Allison Welsh

Analytic Variability in Immunohistochemistry Biomarker Studies

Background: Despite the widespread use of immunohistochemistry (IHC), there are no standardization guidelines that control for antibody probe variability. Here we describe the effect of variable antibody reagents in the assessment of cancer-related biomarkers by IHC. Methods: Estrogen receptor (ER), epidermal growth factor receptor (EGFR) 1, and human epidermal growth factor receptor 3 (HER3) were evaluated by quantitative immunofluorescence. Correlations between ER clones 1D5, SP1, F10, and ER60c, and EGFR monoclonal 31G7, 2-18C9, H11, and 15F8, and polyclonal 2232 antibodies were assessed in 642 breast cancer patients. HER3 was measured by RTJ1, RTJ2, SGP1, M7297, RB-9211, and C-17 antibodies in 42 lung cancer patients. Survival analysis was done with the use of multiple cutoff points to reveal any prognostic classification. Results: All ER antibodies were tightly correlated (Pearsons r2 = 0.94-0.96; P < 0.0001) and western blotting confirmed their specificity in MCF-7 and BT474 cells. All EGFR antibodies but 2232 yielded specific results in western blotting; however, only 31G7 and 2-18C9 were strongly associated (Pearsons r2 = 0.61; P < 0.0001). HER3 staining was nonspecific and nonreproducible. High EGFR–expressing patients had a worse prognosis when EGFR was measured with H11 or 31G7 (log rank P = 0.015 and P = 0.06). There was no statistically significant correlation between survival and EGFR detected by 2-18C9, 15F8, or polyclonal 2232 antibodies. Conclusions: Antibody validation is a critical analytic factor that regulates IHC readings in biomarker studies. Evaluation of IHC proficiency and quality control are key components toward IHC standardization. Impact: This work highlights the importance of IHC standardization and could result in the improvement of clinically relevant IHC protocols. Cancer Epidemiol Biomarkers Prev; 19(4); 982–91. ©2010 AACR.

Co-targeting the insulin-like growth factor I receptor enhances growth-inhibitory and pro-apoptotic effects of anti-estrogens in human breast cancer cell lines.

The insulin-like growth factor I receptor (IGF1R) interacts with estrogen receptor-α (ERα) and HER2. We examined the effect of combinations of IGF1R antagonists (α-IR3, AG1024) and anti-estrogens (4-hydroxy tamoxifen, fulvestrant) in two human ER+ breast cancer cell lines: BT474 (HER2 overexpressing, IGF1R low) and MCF7 (HER2 non-overexpressing, IGF1R high). In BT474 cells, growth was inhibited by anti-estrogens, but not by IGF1R antagonists; however, adding IGF1R inhibitors to anti-estrogens enhanced growth inhibition. In MCF7 cells, growth was inhibited by IGF1R and ER antagonists and more so by their combination. In both cell lines, no single agents could induce apoptosis, but combining IGF1R inhibitors with anti-estrogens induced dramatic levels of apoptosis. IGF1R antagonists enhanced the ability of the anti-estrogens to inhibit ER transcriptional activity in BT474 cells, but not in MCF7 cells. The drug combination synergistically inhibited ER and IGF1R activity. Such combinations may be useful therapy for breast cancer.

Quantitative In Situ Measurement of Estrogen Receptor mRNA Predicts Response to Tamoxifen

Purpose Quantification of mRNA has historically been done by reverse transcription polymerase chain reaction (RT-PCR). Recently, a robust method of detection of mRNA utilizing in situ hybridization has been described that is linear and shows high specificity with low background. Here we describe the use of the AQUA method of quantitative immunofluorescence (QIF) for measuring mRNA in situ using ESR1 (the estrogen receptor alpha gene) in breast cancer to determine its predictive value compared to Estrogen Receptor α (ER) protein. Methods Messenger RNA for ER (ESR1) and Ubiquitin C (UbC) were visualized using RNAscope probes and levels were quantified by quantitative in situ hybridization (qISH) on two Yale breast cancer cohorts on tissue microarrays. ESR1 levels were compared to ER protein levels measured by QIF using the SP1 antibody. Results ESR1 mRNA is reproducibly and specifically measurable by qISH on tissue collected from 1993 or later. ESR1 levels were correlated to ER protein levels in a non-linear manner on two Yale cohorts. High levels of ESR1 were found to be predictive of response to tamoxifin. Conclusion Quantification of mRNA using qISH may allow assessment of large cohorts with minimal formalin fixed, paraffin embedded tissue. Exploratory data using this method suggests that measurement of ESR1 mRNA levels may be predictive of response to endocrine therapy in a manner that is different from the predictive value of ER.

Cytoplasmic Estrogen Receptor in Breast Cancer

Purpose: In addition to genomic signaling, it is accepted that estrogen receptor-α (ERα) has nonnuclear signaling functions, which correlate with tamoxifen resistance in preclinical models. However, evidence for cytoplasmic ER localization in human breast tumors is less established. We sought to determine the presence and implications of nonnuclear ER in clinical specimens. Experimental Design: A panel of ERα-specific antibodies (SP1, MC20, F10, 60c, and 1D5) was validated by Western blot and quantitative immunofluorescent (QIF) analysis of cell lines and patient controls. Then eight retrospective cohorts collected on tissue microarrays were assessed for cytoplasmic ER. Four cohorts were from Yale (YTMA 49, 107, 130, and 128) and four others (NCI YTMA 99, South Swedish Breast Cancer Group SBII, NSABP B14, and a Vietnamese Cohort) from other sites around the world. Results: Four of the antibodies specifically recognized ER by Western and QIF analysis, showed linear increases in amounts of ER in cell line series with progressively increasing ER, and the antibodies were reproducible on YTMA 49 with Pearson correlations (r2 values) ranging from 0.87 to 0.94. One antibody with striking cytoplasmic staining (MC20) failed validation. We found evidence for specific cytoplasmic staining with the other four antibodies across eight cohorts. The average incidence was 1.5%, ranging from 0 to 3.2%. Conclusions: Our data show ERα is present in the cytoplasm in a number of cases using multiple antibodies while reinforcing the importance of antibody validation. In nearly 3,200 cases, cytoplasmic ER is present at very low incidence, suggesting its measurement is unlikely to be of routine clinical value. Clin Cancer Res; 18(1); 118–26. ©2011 AACR.

Quantitative analysis of estrogen receptor expression shows SP1 antibody is more sensitive than 1D5.

Studies comparing rabbit monoclonal SP1 antibody with 1D5 for estrogen receptor (ER) immunohistochemical testing show conflicting results. Here we use a standardized quantitative immunofluorescent (QIF) ER assay to determine the level and significance of discordance between the antibodies. Both antibodies were assessed by QIF on our Index TMA of cell lines and case controls, followed by QIF and immunohistochemical analysis on 2 retrospective cohorts from Yale. On the Index TMA, SP1 displayed stronger signal-to-noise ratio compared with 1D5. On the patient cohorts, the range of discrepancy between the 2 antibodies was 8% to 16.9%, with the majority of discrepant cases being SP1 positive/1D5 negative. Kaplan-Meier analysis of the discrepant cases showed outcomes comparable to those of double-positive cases, suggesting that SP1 is more sensitive than 1D5. A series of cases with high levels of ER-&bgr; shows that neither antibody cross-reacts, suggesting equivalent specificity. Future efforts are needed to determine whether response to endocrine therapies show superiority of either antibody as a companion diagnostic test.

Automated measurement of estrogen receptor in breast cancer: a comparison of fluorescent and chromogenic methods of measurement

Whereas FDA-approved methods of assessment of estrogen receptor (ER) are ‘fit for purpose’, they represent a 30-year-old technology. New quantitative methods, both chromogenic and fluorescent, have been developed and studies have shown that these methods increase the accuracy of assessment of ER. Here, we compare three methods of ER detection and assessment on two retrospective tissue microarray (TMA) cohorts of breast cancer patients: estimates of percent nuclei positive by pathologists and by Aperios nuclear algorithm (standard chromogenic immunostaining), and immunofluorescence as quantified with the automated quantitative analysis (AQUA) method of quantitative immunofluorescence (QIF). Reproducibility was excellent (R2>0.95) between users for both automated analysis methods, and the Aperio and QIF scoring results were also highly correlated, despite the different detection systems. The subjective readings show lower levels of reproducibility and a discontinuous, bimodal distribution of scores not seen by either mechanized method. Kaplan–Meier analysis of 10-year disease-free survival was significant for each method (Pathologist, P=0.0019; Aperio, P=0.0053, AQUA, P=0.0026); however, there were discrepancies in patient classification in 19 out of 233 cases analyzed. Out of these, 11 were visually positive by both chromogenic and fluorescent detection. In 10 cases, the Aperio nuclear algorithm labeled the nuclei as negative; in 1 case, the AQUA score was just under the cutoff for positivity (determined by an Index TMA). In contrast, 8 out of 19 discrepant cases had clear nuclear positivity by fluorescence that was unable to be visualized by chromogenic detection, perhaps because of low positivity masked by the hematoxylin counterstain. These results demonstrate that automated systems enable objective, precise quantification of ER. Furthermore, immunofluorescence detection offers the additional advantage of a signal that cannot be masked by a counterstaining agent. These data support the usage of automated methods for measurement of this and other biomarkers that may be used in companion diagnostic tests.

Abstract ED01-04: Beyond IHC: Quantitative measurement of protein analytes on tissue slides

The large number and elusive nature of analytical and pre‐analytic variables associated with immunohistochemistry (IHC) have relegated the technique to a semi‐quantitative or qualitative status. However, as companion diagnostics become more critical to patient management, there is new motivation to move IHC to a fully quantitative assay. Toward this goal, we have developed the AQUA® method of quantitative immunofluorescence (QIF) to measure critical analytes in breast tissue. Using a series of cell lines which are standardized to recombinant protein quantified on western blot, we have measured estrogen receptor in three separate cohorts of breast cancer cases. The quantitative approach reveals a cutpoint of 50pg/ug total protein and suggests that conventional IHC may have a misclassification rate of around 15%. Furthermore, when comparing the misclassified cases, it appears that QIF classification predicts outcome more accurately than the conventional methods. As accurate as QIF is, it does not address preanalytical variables, most significantly including cold ischemic time, or the time between surgical removal of the tissue sample and fixation. A series of QIF methods for antibody validation may allow neutralization of this variable in assessment of tumor tissue. In summary, measurement of protein on a pathology slide can now be achieved with accuracy and reproducibility of nucleic acid assays or ELISA assays. Combining QIF with rigorous methods of standardization and antibody validation allows companion diagnostic tests to be done on very small tissue fragments. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):ED01-04.

A Quantitative Immunofluorescence Assay (AQUA) Suggests Significant Misclassification (15%) of Estrogen Receptor Status in Breast Cancer.

Introduction: Estrogen Receptor (ER) is arguably the most powerful predictive marker in breast cancer. However, a recent incident in Canada revealed a strikingly high false-negative rate and raised awareness of the current limitations in our measurement of ER. The current clinical standard is both subjective and qualitative. Even though guidelines are about to be issued to standardize this assay, there is very little data on the misclassification rate in current practice in the US.Hypothesis: Our hypothesis is that the use of a quantitative assay for ER on a series of retrospective collections may reveal both the level and significance of the misclassification rate.Method: Cell lines with a range of ER expression levels were analyzed by quantitative western blotting in parallel with IF/AQUA analysis (r2 = 0.865), in order to create standard curves for assessment of absolute ER protein concentration in tissue. The optimized assay was then used to quantify ER protein expression in a large cohort of archival breast cancer samples from Yale (1962-1982, n =617).Results: Using a set of standard curves with recombinant ER and cell line controls, we developed a standardized method for quantifying ER as an absolute concentration (pg ER per μg total protein) in formalin-fixed tissue on TMAs. This ER AQUA assay has a range in sensitivity from 50 pg/μg to 1500pg/μg total protein. Quantification of ER protein expression on the Yale archival cohort revealed a unimodal distribution with 49.8% of cases above the 50pg/ug threshold and thus defined as positive. When compared to pathologist performed conventional ER classification, we found a false negative rate of 6.65% and a false positive rate of 10.2%, for a total misclassification rate of 16.6%. Although no response data is available on that cohort, data is under analysis on 4 other cohorts with endocrine therapy treatment information, including an independent Yale cohort, SWOG 9313, NSABP B14, and the TEAM trial.Conclusion: We have developed a quantitative method to measure absolute levels of ER in breast tissue. Use of this assay on a series of cohorts suggests a misclassification rate in the 15% range. The significance of this level of misclassification with respect to response to endocrine therapy is currently under study. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4068.