Proteomic Testing for Cancer: Protein Analysis to Guide Treatment

Brad Power September 21, 2022 “When you go from DNA all the way to protein, we know there is a loss of information… All your drugs are actually acting against proteins, so the question becomes, should I measure the levels of protein to see what's actually expressed, whether through mass spectrometry or through immunohistochemistry…?

Most cancer patients these days get a genomic analysis (DNA sequencing) of their tumor tissue. Some get transcription analysis (RNA sequencing), even though few oncologists may know how to interpret the RNA results. Few patients get proteomic analysis, so most are missing this opportunity for additional treatment guidance.

There are biomarkers that can be identified through proteomic analysis, such as HER2, that can point to targeted drugs with better patient outcomes than standard treatments. Depending on genomics analysis alone to guide treatment can introduce errors. When you go from DNA to RNA to protein, how much information is lost? When you go from DNA to RNA, let's say 50% of your information is transmitted.

And then from RNA to protein, maybe 30% of the information is translated. So, when you go from DNA to proteins (gene expression), we know there is a loss of information. Yet all drugs are acting against proteins, so the question becomes, should I measure the levels of proteins to see what's actually being expressed, either through mass spectrometry or immunohistochemistry, to know if the proteins are being expressed? This can be super helpful. Dr.

Sheeno Thyparambil is the Senior Director (R&D) of the mProbe Precision Oncology division. He has extensive experience in developing and deploying clinical diagnostics products, especially the use of mass spectrometry for clinical tests.

He describes how mass spectrometry- based clinical proteomics can guide treatment decisions, providing arguments advanced cancer patients can use with their oncologists to liberate some of their tissue (FFPE) for this test. It’s important to be able to distinguish the additional information that is gained from proteomics, especially beyond genomic testing.

A main reason why a lot of oncologists and patients are interested in mass spectrometry- derived proteomic tests is helping with chemotherapy decisions. Many tests help inform targeted therapy decisions, or whether you need chemo or not, but few help decide what type of chemo regimen to choose.

” On the targeted therapy side, the most popular protein biomarkers are HER2 (human epidermal growth factor receptor 2, an important protein in breast and gastric cancer) and PDL1 (programmed death ligand-1, a protein that helps keep immune cells from attacking non-harmful cells in the body), but there are others, especially for prostate cancer, such as AR TROP2 (androgen receptor trophoblast cell-surface antigen-2, an important target for antibody-drug conjugates), which point to new drugs.

Another example: 15% of all glioblastomas tend to have very high levels of the biomarker TOPO1, which you can treat with a drug (irinotecan). Brian McCloskey has a high expression of B7-H3, for which there are targeted treatments. Knowing his TOPO1 levels could be useful.

For patients with metastatic castrate resistant prostate cancer, you should find out what your TROP2 levels are, and if they are high enough, you should consider enrolling in one of two clinical trials of drugs that bind to TROP2. Proteomic identification of biomarkers can also steer treatment to a clinical trial.

For example, a patient showed a very high level of a biomarker (MET, mesenchymal epithelial transition factor receptor), then after a round of chemotherapy, the biomarker jumped. The oncologist decided to switch the patient’s treatment to a phase one clinical trial targeted on the biomarker, which was very successful. HER2 levels can point to treatments outside of gastric and breast cancers.

A patient with pancreatic cancer usually has less than a year of survival. In one case, a pancreatic cancer patient with unusually high HER2 was given an anti-HER2 drug, and this patient is 180 weeks out and still doing very well. HER2 can also be relevant for prostate cancer. In 71 prostate cancer samples, about three to five patients had high levels of HER2. They would want to enroll in an anti-HER2 clinical trial.

Even with low HER2, there is a clinical trial that is going on in prostate cancer, which is a combination of a drug for low HER2 – Enhertu or trastuzumab deruxtecan – and a PARP inhibitor. Mass spectrometry from FFPE tissue can also predict the overall survival of patients. For example, outcomes were accurately predicted in a study of breast cancer patients with high HER2 expression.

The inputs for the test process are relatively straightforward, and the results arrive relatively fast. Once mProbe receives two slides of tumor tissue (FFPE), five days later a clinical report goes to the oncologist with the levels of 72 biomarkers. What’s next for proteomics?

Having examined a few targets in prostate cancer to see what the distribution is, it is surprising to see the number of treatment opportunities, especially for TROP2 and HER2. We can go back at some point and examine the 72 biomarkers to see what else we can find. Not all patients are unique. For every patient, what works? What are the options if something is not working?

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Meeting Notes SUMMARY KEYWORDS patient, drug, protein, samples, cancer, biomarkers, levels, clinical trial, proteomics, tissue, adc, clinical, prostate cancer, oncology, gastric cancer, mass spec, slide, express, presenter mode, chemotherapy SPEAKERS Sheeno Thyparambil (80%), Brian McCloskey (16%), Saed Sayad (2%), Richard Anders (2%), Emma Shtivelman (<1%) Brian McCloskey 0:28 Sheeno Thyparambil is going to talk about mass spectrometry-based clinical proteomics for oncology in regulated environments.

Sheeno is the Senior Director of R&D at the mProbe Precision Oncology Division. He is the co-inventor on 29 US-issued patents related to the use of mass spectrometry for the development of clinical assays.

His specialization is in the integration of molecular oncology, multiomics analysis (analysis of multiple "omes", such as the genome, proteome, transcriptome, epigenome, metabolome, and microbiome), and biomarker data for drug development programs.

He's the site head for mProbe’s Rockville unit, which is a CLIA (Clinical Laboratory Improvement Amendments)-certified, CAP (College of American Pathologists)-accredited lab, where he manages a team of scientists, medical doctors, and clinical and R&D staff that is responsible for delivering clinical reports to oncologists for informed decision-making.

I am particularly excited about this discussion because, as some of you know, I just had a biopsy about a month ago. I have five cores. I've used one of them for whole exome sequencing, but I have four more at my disposal to shed light on my disease. I'm hopeful that I can have some breakthroughs and be able to leverage Sheeno’s technology.

A scientist named Dave Chrisman stepped out from the National Cancer Institute and developed this technology of extracting peptides from formalin fixed paraffin embedded (FFPE) blocks and tissue. Our patent got issued in 2008. I joined in 2009. In 2012 we became a CLIA-certified and CAP-accredited lab. We've been running clinical samples since 2012.

We were acquired in 2015 by NantOmics, and we were able to integrate both genomics and proteomics for patients and doctors. mProbe, which is based in San Francisco, was able to get the proteomics lab from NantOmics. What does this all mean? Over the years we have run possibly 10,000 clinical samples. Half of them are actually the CLIA reports that went out, the other was for R&D, mostly for Pharma.

We have a large collection of mass spec-based tissue proteomics data. I'm preaching to the choir here, but in the process of DNA to RNA to protein to metabolite, our focus at mProbe is on the protein and metabolites side of things. I'll focus today on the protein side of things. Sheeno Thyparambil 6:04 I'll dive directly into the workflow. This is how it all happens. We have a desk requisition form that is signed off by the oncologist.

Our pathology client services team works with the pathology department to get that FFPE tissue block. More often than not, we don't get the block, especially if it is with university systems. So we send our kit to them, which contains a slide that's known as the “DIRECTOR slide”. This is a laser microdissection compatible slide. It's a regular glass slide coated with a proprietary coating so that you can stick it into a laser.

A pathologist marks off the tumor areas. Nothing else needs to be added; no coating, nothing on the tumor sample. Once it is in this format, the key invention that happened about a decade plus ago was how to extract peptides from this fixed tissue that you could shoot straight into the mass spec. There are no additional steps other than spinning them down and adding certain buffers. Then it'll go into the mass spec.

The beauty of mass spectrometry is the fact that it has been used for a lot of testing in the past. For example, for a vitamin D test that you would get from your physician's office, that is a mass spec-based test. Or a drug abuse test is a mass spec-based test. This has been in the clinical world; it’s just that we had not known much about it. What we essentially did was marry this side of the world with the mass spectrometry side of things.

Mass spectrometry comes in two flavors. One is “discovery proteomics”, where you're asking, “What's in my tube? ” In our case we are looking for 72 biomarkers and quantifying the levels of these biomarkers. Five days later, there is a clinical report that is issued. From start to finish, this is a five-day process. It takes two slides. We're looking at sorts of 10 micron thickness.

We're really looking at two to three slides, 20 to 30 micron thickness of slide. Sometimes they ask for a tumor heterogeneity slide. Sometimes we use an H&E (Hematoxylin and Eosin stain) image. Sheeno Thyparambil 9:56 This is the process, from start to finish. This is what happens behind the scenes. A lot of our time is honestly spent sometimes wrangling the tissue from the pathology labs. That's not a pretty process.

But once it is in house, five days later a clinical report goes to the oncologist. ” They can get more depth in the clinical report. We are adding heavy peptides, and then we can quantify exactly the levels of 72 biomarkers in that sample. One thing I want to point out is that for bone mets, this process is compatible. When you have bone tissue, people take an acid, and then it is calcified.

If we take an acid decalcification protocol, we end up destroying the DNA, or we end up destroying the shape of the protein. This makes it incompatible for genomic or immunohistochemistry testing. But with mass spectrometry, we don't have that problem, because we are really looking at the linear sequence of the protein. That's technical speak.