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What are the difference between a diagnostic, a prognostic and a therapeutic/predictive biomarker? Compare and contrast the following four analytic measures: precision, accuracy, sensitivity and specificity. Is there a clinical situation in which specificity is more important than sensitivity? Why?
A biomarker is a biological product that can consist of a cell type, protein, or other macromolecule that exists as a hallmark of a particular disease. Diagnostic biomarkers can diagnose a particular disease or ailment because in many cases, diseases cause the release of specific compounds and metabolites, or may alter the physiological concentrations of compounds that exist naturally. For example, the diagnostic biomarkers monocyte chemoattractant protein 1 (MCP-1) and the macrophage inflammatory protein 1a (MIP-1a) allowed for the diagnosis of chronic prostatitis / chronic pelvic pain syndrome (CP/CPPS). Scientists determined with a 90% accuracy that MCP-1 concentrations exceeding 704 picograms (pg) per mL and MIP-1a concentrations greater than 146 pg/mL were indicative of CP/CPPS.
Prognostic biomarkers are used to determine the course and progression of a particular disease and determine the level of risk an individual has of contracting it. A testament to the efficacy of prognostic markers is the use of c-Met (receptor for the hepatocyte growth factor (HGF)) in determining the progression of Hepatocellular Carcinoma (HCC) in patients afflicted with the disease. Specifically, it is used to determine properties of HCC, where it was determined that patients who have high levels of c-Met/HGF are at high risk for developing metastatic HCC. It has since been used as a therapeutic target in patients who have metastatic HCC.
Therapeutic biomarkers determine how the body is responding to a particular treatment. Circulating levels of various biomolecules can represent disease progression, or remission; ultimately the therapeutic biomarker determines the causal relationship between a treatment and the effect of that treatment on an afflicted patient. In one case in particular, the LDL molecule is a therapeutic biomarker that can be used to indicate the efficacy of statin drugs in patients suffering from familial hypercholesterolemia.
Accuracy and precision are two terms that describe the ability of a diagnostic test to idenitify. Accuracy refers to the percentage of cases that the test records the correct diagnosis per individual, and it can be measured by the area directly under the receiver-operating characteristic (ROC) curve. Precision, on the other hand refers roughly to the number of times that a test will vary in its prediction, i.e. repeatability and reproducibility. A test that is very accurate has a high additive value of true positives and true negatives relative to the total number of positives and negatives (both false and true). A test that is precise will exhibit marginal variation between test results, based on the patients. Accuracy can be determined based on the sensitivity, specificity, and prevalence. Precision refers to the positive predictive value and is correlated with sensitivity.
Sensitivity and specificity are two terms that determine the efficacy of diagnostic tests in disease detection within a clinical setting without a high rate of false negatives and false positives, respectively. Diagnostic tests that are highly sensitive are able to correctly identify the majority of patients that have a particular disease, but only minimally fail to detect the disease in patients who are afflicted (false negative). Tests that are highly specific are able to identify the vast majority of individuals who do not have the disease, while only minimally detecting the disease in patients who are not afflicted (false negative). Ideally, diagnostic tests should be both highly specific and highly sensitive, but there seems to be a bit of a trade-off between the two.
Most often, it is better to have a test that is more sensitive than specific, so that there is a much lower proportion of false negative patients than false positive patients - this is especially true for diseases that have late diagnoses and grim outlooks, such as ovarian cancer. For many other diseases, it is better to have a lower false positive diagnosis, especially in cases where immediate intervention is necessary. For these cases it is beneficial to implement a test that is highly specific following the test that is highly sensitive, so that there is a much lower risk of false positive diagnoses compared to false negative diagnoses.
2. What are the differences in the information provided by the following three major classes of molecules: DNA, RNA, and Proteins? List two technologies that can be used to measure each one of these molecules, and their strengths and weaknesses.
DNA assays can be used to reveal mutations in genes responsible for maintenance of cell integrity. Mutated tumor suppressor genes, oncogenes, and DNA repair genes can cause problems that can eventually lead to cancer development. Using SNPs as a guide, scientists can identify genes containing mutations that will lead to defective gene products. Ideally, patients can be screened based on their DNA profiles to determine which (if any) genes are non-functional that may pose a health risk. Moreover, if a defect exists in a gene that results in poor metabolism of certain toxins, it can be detected before it is too late.
RNA can be used to analyze gene transcription in certain types of cells - it can be used specifically for determining which genes are being transcribed in cancer cells, and possibly help in the development of novel anti-cancer drugs. Specifically within the transcriptome RNA biomarkers can be used to identify genes that are hyperactive in terms of transcription rates and provide prognostic insight and data in patients that have various diseases. Information can be obtained to assess risk and stage determination.
Various assays can be used to determine level of functional protein - this can be especially important in determining how much protein is created or destroyed as a result of a cellular process, either caused by disease or otherwise. Protein biomarker studies can be used in diagnostic, prognostic, and therapeutic applications.
One method to detect DNA biomarkers in the body is to amplify its concentration by performing polymerase chain reaction (PCR), and real time PCR enables scientists to quantitatively measure the levels of DNA biomarkers in solution. Following PCR, gel electrophoresis can be performed in order to separate DNA based on size and weight. The greatest benefit of PCR/electrophoresis is that is a very low cost and speedy method of amplifying target DNA. However, the biggest drawback is that contamination is a major concern. Due to the fact that any ambient nucleic acids can be amplified through PCR, great care must be taken to prevent any cross contamination. Another way to detect DNA is to use the Southern Blot to probe for a specific sequence of DNA within a sample - it can even be used to detect a small fragment of DNA within an entire genome. Southern Blot is particularly useful in producing knockout mice, or knockout stem cells. Since it has been largely replaced by PCR. However, Southern Blot is a complex and time consuming process that only analyzes one fragment of DNA at a time. Even though it provides detail about the presence and location of DNA, it does not provide any information about how the DNA is regulated, nor does it provide information about how it interacts with other genes.
RNA can be measured by utilizing reverse transcriptase-quantitative PCR. This is similar to general PCR with DNA, except that it uses RNA reverse transcribed to cDNA and that it measures the amount of amplification per cycle. First RNA must be extracted from the cell in order to be analyzed. Then the RNA is reverse transcribed into cDNA and hybridized with a target probe, ideally from a gene of interest The benefits of this method include the ability to quantify RNA from tiny sample sizes- the amount of RNA within a single cell would be enough to run the assay. Furthermore, the assay is incredibly accurate and reliably detects levels of gene expression and can diagnose diseases on the basis of biomarkers. One of the main drawbacks however, is that the RNA used must be incredibly pure, which can be detected using a spectrometer - the purification protocol is very meticulous, with the smallest mistake resulting in failure. Furthermore, RNA is extremely unstable and genomic contamination is always an issue.
Another way to detect and locate an RNA sequence is by Northern Blot, whose protocol is virtually identical to that of Southern Blot, except that RNA is used rather than DNA. The same disadvantages exist for Northern Blot that exist for Southern Blot, and the instability of RNA and genomic contamination associated with this procedure add another obstacle. Furthermore, it is not as accurate or sensitive as RT-qPCR. One advantage in using Northern blot is that only partial complementarity is necessary for probes to be effective. It is very effective in determining the size of transcripts and in the detection of transcripts that may be alternatively spliced.
One method of detecting specific proteins is by utilizing the Western Blot. First the proteins within the sample are separated using gel electrophoresis (SDS-PAGE) and then transferred to a thin membrane to be reacted with three solutions: a blocking solution, primary antibody solution, and secondary antibody solution. Finally, the secondary antibody in the membrane reacts with a final solution in order to reveal the protein of interest. Enzyme-linked immunosorbent assay (ELISA) is specifically designed to detect and measure levels of proteins of interest. In this procedure, an antigen is immobilized to a solid surface and hybridized with an enzyme-linked antibody. When this enzyme is incubated with its substrate, detection of the antigen is measurable. The antigen-antibody hybridization must be extremely specific.
Western blot is a protocol designed for denatured proteins, while ELISA works on native proteins, and cannot reveal any data regarding protein purity. Furthermore, ELISA measures concentrations of correctly folded protein, which may be either beneficial or detrimental. However, Western Blot can only be used on one protein at a time and is very time consuming, while ELISA takes less time and can be used for competition and interaction assays. Elisa also consumes less antibody than Western Blot. Furthermore, ELISA can determine the sensitivity of a protein to temperature - at which point it denatures - a feature not shared by Western Blot.
3. Define nanotechnology. Provide three examples of how nanotechnology can be applied to diagnosis, imaging and therapy.
According to the Center for Responsible Nanotechnology (CRN), "nanotechnology is the engineering of functional systems at the molecular scale." In 2000, former President Clinton pushed for the creation of the National Nanotechnology Initiative (NNI). Nanotechnology is so vital to our understanding of physiological processes because most biological and cellular machinery is genetically designed on the nanomolecular level. Therefore, in order to implement successful therapies scientists must be able to do so on the same level. When the research has matured, nanotechnology would be applicable in diagnostic techniques, drugs delivery, and prostheses. Currently several diagnostic techniques have been developed that utilize nanotechnology. Medical sensors are able to aid in the preparation of pharmaceutical grade chemicals that are entirely pure. Furthermore, nanotechnology will apparently be able to sequence whole genomes of humans within seconds. Diagnostic tests that exist on a wafer chip are able to rule out diseases with high sensitivity and specificity. The wafer chip concept is known as "lab on a chip", where tiny amounts of liquids and gases are mixed in reactive cells, and their products are analyzed immediately. One example of this is the Field effect flow switching device known as FLOWFET, which utilizes 100 Î¼m x 25 Î¼m channels and an a 1.5 million (106) V/cm perpendicular electric field in order to regulate osmosis.
One example of biomedical nanotechnology would be a drug-delivery apparatus. This would be most beneficial for delivery of anticancer drugs. One implementation of nanotechnology involves the use of nanotubes and C60/70 Buckyballs. In one example, the anticancer drug paclitaxel was placed in ABI-007, a 130nm protein stabilized nanoparticle and delivered to the target site with success. Another nanoparticle known as N-isopropylacrylamide (NIPAm) complexed with a bait compound would be able to gather up biomarkers within bodily fluids, which would otherwise be undetectable due to low concentrations. This concept has been implemented in testing for hGH in urine samples and for IL6 in sweat samples. This is very beneficial for diagnostics because currently mass spectrometers are only able to detect samples on the nano- scale, and some biomarkers exist on the femto-scale. These biomarkers would otherwise be undetectable, but by gathering enough biomarker through a nanomolecular sieve in order to run mass spec, scientists would be able diagnose diseases much more rapidly. Another application of nanotechnology would be the development and implementation of a skin patch that recognizes proteins and metabolites that would otherwise be too low in concentration to detect. These metabolites and proteins would be amplified and stabilized to protect from degradation. Furthermore, a field test could be implemented for soldiers and occupational hazard workers in order to provide treatment on the spot. One implementation of field test that proves to be innovative is the ability to have a cd player diagnose diseases. Essentially, the molecule would scatter the laser beam, and the scatter pattern would be recorded as different type of music.
4. Define individualized therapy. How is individualized therapy different from conventional therapy? Provide an example of a disease that is now being treated by individualized therapy. Explain, for this disease example, why individualized therapy is expected to provide improvements in the treatment outcome? For this example what is the molecular marker that is used to decide who gets the individualized therapy?
Individualized therapy is a concept that involves using a targeted approach unique to each individual, in order to promote greater treatment efficacy with the lowest toxicity potential. Conventional therapy involves treating all patients suffering from a particular disease in the same way. However, the problem arises when the same disease manifests in patients via different pathways.
Colon cancer is one example of a disease now treated by individualized therapy. By using the UGT1A1 biomarker doctors can determine whether or not the aforementioned gene is intact, and if so then the drug irinotecan (trade name Camptosar) can be administered. Another example involves the drug bendamustine (an anti-tumor drug used in the treatment of Non-Hodgkin's Lymphoma), which can cause toxic side effects in patients lacking a functional copy of the gene CYP1A2. CYP1A2 codes for an enzyme which causes breakdown of harmful metabolites, so by checking for a CYP1A2 biomarker, doctors can avoid prescribing bendamustine and related compounds until it has been ascertained that the patient has a functional CYP1A2 gene. In this case, a proteomic biomarker can be used in order to determine whether or not the gene produces a functional protein, or a metabolite biomarker can be used to detect levels of bendamustine breakdown derivatives within the body.
Patients with Non-Alcoholic Fatty Liver Disease (NAFLD) could benefit from the implementation of individualized therapy. NAFLD often occurs as a result of metabolic syndrome, a combination of various physiological problems including diabetes, dislipidemia, hypertension, and obesity. Over 75% of individuals who are obese also develop NAFLD. In roughly 10%-20% of cases NAFLD progresses to Non-Alcoholic Steatohepatitis (NASH). In NAFLD development, an increase in free fatty acids (FFA) circulation leads to steatosis, which eventually causes an increase in the production of reactive oxygen species (ROS) and necroinflammation. In humans, over a thousand genes code for the expression of miRNAs that exert their effects on other genes through RNA interference (RNAi). miRNAs function to regulate gene expression and are able to inhibit the translation of complementary mRNAs; there are 700 known miRNA that have been discovered to regulate of up to 30% of the human genome. Current studies indicate that there may be a link between miRNA expression and the progression of NAFLD. In particular, a previous study completed in our lab indicates that a change in expression levels of a number of human miRNAs accompany the development of NASH and the fibrosis of the liver.
In this case the specific miRNAs would function as the biomarker, and their levels can be analyzed using RT-qPCR. If the levels of miRNA are elevated relative to healthy individuals, the treatment for NAFLD/NASH can begin. Furthermore, this treatment would be most effective in patients who had not already been diagnosed with either NAFLD or NASH. The problem with this type of biomarker is that it would only be present in pathways between adipose tissue and liver tissue.
5. Explain the stages of cancer invasion and metastasis. Pick one type of cancer (e.g. breast cancer). Define the staging of this example cancer. What is the expected survival probability at each stage? Describe physiologic, biochemical, or physical ways that cancer can contribute to the death of the patient.
When a primary tumor forms in one part of the body and is not benign, there is a risk of it metastasizing to other parts of the body. Typically, when a primary tumor forms there is a high level of cellular activity, partially due to instability of its genome. Therefore, many processes are upregulated, requiring additional levels of nutrients. The tumor obtains these nutrients via angiogenesis, accelerated by the tumor's release of growth factors (such as VEGF). Once angiogenesis begins, the tumor has ready access to the circulatory and lymphatic systems, and it sloughs some of its cells into the circulatory system to set up secondary tumors. Furthermore, certain mechanical factors could potentially increase the rate at which cancer can metastasize. The metastatic cells must be able to move and then implant themselves elsewhere so both transport and viability are factors. Factors that would increase transport of these cells throughout the body and promote second-site implantation.
Prostate cancer is one of the most common forms of cancer to afflict men in the United States. There are four stages of prostate cancer. Stage I and II are both intraprostatic, and only stage II can actually be detected via the digital rectal exam. Both stage I and II can be detected using a prostate specific antigen (PSA) test, though it is very difficult to diagnose stage I. In stage III of the cancer, the prostate cancer cells may have metastasized to the neighboring seminal vesicles, but the damage does not typically extend beyond this point. In stage IV, the cancer has spread to other organs, most notably the bone tissue. There is speculation that prostate and bone stromal cells may hold the key as to why prostate cancer is so quick to spread to bone. Stage IV prostate cancer also readily invades the lymphatic system. In terms of nodal involvement, the cancer receives a score of N1 if the nodal involvement is less than 2cm in diameter, a score of N2 if the nodal involvement is between 2cm and 5cm, and a score of N3 if the nodal involvement is greater than 5cm. The metastasis score clearly shows that bone metastasis occurs just after lymph metastasis.
Cancer can contribute to death of the patient in many ways. There can be various physiological changes - for example, patients with pancreatic cancer may have sudden mood swings, problems with appetite, and weight loss. This is due to the pancreas having a rather large role in digestion, releasing several enzymes into the duodenum. When pancreatic cells become cancerous, they may have trouble releasing their hormones, or may release too many. In some pancreatic cancer cases, patients may develop diabetes, due to improper function of the Î²-acinar cells which release insulin. However, pancreatic cancer is only one example of how cancer contributes to death. Hepatocellular carcinoma (HCC) can cause liver cells to fail, and perhaps cease their detoxification activity. This is especially evident in people who progress to HCC from NASH-induced cirrhosis. Furthermore, osteosarcomas can cause bones to become brittle and break, therefore inhibiting motion and other key functions. This is made worse by the fact that osteosarcomas are second-site metastases of many different types of cancers, such as ovarian cancer and prostate cancer. Even if the cancer does not spread to other tissues, if the integrity of the organ in which the primary tumor resides is damaged, a function necessary to life is destroyed. This is especially true of exocrine organs, such as the pancreas, thyroid, pituitary gland, etc.