Carlos+Medina

=**The Story of Eugene Edwards & His Hepatocellular Carcinoma**=


 * __Aperçu__** - Mr. Eugene Edwards was just like any other middle-aged man you would find at your local grocery store, or at the gym, or working at your company. He had a family, and was the proud husband and father to three children. When diagnosed with hepatocellular carcinoma, his life changed, the same way that it changes for the tens of thousands of people that are diagnosed with this type of cancer every year. Hepatocellular carcinoma is a complex liver cancer which results in extensive liver damage and deterioration. Though treatments can increase survival by a few months, the best way to avoid this cancer altogether is to abstain from excessively drinking alcohol and to remove oneself from a situation conducive to the contraction of Hepatitis B or C.

//__Prologue__:// //Eugene Edwards leaned back and held the large, wooden mug up to his lips and quaffed the beer heartily. There was nothing like a relaxed evening out in the bar with his friends. For a second, he thought back to what his girlfriend had told him just a few days ago. Said that he'd been drinking too much lately, not spending enough time with her. It was not so. Sure, he drank. But not that much.// //"You want to go out and..." the man to his right asked him. But Eugene shook his head vigorously. Lately he'd been going to his friend's house every week to relax the mood even more. But the last time, the needle seemed to hurt him more, it left the area in his forearm redder than usual. The pain had lasted for longer afterwards. It had been time to stop. Enough was enough. Plus, if his girlfriend knew what he was up to...//

//Deep inside of Eugene Edwards, microscopic forces were at play. Though he wouldn't know it till far later, his body had a visitor. A permanent visitor. It was a virus that settled down in his liver. Hepatitis C had arrived and was there to stay.//

**[Part I - General Introduction]** **Of Thieves and Victims: Hepatocellular Carcinoma**
It came quietly, with carefully measured steps. Like a thief that treads in the shadows and steals into the night after seizing treasure, it too lay patiently in wait, in calculating subterfuge. But unlike a thief, it did not come to steal gold and diamonds. Instead, it came to steal the health, and ultimately the life, of Eugene Edwards. Its target was his liver. Hepatocellular carcinoma changed Mr. Edwards’ life forever, from the day of his diagnosis to the marked struggle of his final weeks. The case of Mr. Edwards underscores the forceful resilience with which this cancer strikes and survives. The doctors were certain of Mr. Edwards’ condition when he reported bloating, a loss of appetite, and persistent abdominal pain, among other symptoms [1]. When considering the history of their patient, the doctors were not surprised. The patient contracted Hepatitis C from an earlier incident of intravenous drug use and previously received a blood transfusion that may have been contaminated. Now a fifty-year old man with three children, Eugene Edwards’ chronic Hepatitis C led to cirrhosis of his liver, the point at which his liver was unable to heal itself [2]. A CT scan finally revealed a significant mass on his liver. The liver is essential. Much in the same way that a carbon filter will purify drinking water, the liver is the processing plant that detoxifies blood and breaks down nutrients appropriately. Loss of liver function leads to an accumulation of toxicities and impurities in the blood, leading to an eventual deterioration of health [3].

The last time Mr. Edwards had felt such an intense anxiety was the day he had to give a conference to an audience of hundreds. He felt the same way on the evening of his diagnosis. After the kids had been put to bed, Mr. Edwards and his wife sat down together to talk. “Your AFP levels are really elevated,” the doctors had told him. Serum alpha-fetoprotein was normally made in the liver of unborn babies and was a marker present in pregnant women. In healthy men and non-pregnant women, there was either no AFP or very low levels. A high level of AFP in any other case is often indicative of cancer. In combination with CT scan imaging, hepatocellular carcinoma was confirmed. It came in the form of a fat, foreboding tumor with a class B rating: moderate tumor size, vascularization, and liver function.

Most of what Mr. Edwards understood was what the doctors had told him and what the pamphlet had said. Hepatocellular carcinoma usually manifests itself in patients suffering from either Hepatitis B or C, or those with alcoholism-induced cirrhosis; it is most often secondary to one of these conditions [3-4]. About twenty-thousand new cases arise every year in the United States alone. Men are affected more often than women, usually between the ages of 45 and 60 [3].

But at this time, Mr. Edwards didn't fully understand the nature of his condition; in all likelihood, he never did. He didn't know that the Hepatitis C virus was causing his own immune system to attack his liver. He didn’t understand how his cytotoxic T cells were driving spears into his liver cells, and in doing so damaging them severely, in an attempt to eradicate the virus concealed within. He didn’t know that extensive scar tissue had formed from endothelial cells undergoing fibrosis, which slowly prevented his liver from adequately detoxifying his blood, or that continuous tissue damage led to impaired function and eventually tumor formation.

Over the next year, Mr. Edwards underwent numerous procedures, all with a hopeful longing that the next one would be the one to finally stabilize him and allow him to regain a normal life. First was chemoembolization, wherein anti-cancer drugs (in this case mitomycin, Adriamycin, and carboplatinum [2]) were injected directly into the blood vessel that fed into his liver. Though a surgical resection could have been performed, the location of the tumor provided the opportunity to eliminate the tumor with pharmaceuticals instead. By the middle of the following year, there was a drop in AFP values, closer to normal. The tumor disappeared for some time, but the intense treatment had damaged his liver. Mr. Edwards received a liver transplant [2]. Before long, Hepatitis C returned and the body began to reject the transplanted liver. Then, cirrhosis became evident again and hepatocellular carcinoma returned with a greedy vengeance. Mr. Edwards passed about a year and a half after his initial diagnosis.

Eugene Edwards did not think of cancer in terms of a Hepatitis C virus that was causing his immune cells to attack his liver or the thorny tumor that leeched the life of his liver. Rather, cancer was anxiety and stress. Cancer was pain and suffering. It was fear and uncertainty. Cancer was an obstacle, a threat that could prevent him from ever seeing his wife and kids again. It was because of cancer that he may never be able to play catch with his son again, or manage his daughter’s Girl Scout troop, or have another candlelit dinner with his wife. To Mr. Edwards, hepatocellular carcinoma was a thief, one who was about to steal from him everything that he ever cared about. It was a stubborn tumor defying the will of its host.

Mr. Edwards’ case is not unique. Worldwide, many people suffer and die from hepatocellular carcinoma. Unfortunately for Mr. Edwards, he died in this struggle. Despite everything doctors did for him, the series of treatments and procedures were not enough. Mr. Edwards is survived by his wife and three children, who must now live and find new light in a world without their husband, or father, respectively. Many families must also continue living without a loved one - they have also had loved ones stolen from them by hepatocellular carcinoma, despite all efforts to the contrary.

[Part II - Molecular Basis] Great Complexity & Vexing Questions: Hepatocellular Carcinoma
Mr. Eugene Edwards was not a man to accept anything less than the full picture. Seeking to learn more about his diagnosis, he began to read articles online, ask friends who had majored in molecular biology, and confer with doctors. Eugene arrived at a better understanding of his cancer at the molecular level. Ultimately, hepatocellular carcinoma works partly by inhibiting growth suppressors and upregulating angiogenesis, and partly by maintaining an elusive subterfuge that continues to thwart researchers and the potential for a better targeted therapy.

As in many other cancers, TP53 is the gene that is most prominently mutated in hepatocellular carcinoma. TP53 encodes the p53 protein, which is one of the best known growth suppressors responsible for regulating cell division and apoptosis. Moreover, when receiving apoptotic signals or cellular stress (i.e. oxidative stress), p53 can activate other proteins that will lead to the creation of caspases that will degrade the DNA and lead the cell into apoptosis [6]. In hepatocellular carcinoma, p53 mutations are widely reported and a mutational hotspot has been identified in codon 249 [7]. This hotspot has a higher propensity for cancerous mutations and such hotspot could be explained possibly by its structural location on the outside wherein codon 249 is more exposed than other codons to damage from reactive oxygen species. Because the p53 protein regulates antioxidant genes, DNA damage to TP53 prevents proper DNA repair. Hepatitis viruses can also integrate sequences that code for mutant proteins that bind to and inhibit the function of p53 [8]. The elimination of proper TP53 or p53 activity therefore inhibits a key suppressor – the brake - that normally prevents unfettered cell proliferation. Hepatocellular carcinoma alters both the NF-ĸB and Wnt signaling pathways to promote angiogenesis in the liver. The NF-ĸB pathway is involved in DNA transcription, cytokine production, the inflammatory response and cell survival. It is involved in responses to oxidative stress and other injuries to the cell. Much like EMT’s can quickly respond to local emergencies on campus, so too NF-ĸB is present in an inactive form within the cell, a first responder of sorts, always ready for activation upon the incidence of cellular stresses [9]. Researchers recently found that the up-regulated-gene-4 (URG4) is overexpressed in highly vascularized tumors and that it works directly by inducing angiogenesis through the upregulation of NF-ĸB [10]. These findings suggest that NF-ĸB plays a prominent role in angiogenesis, thereby making it a potential molecular target in future therapies. The Wnt signaling pathway is responsible for the regulation of cell fate, proliferation, and survival [7, 11]. This pathway is altered in the hepatitis-induced forms of hepatocellular carcinoma, either through a deregulation of Wnt signaling or a mutation of the proto-oncogene β-catenin [7]. β-catenin is a co-activator of the Wnt pathway that controls developmental gene expression program. Wnt is required for β-catenin stability and function, otherwise the latter is repressed (see Figure 1) [11]. Mutating the β-catenin, or deregulating Wnt can lead to the inception of cancer. As relates specifically to angiogenesis, research has found that alterations of the overall Wnt/β-catenin pathway lead to an increased vascular morphogenesis and endothelial differentiation [12]. Mutations in this pathway may therefore be involved in the marked hypervascularization that characterizes hepatocellular carcinoma by leading to excessive transcriptional activation of angiogenic genes.

To complicate the general understanding of hepatocellular carcinoma further, numerous paradoxes, of which only one will be described in detail here, have been cited which illustrate the true complexity of this cancer [13]. Shutting down the NF-ĸB pathway seems to negatively impact the progression of hepatocellular carcinoma while having no impact on the early stage of tumor development; it is required for later-stage cancer development but not early stage tumor formation. On the other hand, a different group found that eliminating a kinase (Ikkβ) required for NF-ĸB activation led to a marked increase in hepatocellular carcinoma development, suggesting a potentially anti-tumor effect of NF-ĸB in the liver [13]. A reason for the former observation could be that once a tumor forms, NF-ĸB will only exacerbate the inflammatory response by promoting cytokine activity and angiogenic pathways thereafter. This is why shutting down NF-ĸB after tumor formation will prevent excessive inflammatory molecules from coming to the site and being taken up into the tumor microenvironment. It has been noted that excessive inflammatory factors present within the tumor can promote its growth [14]. Or it could simply mean that the Ikkβ kinase is responsible for activating other defense pathways beyond just NF-ĸB. For the latter part of the paradox, it makes sense that preventing NF-ĸB activation (via inhibition of the Ikkβ kinase) will allow easier development of hepatocellular carcinoma. This could be because at an early or pre-cancerous stage, the absence of the “first responder” NF-ĸB could allow the accumulation of reactive oxygen species and other forms of DNA and cell damage, paving the path for cancer to form. A way to test this would be to test the level of lipid peroxidation, which occurs from reactive oxygen species that damages the cell membrane when exposed to oxidative stress. Another way to test this hypothesis is – depending on whether in vitro or in vivo experiments – to run qPCR on RNA/cDNA from cell or tissue samples to measure the expression of genes related to oxidative stress and/or cell cycle arrest. Another potential option in an in vitro platform would simply be to directly suppress the NF-ĸB gene (i.e. deletion with CRISPR/Cas9 or other genome engineering tools) and then test the effect of adding antioxidants to the culture and compare it to the control. In any case, a full understanding of the molecular mechanism of hepatocellular carcinoma remains tantalizingly out of reach.

So, what now? Firstly, more research is required to understand the molecular basis of hepatocellular carcinoma. At this point, it is evident that this cancer type evades growth suppression while promoting angiogenesis in the liver. Either the NF-kB or the Wnt/β-catenin pathway could be potential targets of molecular therapies. Ultimately, a successful drug will have to consider multiple factors simultaneously, and will have to account for the appropriate conditions for targeting, such as the tumor stage in the patient. Until then, the hepatocellular carcinoma question will continue to vex many researchers.

[Part III - Treatment] The Beginning of the End: Sorafenib & Hepatocellular Carcinoma
When faced with death, the decision between conventional and experimental treatments - between awkwardly limited options and nebulously uncertain trials - becomes a stressful gauntlet in and of itself. At the time when Mr. Eugene Edwards began undergoing treatment for hepatocellular carcinoma, there were already trials occurring to test potential targeted therapies. One drug in particular rose quickly as a leading candidate, years after his death. Sorafenib has shown to have significant benefits over conventional treatments in advanced stage hepatocellular carcinoma and is the first and only successful targeted therapy to date. While several other drugs have been experimented with, none have matched the success of sorafenib.

The current options to treat hepatocellular carcinoma are largely systemic and possess significant limitations. Historically, the standard treatments for patients have been surgical resection and liver transplantation [7]. Unfortunately both of these options come with significant shortcomings. Surgical resection, or the surgical removal of the tumor, works best only in the early stages prior to metastasis; however, both recurrence and metastasis are considerably common in these patients [7]. When hepatocellular carcinoma arises due to a hepatitis C infection, the virus is likely to re-infect the liver and eventually return the patient to his original state. Liver transplantation avoids repairing or eliminating the source of damage, but rather seeks to reintroduce a healthier or newer liver as a way to try to start over; this is a very common option when the liver damage is extensive. There have been good results from transcatheter arterial chemoembolization (TACE), which comprises of an injection of a drug into the artery that feeds directly into the tumor. While this option has been successful at preventing recurrence of the cancer for up to six months, there are considerably poor long-term prospects [15]. The limitations stated above have led to the need for a targeted therapy.

Studies have shown that sorafenib, now the gold standard for treatment of patients with advanced hepatocellular carcinoma, can increase the median overall survival by about three months. Sorafenib, developed and marketed jointly by Bayer and Onyx Pharmaceuticals, is a multikinase inhibitor that targets the vascular endothelial growth factor receptor (VEGFR), the Raf pathway, and the platelet-derived growth factor receptor (PDGFR) [16]. Hepatocellular carcinoma is frequently observed to have extensive vascularization and VEGF is prominent in angiogenic processes. A small molecule like sorafenib can block the VEGF receptor and therefore contribute to the slowing down of angiogenesis in cancer. B-raf and Raf-1 are serine-threonine kinases that contribute to the regulation of pathways related to cell division and differentiation. The platelet derived growth factor is a growth factor prominent in the cell growth and division during angiogenesis. It is believed that sorafenib’s success can be attributed to the fact that it simultaneously targets these three receptors/pathways [16, 17]. The Sorafenib Hepatocellular Carcinoma Assessment Randomized Protocol (SHARP) trial revealed promise for patients with advanced hepatocellular carcinoma. First and foremost, sorafenib increased the overall survival of the patients in the study (n = 602) by about 3 months, from 7.9 months with the placebo to 10.7 months (see Figure 2) [16]. Another significant finding from this trial is that the sorafenib group exhibited a longer median time to radiologic progression; in other words, damage progression to the liver that is detecable by CT scan or radiography in general took longer to appear in the group given sorafenib. Lastly, adverse effects as a result of a sorafenib treatment were classified as “mild to moderate in severity,” which suggests that sorafenib’s side effects of weight loss, skin reactions, and gastrointestinal changes were relatively manageable for most patients in the trial [16]. Since the SHARP trial, sorafenib became (and remains) the best targeted therapy in the market for patients suffering from advanced hepatocellular carcinoma.

Despite the fact that VEGF, PDGFR, and BRAF are all not among the primary mutations in hepatocellular carcinoma according to the COSMIC database, but rather are considerably rare, the success of sorafenib must be explained. There have been many other clinical trials to test potential drugs for late-stage hepatocellular carcinoma and all have failed to outperform sorafenib [17]. For example, a phase III clinical trial was conducted to test sunitinib, which also targets VEGFR and PDGFR, but results indicate that this drug brought about significant toxicity and was overall futile with an overall medial survival of just 7.9 months [17, 18]. Other drugs such as linifanib, which target the same two receptors as sunitinib, were tested in trials but proved to be unsuccessful due to toxicity and inability to improve on overall survival [17]. Most likely, sorafenib’s success can be explained both by the fact that it targets multiple pathways simultaneously and it targets pathways that, though not among the prominent mutations observed for this cancer, are interconnected with molecular pathways that are known to be commonly mutated. In the previous section, the Wnt/β-catenin pathway was implicated in the progression and development of hepatocellular carcinoma, and is one of the most commonly mutated pathways for this cancer. Researchers have demonstrated that the Wnt/β-catenin pathway can contribute to the promotion of angiogenic factors, such as VEGF-A and VEGF-C among others; they found that removing β-catenin from the HCC cell line led to a decrease in angiogenic factors produced [19]. Separate research has also described that PDGF can activate the epithelial to mesenchymal transition (EMT) by upregulating Wnt signaling and by promoting phosphorylation of the p68 protein that leads to the formation of a complex with β-catenin to promote its function in colorectal cancer [20]. Elevated EMT occurrence facilitates metastasis of a cancer. It is likely, therefore, that sorafenib indirectly targets the prominently altered molecular pathways in hepatocellular carcinoma through other interacting pathways.

For now, sorafenib remains the gold standard for treatment of patients with advanced hepatocellular carcinoma. Many other trials have tested drug candidates, but none have surpassed the results of sorafenib. The success of sorafenib could be attributed to several factors, including its multi-kinase inhibiting nature as well as its indirect targeting of prominently mutated pathways. Ongoing research will continue to add to our understanding of the complexities and intricacies of this cancer, which will hopefully pave the way for better targeted therapies in the future. Until then, however, we will have to settle for sorafenib which is nonetheless a great initial targeted therapy for patients with hepatocellular carcinoma.

__**CONCLUSION**__ Hepatocellular carcinoma is the most common form of liver cancer, it accounts for approximately 90% of all liver cancers. It is the second most common cause of mortality from cancer. Interestingly, however, the causes of this cancer are laregly either cirrhosis from excessive alcohol consumption or a hepatitis infection (B or C). This represents a reasonably preventable cancer, and perhaps is the best of all options. Of course, many people often don't realize they even have hepatocellular carcinoma until it is at a very advanced stage. But even before any advanced stage is reached, even an earlier stage may be too late if it's caused by a virus. Hepatitis is a stubborn virus that will cause recurrence of infection even in response to liver transplantation. Mr. Eugene Edwards, a fictitious character based on a case study, is just one of many people who were diagnosed with hepatocellular carcinoma and died from it. It is a cancer that is hard to shake and even the best treatments are laughingly short-lived. That is not to say sorafenib's three month increased survival rate isn't significant, because it is. When a problem is about a loved family member, even a month can be very significant and worthwhile. Now, it's up to the researchers to continue digging deeper for better treatments. Only time will tell where the standard treatments for hepatocellular carcinoma end up.

__**REFERENCES**__
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Tumor classification is used for liver transplantation purposes. Classification is based on how sick a patient is, in what is called a Model for End-Stage Liver Disease (MELD) score. Different categories (among them tumor size, tumor number, tumor vascularization, etc.) area scored and the sum determines the final MELD score and class of tumor. A higher score denotes a worse condition [2, 5]. Surgical resection refers to the removal of the tumor and surrounding liver tissue, while attempting to preserve enough liver to allow for continued normal liver function; this offers the best chance for survival when caught very early [3]. Note to reader: I have had the opportunity to actually run a TBARS assay in the course of performing my senior design research project, though for a different purpose. The TBARS assay is the most common assay used to measure lipid peroxidation.