems may be preferred by Sponsors once an assay has been fully qualified and for use in regulatory decision making e.g. Phase II clinical trial biomarker testing.

Whichever platform is used for performing IHC, it is essential that appropriate care is taken to ensure standardisation of preanalytical and analytical variables to ensure the quality and reproducibility of the IHC staining.

Preanalytical variables that may affect staining include tissue collection (cold ischemia time); tissue fixation; tissue processing and sectioning.

The cold ischemia time is the time from ligation of the last vessel in the organ/tissue to the time in which the sample is put in fixative/frozen. The cold ischemia time has a major impact on the preservation of proteins in the tissue, especially phosphorylated proteins that are highly labile. For analysis of the latter proteins it is recommended that the cold ischemia time is 15-20 minutes or less (REF8). This is not an issue for most biopsies but is a major concern for tissues obtained at surgical resection or for post-mortem specimens. For this reason standard post-mortem tissue is not recommended for immunohistochemistry and biomarker testing.

The second major factor is the period of fixation. Most tissues collected in the clinical setting are fixed in 10% neutral buffered formalin, a solution of 4% formaldehyde in water buffered with phosphate (REF9). Formalin very effectively preserves tissues by cross-linking proteins, but this may also result in the masking of antigenic epitopes. It is therefore vital that tissues fixed for subsequent immunohistochemistry are placed in formalin for 24-48 hours; after this they should either be processed and embedded immediately to paraffin wax blocks or transferred to 70% ethanol. The latter may be necessary if wet tissues are being transported from international investigator sites to a central pathology laboratory where delays in courier delivery.

To see the full whitepaper and references, go to www.pathologydiagnostics.com/blog/