Optimal tumor sampling for immunostaining of biomarkers in breast carcinoma

IntroductionBiomarkers, such as Estrogen Receptor, are used to determine therapy and prognosis in breast carcinoma. Immunostaining assays of biomarker expression have a high rate of inaccuracy; for example, estimates are as high as 20% for Estrogen Receptor. Biomarkers have been shown to be heterogeneously expressed in breast tumors and this heterogeneity may contribute to the inaccuracy of immunostaining assays. Currently, no evidence-based standards exist for the amount of tumor that must be sampled in order to correct for biomarker heterogeneity. The aim of this study was to determine the optimal number of 20X fields that are necessary to estimate a representative measurement of expression in a whole tissue section for selected biomarkers: ER, HER-2, AKT, ERK, S6K1, GAPDH, Cytokeratin, and MAP-Tau.MethodsTwo collections of whole tissue sections of breast carcinoma were immunostained for biomarkers. Expression was quantified using the Automated Quantitative Analysis (AQUA) method of quantitative immunofluorescence. Simulated sampling of various numbers of fields (ranging from one to thirty five) was performed for each marker. The optimal number was selected for each marker via resampling techniques and minimization of prediction error over an independent test set.ResultsThe optimal number of 20X fields varied by biomarker, ranging between three to fourteen fields. More heterogeneous markers, such as MAP-Tau protein, required a larger sample of 20X fields to produce representative measurement.ConclusionsThe optimal number of 20X fields that must be sampled to produce a representative measurement of biomarker expression varies by marker with more heterogeneous markers requiring a larger number. The clinical implication of these findings is that breast biopsies consisting of a small number of fields may be inadequate to represent whole tumor biomarker expression for many markers. Additionally, for biomarkers newly introduced into clinical use, especially if therapeutic response is dictated by level of expression, the optimal size of tissue sample must be determined on a marker-by-marker basis

Comparison of HER2 and Phospho-HER2 Expression between Biopsy and Resected Breast Cancer Specimens Using a Quantitative Assessment Method

<div><p>Background</p><p>HER2/Neu (ErbB-2) overexpression, which occurs in 15–20% of breast cancer cases, is associated with better response to treatment with the drug trastuzumab. PhosphoHER2 (pHER2) has been evaluated for prediction of response to trastuzumab. Both markers are heterogeneously detected and are potentially subject to loss as a consequence of delayed time to fixation. Here, we quantitatively assess both markers in core needle biopsies (CNBs) and matched tumor resections to assess concordance between the core and the resection and between HER2 and pHER2.</p><p>Methods</p><p>A selected retrospective collection of archival breast cancer cases yielded 67 cases with both core and resection specimens. Both HER2 and pTyr¹²⁴⁸HER2 were analyzed by the AQUA® method of quantitative immunofluorescence on each specimen pair.</p><p>Results</p><p>Both HER2 immunoreactivity (P<0.0001) and pTyr¹²⁴⁸HER2 immunoreactivity (P<0.0001) were lower in resections relative to CNB specimens. However, clinical implications of this change may not be evident since no case changed from 3+ (CNB) to negative (resection). Assessment of pTyr¹²⁴⁸HER2 showed no direct correlation with HER2 in either CNB or resection specimens.</p><p>Conclusions</p><p>The data suggest that measurement of both HER2 and phospho- Tyr¹²⁴⁸HER2, in formalin-fixed tissue by immunological methods is significantly affected by pre-analytic variables. The current study warrants the adequate handling of resected specimens for the reproducible evaluation of HER2 and pHER2. The level of pTyr¹²⁴⁸HER2, was not correlated to total HER2 protein. Further studies are required to determine the significance of these observations with respect to response to HER2 directed therapies.</p></div

Expression of HER2 in tumor resections compared to CNBs.

<p>AQUA HER2 scores of 63 pairs of CNB (open bars) and tumor resection (filled bars) were assessed. Average FOVs of CNB: average number of FOVs included in analysis of CNB; Average FOVs of Resection: average number of FOVs included in analysis of tumor resection specimens. The noise cut point is marked with a solid line. Each AQUA score represents the mean ±95% CI. The number of pairs where resections are higher, lower than CNBs, or equal to those of CNBs are listed in inset.</p

HER2 and pTyr¹²⁴⁸HER2 AQUA score comparisons between CNBs and resections.

<p>Note: Abbreviation: CNB. Core Needle Biopsy; R: Resection.</p

Comparison between pTyr¹²⁴⁸HER2 and HER2 AQUA.

<p>Scatter plot of HER2 and pTyr¹²⁴⁸HER2 AQUA scores (CNBs, open diamonds; Resections, filled triangles). Lines in the graph represent the noise cut points of either HER2 or pTyr¹²⁴⁸HER2.</p