Biomarkers+—+The+Future+of+Cancer+Detection.

=Mission Statement=

toc The focus of this Wiki is dedicated to the importance of biomarkers as both a diagnostic as well as a therapeutic tool in the ongoing battle against cancer. Our intent is to present how biomarkers are currently being developed and incorporated as a means to detect and assess the presence of cancer. At the same time we will present how the innovation and power of biomarkers will be harnessed to aid in the actual treatment of various cancers.

=Introduction To Biomarkers=

Cancer is the second leading cause of death in the United States. Sadly, many of these deaths are the result of late detection due to the elusiveness of diagnostic symptoms which only manifest when the tumor has developed into the late stages; which is often common with various types of cancer such as pancreatic cancer. Currently there are only a few methods for diagnosis and prognosis, but they are either invasive or are nondiscriminatory, meaning the treatments effects both normal and cancerous cells. Biomarkers are defined as "a measurable substance in an organism whose presence is indicative of some phenomenon such as disease, infection, or environmental exposure." (dictionary.com). Biomarkers will offer less invasive means for dignosis and prognosis. Biomarkers not only can be implemented to identify the presence of tumors at a very early onset, but they can also be used as a way to assess if the patient is responding to various cancer treatments by being able to visualize the the tumor(s). Biomarkers are more then just a means for targeting and visualizing cancerous cell masses since they can also be used to aid in the actual treatment of cancer. For example, biomarkers can act as a way to guide effective therapeutics directly to the cancerous cells leaving normal cells unharmed. Though there are a wide array as to what can be considered a biomarker for cancer, our focus will be on epigenetic biomarkers, such as DNA methylation patterns, and various cell surface receptors.

=Known Natural Biomarkers: DNA Methylation=

Biomarkers Through DNA Methylation
In our genome, one form of regulated gene expression is achieved through DNA methylation. DNA methylation is the presence of a methyl group attached to a cytosine in CpG island promoters of regulated genes. DNA methyltransferases is responsible for placing these methyl groups onto the newly copied DNA. By attaching a methyl group to a CpG island promoter, the methyl group serves as a "padlock" on the transcription of the gene, effectively silencing the gene. An example of the effect of a silenced gene is an experiment that involves fat yellow mice with the agouti gene that is responsible for their phenotype. By altering the pregnant mothers' diet that induces methylat ion of the agouti gene, the silenced gene results in small brown offspring (9). For a simple understanding of DNA methylation in cancerous cells, we decided to concentrate on pancreatic cancer as an example. Pancreatic cancer's late onset of symptoms prevents an early prognosis and surgical removal of the tumor. The cancer cells make use of the hallmark of evading growth suppressors; tumor suppressor genes are hyper-methylated, thus they are silenced. However, we can make use of the hyper-methylation of the cancer cells to our advantage: DNA methylation patterns can be detected through a methylation-specific PCR (MSP) analysis[|(10)].

Besides MSP, there are many classical clinical ways for the scanning of pancreatic tumors not limited to ultrasound, computed tomography scanning, MRI, endoscopic ultrasound...etc. However, many clinical scans are invasive, often involving the physical intrusion of a endoscope into the body. As with any imaging techniques, there are limitations. With the intake of certain biomarkers, the first few screenings may show accurate scanning. Over time however, the body may adapt to the presence of these biomarkers, causing either a false positive or negative test result. How does one determine whether if the chemotherapy is working if the results are misleading? There are other tests that analyze the samples from the patients, such as cytopathological assessment to accompany the scanning results. For cytopathological assessment, the cells must be intact to provide an accurate reading. Since we are concentrating on the cancer of the pancreas, there are pancreatic proteolytic enzymes in the pancreatic juice. The chances of obtaining intact cells for an accurate reading are not reassuring [|(10)]. So rather than introducing something new to the body and the cancer cells, we can make use of what is already present, the methylation of the CpG islands.

How Hypermethylated Sequences Can Be Used For Cancer Treatment As Part Of Early Detection:
An article by Fukushige and Horii [|(4)] offers 3 reasons why DNA methylation is a promising biomarker:

1. Incidences of aberrant DNA methylation of specific CpG islands are higher than those of genetic defects.
 * There are many CG base pair patterns scattered throughout the human genome. However, CpG islands (repeated sequences of CG base pairs) that are methylated are found only in specific promoters. The methylation of CpG island promoters are tightly regulated by DNA methylatranferases. CpG island promoters are usually unmethylated if the gene was expressed. However, cancerous cells over express DNMT1 (specific DNA methyltransferase) that lead to a gain of function, causing hypermethylation to occur on CpG island promoters. Hypermethylation of CpG island promoters usually lead to silenced or null tumor suppressor genes.

2. Detection of aberrant DNA methylation is technically simple; it can be detected using MSP.
 * Traditional PCR restriction enzyme digests were used to determine hypermethylation patterns. MSP (methlyation specific PCR) offers a more secure and efficient method of testing. MSP does not use restriction enzymes that may lead to a false negative if the enzyme was not successful at cutting the DNA, rather it utilizes the CpG islands (cytosine base) to determine methylation by converting unmethylated cytosines to uracil. After the conversion, through PCR of the bases on DNA can be used to determine methylation.

3. Aberrant DNA methylation seems to occur in early-stage tumors, causing loss- and/or gain-of-function of key processes and signalling properties.
 * If aberrant DNA methylation can be detected in the early-stages, then early detection leads to a higher chance of surgical removal of tumor and can be used as a predictor of when the tumor may metastasize.

**Detecting Cancer By MSP **
As a review, CpG island promoters that are methylated are silenced. Cancerous cells are usually observed to have hypermethylation of genes that result in silenced tumor suppressor genes. Usually, the hypermethylation is due to an excess expression of DNA methyltranserase which is responsible for methylating the promoters by attaching a methyl group to a cytosine base. Rather than using traditional restriction enzymes to cleave distinguished methylated from unmethylated DNA for PCR, MSP (Methylation specific PCR) modifies DNA with sodium bisulfate which converts all unmethylated cytosines to uracil (8). The sodium bisulfate does not affect methylated cytosines, allowing clear distinct comparisons of methylated vs unmethylated DNA.

By converting the cytosine base, we can distinguish the methylated and the unmethylated by PCR. The conversion of unmethlyated DNA base cytosines to uracil are then amplified and copied through PCR. The DNA is then run on gel. On the gel, one would distinguish methylated genes by the presence of cytosine bases and unmethylated genes by the presence of thymidine (after PCR, uracil is read as thymidine). Since there are only specific CpG island promoters in the genome, one doesn’t have to worry if the comparison of thymidine will be invalid with unrelated thymidine bases. Another benefit of using MSP is that you don’t need a lot of DNA samples. As little as 1µ g of DNA would work for MSP (8). By testing the DNA sample directly, such as with stool testing, biomarkers are not introduced to the body through invasive means (penetrating or entering the body). This means that the body is not given a chance to adapt to the biomarkers that may interfere with testing by producing false positives after a number of certain trials. However, with stool testing there are limitations with the types of cancer. Stool samples only apply to cancers that are involved in the digestive system. Brain cancer MSP testing must involve other methods to obtain DNA samples. Traditionally, researchers would distinguish hypermethylation through Southern hybridization approaches. Southern hybridization uses restriction enzymes to cut the DNA and the sample would be run on a gel. However, the restriction enzyme must be recognized as a methylation sensitive enzyme and is limited to the known methylated gene.

With MSP, all CpG island promoters are examined, not just within regions where the restriction enzymes recognize in the DNA (8). Besides the utilization of restriction enzymes (which is not 100% effective), it requires large amount of DNA (5 ug or more) (8). Since PCR with restriction enzymes are not perfect, the uncleaved DNA will turn up as false negatives for methylation. An example is when the DNA is really methylated but since it was not cut, we would have a false negative. MSP does use restriction enzymes however. The difference is the conversion of all unmethylated cytosine to uracil. There are special primers that will not recognize "unmodified" DNA, so there are no false positives for methylation.

MSP allows for specific probing for a certain methylated gene that is commonly abundant in the cancerous cells. The process does not rely on restriction enzymes. The methyl group on methylated DNA prevents the conversion of cytosine to uracil. After conversion of uracil, DNA is amplified through PCR (in this process, uracil is read as thymidine in DNA). An experiment by Herman et al. (1996) tests how effective MSP process was in distinguishing methylated DNA and MSP analysis for a tumor suppressor gene p16 (8). Researchers obtained DNA samples from renal carcinoma cell line and normal tissue as a control. Bisulfite Modification was introduced to DNA which was later re-suspended in water. For means of comparing accuracy, genomic sequencing of both renal carcinoma DNA and normal tissue DNA was performed after the bisulfite modification. The absence of cytosine represents unmethylated cytosines in CpG islands in the DNA. Figure 3 is the genomic sequencing of p16. On the gel, H157 has cytosine bases, indicating the presence of methylated DNA, as expected from the renal carcinoma DNA. H249 is the normal tissue control and shows no methylation on DNA due to the absence of cytosine bases (converted into uracil and read as thymidine). Instead, it is observed to have more thymidine bases than H157. The genomic sequencing allows for the comparison of accuracy of MSP now that expected methylation is obtained. The brackets on the gel indicate a BstUI site where the enzyme will cut for p16 (specific gene probing). The DNA samples were amplified by PCR, were cut by BstUI restriction enzyme, and run on a gel.



For simplicity, we will concentrate on Figure 6a and d. Figure 6a shows MSP results of H157 (renal carcinoma), untreated DNA (without bisulfite modification, normal lymphocytes and H249 (normal tissue control). The DNA in 4a were treated with bisulfite modification and were amplified with PCR. The DNA was not cut with BstUI. Due to modifications of the DNA bases, there are two primers used: methylated & unmethylated specific primers and primers specific to wildtype p16 sequence. The methylated & unmethylated specific primers will only interact with modified DNA (DNA reaction in the presence of bisulfite, even if the DNA is not modified to a uracil). The specific primers for p16 sequence will interact with unmodified DNA (DNA with no bisulfite modification). The primers are selective and will only interact with their specific DNA sequences conditions. The reason the experiment had both methylated and unmethylated specific primers is due to both hyper and hypo methylations in DNA found in cancerous cells. U1752 is lung cancer cell line methylated control; it was a known hypermethylated gene of p16. We see two samples that indicate methylation (under M). H157 is the experimental renal carcinoma, from genomic sequencing we know that it is methylated. We see two bands that indicate methylation. There is also a weak amplification under wild type (under W), indicating incomplete bisulfite reaction. However, this is not a false positive for methylation because the DNA in H157 that has not been modified will not be recognized by methylated specific primers and will not interfere with methylation sensitivity of MSP. Untreated DNA is DNA without bisulfite modification to test if MSP is methylation sensitive by interaction with the modified DNA (to assure no false negatives of methylation by incomplete reaction of bisulfite). We see one band under wild type, indicating that MSP is methylation sensitive. H209 is a unmethylated control (a case of hypomethylated cancer cell line), where we see two unmethylated bands (under U). Lastly, H249 is out control tissue which yield similar results as normal lymphocytes, indicating p16 normal methylation sequence (8).

Figure 6d is the bisulfite modification with restriction enzyme BstUI digest. The purpose of 6d is to indicate that the specific restriction enzyme is only sensitive to methylated promoters. The (+) indicates the presence of BstUI enzyme, while the (-) indicates the absence. From H249 sample which is unmethylated, the presence of the enzyme does not show any differences. However, for H157 which was established to by hypermethylated, the presence of the enzyme is depicted through the result of two small bands. The two bands are the product of the recognition of the BstUI site CGCG. Recall that if the DNA is unmethylated, then the site would be converted into TGTG after PCR. BstUI will not longer recognize the site and the DNA will not be cleaved into two. As for the primary lung cancer sample, there is no difference under unmethylated regions but under methylated regions, two bands are observed. With the restriction enzymes that are sensitive to methylation, CpG islands that are embedded in DNA can also be detected after digest. A final note is MSP does not need genomic sequencing. The only reason the researchers who test this method had done genomic sequencing was to use as a control to compare MSP results. One benefit of MSP is that it does not use radioactivity and its not expensive as genomic sequencing (8).

**MSP and Stool Sampling **
In regards to detecting DNA methylation through noninvasive means, DNA methylation patterns can aid in diagnosis of cancer in external samples such as stool. An experiment done by Kisel and colleagues assessed DNA methylation marker candidates that will aid in diagnosis of pancreatic cancer (12). The target genes are EYA4, MDF1, UCHL1 and BMP3. Stool and tissue sample was obtained from patients with pancreatic cancer. These samples were run through a gel by MSP and compared with normal controls.

In the figure, EYA4 had a higher concentration of methylation in pancreatic cancer stool sample than the control. However, due to a lot of noise, there was no significant difference to indicate whether the sample is cancerous. For MDF1, there is no significant difference. In UCHL1, there is a hypomethylation of the target gene in the pancreatic stool sample. But this target gene could not be used as a biomarker because the amount of methylation did not match the tissue sample. The tissue sample (from the source) is more accurate than the stool sample in terms of the presence of these target genes. For BMP3, there is a significant difference in hypermethylation in pancreatic stool sample than the control. The tissue sample also shows a significant difference.

BMP3 is bone morphogenic protein 3 that plays a role in tumor suppressor genes. From this experiment, BMP3 is hypermethylated in pancreatic cancer samples (tissue and stool). By targeting this gene alone for hypermethylation, 51% of pancreatic cancer patients were detected. When used in combination of target mutant KRAS (oncoprotein that is hypomethylated), gives a more accurate diagnosis of the pancreatic cancer. Hypomethylation of KRAS detected 50% of pancreatic cancer patients. Together, these biomarkers detected 67% of pancreatic cancer patients. The take home message is that these biomarkers that detect hypo or hyper methylation can aid in diagnosis in combination of other means of detection such as clinical scans.

**DNA Methylation Indicates Tumor Prognosis **
The detection of aberrant DNA methylation in the CpG islands is one of the most common molecular characteristics of cancer cells. The hyper-methylation of CpG islands promoters are associated with the over expression of DNMT1 (DNA methyltransferase) [|(6]). Utilizing that idea, the researchers performed an experiment by performing a bacterial artifical chromosome array-based methylated CpG island amplification on tissue samples of normal pancreas, noncancerous pancreatic tissue from patients with ductal adenocarcinomas and cancerous tissue. To be able to differentiate between the tissue samples through the amplification of the CpG islands, not only the epigenetic biomarkers are able to aid in prognosis but in determining of the stages of the tumor.

For the "normal tissue of normal pancreas" control group, 15 tissue samples were obtained from patients without ductal adenocarcinomas, but the patients have a history of other tumors. 32 tissue samples of nearby regions of the dutcal adenocarcinoma tumor but without the presence of contaminating cancer cells were obtained from patients for the noncancerous tissue group. 91 tissue samples were obtained from ductal adenocarcinomas patients for the cancerous tissues, The researchers extracted DNA from the tissues and methylation of CpG islands were analyzed using a custom-made array with 4361 bacterial artificial chromosome clones that can be used to detect cytosine methylation on 23 chormosomes and the sex chormosomes. The arrays were scanned and the signal rations were normalized for each group [|(5)].



The three groups: C as in normal pancreatic control, N as in noncancerous tissue and T as in cancerous tissue were classified by the signal ratio of the bacterial artificial chromosomes and the methylation of DNA from samples. From the data, if there are more signal ratios, then there are more methylations of DNA of the amplified CpG islands promoters. A comparison of the normal control and the cancerous tissue sample shows a significant increase of hypermethylation in the cancerous tissue. If one was to look at the progression from normal to noncancerous, the hypermethylation increases in CpG island promoters [|(5).] In the figure is a bacterial artificial chromosome array-baed methylated CpG island amplification. The samples were digested with methylation specific restrictive enzymes and were run through the PCR. From the information given, this experiment did not use MSP, rather the traditional method with restriction enzymes were used. The red lines in the signal ration graph are used to indicate the barriers of "normal tissue" allowing comparison of noncancerous and cancerous tissues. In N tissue samples (noncancerous) we see an increase of methylation (not one specific target gene mentioned) than normal (C) tissue. In T tissue samples (cancerous) we see a significant increase of hypermethylation compared to both normal and noncancerous cells. Overall, the increase of hypermethylation of genes in the samples allow predictive prognosis of the tumor (by stage) and may aid in monitoring the patient's response to chemotherapy.

**Epigenetic Therapy **
DNA methylation as a biomarker to detect and assess tumor growth is only one part of the cancer equation. New studies show the active role of epigenetic changes in development of a treatment of cancer. Traditionally, clinic treatments involve radiation, surgery or drug poisoning. The introduction of a class of drugs that target some proteins in the cancer pathway seems promising. These drugs are referred as "epi-drugs" in a study done by Juergens and colleages (11). Epi-drugs target the DNA methylation machinery such as inhibiting DNA methyltransferases (DNMTi). By interfering with the methylation, some cancer tumor suppressor genes will not be silenced, thus a epigenetic therapy. The patients whose specific genes were unmethylated responded well to traditional therapies such as radiation after the epigenetic therapy. The demethylation of those genes made the cancerous cells more susceptible to chemotherapy.

The study was an experimental design that tested the toxicity of the drugs and effects in terms of years of survival and the stabilization or nonprogression of the tumor. The article mentions a combined epigenetic therapy, implying that the patients that had recieved the epi-drug with the addition of another drug (inhibits HDACi, which affects histone deacetylases that play a role in transcription of genes). Azacitidine (inhibitor of methyltransferase) and entinostat (histone deacetylation) were the epi-drugs used in the study that involved 45 patients with refractory metastatic non-small call lung cancer (NSCLC). The reason that researchers decided to use a histone deacetylation was because with inhibitors of methyltransferases alone the re-expression of tumor suppressor genes were not expressed. HDAC influences gene expression and when used in combination of inhibitors of methyltransferases, gene expression is reactivated.

Within the experiment, there were 4 genes specifically targeted to de-methylate: CDKN2a, CDH13, APC and RASSF1a. It was correlated that "methylation of any 2 out of the 4 in a tumor conferred to a worse prognosis in patients with stage I lung cancer, similar to patients with stage III disease" (11). However before the effect of epi-drugs on overall survival, researchers had performed an experiment on the toxicity of the drugs on ten patients. 3 patients received 30 mg/m2/d and 7 received 40 mg/m2/d. All patients were given 7 mg/m2/d of entinostat at day 3 and 10 to ensure that demethylation will occur. Based on the observations of side effects such as low grade skin, fatigue, vomiting, constipation, anorexia, electrolyte disturbances and hyperglycemia from entionstat or azacitidine toxicity, the epi-drugs appear to be non-targeted. Traditional chemotherapies have these side effects as well, which lead to the question: are the epi-drugs working? In the results of the toxicity experiment, the 3 patients that recieved a lower dose did not experience dose-limiting toxicity. The maximam dosage of azacitidine was set at 40 mg/m (11).

As for the efficiency of the epi-drugs, 1 patient (out of 45) was observed to be responsive to chemotherapy for 14 months. Another patient was partially responsive for 8 months. Ten were observed to have a stabilized tumor (tumor did not grow) for at least 12 weeks. However, 22 were observed to have a progressive tumor after the epigenetic therapy. Overall, with the exception of 2 very lucky patients, the epi-drugs were not very efficient. This may be due to the fact that the epi-drugs do not target specific methyltranfserases. It also may be due to the fact that the cancerous cells may have adapted to the epi-drugs by over expressing methyltransferases, especially with a low dosage of epi-drugs (11).

However, this experiment targeted 4 genes for demethylation. DNA methylation biomarkers were used to assess the methylation of these genes though blood plasma DNA. In the figure, the red line shows patients with demethylation of 2 or more of the 4 targeted promoter genes. The blue line shows patients that did not indicate a change in hypermethylation of the 4 promoter genes.



From the proportion progressive-free, the patients who had demethylation of the genes were seen to have an overall higher percentage of "stabilized" methylation towards the end of the graph compared to the patients with no change in methylation of the 4 genes. For the proportion surviving, the patients who had demethylation of the genes have a longer "tail" for the amount of months of survival towards the end compared to the patients with stabilized methylation. DNA methylation biomarkers are able to aid in the progression of the tumor of the effects of the epi-drugs.

**Pro/Con of Biomarker **
DNA methylation is relatively stable and will not degrade in or outside the body. Since the DNA methyl groups are already in place, there is no need for an introduction of a fluorescing molecule that will need to bind to certain proteins. Most biomarkers would be introduced to the body and will need to bind to certain target locations. Over time, the body may either adapt to these biomarkers through immune response or these biomarkers may degrade quickly, resulting in false positives and negatives. DNA methyl groups can be sensitively detected by Methylation-Specific PCR (MSP) with no false positives for methylation due to the bisulfite modification and special primers. With MSP, we can also detect DNA methylation outside the body through the means of noninvasive sampling such as stool. Monitoring the increase of hypermethylation or hypomethylation of certain target genes allow predicitive prognosis of the tumor and how the patient is responding to chemotherapy treatment. There are also certain epi-drugs, such as the one mentioned above that inhibits DNA methyltransferases has been observed to make the cancerous cells more sensitive to chemotherapy. Patients with demethylated genes had an overall survival. Another advantage of epi-drugs is the drugs are quite efficient in small doses.

There are some concerns using DNA methylation as biomarkers. For diagnosis of cancer, certain DNA abnormal methylation patterns must be known and identified in both normal and cancerous cells. In stool samples, the combination of the two target genes BMP3 and KRAS only allows 67% of correct diagnosis of pancreatic cancer samples. This is due to pancreatic cancer arising from many different types of mutations and causes. It is the same as treating a breast cancer patient with a drug to inhibit a specific gene, when the patient's cancer is not associated with that specific gene. If we use BMP3 and KRAS as target methylated genes alone, we may be wasting our time and resources. More research about specific DNA methylation patterns is needed. However, one should not throw the baby out with the bath water. Instead, we should look for more methylation markers that associate with the type of cancer and stool sampling can be used as a "pretest" for pancreatic cancer before any imaging or invasive endoscopy is done on the patient.

With stool sampling, we do not know for certain that the precursors found in stool are actually from the pancreatic cancer. In the epi-drugs experiment, DNA blood plasma was used to measure the amount of de-methylation of the target genes. Again, we are not sure if the de-methylation occurred at the tumor site. The epi-drugs' side effects indicate that the inhibition of DNA methyltransferases are non targeted. If the drugs are non targeted, how will we know for certain that the demethylating of target genes are from the tumor? Can we assume that those target genes are unique only to cancerous cells? More research is needed to answer these questions. In regards to stool samples, there are some methyl groups rich foods that will donate or induce the addition of a methyl group in a cascade of reactions (similar to the effect of soybeans in the agouti mice) that might alter the accuracy of the methylation results. Also, increases of DNA methylation as the tumors progress will be different for everyone and only applies to certain genes. In BMP3 and KRAS, as the tumors progressed, there was no significant increase of hypermethylation of these genes. However, as a whole (concentration of all methylated genes in general), we can predict tumor prognosis.

In regards to epi-drugs, low doses are effective when given to patients. However, it worries me that the immune system or the cancerous cells may adapt to the epi-drugs. The immune system may destroy the epi-drug before it has reached its destination. The cancerous cells in response to the epi-drugs, may increase expression of DNA methyltransferase, rendering the drugs useless. A possible solution is to increase the amount of epi-drugs. However, increasing the amount of epi-drugs may do the patient more harm than good. Since the side effects of these drugs are similar to chemotherapy, one might as well suffer under chemotherapy which has yielded some certain positive effects on the cancer cells.

=Known Natural Biomarkers: Cell Surface Proteins=

Cell Surface Proteins and Proteases:
All cells need to communicate with neighboring cells and respond to signals in their soundings as a means to manage and control their environment and have harmonious living conditions. To do this, cells have various types and numerous amounts of cell surface receptors, including proteins, kinases, proteases.. When these receptors have their respective substrate bound, they initiate the corresponding effect on the cell. The types of cell surface receptors that have shown the most potential for use as biomarkers and in cancer therapies are ones derived from proto- oncogenes. These receptors are part of mechanisms involved in DNA synthesis, cell proliferation, cell migration, and other factors that if for some reason mutatated in a certain way could potentially lead to tumor foci formation and tumor progression. Oncoprotiens are the product of these proto-oncogenes that have undergone such mutations and alterations. Oncoproteins are often found in high concentrations on cancerous cells. Although there are countless cell surface receptors that have the potential for biomarker usage our focus will be the mutant form of the epidermal growth factor receptor, EGFR vIII, and uPA uPAR complex.

The biomarker EGFRvIII is a mutant form of the tyrosine kinase receptor EGFR. But why use EGFRVIII as a biomarker? Well, the wild type EGFR, epidermal growth factor receptor, is receptor is transcribed from one the many proto-oncogenes, that if mutated in a certain way couldlead to the cell acquiring cancer like properties. When bound to a ligand, EGFR dimerises eliciting tyrosine kinase signal transduction cascade leading to the signaling and activation of several other downstream proteins such as RAS and MAPK. Ultimately this causes the cell to induce cell proliferation, DNA synthesis, and acquisition of new phenotypes like cell migration. Normally all these properties are kept under tight control, but if this kinase signaling is unregulated it can lead to cancer. This lack of regulation is seen in the EGFR vIII mutant, which was first discovered in a primary human glioblastoma tumor (one of the most common types of brian tumors). EGFRvIII is constituavely activated due to an in-frame deletion in the mRNA causing the extracellular portion of the receptor to be truncated. This allows for the kinase to be continually activated due to the ligand independent binding and an increase in activity of Ras-GTP. This mutation has been shown to enhance the tumorigenicity and apoptosis resistance of cells containing the EGFRVIII mutant. So, why is this important? EGFRvIII is associated with increased invasiveness and growth rate of tumors. The presence of this mutations and the resulting increase in concentration of the mutant epidermal growth factor receptor is almost exclusively found on the cell surface of cancerous cells compared to normal cells, which have virtually none. Mutations in the EGFR, especially the EGFRvIII mutant, are some of the most common mutations found in GMB cells, lung cancer cells, breast and ovarian cancer cells. (6,13)

Tumor metastasis begins with cancer cells invading surrounding tissues; this process is frequently accelerated by cell-surface proteases like uPA and uPAR. Urokinase-type plasminogen activator, otherwise referred to as uPA, is a type of cell surface protease, meaning it is involved in a variety of basic cell activities and possesses influences on growth factors that could have very drastic and rapid effects on the cell. By breaking down the extracellular matrix around the cell, uPA has the potential to activate migration-inducing signal transduction cascades. The binding of uPA to its receptor uPAR, urokinase-type plasminogen activator receptor, increases tissue invasiveness and metastatic potential. In the case of uPA and uPAR complex, it is not necessarily a mutation in the proto-oncoportein directly but an up regulation and over expression of this protease system that allows the complex to be not only tumorigenic but act as a biomarker. “uPA is a serine protease that is secreted in a single-chain inactive form (sc-uPA), which is then cleaved by plasmin into a double-chain active uPA. The uPA receptor (uPAR) binds uPA in both its active and inactive forms and protects it from being inhibited by the plasminogen activator inhib- itor-1 (PAI-1). The active uPA/uPAR complex cleaves plasminogen into plasmin, thus leading to extracellular matrix degradation and tissue invasiveness and metastasis. The uPA system is generally absent on normal cells and is up-regulated only during certain physiologic processes, such as wound healing and tissue remodeling” (2). This explains why uPA and uPAR complex is found at substantially higher levels on metastatic cancer cells, including those of the breast, colon, stomach, and bladder. Due to this, “high levels of both uPA and uPAR are used as a diagnostic markers for metastatic potential and poor clinical outcome in numerous malignancies.”(15)

Both of these biomarkers are commonly found on cancerous growths and have great potential for cancer therapeutics.

**Taking Advantage of Cell Surface Biomarkers As Part of Cancer Treatment**:
With early detection being key for a good prognosis, Biomarkers (substances that can be used as indicators for the presence of particular diseases) are valuable tools that allow for both screening and detection of the early onset of cancer. Unfortunately, detections of cancerous cells or fully formed tumors are often delayed until symptoms of the primary tumor or secondary tumor has arisen and by then it can be too late. But don’t despair cell surface biomarkers present hope for the future of cancer therapies.

**Directly Targeting The Cell Surface Biomarker**:
When considering how to implement cell surface biomarkers as part of cancer therapeutics one prevalent method is finding a way to directly attach the therapy to the receptor. One of the most common solutions to this is by attaching a monoclonal antibody to the treatment. Monoclonal antibody chosen has a high binding affinity for the desired target receptor allowing for specific and direct binding. An example of this procedure is In the article, [|__//Targeting HSV-1 virions for specific binding to epidermal growth factor receptor-vIII bearing tumor cells//__], Paola Grandi and her fellow researchers found a way to effectively target and destroy deadly cancerous cells through the use of this multi-faced Herpes Simplex Virus (HSV).

In a world filled with Purell gel and Lysol wipes, who would of thought that viruses were anything but trouble. This negative connotation of viruses has also been applied to cancer. For the majority of early cancer research, viruses were pinned as the primary cause of cancer. Though now it has been found that there are a select few viruses that actually lead to cancer in humans, viruses are now being looked at in a different light. Ironically the thing that supposedly was the root cause of all cancers is now helping to treat it. With slight modifications to their wild type form, viruses can be designed to act as a tool to mark, target, and attack cancerous cells. Viruses, such as Herpes Simplex Virus, have been sucessfully modified to single out cancerous cells, leaving normal cells unharmed, force the affected tumor cell to secrete the biomarker carried in the virus’s genome. Grandi found a way to modify the HSV viral envelope glycoprotein’s, by swapping out the normal heparan sulfate binding domain with a tumor-specific immunotoxin. This immunotoxin, not only has the ability to directly target this modified virus to the naturally found tumor cell biomarkers, but in other research has also shown the capacity to mediate cell death on its own. This type of immunotherapy is a promising alterative to current methods of treatment, especially with their focus on glioblastoma multiform, one of the “most common primary brain tumors [which] are almost universally fatal despite aggressive therapies, including surgery, radiotherapy, and chemotherapy” [|(]6[|)] .Grandi and her fellow researchers focused on the biomarker EGFR vIII, as menchioned above EGFR vIII is a mutant form of the tyrosine kinase receptor EGFR and a promising biomarker. Also, it has been shown that “several antibodies have been described that are specific to EGFR vIII and do not cross-react with wild-type EGFR” [|(6)] .To target EGFRvIII, Grandi and her fellow researchers, used a modified version of the oncolytic virus HSV. An oncolytic virus is a type of virus that causes the infected cell to lyses. As the virus replicates it not only destroys the host cell but also keeps this destructive cycle going since it infects adjacent cells causing subsequent destruction. Though there are multiple types of viral vector therapies in clinical trials, oncolytic viruses like HSV show the most promise.the Herpes Simplex Virus (HSV) is one of the most common virus type used to treat cancer because of its ability to interact with various cell surface receptors leading to numerous and different possible methods for cell entry. The virus is capable of being produced in sizable quantities, and previous studies have shown it to be safely controlled with current antiviral drugs. Depending on what the virus is set out to target, subsequent mutations allow the for direct targeting and when this is applied to cancer therapeutics it allows the for the targeting cancerous cells only. The key to Grandi research was finding a way to engineer the HSV virus to effectively target onlythe cell specific surface receptors EGFR VIII, unique to cancerous GMB cells, leaving the normal cells unharmed. To generate this tumor selectivity Grandi implemented the single strand monoclonal antibody MR1-1. In previous studies MR1-1 has been shown to have high binding affinity accompanied with long lasting infectivity with EGFR vIII causing it to be extremely potent to cells with this mutation. Instead of having a heparan sulfate binding domain, which allows for the viruses initial binding with the cell surface in general, the virus was modified so it would only bind to the EGFR vIII receptor. Because Grandi created a specific immunotherapeutic target, EGFR vIII, it not only concentrated the viral vectors specifically to the EGFR vIII containing cells but it also increases the safety of the treatment and lessens the needed dose. (6)

What Grandi proposes would increase the potency of these vectors, by enhancing targeting and increasing vector infectivity to tumor cells. To test that attaching MR1-1 to HSV actually does this, Grandi first did a comparative study to see different monoclonal antibodies: MR1-1 (high affinity expected), MRB (low affinity expected), and the normal gC binding domain without the HS-binding domain relative binding affinity. Each of these “were inserted in the pCONG amplicon which also contains an expression cassette for green fluorescent protein (GFP) to monitor amplicon vector infection”[|__(6)_]. Grandi found that the MR1-1 attachment increased the infectivity rate of the human glioblastomas cells with mutant receptor 5x. To test infectivity of the of MR1-1 modified HSV and make made sure the MR1-1 modified HSV would only bind to glioma cells with the mutant form of the epidermal growth factor receptor, Grandi implanted two sets of U87 cells, a human primary glioblastoma cell line, with and without the EGFR vIII in nude mice and let them grow. She then infected the modified virus in each set of mice and monitored the progression over a week as seen in the picture. The infectivity was monitored by using fluorescent or lacZ reporter genes in vivo that were incorporated into a virus. A set of infected mice were injected with the normal gC binding domain lacking the Herparan sufalte binding domain as a control.The MR1-1 replacement was show to have greater infectivity to the glioma cells than compared to the wild type virus. Grandi observed that not only did the tumor shrink but also the modified virus had longer infectivity. This shows incredible promise for future use in cancer therapies. It should be noted that these experiments were preformed at low M.O.I (multiplicity of infection) to imitate the expected in vivo ratio of vectors to tumor cells.This research certainly provides a creative approach to target cancer cells for virus-mediated destruction. GMB is such a fast paced disease, having a median survival rate of 12-18 months after diagnosis, making research into creating effective treatments like this one necessary. This article provides insight into how viruses could actually destroy cancerous cells using cell surface biomarkers as their target.
 * Testing Method and Results: **

**Indirectly Targeting The Cell Surface Biomarker**:
Another way to make use of these cell surface bioarkers is through indirectly targeting them. Unlike the example above where the monoclonal antibody directly attached to the cell surface receptor, other therapeutics target cancerous cells by binging to the ligand for a specific cell surface biomarker. This is seen in the article published in the American Chemical Society, [|__//Reprogramming Urokinase into an Antibody-Recruiting Anticancer Agent//__] by Spiegel and colleges. Using concepts from synthetic immunology Spiegel came up with two biofunctional molecule that target uPA and allows for the immune system to recognize cancer cells that would have otherwise avoided detection. With this technology of biomarker targeting, Spiegel and his fellow colleagues are able to take advantage of natural processes and substances allowing for the body to illicit a native immunological response. Spiegel’s research in targeting the uPA uPAR complex provides a target metastasized cancer cells. This could hold the potential of returning hope to those who thought there was none. To de diagnosed with cancer is bad enough, but to find out it has metastasized is often deemed to be a death sentence. With the survival rate of those with metastatic cancer slim and the fact that, “American men and women have an 38-44% chance of developing invasive cancers in their life time”([|__15__]) there has to be something done. provides insight into research creating a synthetic compound to target the natural biomarker of many metastatic cancers.

Spiegel and his fellow researchers focuse on the biomarkers the urokinase-type plasminogen activator (uPA) and its receptor urokinase-type plasminogen activator receptor (uPAR). As stated above “high levels of both uPA and uPAR are used as a diagnostic markers for metastatic potential and poor clinical outcome in numerous malignancies”([|_15_]). In previous studies that target the uPA- uPAR system cause a reduction in both the size and invasive potential of cancer cells without extensive damage to healthy tissue. This is beneficial since the majority of classic treatments, such as chemotherapy and radiation leave normal tissue harmed by the indiscriminate cancer treatment. To target uPA and uPAR, Spiegel and his felloresearchers used concepts from the field of synthetic immunology. Research in this field attempts to modify biological processes that would otherwise not normally function as in nature through the use of synthetic materials. Spiegel came up with two biofuntional molecules that allows for the immune system to recognize cancer cells that would have otherwise avoided detection. These two molecules “can convert uPA into catalytically inactive, bifunctional constructs (ARM-Us) that are capable of both recruiting antibodies and directing antibody-dependent immune responses against uPAR- expressing cancer cells”([|_15_]). These two molecules posses the power to covalently bond to its active site by either a fluorescein label, which allows for visualization of the cancerous cells, or to 2,4-dinitrophenyl (DNP) moiety. The covalent linkage of DNP offers a way for the body to recruit antibodies to attack the cancer cells because in humans there are natural found anti-DNP antibodies. This means that when the ARM-U binds with high affinity to the cells with high uPAR levels, malignant cancer cells, the simultaneously covalent attachment of DNP triggers the natural immune response of phagocytosis and cytotoxicity because of the signaling of anti-DNP antibodies.
 * What is Spiegel Targeting and Why: **

To perform this experiment the researchers used chloromethyl ketones 1,2. Ketones 1 can simulate ARM-U attached to a fluorescein label while ketone 2 can simulates ARM-U bonded to DNP. Methyl Ketone 3 acted as a negative control because of it lack of ability to form a covalent bond. While ketone 3 showed no bonding to uPA as predicted, the results did show that there was a 97% reduction in uPA activity with the use of Ketone 1 and the fluorescein label when compared to uPA and buffer activity alone. Further testing showed that ARM-Ufluor targets cells via the desired uPAR interactions, and that the attachment of the fluorescence does not affect the initial binding process. After determining that ARM-U actually binds to uPA and can decrease its activity it was time to test the capability of the ARM-U mediated killing via DNP. The ARM-UDNP complex was shown to have high levels of cellular cytotxicity and to be very efficient in the mediated phagocytosis. The subsequent increase in the presence of anti-DNP antibodies in turn decreased the cell viability revealing the importance and actual viability of the attachment of the DNP antibodies. As hoped the negative controls did not elicit an immune response. Lastly, when both the fluorescein label and DNP were combined the immune response increased even more. (15)
 * Testing and results: **

**Pros and Cons of using cell surface biomarkers in cancer therapies**:
Both cancer treatment methods using sell surface biomarkers certainly provide novel set-ups capable of targeting cancer cells for cancer destruction in numerous cancer types and stages. As seen in both studies these method of therapeutic treatment is unlikely to affect other cells, which is definitely a step in the right direction compared to other current methods of cancer treatment. Unlike with chemotherapy, radiation, and even with surgery normal cells are a bystander to the treatment. An added bonus is that the FDA has already approved processes using uPA. This means that if further clinical trials continue to show this progress there will be less time to wait for this technology to come to market. By having other applications targeting uPA, potential side effects can be explored further to minimize and prevent them.Because cancer is such a prevalent and menacing disease, research into creating effective treatments like this one is necessary, however, I did come across some limitations. The main aspects that I feel need further research into are the specific technical aspects allowing for the treatment to be applicable, the possibility of the cancerous cells acclimating to these treatment, and the possible of an immune responses to the therapeutics themselves.

One thing both of these studies fail to mention is the minimum concentration of the biomarker that is needed to act as an effective target and for how long these vectors stay in the body. This would affect not only the window of opportunity for these treatment, but the frequency and amount of treatment is needed. If the therapy is needed in frequent dosages or if it stays in the body for and extended amount of time it needs to be looked into how the cancerous cells will find ways to get around these treatments. Because cancer cells frequently mutate and acclimate to their surrounding, there is a need to further research how these treatments could be effected. Since these process are dependent on the presence of the specific receptors or complexes, if these receptors some how mutate with prolonged exposure to this treatment, the treatment will be rendered either less effetive or even completely ineffective. There have been multiple other drugs that target oncoproteins that after an extended length of time are found to be useless because resistance has been confirmed in the cancerous cells. Two of the most relatable drugs to this are Herceptin and Gleevec. Herceptin also uses a monoclonal antibody to target and block function of the receptor HER2, a type of Epidermal growth factor receptor, by preventing dimerization. But upon tumor relapse, Herceptin elicits no response. This is because the tumor cells that some how survived the first round of Herceptin treatment managed to mutate and developed alternate means to propel growth independent of the HER2 oncogene expression. In the case of Gleevec, the oncoprotein it was designed to bind to mutated causing a change of various amino acids in the binding domain, resulting in the inability for the treatment to bind and target the cell. So what is to say the same thing does not happen to the MR1-1 HSV treatment? If either of these processes occur, finding another path to cause cell growth while avoiding the expression of EGFR vIII or modifying the binding domain of EGFR vIII, this therapy in its current state would be rendered useless. Therefore there needs to further research into other monoclonal antibodies attachment to the virus allowing for the continuation of binding to EGFR vIII and or new cell surface kinase targets for the HSV virus. Also, additional research should explore how the addition of multiple monoclonal antibodies to the HSV virus would accommodate for these changes in the cancer cell’s morphology.

Though this might be avoided through indirect targeting of biomarkers because the receptor still binds to its normal ligand making it less like to mutate the receptor itself but rather decrease the concentration of the receptor. Since these processes are dependent on abnormally high concentration of the receptors, I am left with the question that if cancer cells find a way to either mutate or bypass these cell surface proteases, thus leading to lower levels of the ligand, would this therefore make this model ineffective? As cancer cells try to avoid detection from the immune system it modifies or even down regulates signaling that would otherwise act as an alarm. For example if RasGTD is constantly active due to some mutation, normally the cell would sense this and start to senescence or even apoptosis. To prevent this from occurring cancer cells will try to reduce the concentration of RasGTD. If this same process occurs with uPA and cell surface uPAR, and the cancer cells still manage to spread, then this innovative therapeutic will be rendered ineffective. This is why it is necessary to have further research into at what concentration levels of uPA and uPAR will ARM-U be able to effectively target and activate and immune response. Another follow up research idea is to look into applying this technology to target other cell surface proteases involved in the spread of cancer, subsequently if cancer cell do find a way of flourishing without uPA or uPAR there will be a back up target.

Additionally, since these treatments use substances outside not native to the immune systems, I am left to question how the body’s immune response will affect the efficacy of this therapy. As with increased exposure to any virus the body’s natural immune system starts to acquire, for the lack of a better word, memory of the virus, making the virus a key target for immune systems attack. If this treatment calls for the virus therapy to be administered frequently in a short length of time, I am concerned that the bodys’ own immune system will start attacking the virus and gain immunity to it, greatly diminishing the effects of the treatment. If this happened, it would not matter that the virus can target the cancer; the virus would be prevented from propagating leaving the cancer cell unharmed and the patient in no better condition then before treatment. Also since the study done by Spegial was only preformed on mice it is not known if the synthetic biomolecules, ARM-Us, would elicit an immune response.

=Conclusion and Would We Invest=

Biomarkers provide innovative targets for the diagnosis and treatment of cancers that would otherwise have no means for early detection or treatment, such is the case with pancreatic cancer and metastasized cancer. As described above, DNA methylation is effective at diagnosis of certain cancers and predicting prognosis, however, it is not perfect. For example, methyl rich diets can alter methylation results in stool. Cancerous cells in response to epi-drugs may increase expression of DNA methyltransferases, rendering the treatment useless. Since the epi-drugs were given at a low doses, increasing the dosage will do the patient more harm than good. In regards to using biomarkers as part of the therapeutic process, we find that though the ones mentioned provide great promise they still present some issues of which further research is needed. One of the major advantages to these treatments is the fact that they use these biomarkers to make treatment localized to cancerous cells. Biomarkers allow for cancer cells with metastatic properties to be targeted allowing for growths, no-matter how invasive to be treated, as long as they have the targeted marker. The major concern on the other hand is that since cancer cells are constantly mutating, we fear that the cell surface receptor the biomarker leads the treatment to, will mutate making the treatment useless, leaving the patient no better off. Another issue is the effect of the immune response towards the vector or synthetic property involved in the treatment. Even with these flaws presented we feel that they are outweighed by the benefits. Investing in technologies using biomarkers as either a tool for diagnosis or treatment provides for pioneering methods that with further development could revolutionize and personalize the way cancer is treated. Again referring to MSP, it is not only more affordable than current genomic sequencing but is faster, more efficient, and more accurate.The biomarkers described have not entered clinical trials but do show improvement on current methods. Investing in these therapeutics would allow entrance into clinical trials offering data on what would be applicable to humans, allowing them to reach their full potential. The need for effective cancer treatment is huge, and researching and implementing biomarkers and the technology associated with them is a step in the right direction and can work towards filling this void, thus leveling the playing field of cancer.

=References=
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a) http://www.whitehead.mit.edu/news/paradigm/spring_2007/epigenetics.html b) http://www.cancer.gov/cancertopics/understandingcancer/cancergenomics/page33 c) BioVision at Harvard University: [|__http://sparkleberrysprings.com/innerlifeofcell.html__] d) http://flylib.com/books/en/3.98.1.118/1/
 * Images **