Isabella Del Priore

For cells to grow and divide, they must go through a process called the cell cycle. Under normal circumstances, this cycle is tightly regulated by several molecules and proteins that act as gatekeepers, only letting the cell progress when certain signals are received and criteria are met. CDK4 and CDK6, also known as cyclin-dependent kinase 4 and 6, are two of the many proteins involved in this regulation. In cancer, the cell cycle is often overstimulated and under controlled, leading to unregulated cell growth and the formation of tumors. CDK4/6i work by targeting CDK4 and CDK6, stopping cell growth by halting the cell cycle.

There are three CDK4/6i currently approved to treat ER+/HER2- metastatic breast cancer: ribociclib (Kisqali), abemaciclib (Verzenio), and palbociclib (Ibrance). All three have shown high success rates in patients. However, it is somewhat common for resistance to develop after prolonged treatment. Understanding this resistance is the key to finding new, effective treatments for these patients.

To mimic the resistance that patients gain over time, scientists similarly exposed breast cancer cells in the lab to CDK4/6i over several months, resulting in cells that no longer responded to the therapy. In these insensitive cells, they noticed that the levels of the CDK6 protein were elevated. After analyzing the genetic sequences of patient samples, they identified a handful of genes whose loss correlated with resistance. They modified cells by knocking out these genes, causing the presence of the corresponding proteins to decrease. In these knockout cells, they observed what was expected: high levels of CDK6 and continued growth on treatment, demonstrating resistance. Conversely, the non-altered cells still showed sensitivity to treatment. To further support these results, researchers took patient tumor samples known to be CDK4/6i sensitive and grew them in mice. Upon treating the mice with the drugs, the tumors initially decreased in size; however, over time, the tumors no longer responded to treatment. The resistant tumors had the same characteristics as the resistant cells—loss of one of the genes of interest and high CDK6.

Following the establishment of CDK6 as a key player, researchers next sought to uncover exactly how the observed high levels of this protein led to resistance. Because CDK6 can exist as part of a complex, they began by looking for other components of the complex that were potentially involved. Their investigation revealed that molecules called INK4 proteins were present at much higher levels in resistant compared to sensitive cells. Analysis of physical structures showed that when INK4 proteins attached to CDK6, they altered the shape of the complex. The changes induced by INK4 binding were significant enough to prevent CDK4/6i from binding and thus from inhibiting cell growth.

With a better understanding of the mechanism of resistance, researchers wondered if re-targeting CDK6 in a different manner could recover sensitivity. The development of a new class of drugs, called PROTACs, which degrade instead of inhibit CDK4 and CDK6, offers promise. Instead of binding and blocking CDK4 or CDK6, these drugs recruit a special type of protein to mark them, signaling for machinery in the cell to break them down. After several experiments in multiple breast cancer cell lines, preliminary data showed that CDK4/6i resistant cells were responsive to the degraders. Treatment with PROTACs led to decreased cell growth and shrinking of tumors in mice.

Out of over 1000 estrogen receptor positive breast cancer samples analyzed by the researchers, 17% had mutations that led to high CDK6 and resistance to CDK4/6i therapy, demonstrating the clinical relevance of this type of resistance. The initial results show that PROTACs have significant antitumor effects that are promising for eventual investigation in clinical trials. Similar drugs are already in early-phase trials in other cancer settings, for example those that target the androgen receptor in prostate cancer. The novel re-targeting of CDK4 and CDK6 presents an important potential alternative for breast cancer patients who become resistant to current CDK4/6i therapies.

One component of a liquid biopsy that is of particular interest is circulating tumor cells (CTCs). CTCs are defined as cells that detach from a tumor and circulate in the bloodstream. The idea of cancer cells breaking free and moving through the blood is not a new one. Scientists first hypothesized about the process in the 19^thcentury, including Paget’s 1889 “seed and soil” theory of metastasis. Paget proposed that cells detach from tumors and remain in a dormant state before relocating to other areas of the body and metastasizing. It is even possible for cells to stay dormant for over 10 years before settling into new sites. More recent work over the last few decades has examined the potential for liquid biopsies and CTCs to significantly impact how clinicians approach cancer treatment on a more personalized level. 

In order to use CTCs as a guide for treatment, they first must be successfully identified and isolated from the blood. Despite the fact that a single tumor can release thousands of cells each day, only a small fraction survive and circulate in the blood. Among the millions of blood cells, finding and identifying the few tumor cells is not a trivial task. In a single 7.5 mL sample of blood, there may only be one or two CTCs. Scientists have found ways to detect these rare cells based on characteristics such as size and density, as well as the presence or absence of markers on the surface of the cells that distinguish them from normal blood cells. These markers include three types of proteins: epithelial cell adhesion molecule (EpCAM), cytokeratin (CK), and CD45. CTCs are identified as EpCAM and CK positive and CD45 negative. Currently, the only FDA-approved method of detection, CellSearch, relies on this set of criteria to identify and count CTCs in patient blood samples.

Once detected, analysis of CTCs–both enumeration and biomarker detection–has substantial clinical promise for characterizing a patient’s disease and making treatment decisions. Studies have shown an association between the quantity of CTCs in a blood sample and prognosis. Higher numbers of CTCs (generally five or more per 7.5 mL blood sample) correlate with decreased patient survival, while low CTC counts are associated with more favorable outcomes. In one study, researchers found that among early breast cancer patients who underwent chemotherapy before surgery, those who had fewer CTCs in their blood pre-treatment had a greater chance of survival. In addition to enumeration serving as a source of information prior to therapy, CTC counts following a particular treatment may be useful as well. Though research is ongoing, there is some indication that the number of CTCs present in a post-treatment blood sample could advise clinicians as to whether that treatment is working, or if the patient should be switched to a different one. 

Beyond identification and counting of CTCs, analysis of the cells themselves can tell a deeper story about the characteristics of the cancer. This can influence treatment decisions with the potential to save valuable time and alleviate some of the stress and discomfort of side effects. Currently, primary tumor biopsies serve as the main guide for treatment. Although a standard tissue biopsy is highly informative, cancer is not a static disease. Liquid biopsies have the ability to capture this ever-changing nature, more closely representing the disease both temporally and spatially.

Considering breast cancer as an example, both tissue and liquid biopsies can reveal hormone (estrogen and progesterone) and HER2 (a cell surface protein related to cell growth) receptor status, which is used to guide the course of treatment. However, while tissue biopsies are only taken upon diagnosis or surgery, CTCs can be analyzed with each blood draw, yielding up-to-date results for that specific patient at that particular time. In addition to capturing how a patient’s cancer changes over time, CTCs also provide information about potential differences in primary versus metastatic sites. Since CTCs can originate from any tumor in the body, they offer a more complete picture of the disease. In a single patient, the primary breast tumor may have been hormone receptor (HR) positive and HER2 negative, but a CTC may be positive for both HR and HER2. This distinction could help clinicians make more informed decisions to maximize benefit with treatments that target the appropriate proteins based on the specific patient’s CTC results. One group of studies, the DETECT trials, sought to take advantage of this finding and validate the predictive nature of CTCs. They compared breast cancer patients treated with standard therapy versus adding a HER2-targeted therapy based on CTC analysis. Certain therapies are specifically targeted to HER2, and thus would not be used for HER2 negative patients. However, as researchers now know, even though a patient is designated as HER2 negative from the primary tumor biopsy, a liquid biopsy may reveal HER2 positive CTCs. In this case, it could be beneficial for the patient to receive HER2-targeted therapy, a treatment path that would not have been considered without CTC analysis. 

It is clear that there is much clinical potential for the use of CTCs and liquid biopsies (beyond breast cancer, CTC research has also shown promising results in colorectal cancer, prostate cancer, lung cancer, melanomas, and others). However, there are also significant hurdles that need to be overcome before CTCs become a routine part of cancer care and a guiding force in treatment. A limitation of many studies is the method of CTC detection. Both the FDA-approved CellSearch and other methods in development rely on the presence of EpCAM as a marker to detect CTCs. Though this has proved to be a generally reliable method, it is not all-encompassing and may actually exclude CTCs that are essential to understanding the full scope of disease. For example, CTCs that are in an intermediate state, called epithelial-mesenchymal transition (EMT), lack EpCAM altogether and thus are missed by current detection methods. This is an issue as these CTCs are associated with aggressive disease, which can have a significant impact on metastasis. In addition to improvements in sensitivity and accuracy, detection methods also require standardization before use in the clinical setting. Though further research is necessary before they make their way into standard of care practices, CTCs show significant promise as a prognostic tool, a method for monitoring treatment effectiveness, and a predictive indicator for more personalized treatment decisions that could alter the course of cancer care.

The protection offered by vaccines not only benefits the individual, but also the community as a whole. Getting the vaccine decreases your own chances of getting sick as well as those around you. The goal is to reach herd immunity; once enough people are vaccinated, the disease will no longer have enough susceptible hosts to infect and spread. This is achieved once a large portion of the population (at least 70%) is immune through vaccination or natural exposure to the virus. Thus, it is important that you get vaccinated if you are able.

Vaccines work by taking advantage of the immune system’s ability to fight off unwanted invaders like Covid-19. The body is intentionally introduced to something that it recognizes as foreign, which triggers natural defense mechanisms against it. Specifically, the memory part of the immune system is activated. This involves the creation of antibodies by cells called B cells. Antibodies specifically attach to the introduced foreign particle or protein and facilitate clearing of the intruder. Once the body has created a reservoir of antibodies for that particular pathogen, they are stored in its memory. If you are exposed to the actual disease, the body is ready to launch its strongest attack to eliminate it before you feel sick. This general process applies to all vaccines; however, all vaccines are not completely alike. Although they share in their ability to stimulate antibody production, there are differences between, for example, the familiar flu shot and the seemingly new messenger RNA (mRNA) vaccines. (They are actually not that new; scientists have been developing them for years.) 

Pfizer, Moderna, and Johnson & Johnson—how do they differ from each other, how do they compare to other types of vaccines, and what really is an mRNA vaccine?

Over the past year, scientists have been working to develop vaccines specific to Covid-19; however, the process of vaccine development itself is not novel. Even though there has never been an mRNA vaccine approved for use, the technology has been in development for decades and studied for use against influenza and the Zika virus. With the increase in funding and the large pool of willing volunteers for Covid-19 clinical trials, scientists have been able to rapidly test vaccines and gain emergency use approval from the FDA. There was no science skipped in the development of the vaccine. The three vaccines that are approved in the United States are from the companies Pfizer, Moderna, and Johnson & Johnson. (Update as of 8/23/21, the Pfizer vaccine now has full approval!)

There are several different classes of vaccines, including live weakened (attenuated) vaccines, inactivated vaccines, mRNA vaccines, and viral vector vaccines. Each has certain benefits that may make it more suitable depending on the disease it is meant to protect against and the environment at the time of development. Attenuated vaccines contain live virus. In order to ensure that the live virus cannot infect the patient, it has been altered in a lab to weaken it. The body’s reaction to this type of vaccine generally produces the strongest immunity because it is most similar to natural exposure to the pathogen. Some examples are the measles, mumps, rubella (MMR) vaccine and the chickenpox shot. Inactivated vaccines use a dead version of the pathogen. This can be the whole pathogen or only a part. The flu shot is considered an inactivated vaccine. Booster shots are required with this type of vaccine to sustain continual immunity.

The types of vaccines that have been developed to fight Covid-19 are mRNA and viral vector vaccines. Both of these methods have been extensively studied, but are relatively newer. The mRNA vaccines developed by Pfizer and Moderna use, unsurprisingly, mRNA to generate a response from the immune system. mRNA refers to the messenger that allows the body’s cells to make proteins. It is the essential middleman between the instructions on DNA and the resulting proteins that allow our cells to function properly on a day-to-day basis. The mRNA in the vaccine carries instructions for cells to make a protein found on the coronavirus called the spike protein. The spike protein activates the body’s natural immune response to generate spike protein-specific antibodies. Once the mRNA completes its job of providing the instructions to make the protein, it gets broken down.

Viral vector vaccines use a secondary virus to deliver the DNA of the target virus (the disease you want to protect against) to generate immunity. The virus used as the delivery vessel is modified in a lab so that it cannot infect the recipient and only functions to transport the DNA to your cells. Most recently, this concept was used to develop the Ebola shot and is what is used for the Johnson & Johnson Covid-19 vaccine. To compare viral vector vaccines to mRNA vaccines, the former simply involves one extra step. The DNA in the viral vector vaccine has instructions to make mRNA. The mRNA then acts in the same way as the mRNA in the Pfizer and Moderna vaccines to tell the body to make the spike protein and the corresponding antibodies. There is no risk of infection or becoming sick with Covid-19 from any of the three shots because none inject the actual virus into the body.

Why do some vaccines require two versus one dose, why do we feel sick after getting vaccinated, and why is there a 2-week waiting period before we are fully protected?

In general, the number of vaccine doses is determined by the results of clinical trials. For Moderna and Pfizer, immunity was highest after two doses and for Johnson & Johnson strong immunity was reached with just one. In a two-dose vaccine, the first dose primes the immune system and the second gives it a boost to generate immunity. When the body is first exposed to the viral protein, it creates some antibodies that provide moderate protection. However, when the second shot is given, the reintroduction of the protein triggers the production of a large stockpile of memory B cells that remain in the body. This ensures that if you encounter the real virus, your B cells are ready to quickly make antibodies to defend against the pathogen.

The booster effect also explains why many people experience stronger side effects after the second dose of Pfizer and Moderna. When your body is first exposed to the spike protein, because it has never seen it before, it does not initially know what to do. It attempts to clear out the intruder as efficiently as possible, but since there are no stored B cells, the reaction is relatively small. However, with the second dose, the body has a memory of the spike protein and can launch a stronger attack. This larger reaction manifests as more intense side effects. The degree of side effects differs from person to person depending on factors such as the strength of your immune system and age. Feeling sick after the vaccine is not only normal, but also a good sign because it means the body is doing its job and making antibodies. (However, even if you do not experience side effects, that does not mean you are not making antibodies.) It takes time for your body to fully generate the memory B cells and corresponding antibodies specific for Covid-19. Though it varies, in general, giving the body two weeks is a sufficient amount of time to build up immunity.

For Johnson & Johnson, clinical trials showed that the response is already relatively strong after one dose, which is why people feel side effects following the one shot and why only one dose is necessary. Though there are differences in the effectiveness of the Moderna and Pfizer vaccines versus the Johnson & Johnson vaccine (94-95% versus 70% effective, respectively), these statistics do not necessarily tell the full story. A vaccine that is 70% effective at preventing illness still offers significant protection. For comparison, the flu shot is generally around 50% effective. All three Covid-19 vaccines prevent death and significantly decrease the chances of becoming severely ill. Importantly, because Johnson & Johnson requires only one shot, it is easier and quicker to get more people fully vaccinated, thus bringing us closer to reaching herd immunity.

So, how does the Covid-19 pandemic end?

Timing is everything when it comes to beating the pandemic. Viruses rely on hosts to survive and spread. Each time the virus spreads, it replicates. And with each replication, there is an opportunity for mutation. The mutations that make the virus better at spreading or infecting stick around and eventually dominate the population, resulting in new variants. This has already been seen with the UK B.1.1.7 and South Africa B.1.351 variants. For the pandemic to end, enough people must have antibodies against the virus before it has time to accumulate mutations that would render the current vaccines ineffective. This requires not only rapid vaccination of large numbers of people, but also that regulations on social distancing and masking not be lifted too prematurely. It is important to make it as difficult as possible for the virus to find vulnerable individuals so that it dies out. If you are able to get vaccinated, you are highly encouraged to do so, as vaccines are a safe and effective way to protect yourself and those around you from Covid-19.