Equivalency of RT-PCR and immunohistochemistry: fact or factoid.

Management of breast cancer relies heavily on determination of predictive and prognostic markers. Analysis of estrogen and progesterone receptors was originally performed using ligand-binding assays (LBA); this has been replaced by immunohistochemistry (IHC) in the last couple of decades. In recent days, RT-PCR assays for ER and PR have been proposed as a superior alternative to IHC. Several studies have attempted to analyze the concordance between these different types of assays. In this issue, investigators from the Hellenic Oncology Group examines the expression and predictive significance of ER, PR and microtubule-associated protein tau (MAP-tau) by quantitative RT-PCR (qRT-PCR) in a series of 274/279 breast cancer patients enrolled in clinical trial HE 10/97 [1]. In these analyses, MAP-tau strongly correlated with ER and PR mRNA status (Spearmann r = 0.52 and 0.64, P \ 0.001). However, they find only a modest correlation between RT-PCR and IHC results for ER (kappa = 0.41) and a fair correlation for PR (kappa = 0.33). Significantly, no correlation was observed between increasing ER and PR mRNA and increasing benefit from endocrine therapy in 203 ER and/or PR IHC-positive patients receiving adjuvant hormone therapy (Wald P = 0.54 for ER, 0.51 for PR). As has been acknowledged by the authors, the findings in this study are at variance with prior reports [2–4] with regards to concordance between IHC and qRT-PCR. These differences seen in the qRT-PCR results may be ascribed to a variety of pre-analytical and analytical parameters, which are briefly discussed below. Analysis for both these assays was performed on formalin fixed paraffin-embedded (FFPE) tissues obtained from multiple institutions; it can be safely presumed that these were associated with a variety of fixation and processing protocols. The time duration of warm ischemia, length of exposure to formalin, and exact temperatures used in tissue processing and embedding are thought to be important for intactness of both RNA and protein. In addition, the age of the paraffin block has been shown to directly correlate with amount of fragmentation of RNA [5]. Akin to the choice of antigen retrieval methods available for IHC, multiple kits are available for extraction of RNA from FFPE materials. Abramowitz et al. [6] have recently performed a comparative analysis of different commercially available kits and demonstrated significant differences in quantities of RNA obtained and reproducibility of results. In their study, variation in the digestion/ incubation time (3 h versus overnight) resulted in significant differences in reproducibility even when using the same kit. Tumor tissue is a composite mixture of tumor cells, tumor microenvironment and non-malignant elements, which include normal breast epithelium and in situ carcinoma. As morphologic assessment is a critical part of IHC methods, expression is only analyzed in tumor cells. The pathologist has the capacity to exclude expression in normal elements and ductal carcinoma in situ, which therefore do not contribute to the final results. Assays such as LBA and qRT-PCR that require tissue digestion, do not permit morphologic distinction of tumor cells from nonmalignant This is a commentary to doi:10.1007/s10549-008-0144-9.