Questions Arise about TMB as a Predictive Biomarker for Immune Checkpoint Inhibitor Therapy

Artist rendition of immune cells attacking a cancer cells. Immune checkpoint inhbitor therapy is a relatively new therapy for certain cancers.

Immune checkpoint inhibitor (ICI), or immune checkpoint blockade, therapies are a revolutionary, and relatively new, approach to treating cancer. These therapies work by blocking immune checkpoint proteins that act to negatively regulate the immune system through the PD-1 pathway. Some tumors express immune checkpoints to prevent the immune system from producing a strong enough immune response to kill the cancer cells. When these checkpoint proteins are blocked by an ICI, the body’s T-cells can recognize and kill the cancer cells. ICI therapies show tremendous promise. Unfortunately, not all tumors express immune checkpoint proteins, and so, not all tumors will be effectively treated with ICI therapies. The challenge is differentiating between the tumors that will respond and tumors that won’t.

DNA Mismatch Repair Deficiency Status as Detected by Microsatellite Instability or Immunohisotchemistry are Important Biomarkers for ICI

Biomarkers are measurable indicators of a clinical condition that can be found in tissue, blood, or other fluids. Predictive biomarkers for ICIs can help determine if these therapies are a suitable choice for treatment. Some tumors have deficiencies in their DNA mismatch repair mechanisms. Mismatch repair deficiency (dMMR) leads to the accumulation of mutations across the genome, particularly in microsatellites, which over time can result in higher levels of neoantigen production, rendering the tumors susceptible to the ICI therapy (1–5).

In 2017, Le et al. demonstrated that dMMR status reliably predicted response to an ICI therapy targeting the PD-1 checkpoint protein (6). Following this discovery, ICI based on dMMR  determined using either microsatellite instbility (MSI) or immunohistochemistry (IHC), gained clearance from the US Food and Drug Administation (FDA) for microsatellite instability-high (MSI-H) or dMMR by IHC solid tumors. This was the first time a cancer treatment was cleared based on a biomarker regardless of cancer origin (1,7).  Since then, MSI-H and dMMR, have become some of the most recognized tissue agnostic biomarkers for improved survival following ICI therapy of solid tumors (6,8,9).

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Bringing Cutting Edge Technologies to Academic Researchers Through the Academic Access Program

This post was written by guest blogger Iain Ronald, Director Academic/Government Market Segment at Promega.

My back story is similar to most of you reading this blog, high school education, undergraduate degree then onto a postgraduate degree. However, over 25 years ago during my undergraduate study, I was fortunate enough to work in the lab of Professor Ray Waters studying DNA damage in S. cerevisiae as a model organism and at the time PCR was cutting-edge technology and the PCR license was in full effect. However, there was one company that was fighting the good fight to democratize PCR for the good of the scientific community, Promega.

I became enamored with Promega then, and the next steps in my career were taken with a view to working at this company who, for all intents and purposes, seemed to really care about the progression of science beyond self-aggrandizement.

Now that I am working at Promega in a position where I can bring benefit to our academic community, I have pondered what I can do to equal the disruptive attitude I observed in this company all those years ago when they were fighting the then “big tech” for the enablement of the scientific community. 

Reporter bioassays are one of hte many offerings of the academic access program.
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How Promega Helped Our Lab Scale Up Drug Discovery for Bloodborne Pathogens

This blog was written by Sebastien Smick, Research Technician in Dr. Jacquin Niles’ laboratory at Massachusetts Institute of Technology (MIT)

Our lab is heavily focused on the basic biology and drug discovery of the human bloodborne pathogen Plasmodium falciparum, which causes malaria. We use the CRISPR/Cas9 system, paired with a TetR protein fused to a native translational repressor alongside a Renilla luciferase reporter gene, to conditionally knock down genes of interest to create modified parasites. We can then test all kinds of compounds as potential drug scaffolds against these gene-edited parasites. Our most recent endeavor, one made possible by Promega, is a medium-low throughput robotic screening pipeline which compares conditionally-activated or-repressed parasites against our dose-response drug libraries in a 384-well format. This process has been developed over the past few years and is a major upgrade for our lab in terms of data production. Our researchers are working very hard to generate new modified parasites to test. Our robots and plate readers rarely get a day’s rest!

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Toxicity Studies in Organoid Models: Developing an Alternative to Animal Testing

Alternatives to animal testing have long been explored when it comes to studying the safety of various chemical compounds for use in food, medicine and cosmetics. With the advent of three-dimensional (3D) cell culture to create organoids, more relevant human organoid models are being explored as one way to provide safe and effective compound testing while minimizing the need for testing in animals. The international project Physiologically Anchored Tools for Realistic nanOmateriaL hazard aSsessment (PATROLS) led by the Swansea University Medical School aims to establish a battery of innovative, next-generation safety testing tools that can more accurately predict the effects of engineered nanomaterial (ENM) exposure in humans and the environment.

One project being researched by Samantha Llewellyn, a research assistant at Swansea University, is developing predictive physiologically relevant 3D liver models for ENM safety assessment. By having a model to evaluate realistic ENM exposures, a researcher can study liver function, hepatic metabolism and microtissue cell viability after acute (24 hours) or prolonged (several days) exposure. A microtissue model for assessing ENM hepatotoxicity needs to mimic primary hepatocytes and be amenable to assays used to test cell viability and metabolism.

The right tools for testing this 3D liver model include the bioluminescent-based CellTiter-Glo® 3D Viability and P450-Glo® Assays. When creating organoids, having reagents that can penetrate to the center of the dense and complex 3D liver spheroids is important so that the cell viability readout encompasses the entire microtissue. The CellTiter-Glo® 3D Viability Assay accomplishes this task, providing accurate assessment of 3D tissue cell health. Measuring cytochrome P450 (CYP450) activity is necessary for studying liver function. The P450-Glo® Assays have the flexibility to assess CYP450 activity while preserving the liver spheroids; thus, researchers can gather more data from a single experiment.

The importance of Samantha Llewellyn’s research as part of PATROLs is establishing a 3D liver model that could evaluate realistic ENM exposures and reduce the need for animal testing. Bioluminescent assays for assessing cell health and liver enzyme function are necessary to reach this goal.

luciferse technologies are allowing researchers to develop predictive 3D organoid models for ERM testing

To learn more about the last 30 years of bioluminescent innovations and the discoveries they’ve enabled, please visit our 30th anniversary celebration page.

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Improving SARS-CoV-2 Antibody Detection with Bioluminescence

3D artistic rendering of the Lumit SARS-CoV-2 antibody test

Science is the practice of figuring out how things work and then using that knowledge to further our understanding or to create tools that can solve problems facing the world. Bioluminescent tools and assays are examples of science doing all these things. Bioluminescence is the light-yielding (luminescence) chemical reaction that is used by many lifeforms. When fireflies flicker in the twilight, they are using bioluminescence to flash on and off.  Chemically, bioluminescence happens when an enzyme called luciferase acts on a light-emitting compound, luciferin, in the presence of adenosine triphosphate (ATP), magnesium and oxygen.

For scientists, bioluminescence can serve as a tool to help them understand many cellular functions. Since few animal or plant cells produce their own light, there is little to no background signal (light) to be concerned about. This lack of background means that all light coming from the sample can be measured. In fact, bioluminescence is often a preferred tool for scientists because it does not require an external light source or special filters, which are required for fluorescence-based technologies.

Promega scientists have developed bioluminescent tools and assays to support leading edge scientific research for decades, beginning in 1990 with the Luciferase biosensor technology based on firefly luciferase. Luciferase is a wonderful tool for studying how enzymes work because its output (light) is so easy to measure: samples are placed into a special instrument called a luminometer, and the amount of light being produced (Relative Light Units) is recorded. Bioluminescence technology can be configured to measure a variety of cellular biology, ranging from cell health to enzyme activity down to the specific event of turning a gene on or off. The advent of new techniques for genetic manipulation, along with an enhanced understanding of bioluminescence and the discovery and engineering of better luciferases, enables science to use bioluminescence in even more unique ways.

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Endpoint PCR in 15 Minutes with New Master Mix and Thermal Cycler Combination

Since its invention in 1983, the polymerase chain reaction (PCR) has become a fundamental technology in life science laboratories across the world. Much of the technological innovation is driven by quantitative PCR and digital PCR (1); however, endpoint PCR remains a workhorse technology for applications such as gene cloning, mutagenesis and detection of microbial pathogens. Variations on the basic endpoint PCR method—for example, the use of multiplexed, fluorescently labeled primers followed by capillary electrophoresis to analyze the amplified DNA fragments—are popular in forensic DNA analysis and cell line authentication.

The COVID-19 pandemic has created an urgent need for PCR-based diagnostic testing for SARS-CoV-2. Most of these diagnostic tests use real-time, reverse-transcription quantitative PCR (RT-qPCR). However, RT-qPCR can be challenging for routine use in developing countries and in laboratories with limited access to real-time PCR thermal cyclers. A recent study described an endpoint PCR method for SARS-CoV-2 detection to address these limitations (2).

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Understanding Inflammation: A Faster, Easier Way to Detect Cytokines in Cells

Inflammation, a process that was meant to defend our body from infection, has been found to contribute to a wide range of diseases, such as chronic inflammation, neurodegenerative disorders—and more recently, COVID-19. The development of new tools and methods to measure inflammation is crucial to help researchers understand these diseases.

This diagram shows how the Lumit™ Immuno assay can be used to detect cytokines.

Cytokines—small signaling molecules that regulate inflammation and immunity—have recently become the focus of inflammation research due to their role in causing severe COVID-19 symptoms. In these severe cases, the patient’s immune system responds to the infection with uncontrolled cytokine release and immune cell activation, called the “cytokine storm”. Although the cytokine storm can be treated using established drugs, more research is needed to understand what causes this severe immune response and why only some patients develop it.

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RNA-Protein Interactions: A New Frontier for Drug Discovery

Almost 90% of the human genome is transcribed into RNA, but only 3% is ultimately translated into a protein. Some non-translated RNA is thought to be useless, while some play a significant yet often mysterious role in cancer and other diseases. Despite its abundance and biological significance, RNA is rarely the target of therapeutics.

“We say it’s undruggable, but I would say that ‘not-yet-drugged’ is a better way to put it,” says Amanda Garner, Associate Professor of Medicinal Chemistry at the University of Michigan. “We know that RNA biology is important, but we don’t yet know how to target it.”

Amanda’s lab develops systems to study RNA biology. She employs a variety of approaches to analyze the functions of different RNAs and study their interactions with proteins. Her lab recently published a paper describing a novel method for studying RNA-protein interactions (RPI) in live cells. Amanda says that with the right tools, RPI could become a critical target for drug discovery.

“It’s amazing that current drugs ever work, because they’re all based on really old approaches,” Amanda says. “This isn’t going to be like developing a small molecule kinase inhibitor. It’s a whole new world.”

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A Day in the Life of a Technical Services Scientist

Technical Services Scientists answer questions such as "Which product is right for me?" "How does this product work?" and "Help! Something went wrong!"

As a student, I wasted so much time wondering which assay would work best or muddling through problems on my own. I wish I had known I could reach out to get help from another knowledgeable scientist: A Technical Services Scientist!

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Shifting Conservation Status: Endangered Species Get a Second Chance

On May 21st, 2021 we celebrate National Endangered Species Day. This day helps raise awareness and increase knowledge of endangered species and wildlife, in hopes to save them. We have been lucky enough to collaborate with organizations and partners to help save species that were on the brink of extinction. Take a look at some species that are hoping for a second chance to survive and thrive.

Kit Elizabeth Ann the Black-Footed Ferret

Picture of black footed ferret Elizabeth Anne, one of the endangered species that Revive & Restore is working on.

In February 2018, resurrection efforts began for the then endangered black-footed ferret. With the help of the U.S. Fish and Wildlife Service, Revive and Restore, partners ViaGen Pets & Equine, San Diego Zoo Global, and the Association of Zoos and Aquariums, the successful cloning of a black-footed ferret was announced in February 2021. “Elizabeth Ann” was cloned from Willa, a female ferret that died in 1988, using somatic cell nuclear transfer (SCNT). Elizabeth Ann’s genetic variants reveal a lot of much-needed hope for the genetic diversity of wild ferrets. Check out the full story on Elizabeth Ann’s journey here!

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