Diagnostics' Role in Medicine Today PDF

PROPRIETARY. DO NOT SHARE. Transcript: Diagnostics’ Role in Medicine Today Section 1: Use of Diagnostics Welcome Welcome to our course on Diagnostics’ Role in Medicine today. This course will be broken into three sections: uses of diagnostics, types of diagnostics, and companion diagnostics. Sectio...

PROPRIETARY. DO NOT SHARE. Transcript: Diagnostics’ Role in Medicine Today Section 1: Use of Diagnostics Welcome Welcome to our course on Diagnostics’ Role in Medicine today. This course will be broken into three sections: uses of diagnostics, types of diagnostics, and companion diagnostics. Section 1: Use of Diagnostics Objectives At the end of this section, you should be able to: ● Define diagnostic. ● Define biomarkers. ● Describe different uses for diagnostics. What is a Diagnostic? So, first of all, what is a diagnostic? Diagnostics detect the disease state of an organism. So, a diagnostic is a measurement that gives us information about your health. Things like your height, your weight, and your blood alcohol, can be determined by diagnostics. Diagnostics can also be used to tell us whether or not a patient has a particular disease. For example, a biopsy where we take a tissue sample, a little scoop of tissue, and analyze or examine that tissue, and see if it's normal or not. Or we can take other types of measurements, like an EKG, which are electrical measurements to determine if you've had a heart attack or not. And diagnostics can be a means to identify the nature or cause of a particular health state. For example, tremors or weight gain or loss could be reflective of thyroid conditions, or it could be reflective of a brain issue like Parkinson's disease, so diagnostics can be used to determine the cause of the symptoms so the correct treatment can be administered. So, the idea here is we are measuring some aspect of a patient's cells, molecules, or tissues, and determining if they appear, quote, unquote, "normal" Diagnostic Tests are Measurements So, we just want to make sure that we establish the idea that diagnostic tests are measurements. And what is a measurement? A measurement is just a recorded observation. 1 Copyright 2024 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. And there are two types of measurements that we could make, or two types of observations: qualitative measurements, qualitative observations, and quantitative measurements. Qualitative measurements are subjective. So, it could be "I'm hot." A quantitative measurement would be "What is your exact temperature? Is it 101 or is it 110?" And based on that, you would make some different decisions. So, whenever possible, we'd like to try to make our measurements quantitative. What are some of the types of quantitative measurements that we can make? We could do size, shape, and weight. Numbers: how many cells? Volumes: how big are the cells? Density: how much essentially does it weigh relative to water? We could do electrical measurements. We can measure voltage or current, like EKGs. Optical or radiation. Such as colors or density absorbance. This would be part of an assay. They could be chemical measurements such as an ion count. That is, what's your sodium level? What's your chloride level? Viscosity: that would be, for example, for your urine. How thick is it? Or precipitates: what is or is not dissolved in there? So, single or multiple measurements can be evaluated by algorithms. Algorithms are simply mathematical expressions designed to do repeated calculations, and designed to do comparisons. This way we can compare large amounts of data from large amounts of measurements. What kind of results might we get that would be determined by algorithms? It could be a value of concentration. Such as how many milligrams per deciliter of cholesterol are present, or how many white blood cells you have. It could just be a positive or a negative result. Is this protein present or not? And we're not even going to worry about how much. Just a yes or a no. Biomarkers The key phrase that we now hear in diagnostics is "biomarker". A biomarker is simply a biological molecule that allows us to infer something about the health status of an organism. So, it is simply a molecule that we can detect and measure. And based on the presence or absence of that particular molecule, or the amount of that molecule present it will give us information about the potential health status of an individual. 2 Copyright 2024 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. So, ideally, we look at biomarkers that we can obtain from body fluids. This could include blood, urine, or saliva. What we have to do, though, is then link this molecule or groups of molecules to a specific disease. And that's the other trend in biomarker research, and why the idea of algorithms, the concept of being able to handle large datasets with multiple components, becomes very important. Typically, we're looking at multiple molecules which together help us get an accurate picture of what's happening in the patient. Biomarkers include things like LDL, low-density lipoprotein, for cholesterol. We know that we have LDL and we have high-density lipoprotein or HDL. We'd like to have not much LDL and lots of HDL, and that tells us that our cholesterol levels should be pretty good. PSAs: prostate-specific antigen. CRP is a C-reactive protein. This is a protein that increases inflammation, and there are normal levels and abnormal levels. CK-MB is a cardiac enzyme that appears if you have a myocardial infarction or a heart attack. BNP is also related to heart failure. T3/TSH is related to thyroid disease. And then genetic markers, these are genes, DNA sequences, that may be associated with particular inherited diseases. 3 Copyright 2024 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. Section 2: Types of Diagnostics Section 2: Types of Diagnostics Objectives At the end of this section, you should be able to: ● Describe the different types of diagnostics that exist. ● Give examples of when these types of diagnostics are useful in the field of medicine. Types of Diagnostic Tests: General Chemistry The first type of diagnostic test that has been done for years and years is general analytical chemistry tests for molecules, and these are not necessarily biomolecules. Some of these are just simple chemicals. An example of this is urinalysis test strips. Urinalysis frequently tests for the viscosity of your urine. We can look at calcium. It's a bone test. Or an HbA1c test, again, for diabetes. Or a fecal occult blood test for colorectal cancer. Types of Diagnostic Tests: Immunochemistry Another type of diagnostic test is called an immunochemistry diagnostic. In this case, we are using an antibody to detect a protein, and a protein antigen, that is specific to a particular marker for a diseased state. So, for example, the protein troponin is associated with cardiac diseases, diseases of the heart. If you have a heart attack, some of the heart cells die, and they release troponin into the blood. And by taking a blood sample, you can detect the presence of elevated levels of troponin using an antibody. And so, on the right side of the screen, we see a schematic of an antibody binding to the troponin protein. So, once again, the antibody is our detection reagent, that is the molecule that detects a particular marker. And troponin is the marker for, again, cardiac disease. The antibody is mixed in with a sample of blood. And then, we try to detect the presence of an increased amount of troponin. 4 Copyright 2024 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. Alpha-fetoprotein is a protein that becomes elevated in certain cancers. Therefore, the presence of increased levels of alpha-fetoprotein is an indication that you may have a particular type of cancer. And again, there is an antibody that we make that will detect alpha-fetoprotein. So, just to be clear about this, for each of these different proteins, we have to produce a different antibody to detect it. So, each protein or each biomarker for a particular disease has to have its specific antibody, which we have to make in the lab. And of course, we've used antibodies to detect HIV now for a couple of decades. And the advantage of using an antibody as a diagnostic is the fact that it is exquisitely specific. It's less likely to give us a false positive test. It will recognize a specific protein. And in some cases, if done correctly, this can also give us some quantitative information. We can also use antibodies to detect substance abuse. While these are not proteins, we can detect heroin, cocaine, and steroids, by producing antibodies against particular small molecules. It just takes a little more work to do that. But again, these tests are highly accurate. It's difficult to question the result of an antibody test. And they can give us quantitative information if we need them to. Types of Diagnostic Tests: Hematology and Cytology Two more types of diagnostic tests are hematology and cytology. Hematology refers to blood work. Cytology is looking at particular cells. These types of tests have been around for decades, and focus on the blood and blood cells. And so, an example here would be to do a complete count of all the different cells in your blood. So, we're looking here at two things: we're looking for the presence or absence of particular cells, which are shown here in the slide. These are all white blood cells. And so, from previous data, just by sampling hundreds of thousands of patients, we know what the normal levels of these cells should be. So, these are then counted, and the counts of each of these types of cells are compared to what the normal counts are. And based on that, we have a diagnosis as to whether or not you may have a disease. Coagulation tests. These are blood clotting tests. This could be, for example, if you have hemophilia. Additionally, people who have heart arrhythmias are more likely to crush the 5 Copyright 2024 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. platelets in their hearts, which could lead to a blood clot. So, we take an anticoagulant, and then we just check to make sure that your blood is clotting correctly. Types of Diagnostic Tests: Microbiology and Infectious Disease Another set of diagnostic tests is for microbiology and infectious diseases, with these tests we can detect infectious diseases, as we said earlier, using immunochemistry. But sometimes, you don't have an antibody for the particular disease, or it's not cost-effective. So, alternatively, what we can do is use some of these test kits. For example, streptococcal testing is an antibody test kit. We can test for strep throat right in the doctor's office. So, the doctor takes a swab out of your throat, gets some of the mucus that's in the back of your throat where the bacteria is growing, puts it on a little strip, and after a certain number of minutes, the reaction between the streptococcus and the antibody has occurred, and you get a result that says, "Yes, you have strep throat.", or "No, you don't." In other cases, we need to do a bacterial culture. And in this case, we obtain a sample of body fluid. It could be saliva, it could be urine, or it could be blood. If we think we have something like meningitis, we might have to do a cerebrospinal fluid tap, and then this is smeared onto a Petri dish, put into an incubator, and then you wait for the bacteria to grow. And so, the main disadvantage of culturing is the fact that it may take several days before you get a result. The advantage is, by doing cultures, you can detect things that there may not be an antibody for. Types of Diagnostic Tests: Anatomic Imaging A hot area right now is imaging, and imaging allows us to scan your whole body to detect anatomic abnormalities. X-rays: classic for broken bones. Magnetic resonance imaging, or MRI: is primarily for blood flow in the brain, but it's sometimes also used to image heart functions. This allows you to see what's moving normally in terms of blood and what's not moving normally. 6 Copyright 2024 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. PET scans and CAT scans measure, usually, the accumulation of specifically labeled molecules within your different tissues. So, these give us a whole tissue image and tell what is happening where. Things like broken bones, tumors, and even inflammation, all of this can be seen inside. These tests may then be coupled with other diagnostic tests to gain further information. Types of Diagnostic Tests: Molecular The newest type of test is the molecular test. And mostly, these are focusing on DNA and/or RNA, and we're trying to detect a specific sequence of DNA or RNA that has a specific order of nucleotides, our ACT versus CAT sequence, because these sequences can give us information about a particular disease. (pause) It may indicate the presence of a disease. For example, we could look at sequences of viruses or bacteria, or even cancerous cells, or we could predict, if it's a genetically inherited disease, we may look at a DNA sequence and predict how susceptible you are to that particular disease. The classic example is BRACA-1 and BRACA-2 testing, and this indicates the risk of developing breast or ovarian cancers. And so, the idea is all of us have a BRACA-1 and a BRACA-2 gene. But in some individuals, there is a change in the DNA sequence of the BRACA-1 or BRACA-2 gene; and that sequence may put the person at a higher risk of developing breast or ovarian cancer. So, it doesn't mean that you're going to get it, but it means that instead of having a 5% chance, you may go up to as much as an 80% chance of getting that cancer. Section 2: Types of Diagnostics Summary In summary, in this section, we learned that: ● The major categories of diagnostic tests include blood tests, physiologic tests, anatomic tests, histological tests, and DNA tests. ● Examples include urine pregnancy tests, blood glucose tests, HIV antibody tests, complete blood counts, coagulation tests, X-rays, BRCA tests for breast cancer, and many more. 7 Copyright 2024 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. Section 3: Companion Diagnostics Section 3: Companion Diagnostics Objectives At the end of this section, you should be able to: ● Describe the concept of genetic variation. ● Describe different types of DNA mutations. ● Discuss the use of genetic variation in developing targeted therapeutics and diagnostics. Genetic Variation If we look at this concept called genetic variation, it's kind of reassuring because nobody is identical. In a sense, we are all mutants. So, between all the individuals in a particular room, there is only a .1% difference in the DNA sequence between everybody in the room. And so, this translates into being a one base A, G, T, or C difference roughly, on average, every five thousand base pairs. Because we call these single-base changes, these are given the name "single nucleotide polymorphisms". And again, what that means is, in position 299, you may have an A, and everyone else in the room may have a C. But instead of saying "single nucleotide polymorphism", we call it an "SNP" (pronounced Snip). This difference in DNA sequence between people accounts for subtle differences. Things like eye color and hair color, are obvious, what we call phenotypic, physically observable traits that make us all slightly different from one another. And that's nice, but from a healthcare point of view, these SNPs, these one nucleotide changes that occur between people, have much more profound effects. They can, for example, impact our susceptibility to disease, our response to different environmental factors, whether we’re allergic to latex or not, or our response to different drugs and different vaccines. Genetic Basis of Disease So, if we look at the genetic basis of a disease, here we are discussing inherited. These are diseases where a gene is responsible for the disease, and this gene has been inherited from Mom and/or Dad. We broadly categorize genetic diseases into two classes: monogenic and polygenic. 8 Copyright 2024 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. In a monogenic disease, one gene causes the disease. We know exactly what causes the disease. It's a mutation, a change in the DNA sequence in one particular gene. Examples of this would be sickle cell anemia. And here, we simply have a substitution. It's a SNP. An A is changed to a T, and that one base change in all three billion letters in your body, that's enough to cause hemoglobin to change its shape, and so that it cannot carry oxygen, and so that it forms what are called aggregate, clumps of hemoglobin molecules in the red blood cell, which then causes the red blood cell to become deformed, to change its shape into that characteristic sickle shape. In cystic fibrosis, we have a deletion: three base pairs were omitted during replication. So, three base pairs are absent, and that changes all of the other amino acids in the protein, and their position. And now this particular protein cannot pump the chloride ion into and out of the lung cells. And then, in Huntington's disease, we have what's known as an insertion. Extra nucleotides are inserted into the particular gene. And in this case, we call it a trinucleotide repeat. "Tri" just means "three nucleotides". And in this case, the repeated sequence is CAG. And so, what that means is we have CAG, CAG, and CAG all in a row. And if you have too many CAGs in a row in the particular gene that's responsible for Huntington's disease, then you will get the disease. So, by looking at your DNA sequence, we can tell whether or not you have this sequence, the DNA sequence that makes you susceptible to the disease, or that says you're not going to get the disease. In a polygenic disease, the situation is a little different. Here, many genes contribute to the disease. It's not one gene, but a combination of genes that contribute to the disease. And this is the case with cancer. In some tumors, as many as 50, sometimes as many as 200 genes have an altered DNA sequence that gives rise to the cancer. Same thing in heart disease, same thing in Parkinson's disease, and numerous other diseases. So, the idea here is that these diseases have susceptibility to genes. These are genes that if there is a change in their DNA sequence, the probability of you getting the disease increases. 9 Copyright 2024 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. So, there are two implications here. One is whether this is partially diagnostic, or partially predictive. So, if I have a mutation of the BRACA gene, it says that my risk, my susceptibility of getting breast cancer or ovarian cancer is greatly increased. But it doesn't mean 100% that I'm going to get that disease. It just means the probability has increased that I will get the disease. So, I'm more susceptible to the disease, but there are at least one other, potentially many other genes, that also must be mutated to give rise to the disease. Monogenic Disease: Sickle Cell Anemia Let's look at sickle cell anemia in a little more detail. At the top of the screen, we see the sequence for the DNA, then the corresponding RNA, and then the translated protein product with the amino acids there. If you look at the bottom of the screen we see the sickle cells, we see that second codon, the second set of third bases from the right, and we have CTT to CAT. So, an A has replaced a T in there, and that has changed us from glutamine to valine. So, we've changed one single amino acid in hemoglobin, but that change of one amino acid is enough to change the shape of the hemoglobin so that it no longer stays dissolved in the cell. And instead, it interacts with other deformed hemoglobin to form big clumps, these hemoglobin aggregates, which then ultimately cause the cell to become deformed in its shape, and form the sickle cell. And now those sickle cells do not roll easily through the blood vessels. They collide with one another where we have branch points in the blood vessels. Then those red blood cells break apart, they release iron, and that causes damage to the blood vessel that it's near. Polygenic Disease: Cancer Cancer is an example of a polygenic disease. Cancer is a disease that, in many cases, evolves, frequently due to exposure to something in the environment. We're starting here with normal cells. That is, none of the cells have any kind of damage to the DNA that would cause them to grow out of control. 10 Copyright 2024 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. We get what's called an "initiating event". An initial mutation. This could be exposed to environmental pollutants. This could be exposure to radiation, X-rays, radon, or something like that. Or it could be something like cigarette smoke. But in any case, one of the cells gets an initial mutation. That is, there's a change in the DNA sequence, and this would be a change in the sequence of DNA in a gene that controls cell growth. So, this mutated gene causes one cell to begin to divide a little faster than all of the surrounding normal cells. So, that cell has a growth advantage. And initially, this won't be noticeable. Maybe the cell divides a couple of hours sooner than any of its nearby neighbors. So, for many years, you won't even notice that anything is happening. But over time, this cell that has the initial mutation will then acquire a second mutation. And right now, this is believed to happen randomly. And so, just because the cell is dividing faster, it has less time to repair any mistakes it makes in copying the DNA, and so, we get a second mutation. The second mutation could either make the tumor cells grow even faster, or it might do something to ensure their survival. But in any event, over time, we get more and more mutations. And so, where we may have started with a single initial mutation, we now have many different mutations. Many genes are now mutated, and so we have a polygenic disease. It's interesting to note that by the time we have multiple mutations occurring that our population of tumor cells is not necessarily identical. And so, you can see here in the illustration, we have two different shapes of tumor cells. And that's because not every mutation is identical. So, we may have two, three, or four different new populations of tumor cells, each having its subset of unique mutations. And so, that's what makes this difficult to treat, because while one therapy may kill one population of tumor cells, the other may survive. Ultimately then, this morphs into the tumor that we can detect through imaging. Diagnostics: Selecting a Treatment More and more, in the course of modern medicine, diagnosis is not only about detecting the disease but also in determining what the best course of therapeutic intervention is. That is, do we use chemotherapy? Do we use radiation? Do you use a biological molecule? So, frequently, the diagnostic is a companion to the therapeutic. 11 Copyright 2024 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. As we've established, not all breast cancers are the same. Some tumors may respond to one type of treatment. Other tumors respond to a different type of treatment. So, we'd like to know exactly what type of cancer we have. Same thing if we have an infection. If it's a viral infection, we're not going to use antibiotics. Certain bacteria are susceptible to certain types of antibiotics. So, knowing, 1. Whether it's a bacterial or a viral infection, and 2. What the nature of the bacterial infection is, helps us to select the appropriate course of treatment. HER2 Receptor Causes Cells to Divide A fantastic example of a companion diagnostic is a diagnostic that is performed to determine if Herceptin, a treatment for breast cancer that targets the HER2 protein, will be successful. To understand this diagnostic, we must first understand what HER2 does and how its overexpression leads to breast cancer. HER2 stands for “human epidermal growth factor receptor”. So, growth factors and growth factor receptors interact with one another to control cell division. A growth factor comes in and binds to the growth factor receptor and that then results in the growth factor receptor sending a signal to the cell to divide. The growth factor interacts with the growth factor receptor and that triggers cell division. HER2 Over-Expression Leads to Tumor Growth You can see that if we have one growth factor receptor, the cell won’t divide very often. But if we have hundreds of the same growth factor receptor present on the surface of the cell, the cell is going to get a continuous signal to divide and it’s going to keep dividing even when it shouldn’t just because it has too many growth factor receptors, and there’s too much growth factor interaction. Customizing Therapy: Herceptin Herceptin is an antibody that recognizes HER2, the growth factor receptor, and binds to it. And you can sort of think of this as the growth factor interacting with the growth factor receptor is much like a key interacting with a lock. The growth factor is the key, and the growth factor receptor is the lock. For anything to happen, the key has to be inserted into the lock. That is, for the cell to divide, the key has to be inserted into the lock. 12 Copyright 2024 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. So, if I block that interaction from occurring if I prevent the growth factor from binding to the growth factor receptor, then the cell will not get a signal to divide. It will stop dividing, and the tumor will then, at least, not get any larger. And so, this is exactly what the antibody Herceptin does. Companion Diagnostics: Herceptin As this therapy was brought out and used in patients, it turned out that three categories of patients responded to the HER2 or did not. In the first case, we have what we call "HER2". And all that means is that when we did not do the biopsy and checked for the presence of over-expression of HER2. These people did not over-express HER2. Even though they had a breast tumor, they did not make extra copies of the HER2. So, treating them with Herceptin will not work because there's nothing for the Herceptin to bind to and cover up. Their tumor is being caused by something else, so we won't even treat those people with Herceptin. We'll go to more traditional radiation and chemotherapy treatments. Then we have patients who are HER2+ and many of these people respond, but sometimes the response is temporary, or sometimes it’s just a maintenance therapy. And then we have the third category, which is HER2+ and they also express or make another protein called PTEN, which is a tumor suppressor protein. And if the patients have both of these proteins present, then they are much more likely to have a good outcome. Companion Diagnostics: Choosing a Dosage Another application for diagnostics is using diagnostics to choose not only the correct medication but the correct dosage for a particular medication. And this is a pharmacogenomics example, and it deals with a class of enzymes found primarily in your liver called CYP enzymes. And CYP stands for cytochrome P450s. Cytochrome P450s are enzymes that naturally evolved to break down small molecules. They are here in your body to protect you from toxins. There are 50 to 100 different cytochrome P450s in your liver. Each one is designed to break down different classes of molecules. So, for any drug that you take, there's a P450 to break it down. 13 Copyright 2024 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. While we all have P450s, the P450s are slightly different from one person to the next. And what that means is that for some of us, our P450s are very efficient, and they easily metabolize or break down the drugs that we take. And for others of us, they are not so efficient, and it takes longer to break down the drugs. The drug stays in your system longer, effectively giving you a higher dose. So, there is an impact here that, depending on which particular version of a P450 you have, that is, which SNP you have, there is an impact on both the drug safety and the drug efficacy of this. Let’s look at, for example, a particular type of cytochrome P450 called 2D6. The numbers there just refer to a particular class of P450s. There's an A, B, C, D, and E class. And then within each class, there are different families. So, there's 1A, 2A, 3A, and so forth. And the same thing with the D's: 1D, 2D, 3D, and so forth. And then the 6 is a particular subtype of that. 2D6 is a type of P450 that will break down medications that are used to treat depression. The idea here is, for antidepressant medications, and even for blood pressure medications, it frequently takes a long time to get to the right dose. The idea is, if you are suffering from depression, and you get treated with an antidepressant, if you're a good metabolizer, that is, if you break down the depressant very quickly, then you'll need a higher dose because the P450 will break down what you're being given, you'll eliminate it quickly, and you won't get a therapeutically effective dose in your bloodstream to treat the depression. And so, we need to increase the dose. On the other hand, if you're a poor metabolizer, if your P450 doesn't break down the medication very quickly, then you'll have an elevated dose, and this may have some unwanted side effects. For example, you're very, very lethargic; you may even be a little bit dizzy. And so, we need to give you a lower dose. Normally, the dosage is just figured out by trial and error. They start you off on the normal dose. If it doesn't work, they give you a little bit more. If it makes you too tired, they give you a little bit less. 14 Copyright 2024 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. With diagnostics, the idea is that we could, by looking at the particular DNA sequence of the P450 you have, determine if you are a good metabolizer, a normal metabolizer, or a poor metabolizer. Just by looking at the DNA sequence of your P450. Section 3: Companion Diagnostics Summary In summary, in this section, we learned that: ● SNP changes between individuals can have a profound impact on their susceptibility to disease, their response to different environmental factors, or their response to different drugs and different vaccines. ● DNA mutations may cause disease because they can change proteins’ structure and function. ● By characterizing different genetic variations and their association with diseases, scientists can develop customized diagnostics and therapeutics for patients. 15 Copyright 2024 Biotech Primer, Inc.