Pre-analytic variables and phospho-specific antibodies: the Achilles heel of immunohistochemistry

Immunohistochemistry is the most common method for companion diagnostic testing in breast cancer. The readings for estrogen receptor, progesterone receptor, and Her2 directly affect prescription of critical therapies. However, immunohistochemistry is highly sensitive to innumerable pre-analytic variables that result in loss of signal in these assays. Perhaps the most significant pre-analytic variable is cold ischemic time. The work of Pinhel and colleagues in the previous issue of Breast Cancer Research examines the effects of cold ischemic time and finds a chilling result. The authors show that while the classic markers may be only mildly affected, phospho-specific markers are highly sensitive to this artifact. As a result, it is likely that future companion diagnostic tests that include phospho-specific epitopes will be reliably done only in core needle biopsies that minimize ischemic time.

variables, eff orts to characterize their eff ects are under way.
In the previous issue of Breast Cancer Research, Pinhel and colleagues [1] examined the eff ects of time to fi xation by the measurement of protein expression of traditional breast cancer biomarkers in a timed series of core needle biopsies and the conventional resection specimens. Th e authors compared the expression of estrogen receptor (ER), Her2, progesterone receptor (PgR), ki-67, p-Akt, and p-Erk in each set and used conventional IHC with semi-quantitative readouts and found no signifi cant diff erence between the expression of ER, Her2, PgR, and ki-67 in the core needle specimens with a median time diff erence of 30 minutes. Comparison with tumor resection samples from the same patient also showed no signifi cant diff erence except that a lower ER value was seen in the conventional resection compared with the biopsy. Th ese fi ndings suggest that if epitope degradation occurs during the ischemic time prior to fi xation, its impact may be limited to borderline cases.
Th e impact for phospho-epitopes is not so subtle. Here, Pinhel and colleagues show a dramatic loss of antigenicity for both p-Akt and p-Erk1/2 when comparing the cores with conventional resections. Although these markers are not routinely used in current diagnostic testing, their potential for sensing pathway activation has made them extremely popular candidate markers for companion diagnostics for kinase inhibitors [2]. Th e work of Pinhel and colleagues shows signifi cantly lower levels of the phospho-epitopes in tumor resection samples with longer time to fi xation. Th is work suggests that the timing and tissue handling are critical for biomarker assessment of phospho-proteins in clinical specimens. Since delayed time to fi xation can alter the phosphorylation status in resection specimens, use of these epitopes in companion diagnostic tests will likely have to be limited to core needle biopsies.
Th e work of Pinhel and colleagues validates data seen in other studies that have shown the decrease in phospho-protein biomarker expression as a function of Abstract Immunohistochemistry is the most common method for companion diagnostic testing in breast cancer. The readings for estrogen receptor, progesterone receptor, and Her2 directly aff ect prescription of critical therapies. However, immunohistochemistry is highly sensitive to innumerable pre-analytic variables that result in loss of signal in these assays. Perhaps the most signifi cant pre-analytic variable is cold ischemic time. The work of Pinhel and colleagues in the previous issue of Breast Cancer Research examines the eff ects of cold ischemic time and fi nds a chilling result. The authors show that while the classic markers may be only mildly aff ected, phospho-specifi c markers are highly sensitive to this artifact. As a result, it is likely that future companion diagnostic tests that include phosphospecifi c epitopes will be reliably done only in core needle biopsies that minimize ischemic time.
time to fi xation [3][4][5]. Work in our lab has also quantifi ed this loss for these and other phospho-epitopes while show ing less change in the non-phospho-sensitive epitopes of the same proteins (Yalai Bai and colleagues, Depart ment of Pathology, Yale University School of Medicine, New Haven, CT, USA, manuscript submitted). Th us, although phospho-proteins are a tempting target, they present unique challenges. Most likely, this is explained by the fact that phospho-epitopes are highly sensitive to phospha tases. In fact, it is the transient balance between kinases and phosphatases that is critical in cell proliferation, cell migration, and other pathways in tumor progression [6]. Most likely, the loss of the phospho-epitopes is due to the unregulated phosphatase activity seen in ischemic conditions or early-stage tissue degradation that occurs prior to fi xation.
Can anything be done to address this issue of preanalytic variability? Eff orts have been made to standardize tissue management in the American Society of Clinical Oncology/College of American Pathologists guide lines for Her2 [7] and for ER and PgR [8]. Th e guidelines stipulate a maximum of 1 hour between resection and fi xation and a minimum of 6 hours in fi xative. Clinical trial groups have also set guide lines for specimen handling [9]. However, the concept of biospecimen science is still relatively new. Historically, work characterizing time to fi xation and other pre-analytic variables was not recognized as important and was diffi cult to publish. As a result, the issued guidelines are based on a very limited body of literature [10][11][12]. Th e paper of Pinhel and colleagues and similar studies are welcome additions to the literature. No doubt, there will soon be others since the National Institutes of Health has recently established the Offi ce of Biorepositories and Biospecimen Research and has funded a series of studies on biospecimen science. Th e work of the funded investigators and works like this article by Pinhel and colleagues are likely to be cited as key evidence to form the basis of future guidelines for companion diagnostic tests.

Competing interests
DLR is a co-founder of, consultant to, and stockholder in HistoRx Inc. (Branford, CT, USA) and Metamark Genetics Inc. (Cambridge, MA, USA). However, this editorial does not refer to any technologies related to those companies. SS declares that he has no competing interests.