The+efficacy+of+therapies+for+the+BRAF+gene+in+Melanoma.

= = Ellie Pringle and Brendan Burke

There are a three types of skin cancer. The other two beside melanoma are basal cell skin cancer and squamous cell skin cancer. Skin cancer that originates in melanocytes is melanoma. Melanocytes (pigment cells) are located throughout the lower part of the epidermis. Because most melanoma occurs in a location where the patient can see the tumors, patients often first detect this form of cancer. Early detection of melanoma can be cured with relatively minor surgical treatment. The focus of our project is to discuss the efficacies of chemotherapeutic treatments. A knowledge of melanoma development and the biology of the mutations associated with this cancer has lead to recent advances in drug therapies. Therapy techniques have transformed from surgery as the primary treatment for the localized melanoma to an era of targeted chemotherapy. The different stages of melanoma and their properties are important in the determining the selection of target chemotherapies. The stages of melanoma and their characteristics are summarized [|here] in my first blog.
 * __An Introduction to Malignant Melanoma__**

__History of Chemotherapy for Metastatic Melanoma__
Dacarbazine(DTIC) was the standard chemotherapeutic therapy for melanoma starting in 1975. DTIC has proven to show a measurable response fighting against melanoma. As an alkylating agent, DTIC acts by inhibiting the transcription of DNA into RNA into protein synthesis. Alkylating agents substitute alkyl groups for hydrogen atoms on DNA which results in the formation of cross links with the DNA chain. This drug interferes with the growth of cancer cells which eventually are destroyed. [|Table 1] shows eight randomized control trials in which DTIC was used as the control arm. Patients from the eight trials experienced a 13.4% average response rate to DTIC. The median overall survival of these treated patients ranged from 5.6 to 11 months. The inconsistency of median overall survival suggests a lack of randomization among patients. Patients from different trials could have been treated with DTIC at different developing stages of melanoma. The low response rate also suggests that it is unlikely that DTIC has an actual effect on median survival. These trials started in 1992, in the future chemotherapeutic research would try to find target agents with a much higher response rate than experienced with dacarbazine treatment trials (Yang).

Temozolomide(TMZ) is another orally administered prodrug synthesized in 1984 that can be compared and contrasted to DTIC. This agent has proved promising results in brain tumors as well as solid tumors found in malignant melanoma cases. This [|journal] provides no particular difference in the response rate or survival rate between TMZ and DTIC but the chemical structure of TMZ gives it an advantage. The small molecular weight of TMZ and its ability to dissolve in non-polar solvents allows the agent to cross the blood-brain barrier with ease. Essentially 100% of the administered dosage enters the bloodstream of the patient. Stable at an acidic pH in the stomach, once contacting the basicity of the blood stream, TMZ undergoes hydrolysis. This hydrolysis eventually leads to the formation of methyldiazonium ion. The structure of TMZ before hydrolysis and post can be seen [|here] (Livertox). The structural formation of O6-methylguanine leads to apoptotic pathways in the cancerous cells. These compound properties allow TMZ to successfully treat central nervous malignancies. TMZ was majorly pursued as a treatment that prevented brain metastases in melanoma patients. Particularly in metastatic melanoma, the activity of TMZ was observed in several studies conducted by the Cancer Research Campaign. A large randomized phase III trial was used to compare the efficacies of TMZ to DTIC. [|Table 2]. Overall TMZ compared favorably to DTIC. With very similar response rates 13% compared to 12%,TMZ proved to have a better overall survival percentage at 6 months, 61% compared to 51% (Yang) With only approximately 150 patients treated with each type of targeting agents, a larger trial needs to be performed to confirm the efficacy of TMZ compared to DTIC. In the case of side-effects, a larger percentage of patients treated with TMZ maintained scores for physical functioning.

For the most part, DTIC and TMZ remained the standard chemotherapeutic treatments associated with metastatic melanoma. Although not proven in many trials, combination therapy with these two agents have shown an improvement in survival rates. It remains unlikely that trials conducted on melanoma will be large enough to prove the efficacy of combination therapy compared to traditional chemotherapeutic options.

__**Function of BRAF**__ One of the first discoveries from the genome sequencing screening project conducted by Davies et al. was a BRAF somatic missense mutation in 66% of patients with malignant melanoma. Of these patients, 80% had a single substitution of glutamic acid (E) for valine (V) in codon 600. Other mutations include BRAFV600K, which is a lysine substitution of valine occurring in 10-30% of patients, and BRAFV600R, which is an arginine substation of valine occurring in 1-7% of patients.

The BRAF protein kinase is in the MAPK signaling pathway (mitogen-activated protein kinases), which is used in communicating to cells through phosphorylation. BRAF codes for a serene/threonine kinase oncogene that transmits signals downstream of Ras to transduce regulatory signals from Ras to MEK1/2. This can be seen in



A mutation in BRAF causes a 500-fold increase in activity compared to wild-type BRAF. The mutation mimics the phosphorylation needed to increase cell proliferation and to prevent cell apoptosis. This eventually leads to tumor growth.

A major tool leading to the development of target therapies for oncogenic BRAF melanoma patients is the Cobas 4800 BRAFV600 mutation test made by Roche Molecular Systems Inc. It was pivotal in clinical trials for vemurafenib in order to select patients which tumors contain the V600 mutation in the BRAF gene. This mutation test has a 97% positive agreement in detecting the BRAF V600 mutation.

a component of the RAS-RAF-MEK-MAPK signal transduction pathway __**Mutated BRAF gene in Melanoma and Staging**__ As we have learned in class, many active mutations are restricted to particular cell pathways and in turn are linked to certain types of cancer. A perfect example of this are mutations in the BRAF gene, as discussed above, deal with cell growth and proliferation. The identification of point mutations of the BRAF gene has been proven to be a milestone contributing to our knowledge of melanoma development and clinical treatments for the disease. A deregulation of BRAF regulation can be associated with more than half of the tumors associated with malignant melanoma.

The oncogenic BRAF signaling can be associated along with various steps in developing stages of melanoma. That being said, mutations in BRAF alone is not sufficient to initiate oncogenic signaling in melanoma. Mutations in the BRAF gene can still be found in the different stages of the disease. First, BRAF mutations have been observed in the initial stages of melanoma. Complications with the RAS-RAF signaling pathway have resulted in the initiation of melanoma. In the case of very early stages of melanoma development, BRAF mutations are viewed as markers for future metastatic melanoma issues in patients. In these early stages, treatment options usually include wide excision surgery to remove the melanoma as well as no more than 2 cm of skin around it.

Oncogenic BRAF can also be associated with the tumor progression stage of melanoma. BRAF is involved with the activation of tumor progression due to related genes located down the affected pathway. For example, BIM is located downstream the oncogenic BRAF gene. BIM is a protein that promotes further tumor cell survival rather than apoptosis. Mutated BRAF also contributes to excessive angiogenesis by inducing an autocrine vascular endothelial growth factor secretion. Before reaching the lymph nodes, stage II melanoma patients are usually treated with a more invasive wide excision surgery. A sentinel lymph node biopsy is usually suggested to make sure the cancer has not spread to nearby lymph nodes.

Finally in the metastatic phases of melanoma when cancerous cells have spread to lymph nodes or other areas of the body, oncogenic BRAF usually activates transcription factors that that promote cell invasion/metastasis and also the promotion of cytokine-mediated signaling. Both of these promote invasive cell behavior and contribute to the development of metastatic melanoma in humans. Metastases in internal organs are sometimes removed. Organs that experience metastatic symptoms but cannot be treated must resort to targeted therapy, chemotherapy or immunotherapy. This type of late stage treatment is what we will be focusing most of our cancer project on. (http://www.biooncology.com/research-education/braf/metastatic-melanoma/melanoma-development)

__Target Chemotherapies__
__Vemurafenib__ Vemurafenib is an orally administered chemotherapeutic drug developed mainly to treat late stage malignant melanoma. It was officially approved by the [|U.S. Food and Drug Administration] in August 2011. Zelboraf is the commercial name of this drug. The name Vemurafenib is derived from V 600 E mu tated B RA F inhib ition. Vemerafenib was found by using fragment-based drug discovery (FDA). The mechanism of this drug as a BRAF enzyme inhibitor can be found on the Zelboraf official [|website]. In short, the video describes the function of Zelboraf (Vemurafenib) in the oncogenic signaling pathway to stop the mutated BRAF protein from continuing the signaling pathway to MEK and ERK. This pathway will eventually lead to excessive cell proliferation and survival, just like most other cancers. As discussed above, confirmation of BRAFV600 mutation-positive is required before treatment with vemurafenib.

In 2011, the [|New England Journal of Medicine] published a randomized phase 3 trial to determine whether vemurafenib would prolong the rate of overall or progression free survival. They compared these results with patients treated with only DTIC. All patients had previously untreated stage III or IV melanoma and also tested positive for the BRAF V600E mutation. 672 patients were evaluated for overall survival. At 6 months, overall survival for the vemurafenib was at 84% while it was 64% for the DTIC group (Chapman). Progression-free survival was measured in 549 patients. The median progression-free survival for the patients treated with vemurafenib is 5.3 months and 1.6 months in the DTIC group. From this trial, benefit was experienced in all subgroups of patients who were included in this analysis. Overall, 48% of patients treated with vemurafenib experienced tumor shrinkage. With DTIC only having a confirmed response rate of 5%, this suggests the possibility that patients with a BRAFV600E mutation have a more aggressive type of melanoma compared to wild-type BRAF melanomas.

In 2012,the [|National Institute of Health and Clinical Excellence] did a random, open-labeled and active controlled trial called BRIM3. They focused on vemurafenib for treating advanced stage IV cases of metastatic melanoma and the cost effectiveness of treatment as well as potential side effects. The recommended dosage is 960mg taken orally twice a day. With a one week supply of a vemurafenib supplement being 1750 Euros(2380$), is it logical to take this target chemotherapy with the price and side effects? Side effects include nausea, fatigue, rash and arthralgia. Cutaneous squamous cell carcinomas of the skin were found in 24% of patients treated with vemurafenib. The only outcome of this study that could justify the price and uncomfortable side effects would be a longer progression-free survival and a high response rate associated with vemurafenib. This study presented three analyses of overall survival at three different cut-off points. Results from the first cut-off showed average progression-free survival with vemurafenib to be 5.32 months compared to 1.61 months when treated with only DTIC. 48% of patients treated with vemurafenib experienced a confirmed response while only 5% of patients treated with DTIC experienced a partial response (NIHCE). Overall from the first response and the other two, results showed a statistically significant progression-free survival benefits. Even with the side effects associated with vemurafenib, I believe it is worth it to take the standard dosage of this target therapy when diagnosed with late stage melanoma.

__Dabrafenib__ Dabrafenib is a newer chemotherapeutic drug that, like vemurafenib, targets the BRAF protein to decrease phosphorylation in the MAPK signaling pathway and causes cell cycle death to inhibit cell growth. It is a selective and reversible ATP-competitive inhibitor. Clinical trials for dabrafenib began in 2009, final results were published in 2012, and it was first approved by the FDA in 2013 for single-agent treatment. In Phase I trials, a dose of 150mg twice daily was established to minimize toxicity effects and maximize efficiency. The most common side effects associated with the drug include cutaneous squamous cell carcinoma, pyrexia, fatigue, and nausea, but these were rarely severe. In Phase II trials the results were promising. Patients with V600E BRAF-mutation responded at a rate of 57% and patients with V600K mutations, which is the second most prevalent gene substitution, responded at a rate of 37%. Although 57% seems low, it is higher than the response rate of 48% found in vemurafenib which is a similar drug that has been used in treating metastic melanoma. In Phase III trials, dabrafenib was compared to dacarbazine, which is a chemotherapy drug commonly used in treating melanoma. The response rate in Phase III trials was 59% with a median progression-free survival of 6.9 months, compared to a PFS of 2.7 months with dacarbazine. The question about this drug is: how does it compare to vemurafenib and which one should continue to be used? In comparing demurafenib and vemurafenib, the drugs are not very different. The response rates are similar (between 55-60%) and the progression-free survival is 6-7 months for both of the drugs. Dabrafenib is 20 times more selective for inhibiting V600E BRAF mutants than wild-type BRAF, compared to vemurafenib, which is 10 times more selective. The more selective a drug is for the mutation, the better it is because the less it targets non-cancerous cells. The main difference between the drugs, though, is toxicity. Vemurafenib reached maximum toxicity levels in Phase I and has more adverse severe side effects than dabrafenib, which did not reach maximum toxicity levels in Phase I.

__Trametinib__ Trametinib is a drug that inhibits MEK1 and MEK2 downstream of BRAF in the MAPK signaling pathway. The location of MEK and BRAF and the drugs that target those locations can be seen in Figure 4. The FDA approved Trametinib in 2013 as a single-agent drug therapy for BRAF mutant melanoma. In a comparison during Phase II trials between trametinib and other older methods of chemotherapy(dacarbazine or paclitaxel), the response rate is 22% with a median PFS of 4.8 months compared with 1.4 months in the chemotherapy arm. Although trametinib seems like it has a low response rate and PFS, its strength is not in monotherapy.

__Combination Therapy__ The aim of combination therapy of BRAF and MEK inhibitors is to delay the onset of resistance and increase cancer cell apoptosis in comparison to BRAF inhibitor monotherapy. Another interesting benefit is that the toxicity of the drugs decreases when in combination with each other. Dabrafenib and trametinib, called CombiDT, is the first combination of BRAF and MEK inhibiting drugs to undergo clinical trials. In Phase II trials, CombiDT had responses rates of 76% compared to a 54% response rate of monotherapy with dabrafenib. The progression-free survival of CombiDT is 9.4 months compared to 5.8 months in monotherapy and the median overall survival is 23.8 months. The response rate of patients who switched from dabrafenib to CombiDT is only 9%, which suggests that CombiDT is best used up-front and not after other treatment. CombiDT was approved by the FDA in January 2014 at an accelerated rate due to such impressive Phase II data. Phase III trials are still taking place and results should come out in the next few years. In this Figure that compares monotherapy with combination therapy, the highest dosage of dabrafenib and trametinib of 150mg and 2mg twice a day respectively, yielded a response rate of 76%.

**__BRAF inhibition combined with immunotherapy__ **
In 2012, studies have emerged suggesting that the combination of BRAF inhibition chemotherapeutic agents like Vemurafenib and immunotherapy can decrease tumor size in metastatic melanoma cases. In general, immunotherapy is the use of medicines to stimulate a patients immune system to recognize and destroy cancer cells effectively. A study out of [|UCLA's Jonsson Comprehensive Center] have shown that engineered T-cell immunotherapy with Zelboraf has resulted with increased response rates and survival rates in animals. Animals treated with this combination therapy are living twice as long as animals treated with each therapy separately. In human trials, researchers are hoping that Zelboraf combined with immunotherapy will maintain response rates of previous treatments but increase the length of disease-free progression time. The immunotherapy portion has engineered lymphocytes to express a receptor that recognizes cancer cells and creates an immune response (Ribas). Combination therapy would simultaneously inhibit BRAF while adding T-cells to start an anti-tumor immune system response.

Another study by published in 2013 by the [|Clinical Cancer Research] website tests whether BRAF inhibition combined with adoptive T-cell transfer can be more effective in inducing a longer disease-free progression time. Common BRAF inhibition therapies show a 50% response rate to patients with oncogenic BRAF but complete and durable remissions are rarely seen in these treatment options (Chenweng). Unfortunately, combination therapy, along with monotherapy, has a resistance rate. 50% of patients using CombiDT have resistance within 6-7 months after initiation of treatment. This high resistance rate makes these melanoma patients extremely hard to treat. Combination therapy and the current target therapies are important positive steps in the right direction towards improving progression-free survival and overall survival of melanoma.
 * __Conclusion__**

Work Cited

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Davies, Helen. et al. "Mutations of the BRAF gene in human cancer". Nature Publishing Group, 2002. Print. Kainthla, Radhika, et al. Dabrafenib for treatment of BRAF-mutant melanoma.Pharmacogenics and Personalized Medicine. Dovepress. 2014.

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Sarange-Perry MD, VIta, et al. Recent developments in the medical and surgical treatment of melanoma. A Cancer Journal for Clinicians. 2014.

Yang, Arvin, Dr. "The History and Future of Chemotherapy for Melanoma."Hematology/Oncology Clinics of North America. N.p., n.d. Web.

Chenweng, Liu. "BRAF Inhibition Increases Tumor Infiltration by T Cells and Enhances the Antitumor Activity of Adoptive Immunotherapy in Mice." //Clinical Cancer Research //<span style="background-color: rgba(255,255,255,0.0980392); font-family: 'Times New Roman',Times,serif; font-size: 16px;">. N.p., 2012. Web.

<span style="background-color: rgba(255,255,255,0.0980392); font-family: 'Times New Roman',Times,serif; font-size: 16px;">"FDA Approves Zelboraf and Companion Diagnostic Test for Late-stage Skin Cancer." //<span style="font-family: 'Times New Roman',Times,serif; font-size: 16px;">FDA.gov //<span style="background-color: rgba(255,255,255,0.0980392); font-family: 'Times New Roman',Times,serif; font-size: 16px;">. N.p., 17 Aug. 2011. Web. 11 June 2014.