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Fred Hutch Team Develops Mass Spec Assay for HER2 Scoring


NEW YORK – Researchers at the Fred Hutchinson Cancer Research Center have developed a mass spectrometry-based assay for the cancer protein biomarker HER2.

Detailed in a paper published last month in Clinical Chemistry, the assay could allow researchers to better identify patients who express low levels of HER2 but who may nonetheless respond to anti-HER2 therapies, said Amanda Paulovich, a professor at Fred Hutch and senior author on the study.

Fred Hutch offers the assay out of the institute's CLIA Targeted Proteomics Laboratory, which Paulovich directs. Paulovich and her colleagues launched the lab last year with the aim of developing targeted mass spec assays for use in clinical trials and, ultimately, the clinic. The HER2 assay is the first test that the lab has developed and begun offering. Paulovich said that the lab is currently testing it as part of clinical trials incorporating HER2-targeting therapies, though she declined to say for which pharma firms.

Anti-HER2 therapies are commonly used to treat patients with HER2-positive breast cancers. Typically, patient tumors are evaluated using immunohistochemistry to determine whether they express enough HER2 protein to make them likely to respond to such drugs. These IHC assays provide only limited quantitative information, though, typically returning a score of 0 to 3+, with 0 to 1 score being HER2-negative, 2 being borderline, and 3+ being HER2-positive, indicating that a patient is likely to benefit from anti-HER2 therapy.

Additionally, a subset of patients scored as HER2-negative have showed response to anti-HER2 drugs, especially newer agents targeting the protein. In one study cited by the Fed Hutch researchers, 37 percent of HER2-negative patients responded to an antibody-drug conjugate anti-HER2 treatment.

Paulovich and her colleagues believe their mass spec assay could provide more quantitative HER2 scoring that would allow doctors to distinguish between responders and non-responders in the HER2-negative population.

In the Clinical Chemistry study, the Fred Hutch researchers demonstrated that the mass spec assay "enables robust quantification of HER2 even in those tumors that are typically scored as HER2-negative," Paulovich said, noting that she and her colleagues detected a seventyfold range of HER2 expression within patients scored as negative by conventional IHC assays.

"Given that now there is an assay that can be quantitative in those low HER2 expression [ranges], does that help select patients that respond to these newer [anti-HER2] therapies?" she said. "That is what we will be testing."

The Fred Hutch test uses immunoaffinity enrichment of a target HER2 peptide followed by quantification of that enriched peptide by multiple-reaction monitoring mass spec, or MRM-MS. In the Clinical Chemistry study, the researchers characterized the performance of the assay in 96 frozen and 119 formalin-fixed paraffin-embedded breast cancer biopsies, finding that the MRM-MS measurements strongly correlated with predicate IHC and in situ hybridization, or ISH, assays, with coefficients of variation under 8 percent.

The researchers also explored whether including measurements of proteins characteristic of different cell types (epithelium, stroma, blood vessels, lymphocytes, red blood cells, and adipocytes) could help the assay account for tissue heterogeneity and the varying tumor cellularity of the different samples.

While IHC assays retain information on tumor cellularity, the Fred Hutch mass spec assay is a bulk extraction assay, making variability in tumor cellularity across biospecimens a potential challenge. Paulovich and her colleagues found that normalizing their HER2 measurements to expression levels of glyceraldehyde-3-phosphate dehydrogenase improved the assay's concordance with the predicate IHC and ISH assays.

She added that her lab was continuing to explore how they might use additional normalization markers to improve the assay.

"I think it is going to get more challenging down at those low levels of HER2 expression, and it will be interesting to see how our panel of normalization markers come into play in that setting and whether there is a reliance on more of them," she said. "I don't think we know the answer to that yet."

In addition to HER2, the Fred Hutch researchers are also developing an MRM-MS assay that aims to improve upon existing PD-L1 testing to select patients likely to respond to cancer immunotherapies.

Despite being widely used, PD-L1 IHC assays have struggled with accuracy and harmonization, with some mass spec-based studies finding that IHC testing masks significant heterogeneity in PD-L1 expression.

Paulovich said her lab aims to multiplex a range of proteins including immunomodulatory proteins, immune checkpoint targets, and mismatch repair proteins in a single assay.

"Being able to put all of those together in one quantitative assay should provide an advantage," she said.

She said her lab currently has around 300 tumor samples that they will be analyzing by MRM and comparing results to those from a predicate PD-L1 IHC assay.

The researchers are also developing MRM-MS assays to the protein targets of a variety of antibody-drug conjugate agents with the idea being that such assays could improve precision oncology trials by facilitating patient selection, a bottleneck with current methods. They have also developed under the National Cancer Institute's Moonshot program several MRM-MS assay panels to quantify immunomodulatory proteins, and the lab has begun running these assays as part of clinical trials for cancer immunotherapies. The panels include mismatch repair proteins and could also potentially be useful for diagnosing patients with Lynch syndrome-related cancers, which may respond better to immunotherapies due to their high tumor mutational burden.