Date of Award

January 2011

Document Type

Open Access Thesis

Degree Name

Medical Doctor (MD)



First Advisor

Annette M. Molinaro

Subject Area(s)

Statistics, Pathology


OPTIMAL TUMOR SAMPLING FOR IMMUNOSTAINING OF BIOMARKERS IN BREAST CARCINOMA. Juliana Tolles, Yalai Bai, Maria Baquero, Lyndsay N. Harris, David L. Rimm, Annette M. Molinaro. Division of Biostatistics, Yale University School of Public Health, New Haven, CT.

Biomarkers, 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 purpose of this study is 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: estrogen receptor (ER), human epidermal growth factor receptor 2 (HER2), AKT, extracellular signal-regulated kinase (ERK), ribosomal protein S6 kinase 1 (S6K1), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), cytokeratin, and microtubule-associated protein-Tau (MAP-Tau).

Two collections of whole tissue sections of breast carcinoma were immunostained for biomarkers. Expression was quantified using Automated Quantitative Analysis (AQUA). Simulated sampling of various numbers of fields (ranging from 1-35) 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.

The optimal number of 20X fields varied by marker, ranging between 3-14 fields. More heterogeneous markers, such as MAP-Tau, required a larger sample of 20X fields to produce representative measurement. The clinical implication of these findings is that small core needle breast biopsies may be inadequate to represent whole tumor biomarker expression for many markers. Also, 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.