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Erasmus Researchers Develop Immuno-MRM Assay for Predicting Tamoxifen Resistance


NEW YORK (GenomeWeb) – A team led by researchers at Rotterdam's Erasmus University Medical Center have developed a mass spec-based assay for predicting tamoxifen resistance in estrogen receptor positive breast cancer.

Detailed in a study published last week in the Journal of Proteome Research, the assay uses immunoenrichment multiple-reaction monitoring mass spec to measure levels of four proteins in either tumor tissue or patient sera to identify recurrent ER-positive breast cancer patients likely to develop resistance to tamoxifen treatment.

The work builds on a previous study by EUMC researchers in which they used a combination of laser capture microdissection and high-resolution mass spec in looking for a protein signature capable of predicting patient response to tamoxifen treatment. That work identified a four-analyte panel consisting of the proteins PDCD4, CGN, G3BP2, and OCIAD1.

Looking to further validate their signature, they turned to immunohistochemistry. However, said Tommaso De Marchi, an EUMC researcher and first author on the study, he and his colleagues were unable to get quality quantitative data on their markers using IHC and so turned, instead, to targeted mass spec, developing assays in collaboration with Eric Kuhn, a researcher in the lab of Steven Carr at the Broad Institute, both of whom were also co-authors on the JPR paper.

De Marchi noted that while IHC was an attractive format due to its broad clinical acceptance, MRM mass spec has recently emerged as a clinical technique, particularly in the US. This, he said, meant that the researchers could keep their panel in a mass spec format while also maintaining future clinical viability.

He and his colleagues developed both conventional MRM assays and immuno-MRM assays to their proteins, comparing the performance of each. Immuno-MRM assays use antibody-based enrichment of target peptides prior to mass spec analysis in order to improve sensitivity. Such approaches also have the advantage of greatly simplifying the sample being run on the LC-MS system, which reduces background and can improve throughput by enabling shorter chromatography runs.

In the case of the JPR study, the researchers also hoped that use of immuno-MRM would, in their tissue-based assays, allow them to use whole tissue lysates as opposed to tumor tissue prepared using laser-capture microdissection (LCM). In LCM, a laser is used to select only tumor tissue for analysis, the idea being that enriching for this tissue provides a better picture of the tumor environment compared to a tissue sample containing both tumor and stromal cells.

In their initial discovery work, EUMC team found that LCM was necessary to obtain a signature with enough power to identify patients who would go on to develop tamoxifen resistance. However, because LCM adds time to the assay, they hoped that immune-MRM would deliver enough discriminating power that the tissue samples could be analyzed without LCM.

The approach proved effective in the whole tissue lysate samples, identifying patients developing resistance with an area under the curve of .82, similar to the performance of the classifier in the original experiments using high-resolution mass spec on LCM-prepared samples.

They also applied their assays to measurement of the proteins in serum, with the immuno-MRM method outperforming the standard MRM approach. The serum-based immune-MRM assay was able to identify patients who developed resistance with an area under the curve of .83.

Room remains for improving the assay, De Marchi said, particularly on the antibody side where better reagents could further enhance performance. He added that he and his colleagues might move to monoclonal antibodies in the future.

Additionally, he noted that use of a newer mass spec system could also improve the assay. In the JPR paper the researchers used an AB Sciex QTRAP 4000 for their MRM work.

The researchers are also working to validate the signature in a collection of patients previously treated with aromatase-inhibitor therapy, which, De Marchi said, is an important step from a clinical perspective. This, he noted, is because aromatase inhibitors are more commonly used than tamoxifen as first-line treatment for recurrent ER-positive breast cancer. Therefore, in actual clinical practice, such an assay would most likely be used in patients who had already been treated with aromatase inhibitors.

De Marchi said the researchers are currently involved in this analysis and "will know in a few months if our proteins have value in this kind of clinical group."