Biomarkers and CRC: An introduction (2020)

Manju George 0:06
Hi, everyone. Welcome to a short video on CRC Biomarkers. As we all know, our bodies are made of tissues and organ systems and the basic functional unit of our body is a cell. Inside the cell is a nucleus, which is the command center of the cell, and houses the DNA, the genetic material that encodes a blueprint of the entire cell. The tissues and organ systems are composed of tight communities of cells, where each cell has specific roles and follow very strict rules to exist as part of the community. A tumor biomarker is anything that can inform us about the tumor. In my talk, I will cover germline testing, tumor testing and liquid biopsies. Through these tests, we’re looking at DNA. And why are we interested in DNA? Normal cells are normal because their DNA is normal. And when normal cellular DNA becomes damaged and changes, it can cause changes in proteins that it encodes. And these cells that have abnormal proteins can behave abnormally and fail to follow the rules in the community, that make up the tissue and this can give rise to a tumor. So, first, why are these changes important? These changes are important because knowing what is altered in a tumor cell can help us know how to kill it. And clinically these changes may have important implications, and also help us classify the tumor. So let’s first look at germline testing.

Manju George 1:59
So by germline I mean the DNA in sperm or eggs. Changes in the germline DNA are heritable and can be found in all cells in the body, including the tumor cells. People with germline mutations may be susceptible to other cancers other than CRC and may need additional screening and surveillance. Since germline mutations are heritable, there may be implications for family, like siblings and children, and they may also need to get tested if an individual is found to have germline mutations.

Manju George 2:39
Here is a list of common inherited cancer syndromes and associated genes. Here you can see familial adenomatous polyposis. That condition is caused by mutations in the APC gene and can cause colorectal, brain, stomach, bone, and skin cancers. Another common inherited CRC is because of Lynch Syndrome, where you have mutations in these four proteins, and commonly can cause colorectal and endometrial cancers.

Manju George 3:18
Heritable CRC, that means colorectal cancer because of inherited mutations, are less than about 5% of the total CRC. There are two kinds. One is known as hereditary nonpolyposis CRC and that includes Lynch Syndrome, which we’re all familiar with. And the other one is known as polyposis, which is characterized by a large number of polyps found in the intestine of affected individuals.

Manju George 3:51
I’ve listed a few conditions known as FAP and AFAP, which is due to germline mutations in APC. Another condition is known as MAP or MUYTH-associated polyposis, which is due to mutations in the MUYTH gene. There are other conditions. For example, due to mutations in the POLE or POLD1. I’ve listed these mutations, or gene names, basically to show that some of the same genes are involved in sporadic CRC which is what the majority of us have, that is the rest of the 95% of CRC which is not heritable.

Manju George 4:39
So next slide. The second kind of inherited CRC is known as Lynch Syndrome. And this is due to germline pathogenic variants in the DNA mismatch repair genes, which are MLH1, MSH2, MSH6 and PMS2. So usually Lynch Syndrome is due to germline mutations in these four genes. And less commonly, Lynch Syndrome is also caused by mutations in the EpCAM gene.

Manju George 5:10
So now that we have seen what germline testing is, let’s go find out what tumor testing is.

Manju George 5:18
Tumor testing is what most of us that do not have heritable or inherited colorectal cancer, that is what we all have is known as sporadic colorectal cancer. And we are concerned about tumor testing. So tumor testing looks at changes in the somatic, which means only in the tumor. And you can find the changes in tumor cells by using very low tech tests such as immunohistochemistry, that is IHC. And using that you can look for mismatch repair proteins, you can also look at another common mutation, which is found in about 10% of colorectal cancer, which is a BRAF V600E mutation. So, if there are mutations in the mismatch repair proteins, then your tumor is classified as MSI-high and MSI-high tumors comprise about 15% of all CRC and about 4% of metastatic colorectal cancer. BRAF V600E mutations are found in about 10% of colorectal cancer. Another mutation that is of interest to most of CRC patients is RAS mutations. There are three RAS genes that are important, which are KRAS, NRAS and HRAS. This is because if your tumor has mutations in the RAS genes, then you are not eligible for treatment using a class of targeted therapy known as EGFR inhibitors. So, for anybody who has colorectal cancer, it is very important that they know what stage they are, and if they’re stage IV, where their primary tumor is and where the mets are. And then the other important thing to know is the status of mismatch repair proteins. So, if you have unmutated mismatch repair proteins, then your tumor is called microsatellite stable or MSS. And if your tumor has abnormal or mutated mismatch repair proteins, then your tumor is MSI-high or microsatellite instable high. So, this is the basic kind of testing that everyone should have. And there is like different degrees of tumor testing.

Manju George 8:11
So the other kind of tumor genomic profiling which is really extensive and needs more sophisticated equipment is the tumor genomic profiling. This uses resected tumor tissue and it looks at various kinds of gene changes. So, it can look at gene mutations, deletions, inversions, some kinds of chromosomal translocations, amplifications, etc. And many of the newer tests, they will also look at RNA profile, and they give what is known as the TMB, which is tumor mutational burden. You can get the value of TMB also by using tumor genomic profiling. There are many tests available. The common ones are Foundation One, Caris, Tempus, etc. The most extensive profiling is done by MSKCC. The test is called MSKCC Impact. In these commonly used tests like Foundation One, Caris, Tempus, etc., they look at about 300 different changes on over 72 different cancer relevant genes. So, in general, the mutations that are identified, like you’ll get a list of mutations, some kind of an alphabet soup and they are, in general, categorized into two. One is actionable mutations. That means those mutations that there is targeted therapy against or for example, with the NRAS and KRAS mutations, if you have those mutations, like I said before, you are not eligible for EGFR inhibitor antibodies. But for the BRAF V600E, there is targeted therapy against that particular mutation. For mutations in the MMR genes, then that makes your tumor MSI-high, in which case, you are eligible for immunotherapy. Some other mutations that are actionable or POLE mutations, and NTRK fusions, and also HER2 or ERBB2 amplifications. So I kind of want to bring your attention here to show that some of the genes that we talked about in the inherited or heritable CRC, those same genes are also seen to be mutated in sporadic CRC. Some of the genes are not, or mutations are not actionable. For example, APC, TP53, etc. Mutations in these genes are important for the change in a normal cell to become a colorectal cancer. So right now, we don’t have any targeted therapies against them.

Manju George 11:02
That brings us to the next topic, which is liquid biopsies.

Manju George 11:19
So recently, I heard this talk from Dr. Marshall and he called ctDNA, tumor trash in the bloodstream. So all cells turnover in our body. And as a result of it, they shed pieces of their cellular DNA into the bloodstream. So tumor cells also do this. But because tumor cells have mutated DNA, because of that property, we can now find ctDNA by virtue of their mutations, and then use it as an indicator for the presence of tumors in our body. So liquid biopsies, actually look for ctDNA or circulating tumor DNA. And as I said, the assumption is that circulating tumor DNA is abnormal DNA and therefore we can detect it.

Manju George 12:10
So here is a diagram showing the uses of liquid biopsies for treatment in various stages of cancer. In this figure, they’re talking about circulating tumor cells. But in colorectal cancer, in general, we usually look at circulating tumor DNA, not so much the circulating tumor cells. So using that you can look at DNA abnormalities, amplification and deletions, translocations, chromosomal abnormalities and point mutations. Circulating tumor DNA can be used for early intervention. So, there are some trials going on to find out if you can look at circulating tumor DNA levels to detect the presence of a tumor. So, in say screening. The other use of liquid biopsies is in localized disease to determine the risk of recurrence after treatment. And liquid biopsies are also important in metastatic disease because it can enable treatment selection based on the presence of certain specific biomarkers. The other use of liquid biopsies is to see once you’re on a certain treatment, to find out if there is an emergence of resistant clones, and so basically looking at treatment response.

Manju George 13:39
So, there are two different kinds of liquid biopsies. One is tumor-naive and the other one is tumor-informed. Tumor-naive basically means that you have the same panel for everyone. An example of tumor-naive liquid biopsies or Guardant360, Foundation One liquid, etc. Here, they look at a panel of about 300 common mutations and other changes. The test only needs a blood sample, and the results usually include mutational info. The other class of liquid biopsies is tumor-informed. An example is Signatera. In this case, this is a liquid biopsy that is individualized to each person’s tumor. So for this, what you do is that you need a tumor or biopsy tissue and Next Gen sequencing of the tumor tissue is done and then based on the mutations that are present in the tumor, a panel of about 16 different genes are selected for each individual patient, and a liquid biopsy test is designed. So the disadvantage is that it does not contain mutational info. They give you a total value, which is mutations per ml. And the result of this test can be almost used like that of CEA.

Manju George 15:00
So, then in the last slide, I want to go over a comparison between tissue biopsy and liquid biopsy. So with respect to tumor genomic testing, you obviously need a tumor sample or biopsy. And the disadvantage is that it is a very small portion of the entire tumor. So there is this idea of tumor heterogeneity, which means the whole tumor is composed of a bunch of clones. So when you’re sampling only a part of the tumor, and you get those mutations, and you assume that the rest of the tumor is composed of the same mutations, which may or may not be true. And then frequent sampling is not possible with tumor testing, because your mets may be in places which are not easily accessible. And, you cannot go every month to do a biopsy. Using tumor testing, you can calculate TMB. And the data that you get from tumor testing is considered historic, not real time. Because if you say, for example have a surgically resected tissue, then that issue is no longer in your body. So it’s giving you historic information. You compare liquid biopsy, it does not need a tumor biopsy, except in the case of like a tumor informed test such as Signatera, where for the very first test, you need a sample of the tumor. A liquid biopsy is supposed to be more representative of the whole tumor because it is thought that different parts of the tumor can shed ctDNA and so therefore, when you look at blood to find ctDNA, then you assume that you’re getting a representation of the entire tumor. Frequent testing is possible because all you need is a blood draw. For now, a liquid biopsy cannot be used to calculate TMB. But maybe in the next few years, it may be possible to calculate TMB from liquid biopsies. You know, serial liquid biopsy testing can give you real time info on response to treatment. So, for example, if you did a liquid biopsy before you started treatment, and then you got on, like a targeted therapy like EGFR inhibitors, or if you have a BRAF mutated tumor, then you are on targeted therapy against the BRAF mutation, then over time, if the tumor starts over growing, you could do another liquid biopsy and then see what are the mutations? Are there new mutations? And is that the mechanism of how the tumor has become resistant to this particular treatment? So this is a direct comparison between the advantages & disadvantages of tumor testing versus liquid biopsy. So, with that, I’m going to stop sharing my screen. So, the idea is that this video will be deposited in a common place, and that everyone who is part of the EPL can watch it at their own convenience. And during our first session, I hope to answer any questions you have. And, like I said before, in my introductory video, there will be a short quiz. So I hope you guys find this video informative. So thank you very much. Bye