As protein biomarkers are now being used to determine therapy, the importance of accurate measurement has increased. This has led to the observation that many events that occur prior to fixation of the tissue (pre-analytic variables) can be critically important. Even the best tests can only measure epitopes that are present on the slide, so the stability of these epitopes during the time between loss of circulation and fixation may be a key factor in their accurate measurement. Variables that can affect the stability of these epitopes include local pH changes, anoxic or ischemic biological reactions, loss of specificity of proteases or phosphatases. All these may result in alterations of post-translational modification and possible transcription of apoptotic factors beginning at the time the blood supply is clamped off.5, 25-28
Although this list of pre-analytic variables is incomplete, in this work we focus specifically on signaling protein phosphorylation. There is evidence that intrinsic enzymatic activity from slowly fixed endogenous phosphotase in large specimens results in the dephosphorylation of biomarkers.29-30
The papers and findings reported here suggest that measurement of phospho-modification is essentially impossible in routinely collected surgical resection specimens.
In this work, we define core needle biopsies as a standard with the assumption that the procedure allows rapid fixation and prevents pre-analytic artifacts. While we believe this is the closest we can come to assessment of live tissue, we have no evidence that artifacts do not occur in the short time between obtaining the core and cross-linking by formalin. However, the fact that measurement of phospho-proteins spans a range of expression in our small case series suggests that at least some of the activity of signaling proteins can be assessed using this technique. This suggests that if phospho-specific markers are critical to companion diagnostic testing, surgical protocols should be altered to include a core needle biopsy of the lesion immediately prior to surgery for diagnostic testing purposes.
If core needle biopsies' assessment is considered to reveal the “truth”, it would be interesting to determine if some protein expression marker could be used to normalize or adjust for degradation due to pre-analytic variables. As cytokeratin staining was done on each specimen as part of the AQUA process, we tested whether normalization of the phospho-protein AQUA scores by the degradation seen in cytokeratin in the tumor resections would more accurately reflect the levels of phospho-protein seen in the core needle biopsies. Our data indicated that AQUA scores of pAKT, pERK, pS6K1 and ER were still lower after pan-CK normalization (data not shown). While it may be possible in the future to find a method of normalization or qualification of tissue for companion diagnostic assay assessment, we believe it will not be possible to normalize for post-translational modifications. We are currently working on intrinsic controls for assessment of tissue quality.
Of the targets studied in this work, only ER is in clinical usage today. The current standard of practice for ER has recently been reviewed and an ASCO/CAP committee has issued standards that include issues related to cold ischemic time. 18
. Our work supports the need for those standards as, shown here, the levels of ER in tumor resection specimens was significantly lower than in core needle biopsy specimens. It is common practice to assess ER and PR on core needle biopsy specimens; nonetheless, it is not required. Although none of the cases in this study changed from a designation of ER-positive to a designation of ER-negative between the core needle biopsy and the tumor resection, three cases were very close to negative in the tumor resection while clearly positive in the core needle biopsy (). In a broader collection of cases, it is highly likely we would see changes in patient ER status resulting in changes in endocrine therapy as a function of which specimen was assayed.
While this work shows quantitative loss of detection for a range of biomarkers, there are several limitations. Specifically, in each setting, we sampled specimens from only 10 to 14 patients. While statistical significance is observed, it would be valuable to validate these results in other labs. Importantly, our power calculations demonstrate that, for every epitope without a significant difference between core needle biopsy and tumor resection, the sample size was insufficient. Thus, further investigation is needed to establish the affect of pre-analytical variables on measurement of non-phospho markers, such as Ki67. This data has led to funding that will assess larger cohorts in the future, including measured time to fixation, which is not available for these specimens. An additional weakness of this work, and essentially all work associated with assessment of core needle biopsies, is representation. In each case we were able to quantify only a limited number of FOVs from each core needle biopsy. Furthermore, we have no way of determining whether a core needle biopsy was representative of an entire tumor. Our previous work suggests that there is substantial heterogeneity in ER expression when multiple tissue blocks are assessed in larger cases.31-32
Heterogeneity may explain the observation that rare cases show higher average expression in the resection than in the core biopsy. For example, case 14 in shows higher average levels in the resection, even though all other cases trend in the opposite direction. Finally, another weakness of this work is the fact that that the core needle biopsy was not taken at the same time as the tumor resection. As this was a retrospective collection of tissue, we collected core needle biopsies that were taken as part of routine patient management and which were followed by resections. It is possible that assessment of tumors where the core needle biopsy and tumor resection are taken simultaneously would show different results. A further limitation is that while AQUA is now used in one clinical laboratory, it is not the common standard for assessment of antibody/antigen interactions. However, the immunofluorescence method is identical to DAB based chromogenic studies in detection of antigen. That is, the primary antibody step is the same. The difference is in visualization, where the use of fluorescent visualization methods is easier to accurately quantify. Thus it is important to emphasize that the conclusions we draw from this work are a function of the tissue assessed, not a function of the method of assessment. This is confirmed by work published while this manuscript was under review that showed qualitative assessment of loss of pAKT and pERK using chromogenic stains 33
Nonetheless, work is underway toward the goal of developing quantitative methods that work for chromogenic visualization methods that can be used to assess this and other expression patterns in tissue.
In summary, this work quantitatively assesses the effect of delayed formalin fixation on surgical resections by using biopsies as standardized controls. Although it is a limited set of experiments, all three phospho-antibodies tested showed significant degradation in the resection specimens. These results indicate that conventional resection tissues with uncontrolled cold ischemic time should not be used for companion diagnostic testing, at least for the five antibodies shown to be affected here. This work also suggests that any antibody planned for use on resection specimens should be validated for resistance to cold ischemic time.