	Stage IV melanoma is an aggressive malignancy with a median survival shorter than 1 year. The identification of activating mutations in a component of the mitogen activated protein kinase (MAPK) pathway called BRAF in 40% to 60% of melanomas has allowed for the development of efficacious therapies that inhibit the activity of the pathway. Targeted inhibition of BRAF with vemurafenib or dabrafenib or direct inhibition of MEK with trametinib confers high response rates and statistically significant survival benefits relative to treatment with traditional chemotherapy. The FDA has approved these agents as monotherapies for the treatment of metastatic melanoma containing a V600 BRAF mutation. Phase II data suggest that treatment with the combination of a BRAF and a MEK inhibitor, dabrafenib plus trametinib, can further increase the rate and durability of treatment responses and lengthen the survival benefit conferred by single-agent treatment. Combined treatment with dabrafenib and trametinib was conditionally approved by the FDA in January 2014 for the treatment of unresectable and metastatic melanoma expressing a V600E or V600K BRAF mutation.

 It is estimated that in 2014 more than 76,000 people will be diagnosed with melanoma and approximately 9700 people will die from the malignancy.

 When melanoma metastasizes to distant sites, the prognosis is poor with a median survival of less than 1 year. The current American Joint Committee on Cancer (AJCC) staging system for melanoma uses histologic features to predict the outcome of patients with localized or regionally advanced disease. In distantly metastatic melanoma, the location of metastases and level of lactate dehydrogenase (LDH) in the blood are predictors of outcomes. Until 2011, there were only 2 systemic therapies FDA approved for the treatment of stage IV melanoma—the chemotherapy dacarbazine and the immunotherapy high dose interleukin-2 (HDIL2). Dacarbazine treatment confers a 5% to 20% response rate that does not translate into a survival benefit.

	HD-IL2 offers a 5% chance for durable benefit but requires in-patient management, and the toxicity profile limits the patients who are eligible for this treatment.

 Neither of these therapies target defined molecular abnormalities selected by the tumor cells. Identifying driver mutations offers the potential to define targets for more effective therapeutic interventions. The MAPK pathway (

) relays signals from activated cell surface receptors to RAS proteins leading to the dimerization and activation of A-, B-, and C-RAF.

 Active RAF activates MEK1 and MEK2, leading to ERK activation and cell proliferation. Using a genome-wide screen to systematically identify genes mutated in human cancer, researchers at the Wellcome Trust Sanger Institute identified activating mutations in BRAF in the majority of melanomas sampled.

 More than 90% of the BRAF mutations are located at position 600 with the substitution of valine by a negatively charged glutamate (V600E BRAF mutation).

	This mutation causes constitutive activation of BRAF. Other rarer activating mutations also have been identified at position 600, including V600K and V600R mutations.

 The location from which a primary melanoma arises predicts for the likelihood of a BRAF mutation.

	Vemurafenib (Zelboraf) is a potent inhibitor of BRAF activity.

 In preclinical models, an analogue of vemurafenib inhibited the proliferation of V600E BRAF-expressing melanoma cell lines.

 A phase I study evaluated the safety of oral vemurafenib in patients with advanced melanoma and established a recommended phase II dosage of 960 mg twice daily.

	Responses were detected in melanomas expressing V600E BRAF mutations but not in those expressing wild-type BRAF. Rapid treatment responses were detected as evidenced by decreases in metabolic activity of metastatic deposits seen on FDG-PET imaging performed 15 days after treatment initiation. A multicenter phase II trial (BRIM-2) enrolled patients with stage IV V600E BRAF-expressing melanoma who received at least 1 prior systemic therapy; the trial assessed the response rate to vemurafenib.

 The study enrolled 132 patients; 53% developed a response to treatment (6% complete and 47% partial responses). The median duration of response was 6.7 months and the median progression-free survival (PFS) was 6.8 months.

	A randomized phase III study (BRIM-3) was conducted to compare survival in patients with untreated stage IV V600E or V600K BRAF-expressing melanoma treated with vemurafenib versus dacarbazine chemotherapy.

 The response rate for vemurafenib was 48% as opposed to 5% for dacarbazine. Treatment with vemurafenib conferred a significant improvement in both PFS and overall survival (OS). The median PFS was 6.9 months following vemurafenib treatment as compared to 1.6 months following chemotherapy. The median OS increased from 9.7 months in the dacarbazine arm to 13.6 months in the vemurafenib arm. Following the first interim analysis, the Data and Safety Monitoring Board recommended that patients randomized to dacarbazine be crossed over to vemurafenib. This crossover confounds subsequent OS analysis.

	The survival benefit seen with vemurafenib is not drugspecific, as evidenced by activity seen in clinical trials with another oral BRAF inhibitor, dabrafenib (Tafinlar). In a phase I study, 50% of patients with V600 BRAF-mutated melanoma treated with dabrafenib developed a response.

 A phase II (BREAK-2) study treating patients with dabrafenib at the maximum tolerated dose of 150 mg twice daily demonstrated a 59% confirmed response rate in subjects with V600E-mutated melanoma and a 6.3 month median PFS.

	The phase III BREAK-3 study randomized in a 3:1 fashion 250 patients with V600 BRAF-mutated stage IV melanoma to dabrafenib (187 patients) or dacarbazine (63 patients).

 Dabrafenib conferred a significant improvement in median PFS: 2.7 months in the dacarbazine arm versus 5.1 months in the dabrafenib arm.

	Targeted inhibition of V600-mutated BRAF in patients with advanced melanoma leads to high response rates and to survival benefits. However, PFS remains limited due to selection for resistance to treatment in the tumor cells. Multiple resistance mechanisms have been identified,

 including but not limited to tumor selection for activating NRAS mutations, increase in CRAF expression, and activation of COT kinase, which activates MEK independent of BRAF.

 Certain resistance mechanisms remain MEK dependent.

	Trametinib is a selective inhibitor of MEK with a recommended oral dosage of 2 mg daily.

 Treatment with trametinib demonstrated a 33% response rate in a phase I study consisting of 30 patients with BRAF-mutated melanoma.

 A phase II study enrolled 2 cohorts of patients with BRAF-mutated melanoma and demonstrated a 25% response rate to trametinib treatment with a median PFS of 4 months in a BRAF inhibitornäive cohort. The response rate in a cohort of patients previously treated with a BRAF inhibitor was 0%.

 As such, sequential treatment with a MEK inhibitor after progressing on treatment with a BRAF inhibitor does not appear to be a promising strategy, at least with trametinib.

	The METRIC phase III study randomized BRAF inhibitornaïve patients with V600 BRAF-mutated melanoma to trametinib or chemotherapy (dacarbazine or paclitaxel).

 Trametinib significantly improved PFS (4.8 months vs 1.5 months) and OS despite a high crossover rate in the dacarbazine arm to trametinib upon progression. The hazard ratio for death was 0.54 favoring the trametinib-treated patients. The 6-month OS in the trametinib arm was 81% compared with 67% in the dacarbazine arm. Similar to that seen with BRAF inhibition, trametinib can significantly improve survival but the extent of the median PFS benefit remains limited.

	Another limitation of BRAF or MEK targeted therapy is toxicity. In the BRIM-3 study, 38% of patients treated with vemurafenib required dose interruptions or reductions.

 The most common high-grade adverse events (AEs) were cutaneous toxicities, arthralgias, photosensitivity, and fatigue. Cutaneous squamous cell carcinomas or keratoacanthomas developed in 18% of patients. A grade-2 or higher rash developed in 18% of patients. The development of cutaneous squamous cell carcinomas (SCCs) appears to be related to paradoxical activation by BRAF inhibitors of the MAPK pathway in the setting of RAS mutations present in the squamous cells.

 In the METRIC study, 27% of patients treated with trametinib required dose reductions. The most common AEs were rash, peripheral edema, diarrhea, and fatigue.25 Rash developed in 57% of treated patients, with 8% developing a grade-3 or -4 rash. No cutaneous SCCs or hyperproliferative cutaneous lesions were diagnosed.

	While single-agent inhibition of either BRAF or MEK confers survival benefits, there are several limitations to approaches that target a single component of the MAPK pathway. These include only a few months’ improvements in median PFS, the development of resistance, and the development of toxicity, including SCCs associated with BRAF targeted therapies.

	In theory more complete inhibition of the MAPK pathway using the combination of dabrafenib and trametinib could potentially lead to more durable clinical benefit, delayed time to treatment resistance, and decreased incidence of SCCs.

	A phase I/II trial assessed the safety, clinical efficacy, and pharmacokinetics of combined therapy with dabrafenib and trametinib in patients with V600-mutated BRAF melanoma.

	With a median follow-up of 14.1 months, the median PFS of patients randomized to the 150 mg/2 mg arm was 9.4 months compared with 5.8 months in the dabrafenib monotherapy arm. The response rate of 76% in the 150 mg/2 mg arm was higher than the 54% seen with dabrafenib. The median duration of response in the 150 mg/2 mg arm (10.5 months) almost doubled the 5.6 months with dabrafenib monotherapy.

	In concordance with preclinical data, the incidence of cutaneous SCCs or other hyperproliferative skin lesions was less in the combination therapy group. The most frequent AEs in the 150 mg/2 mg arm were fever and chills developing in the majority of patients. MEK-inhibitor—related peripheral edema, cardiac toxicity, hypertension, and ocular toxicity occurred more frequently in the combination arm. A total of 58% of patients in the 150 mg/2 mg arm required dose reductions, most commonly because of the development of fever.

	Randomized phase III studies directly comparing the clinical efficacy and safety of BRAF inhibition alone to the combination of BRAF and MEK inhibition have completed enrollment. The MEK115306 (COMBI-D) study randomized patients with V600E/K-mutated melanoma to dabrafenib plus placebo (D+P) versus dabrafenib plus trametinib (D+T), while the MEK 116513 study randomized patients to vemurafenib versus dabrafenib plus trametinib. Results of the COMBI-D study were recently presented.

 Patients with untreated stage IV V600E or V600K BRAF-expressing stage IV melanoma were randomized in a 1:1 fashion to receive treatment with either D+T or D+P.

	The study met its primary end point of improvement in investigator assessed PFS at 9 month median follow-up with a HR for recurrence of 0.75 (P= .035) favoring the combination therapy arm. The median PFS increased from 8.8 months in the D+P arm to 9.3 months in the D+T arm. Six-month PFS increased from 57% to 70% of patients. Response rate also improved from 51% in the D+P arm to 67% in the D+T arm. A preplanned interim analysis of OS demonstrated an improvement in 6-month OS from 85% in the D+P arm to 93% in the D+T arm. However, this difference did not cross the stopping boundary for the study and an updated OS analysis is planned following 70% of death events. The addition of trametinib to dabrafenib led to fewer cutaneous SCCs and cutaneous hyperproliferative events. The incidence of pyrexia and chills increased with combination therapy. Adverse events led to a higher frequency of dose interruptions and dose reductions in the D+T arm (33% vs 49% of patients for dose interruptions and 13% vs 25% for dose reductions). This study is the first study to demonstrate in a randomized fashion that the addition of a MEK inhibitor to a BRAF inhibitor leads to survival benefits. The OS data remains immature and longer follow-up is necessary.

 compares the efficacy demonstrated in phase II and III clinical trials following treatment with single-agent or combined BRAF and MEK inhibitory approaches.

	Single-agent vemurafenib was approved by the FDA in 2011 for the treatment of V600E BRAF—mutated metastatic melanomas. In 2013, dabrafenib and trametinib received FDA approval as monotherapy treatments of V600 BRAF–mutated melanomas. In 2014, the FDA granted accelerated conditional approval for dual therapy with the combination of dabrafenib and trametinib (

) in melanoma expressing a V600E or V600K BRAF mutation. Combined therapy may create more durable and frequent responses with longer PFS when compared with single-agent treatment. Combining BRAF and MEK inhibition appears to decrease the incidence of certain cutaneous toxicities.

			Table 1. Comparison of Efficacy in Phase II and Phase III Trials With Dabrafenib, Trametinib, and Combination Therapy in Patients With V600 BRAF-Mutated Unresectable or Stage IV Metastatic Melanoma

Articles

It is estimated that in 2014 more than 76,000 people will be diagnosed with melanoma and approximately 9700 people will die from the malignancy.

Combination BRAF-MEK Inhibition With Dabrafenib and Trametinib for BRAF-Mutant Melanoma

		Pancreatic cancer has one of the poorest prognoses out of all human malignancies. Only 20% of patients who present with this disease have surgically resectable tumors at the time of diagnosis, and of these 20% of patients, the vast majority will experience recurrence within the first 2 years. Chemotherapy is the mainstay of treatment. Unfortunately, little improvement has been made in the past few decades with regard to overall survival (OS) with chemotherapy alone. Of note, the immune system’s involvement in cancer development and progression has sparked much interest in recent years. The model of the cancer-immunity cycle suggests an interplay of immune-suppression and immune-stimulation. In normal individuals, a state of immunosurveillance is in place. However, within the tumor microenvironment, inhibitory signals and immunosuppressive cells are present and tip the scale in favor of immune suppression. This enables cancer cells to evade the immune system and allows tumors to grow. Studies in a variety of solid tumors have shown that many chemotherapies, including those used as standards of care in pancreatic cancer, have immune-modulating functions, including inhibition of immune suppression and stimulation of immune function. This article reviews up-to-date clinical studies combining chemotherapy with immunotherapy; it also explains the rationale for this combination in treatment of pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDA) has one of the most dismal prognoses out of all human malignancies, currently ranking as the fourth most common cause of cancer-related death in the United States.1 Although advances have been made in both understanding the molecular biology of the disease as well as its imaging, the current 5-year survival rate for all stages combined is only 6%, which is one of the lowest out of all cancer diagnoses.

 Currently, the only hope of cure for PDA is surgical resection.

However, even after an R0 resection with node-negative disease, the long-term survival rate is lower than 10%.2 This is due to a high recurrence rate, with 80% of those patients experiencing recurrence within 2 years of surgical resection. Over the last few decades, new chemotherapies have been discovered; still however, the effect on OS in patients with metastatic tumors has been minimal. For example, in 1997 gemcitabine was shown to improve the median OS by 1.2 months when compared with fluorouracil.

 More recently, 2 sets of chemotherapy combinations have also been shown to improve OS in this disease. The chemotherapy cocktail of fluorouracil, oxaliplatin, irinotecan and the vitamin leucovorin (FOLFIRINOX) used in the first-line setting was shown to improve OS to 11.1 months when compared with gemcitabine alone.

In October 2013, nab-paclitaxel was shown to achieve an OS of 8.5 months compared with 6.7 months achieved with gemcitabine alone.

 The fact remains that, although we have seen advances in the treatment of this disease, the clinical effect on the overall prognosis has been slight, with 5-year survival essentially unaffected. In addition, the trial that achieved the greatest improvement in OS with FOLFIRINOX was accompanied by substantial toxicities and, therefore, is only recommended for robust patients with an Eastern Cooperative Oncology Group (ECOG) performance status of 0 to 1.4 The meager improvement in OS is thought to be due to the disease’s resistance to both chemotherapy and targeted therapy.

Today, many studies have focused on the immune system’s role in cancer and how it can recognize both cancer cells and tumor-associated antigens. Therefore, many investigators have turned their focus to immunotherapy as a potential treatment for PDA.

Chen and Mellman have delineated the cancer-immunity cycle, which depicts the immune system’s role in controlling tumor growth in normal individuals. Understanding this cycle provides insight into how tumors can evade it. As cancer develops, oncogenic proteins are produced. In the normal individual, these cancer-associated antigens are released into the microenvironment during necrotic or immunogenic cell death. The antigens are captured by antigen-presenting cells (APCs), such as dendritic cells, processed into peptides, and presented on the cell surface bound to major histocompatibility complex I (MHCI) molecules.

Next, the APCs recruit T cells by the release of proinflammatory cytokines such as TNF-alpha, IL-1, and IFN-alpha. The binding of the T cells to the MHCI/ antigen complex on the APC is the first step of T-cell priming and activation. Following activation, T-effector cells travel to the tumor site, infiltrate the tumor and, ultimately, the T-cell receptor binds to a tumor cell and kills tumor cells via the cytotoxic T-cell response (CTR). At this point, the cycle restarts as the immunogenic death releases more cancer cell antigens; therefore, the cycle propagates itself and potentiates its own response (

	The Effects of Chemotherapy on the Cancer- Immunity Cycle

The idea of the cancer-immunity cycle proposes that, for a cancer immune response to be generated, the net balance between immune stimulation versus immune suppression must be tipped in favor of the former. Studies in various cancers have suggested that tumors evade the immunogenic process mostly by factors that promote immunosuppression. In pancreatic cancer, these evasion mechanisms include: 1) inhibition of antigen presentation by downregulation of HLA/ tumor antigen complexes on tumor cells

;2) upregulation of immune cell inhibitors, such as programmed death ligand (PD-L1), upon which binding to the programmed death (PD- 1) receptor on T cells results in T cell apoptosis and immunosuppressive cytokine release

; 3) receptor inhibition of costimulatory interactions between CD28 and B7 proteins by CTLA-4, which is further suggested by CTLA-4 blockade slowing tumor growth

; and 4) infiltration of tumors by leukocytes that promote immune suppression and immune tolerance such as regulator T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) (

 Of particular interest, from a medical oncology standpoint, are the effects of chemotherapy on the cancer-immunity cycle and, ultimately, how chemotherapies may be used in treatment to synergize with immunotherapies.

Two general ways chemotherapy may affect the cancer-immunity cycle are improving immune stimulation and inhibiting immune suppression. On the immune stimulation side of the equation, some chemotherapies may improve the immune system’s recognition of malignant cells. For example, gemcitabine was shown to increase the spectrum of epitopes that are presented on the surfaces of mesothelioma cells.

		Figure 1. Neoplastic Growth Control Via Normal Immunosurveillance and the Effects of Various Chemotherapies

. Neoplastic growth control via normal immunosurveillance and the effects of various chemotherapies. The cancer immunity cycle starting at cancer cell death with release of antigens into the surrounding microenvironment. The antigens are captured by APCs, processed and presented on the APC surface. The APCs, with antigens on their surface, then prime and activate T cells. T cells then move to the site of the tumor and are now “programmed” to bind to and kill tumor cells. Certain chemotherapies potentiate the immune response (green arrows).

Furthermore, gemcitabine and oxaliplatin have been shown to upregulate HLA molecules on tumor cells.

 These molecular mechanisms increase the chances of activated T cells recognizing malignant cells. In addition to this mechanism, some chemotherapies have been shown to improve the ability of the tumor cells to induce an immune response, essentially improving the immunogenicity. For example, oxaliplatin has been shown to upregulate cancer death associated molecules (CDAMs) on the surfaces of colon cancer cells.

CDAMs essentially tag dying tumor cells for phagocytosis. Additionally, some chemotherapies can also induce tumor cells to secrete chemical signals that promote dendritic cell maturation. For example, when lung cancer cells are treated with gemcitabine or oxaliplatin in vitro, the resulting supernatant has the capacity to induce dendritic cell maturation.

 Cyclophosphamide was also shown to induce dendritic cell differentiation as well as release CDAMs in various cancer cell lines in vitro.

 Furthermore, some chemotherapies upregulate the expression of costimulatory molecules on malignant cells, such as CD80/B7-1, which is required for generating an immune response.

Additional examples of chemotherapy improving the immune response include increasing the permeability of the cell to cytotoxic granzyme B and upregulating antigen presenting complexes on dendritic cells, both of which are promoted by paclitaxel.

 Finally, as a class, taxanes can increase the production and local release of cytokines that stimulate T-cell priming and activation, such as IL-1 and TNFalpha.

 All of these are examples of how chemotherapies can augment the immunogenicity of malignant cells.

 The other side of the equation addresses immune suppression. Studies in a variety of cancers have shown that many tumors are infiltrated with immune-suppressive cells. In a pancreatic cancer mouse model, for example, it has been shown that these tumors are infiltrated with suppressive regulatory T cells (Tregs) and MDSCs (

	Changing it up: Immunotherapy Combined With Chemotherapy

Treatment of these mice with single agent gemcitabine decreased immunosuppressive MDSCs. Additionally, tumor regression and improvement in OS were seen in mice treated with gemcitabine in combination with a dendritic cell vaccine. These results showed that hindering the tumor’s endogenous immunosuppressive function, in turn, allowed the dendritic cell vaccine to propagate its immunogenic function. The same study showed that pancreatic cancer patients had higher levels of MDSCs in their peripheral blood compared with healthy donors and these levels were significantly decreased in the majority of these patients after 1 cycle of single agent gemcitabine. In light of these findings, further studies are being pursued in human clinical trials.

Clinical trials testing pancreatic cancer vaccines started in the late 1990s (

 GVAX was the first pancreatic cancer whole cell vaccine that was combined with chemotherapy (cyclophosphamide).

 The GVAX vaccine consists of pancreatic cancer cells genetically modified to secrete granulocyte macrophage colony stimulating factor (GM-CSF). Cyclophosphamide is thought to promote immune stimulation by promoting dendritic cell maturation and upregulating HLA molecules on tumor cells. Cyclophosphamide is also thought to negatively regulate the immune suppressive functions of Tregs. Therefore, the rationale of this combination is quite clear as pancreatic cancer cells secreting GM-CSF is immune stimulating and cyclophosphamide both stimulates immunogenicity and relieves immune suppression. It should be noted that this study was the first vaccine study in patients with metastatic pancreatic cancer. Importantly, the median survival more than doubled when the vaccine was combined with cyclophosphamide compared with the vaccine arm alone (4.7 months vs 2.3 months).

. Tumor microenvironment. Many solid tumors are infiltrated by immunosuppressive cells, such as MDSCs and Tregs. Certain chemotherapies inhibit these suppressive functions.

As previously mentioned, single-agent gemcitabine has long since been the first-line standard of care for pancreatic cancer across all stages. Gemcitabine has been found to have multiple effects on the cancer-immunity cycle. It improves immune stimulation by inducing dendritic cell maturation, increasing epitope presentation on tumor cells, and decreasing tumor infiltrative MDSCs.

 These studies show that gemcitabine also augments the immune response in favor of generating immunity. Therefore, the data lay the foundation for investigating combinations of this chemotherapy with immunotherapy.

The first example of such a combination is a phase I study in Japan investigating the combination of single-agent gemcitabine with a vascular endothelial growth factor receptor 2 (VEGFR2) peptide vaccine. VEGFR2 is highly expressed in the new vasculature of tumors, but not in normal blood vessels. The rationale for this study was to utilize the immune modulating characteristics of gemcitabine to generate an immune response to the VEGFR2 peptide. Twelve of the 18 patients enrolled in the study had either a partial response or stable disease and the median OS was 7.7 months, which was greater than treatment with gemcitabine alone.

 Two other studies have also investigated peptide vaccines, 1 using a personalized peptide and the other using the Wilms tumor peptide. The personalized peptide showed a median survival of 9 months when combined with gemcitabine, which is an improvement over chemotherapy alone.

The algenpantucel-L hyperacute pancreas vaccine trials are currently investigating the benefit of adding a pancreatic cancer vaccine to the standard adjuvant treatment chemotherapy and chemoradiotherapy (known as the RTOG-9704 standard) for resected pancreatic cancer. The algenpanctucel-L vaccine consists of human pancreatic cancer cells that express α-gal epitopes, which are not present on human cells; however, they are present on cells of most other mammalian species. Bacterial gut flora within the human gastrointestinal tract continuously stimulate production of antibodies to α-gal, as the epitopes are frequently introduced through the human diet. Upon administration of the vaccine, the human immune system already has humoral immunity in place and antibodies recognize the α-gal epitopes on the vaccinated pancreatic cancer cells. These antibodies then fix complement and activate complement mediated lysis, starting the cancer-immunity cycle.

 Phase II data from this study showed an added benefit when combining algenpantucel-L with the RTOG-9704 standard adjuvant chemotherapy; 12-month disease-free survival was 62% with the vaccine compared with less than 50% in the initial study of the adjuvant standard of care (RTOG-9704).

		Table. Pancreatic Cancer Vaccines Combined With Chemotherapy

					FOLFIRINOX Gemcitabine + nab-paclitaxel

		Cy indicates cyclophosphamide; DCR, disease control rate; DFS, disease-free survival; MS, median survival; OS, overall survival; PD, progressive disease; PFS, progression-free survival; PR, partial response; SD, stable disease; TTP, time to progression.

The median disease-free survival was 14.1 months with the vaccine compared with 11.4 months with RTOG-9704. The 12-month OS was 86% with the vaccine compared with 69% in the standard adjuvant study. Phase III study of algenpantucel- L is still ongoing and will enable appropriate comparison to the standard of care. In the algenpantucel-L phase II study, 3 patients who had recurrence after immunotherapy had a resultant complete radiographic response to subsequent chemotherapy. This raises the possibility of increased chemosensitivity following immunotherapy.

As previously mentioned, the recent advances in pancreatic cancer include the combination of gemcitabine and nab-paclitaxel (Abraxane) or the chemotherapy cocktail FOLFIRINOX as first-line treatment, depending on performance status, in stage 4 disease. Either combination is used at various cancer centers in the setting of locally advanced disease. Pertaining to the gemcitabine/nab-paclitaxel combination, we have already discussed the effects of gemcitabine on immunogenicity and immune suppression. Again, taxanes augment the cancerimmunity cycle by improving immunogenicity. Furthermore, paclitaxel reduces immune suppression by antagonizing Treg functions.

Similarly, the chemotherapies that make up FOLFIRINOX augment the immune system both by improving immunogenicity as well as prohibiting immunosuppression. With these immune-modulating functions, another hyperacute pancreas vaccine study with algenpantucel-L is presently under way for locally advanced pancreatic cancer. This study has recently opened and has several arms including gemcitabine and nab-paclitaxel alone, FOLFIRINOX alone, and each standard chemotherapy in combination with the vaccine. Another immunotherapy option that has been explored is agonist monoclonal antibodies to CD40.

CD40 is a receptor of the tumor necrosis factor (TNF) family and is usually expressed on B cells, dendritic cells, and monocytes. Its ligand, CD40 ligand, is mainly expressed on activated T cells. Binding of CD40 ligand to the CD40 receptor is essentially immune-stimulating, resulting in the upregulation of MHC and costimulatory molecules, the release of proinflammatory cytokines and the further enhancement of T-cell activation. Vonderheide and colleagues

 investigated the stimulation of the CD40 receptor using an agonist monoclonal antibody in combination with gemcitabine.

They found that stimulating CD40 in combination with gemcitabine induced tumor regression in 80% of the mice, whereas none of the mice treated with gemcitabine alone saw tumor regression. Further studies showed the CD40 stimulated tumor regression was controlled by both T-cell dependent and T-cell independent mechanisms.

 Beatty and colleagues then went on to test this phenomenon in humans in a phase I clinical trial investigating agonist CD40 monoclonal antibody, CP-870,893, combined with gemcitabine in pancreatic cancer patients. In this study 10 patients were in the mean tolerated dose expansion cohort. Eight of these 10 received at least 2 cycles of chemotherapy.

Two of the 8 achieved a partial metabolic response as evidenced by >25% reduction of standard uptake value (SUV) on positron emission tomography (PET) scan. They also found an average SUV reduction of 26.9% within the primary lesion and all patients did have some reduction.

The cancer-immunity cycle is an ideal model to envision how tumor cells evade immunosurveillance as well as where future modalities may intervene with hopes of potentiating tumor cell death. The cancer-immunity cycle together with the immunemodulating functions of chemotherapies that are used in pancreatic cancer creates a rationale for investigating vaccinechemotherapy combinations.

Studies to date have suggested benefits of adding immunotherapies to standard chemotherapy regimens. Additional benefits are also suggested by the indication that immunotherapy may render improved chemosensitivity at later dates. In addition, vaccines are often well tolerated with minimal toxicities, which make them a favorable approach. The hope is that we can identify the appropriate combination of vaccine and immune-modulating chemotherapy that will eradicate the disease. There is also likely to be a role for immune checkpoint therapy with inhibitors of PD-1 and PD-L1. Such phase I single-agent studies are currently in progress for pancreatic cancer. The results of studies so far create hope that the combination of chemotherapy with immunotherapy may be a game changer in the treatment of pancreatic cancer.

The path to integrating immunotherapy with chemotherapy will likely be bumpy and afflicted by setbacks, many of which we have already seen. The only hope for changing the paradigm of treating pancreatic cancer is to maintain persistent clinical trials. Any pancreatic cancer patient should be presented with an opportunity to enroll in a trial, or should be referred to a center that offers them. These patients should be given a chance at tomorrow’s drugs as well as an opportunity to contribute to advancing the treatment of this disease.

 Kara D. Forinash, MS, PA-C, is a physician assistant, Shari Pilon-Thomas, PhD, is a professor of medicine, and Gregory M. Springett, MD, PhD, is a professor of medicine at Moffitt Cancer Center, Tampa, FL.

 Kara Forinash, and Drs Pilon-Thomas and Springett report no relevant conflicts of interest to disclose.

 Kara Forinash, PA-C; 12902 Magnolia Dr. Tampa, FL 33612; phone: 813-745-3346; fax: 813-745-7229.

			Siegel R, Naishadham D, Jemal A. Cancer statistics, 2012. CA Cancer J Clin. 2012;62(1):10-29.

			Antoniou G, Kountourakis P, Papadimitriou K, Vassiliou V, Papamichael D. Adjuvant therapy for resectable pancreatic adenocarcinoma: review of the current treatment approaches and future directions. Cancer Treat Rev. 2014;40(1):78-85.

This article reviews up-to-date clinical studies combining chemotherapy with immunotherapy; it also explains the rationale for this combination in treatment of pancreatic cancer.

Immunotherapy Combined With Chemotherapy for Pancreatic Cancer: A Game Changer?

	Chimeric antigen receptor (CAR) T-cell therapy is an immunotherapy in which the patient’s own T cells are isolated in the laboratory, redirected with a synthetic receptor to recognize a particular antigen or protein, and reinfused into the patient. Clinical trials of CAR T cells directed to the CD19 antigen have shown impressive results in advanced B-cell malignancies at multiple academic centers over the last 3 years. We describe the technology of CAR T cells, findings at 5 academic centers, and toxicities associated with CAR T cell-treatment and their management. Although CAR T cells for B-cell malignancies are the most advanced in terms of clinical testing, CAR T cells are the basis of a new platform technology that is poised to be expanded to other hematologic and nonhematologic malignancies, especially as new targets are identified and manufacturing processes are streamlined.

Chimeric antigen receptor (CAR) T-cell therapy is an immunotherapy in which the patient’s own T cells are isolated in the lab, redirected with a synthetic receptor to recognize a particular antigen or protein, and re-infused into the patient (

). Over the last 5 years, at least 15 clinical trials of CAR T cell therapy have been published. A new wave of excitement surrounding CAR T cell therapy began in August 2011, when investigators from the University of Pennsylvania (Penn) published a report on 3 patients with refractory chronic lymphocytic leukemia (CLL) who had long-lasting remissions after a single dose of CAR T cells directed to CD19.

 Since then, the enthusiasm has only increased as investigators from the National Cancer Institute (NCI),

 Memorial Sloan Kettering Cancer Center (MSKCC),

 which allowed us to transition most of these patients to a standard- of-care allogeneic hematopoietic stem cell transplant (allo- SCT), and Penn

have published impressive results in lymphoma and acute lymphoblastic leukemia (ALL). At large meetings, including those of the American Society of Hematology (ASH) and the American Association for Cancer Research (AACR), new sessions devoted to CAR T cell therapies have been organized and well attended, and all the major centers developing CAR T cells, including also Baylor College of Medicine, Fred Hutchinson Cancer Research Center, City of Hope National Medical Center, and MD Anderson Cancer Center, have discussed their latest results. Tellingly, what was considered a fringe field only 5 years ago has recently attracted investors, and several new partnerships and biotechnology companies have been formed around CAR T cell therapies (

CARs are synthetic molecules that minimally contain: (1) an antigen-binding region, typically derived from an antibody, (2) a transmembrane domain to anchor the CAR into the T cells, and (3) 1 or more intracellular T cell signaling domains.

 A CAR redirects T cell specificity to an antigen in a human leukocyte antigen (HLA)-independent fashion, and overcomes issues related to T cell tolerance. Thus far, CAR T cells directed against CD19 are the only ones that have achieved a clinical proof-of-concept and are in later stages of clinical development. However, CAR-modified T cells are a platform for a therapy that could be extended to other hematologic and nonhematologic tumors, provided the CAR is specific to the desired proteins on the surface of the tumor cell. The CAR can be introduced into the T cells by a variety of techniques, some of which permanently modify the genome of the T cell (integrating vectors) and have the potential for long-term effects, and some of which are transient (nonintegrating). Examples of integrating methods are retroviral, lentiviral, and transposase-based vectors; examples of nonintegrating methods are adenoviral vectors and plasmid or RNA electroporation. Each method carries unique benefits and concerns related to cost of development, safety, and the durability of CAR expression. The intracellular domains included in the CAR (ie, CD28-z or 4-1BB-z) can modulate T cell properties, such as their ability to survive and proliferate.

Hematologic malignancies are a natural setting for initial testing of CAR T cell therapy: There is extensive knowledge of the surface antigens on leukemic cells (some of which are exclusively expressed in specific hematopoietic cell populations), T cells naturally home to hematologic organs and tissues, and investigators can reasonably and repetitively sample blood and marrow to monitor disease and the infused CAR T cells. To date, most studies have focused on the treatment of advanced B-cell malignancies, including CLL, ALL, and non-Hodgkin lymphoma (NHL), such as diffuse large B-cell lymphoma (DLBCL), using CAR T cells directed to the CD19 antigen found uniquely on B cells. The majority of these studies have utilized second-generation CARs containing a CD28 intracellular signaling domain (CD19-28z), while some studies have utilized a second-generation CAR with a 4-1BB intracellular domain (CD19-BBz). There are currently 5 centers conducting advanced clinical trials of CD19 CAR T cell therapy. Each center uses its own type of CAR, method of introducing the CAR into the T cells, and clinical study design.

		Table. Academic-Industry Partnerships Around CAR T Cells

					Memorial Sloan Kettering and Fred Hutchinson Cancer Centers

					Baylor Center for Cell and Gene Therapy

The University of Pennsylvania has open trials to test CAR T cell therapy for the treatment of pediatric and adult ALL, as well as CLL and NHL (NCT01626495, NCT02030834, NCT02030847, NCT01551043). The first report from the university came from a study of 3 patients with relapsed/refractory CLL who were treated with CAR T cells; 2 of the patients achieved a complete response (CR), and 1 had an impressive partial response (PR).

Notably, the CAR T cells expanded inside the patients and mediated their effects, both responses and toxicities, in a relatively delayed fashion (several weeks after infusion).

 In pediatric ALL, the first 2 patients reported on achieved complete remissions; one was durable (and remains ongoing) and one was transient due to outgrowth of a subpopulation of tumor cells that did not express CD19, known as “antigen escape.”6 Importantly in this study, the major toxicity of cytokine release syndrome was aborted rapidly and completely with administration of tocilizumab, an anti-IL-6 receptor antibody.

The Penn group presented 4 abstracts updating the results of their ongoing clinical trials at ASH 2013. In 1 report, 22 pediatric and 5 adult patients with resistant/recurring (r/r) ALL were treated with CD19-BBz CAR transduced T cells.

 This cohort experienced an 89% CR rate to the therapy, although there were 6 who relapsed following an initial response. The CAR T cells were found to persist in the blood, marrow, and cerebrospinal fluid of patients, and the persistence correlated with response. Two abstracts reported the results of 2 separate CAR trials for patients with r/r CLL: in a dose-escalation trial, 18 patients were treated and an overall response rate (ORR) of 39% (3 CR and 4 PR) was seen; the infused dose did not seem to have an impact on efficacy or toxicity.

In the second trial, an ORR of 57% was reported, and all of the responding patients experienced a cytokine-release syndrome and B cell aplasia.

 Finally, in an abstract presenting the immune monitoring of all patients treated at Penn, the investigators reported that robust in vivo expansion of CAR T cells correlated with responses and B cell aplasia, and cytokine release syndrome was concurrent with peak CAR T cell expansion.

The NCI currently has 3 trials testing CD19-28z CAR T cells for the treatment of B cell malignancies, including relapsed/ refractory B cell malignancy (NCT009243264), relapsed B cell malignancy post-allogeneic stem cell transplantation (SCT; NCT01087294

), and pediatric B-cell malignancy (NCT01593696). Complete and partial responses lasting several months have been observed in the 30% to 67% range in all 3 trials, with the highest response rates in ALL. In the relapsed/ refractory setting, patients receive lymphodepleting chemotherapy with high-dose cyclophosphamide and fludarabine before CAR T cell infusion, and IL-2 after CAR T cell infusion; both pretreatment with lymphodepleting chemotherapy and posttreatment cytokine therapy are thought to improve the persistence, and therefore efficacy, of the CAR T cells. Pediatric patients receive lymphodepleting chemotherapy prior to CAR T cells, but no IL-2 after CAR T cells. Because of safety concerns regarding graft-versus-host disease, post-allogeneic patients receive donor-derived CAR T cells without lymphodepletion or IL-2.

The first MSKCC report of CAR T cells was mixed but telling in that the single patient to achieve a clinical response was the patient with ALL; the other 8 patients reported all had refractory CLL and none of them achieved a CR.

 Most patients tolerated CAR T cell infusions well, while 1 (a patient with CLL) died shortly after transfusion (possibly caused by sepsis). In the first follow-up report of the MSKCC ALL trial, rapid and deep remissions were observed: 5 relapsed patients with ALL received treatment with CAR T cells and all of them responded.

 The duration of response to CAR T cells was not evaluable because the patients were treated with allogeneic SCT when they achieved negative minimal residual disease (MRD) status. In the second follow-up report on the MSKCC ALL trial, reporting on 16 patients, a CR rate of 88% was observed; again, those who were eligible went on to allogeneic transplant (NCT01044069).

 At ASH 2013, the investigators reported on a new study of previously untreated CLL patients, where CARs are administered as consolidation after pentostatin/cyclophosphamide/ rituximab chemotherapy. Of the 7 patients treated to date, 3 who had PR after chemotherapy achieved CR, 2 maintained PR, and 2 had progressive disease for an ORR of 43%.

 Additional CAR T cell trials at MSKCC are investigating the efficacy of CAR T cells in the post-autologous SCT setting for lymphomas (NCT01840566), in the setting of relapsed ALL after allogeneic SCT (NCT01430390), and in pediatric relapsed ALL (NCT01860937).

Investigators at Baylor College of Medicine in Houston have 3 ongoing clinical trials of their CAR T cells. Unlike the other groups discussed here that are using bulk T cells to make CAR product, the Baylor group is also using virus-specific T cells for their CAR T cells; in addition to carrying the CAR construct, the T cells are specific to adenovirus, Epstein-Barr virus (EBV), cytomegalovirus (CMV), and/or BK virus, all of which can cause complications in the posttransplant setting. In addition to their potential ability to control viremia, the rationale for using virusspecific T cells includes enrichment of long-lived central memory T cells and continued antigen stimulation of these T cells through their virus-specific T cell receptor. One difficulty with virusspecific CAR T cells is that it takes longer to generate a product for a patient. In an update presented at ASH 2013, the group reported that their post allogeneic SCT trial (NCT00840853) had enrolled and infused 9 patients with ALL or CLL with the dualtropic CARs and a 22% ORR was achieved, with 1 CR and 1 PR.

The CARs were also found to exhibit antiviral activity in vivo. The CR lasted 3 months, the PR lasted 8 months, and stable disease lasted 8 months. Other open CAR trials at Baylor include a competitive repopulation study in which patients with NHL and CLL are being treated with 2 CARs at the same time to evaluate differences in the CAR signaling domains (NCT01853631) and a trial combining CAR T cells with checkpoint blockade (ipilimumab) (NCT00586391). Initial results from this type of competitive repopulation design have been published and demonstrated that second-generation CARs engrafted and expanded better in patients than first-generation CARs.

MD Anderson Cancer Center in Houston is conducting 4 phase I clinical trials to test CAR T cells for treatment of B-cell malignancies. Abstracts describing the progress of these studies were recently presented at ASH 2013.

 A total of 32 patients have been enrolled into the 4 trials, most having ALL or NHL; although the numbers are small, the remission rates thus far appear to be high (37% - 50%) and durable for at least 6 months. In 1 trial, CAR T cells are administered with standard chemotherapy for CLL (NCT01653717); in the other 3 trials, CAR T cells are administered after autologous SCT for lymphoma (NCT00968760), or after allogeneic SCT (NCT01497184), or umbilical cord SCT (NCT01362452) for leukemias.

Although it is early in the clinical experience with CAR T cells, there appear to be 3 major types of toxicities: B cell aplasia, cytokine release syndrome (CRS), and tumor lysis syndrome. Of note, all of these are directly related to the efficacy of CAR T cells as a therapeutic agent, as most patients who respond to CAR T cells experience these toxicities to some degree.

B cell aplasia is a consequence of the effective targeting of the CD19 antigen. Fortunately, B cells are not a life-sustaining type of tissue, and it is for this reason that most investigators have used CD19 as the initial target for CAR T cells. Targeting normal tissues is probably the largest hurdle in expanding CAR T cells to other tumors. The fact that CAR T cells discriminate only on the basis of target expression and not on tumorigenic mutations or aberrant signaling pathways is both their strength and weakness, as it is probably not necessary to target driver mutations, but it is important to rule out expression of the antigen on life-sustaining tissues before administration of CAR T cells.

Expansion of the CAR T cells in the body is associated with CRS. Manifestations of CRS include fever, hypotension, hypoxia, and neurologic changes. Additional clinical and biochemical changes may occur, such as disseminated intravascular coagulation and/or transaminitis associated with marked elevations in ferritin and C-reactive protein, a constellation of findings similar to macrophage activation syndrome or hemophagocytic lymphohistiocytosis (HLH). Neurologic complications, including seizures, aphasia, and mental status changes have also been reported, and appear to be reversible in the vast majority of cases.5. Cytokine release syndrome can occur regardless of the tumor subtype, and has been observed in ALL, CLL, and lymphomas. Initially, investigators encountering severe CRS managed it with high-dose steroids (>100 mg daily of prednisone equivalent); however, aborting the CRS with steroids is not an ideal intervention because steroids impair CAR T cell function and may be insufficient to manage the syndrome.6 Based on the results of immune monitoring studies that linked CRS to significant elevations in multiple serum cytokines, including IL-6, subsequent patients who exhibited severe CRS have been treated with tocilizumab, an IL-6 receptor—blocking antibody. Tocilizumab dramatically improves the CRS syndrome and thus far does not appear to affect CAR T cell function or tumor responses.

Because CAR T cells have mostly been developed in the academic setting, each group has developed its own manufacturing processes and CAR designs. One of the big challenges moving forward will be optimizing the complex manufacturing and distribution of CAR T cells such that patients all over the world can receive this promising therapy.

 It will also be interesting to learn with time which construct of CAR T cells allows for the greatest responses and least toxicity. CAR T cells may someday be effective enough to challenge allogeneic bone marrow transplant as definitive therapy for lymphoid malignancies, and perhaps even other hematologic malignancies as new targets for myeloid and plasma cell malignancies are defined and tested in clinical trials.

: Sagar B. Kudchodkar, PhD, is scientist, and Marcela V. Maus, MD, PhD, is assistant professor and director of translational medicine and early clinical development, from the Translational Research Program, Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA.

 Marcela V. Maus, MD, PhD, Translational Research Program, Abramson Cancer Center, University of Pennsylvania, Smilow Center for Translational Research, 3400 Civic Center Blvd, Bldg 421, 8th Fl, Rm 154, Philadelphia, PA 19104-5156; phone: (215) 746-5554; fax: (215) 573-0077; e-mail: mausm@uphs.upenn.edu.

 Drs. Kudchodkar and Maus have received sponsored research support from Novartis; Dr. Maus is supported by NCI K08CA166039.

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			Kochenderfer JN, Dudley ME, Kassim SH, et al. Effective treatment of chemotherapy-refractory diffuse large B-cell lymphoma with autologous T cells genetically-engineered to express an anti-CD19 chimeric antigen receptor. Blood. 2013;122(21):168.

			Kochenderfer JN, Dudley ME, Feldman SA, et al. B-cell depletion and remissions of malignancy along with cytokine-associated toxicity in a clinical trial of anti-CD19 chimeric-antigen-receptor-transduced T cells. Blood. 2012;119(12):2709—2720.

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			Grupp SA, Frey NV, Aplenc R, et al. T cells engineered with a chimeric antigen receptor (CAR) targeting CD19 (CTL019) produce significant in vivo proliferation, complete responses and long-term persistence without GVHD in children and adults with relapsed, refractory ALL. Blood. 2013;122(21):67.

			Kalos M, Frey N V, Grupp SA, et al. Randomized, phase II dose optimization study of chimeric antigen receptor modified T cells directed against CD19 (CTL019) in patients with relapsed, refractory CLL. Blood. 2013;122 (21):873.

			Porter DL, Kalos M, Frey NV, et al. Chimeric antigen receptor modified T cells directed against CD19 (CTL019 cells) have long-term persistence and induce durable responses in relapsed, refractory CLL. Blood. 2013;122(21):4162.

			Kalos M, Nazimuddin F, Finklestein JM, et al. Long-term functional persistence, B cell aplasia and anti-leukemia efficacy in refractory B cell malignancies following T cell immunotherapy using CAR-redirected T cells targeting CD19. Blood. 2013;122(21):163.

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			Brentjens RJ, Rivière I, Park JH, et al. Safety and persistence of adoptively transferred autologous CD19-targeted T cells in patients with relapsed or chemotherapy refractory B-cell leukemias. Blood. 2011;118(18):4817—4828.

			Brentjens RJ, Davila ML, Rivière I, et al. CD19-targeted T cells rapidly induce molecular remissions in adults with chemotherapy-refractory acute lymphoblastic leukemia. Sci Transl Med. 2013;5(177):177ra38.

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Chimeric antigen receptor T-cell therapy is an immunotherapy in which the patient's own T cells are isolated in the laboratory, redirected with a synthetic receptor to recognize a particular antigen or protein, and reinfused into the patient.

Chimeric Antigen Receptor (CAR) T-Cell Immunotherapy for Leukemia and Beyond

	The V600E BRAF mutation occurs in 5% to 15% of colorectal cancer patients and is a negative prognostic feature. BRAF-directed therapies in metastatic melanoma have shown great promise. However, unlike BRAF-mutated melanoma, single-agent BRAF inhibition has been an ineffective strategy in BRAF-mutated colorectal cancer. In recent years, preclinical experiments have increased our knowledge of BRAF-mutated colorectal cancer. This review highlights that preclinical data and discusses several ongoing clinical trials that are leveraging this information to explore new therapeutic strategies in targeting BRAF-mutated colorectal cancer.

	Pathologic and Prognostic Characteristics of BRAF-Mutated Colorectal Cancers

In recent years, one of the most encouraging developments in oncology has been the success of BRAF inhibitors in BRAF- mutated melanoma. In addition to BRAF-mutated melanoma, there have been reports of significant responses to BRAF inhibitors in patients with BRAF-mutated lung cancers, thyroid cancers, and hairy cell leukemia.

 Unfortunately, to date, attempts to target BRAF-mutated colorectal cancer have been largely unsuccessful. In this article, we will review the molecular and clinical characteristics of BRAF-mutated colorectal cancers and ongoing research efforts to target this aggressive molecular subtype.

Colorectal adenocarcinoma is classically thought to develop from adenomatous polyps.

 Recent work has demonstrated that colorectal cancers can also arise from serrated polyps.

 Serrated polyps, which include sessile serrated adenomas and traditional serrated adenomas, differ from traditional adenomas in that they have a sawtooth (ie, serrated) appearance. Colorectal cancers harboring BRAF mutations predominantly develop from these serrated polyps.

 Many tumors arising from serrated polyps demonstrate the CpG island methylator (CIMP) phenotype.

	BRAF Signal Transduction and Murine Models of BRAF-Mutated Colorectal Cancer

BRAF mutations occur in approximately 5% to 15% of colorectal cancers.

 BRAF-mutated colorectal cancers are located predominantly in the right side of the colon and are typically poorly differentiated.

 In addition, BRAF-mutated colorectal cancers occur more commonly in older patients, and predominantly in females.

 Multiple studies have demonstrated that BRAF-mutated tumors carry a poor prognosis. This is especially true in microsatellite stable (MSS) colorectal cancers that harbor a BRAF mutation.

 Roth et al and Popovici et al found that V600E BRAF mutations were a negative prognostic feature in MSS stage II and III colorectal cancers.

 The prognosis of BRAF-mutated colorectal cancers with high levels of microsatellite instability (MSI-H) is still unclear. Two recent studies suggested that the prognosis of BRAF-mutated MSI-H colorectal cancers is favorable compared with BRAF-mutated MSS colorectal cancers.

The RAF oncogene was discovered in 1983 when it was observed that retroviruses carrying the RAF oncogene transformed murine and avian cells in vitro and in vivo.

 Mutations in the BRAF oncogene have now been described in multiple cancers including melanoma, adenocarcinoma of the lung, papillary thyroid cancer, testicular cancer, and hairy cell leukemia.

 The V600E BRAF mutation is the most common form of BRAF mutation and results in the constitutive activation of the BRAF kinase domain.

V600E BRAF mutations drive colorectal cancer by causing sustained activation of the MAPK pathway. BRAF is serine/threonine kinase that is downstream of KRAS and is immediately upstream of MEK (Figure). Demonstrative of the importance of the MAPK in colorectal cancer, BRAF and KRAS mutations are mutually exclusive in colorectal cancer and only a few exceptions to this mutual exclusivity have been described.

In colorectal cancer, the V600E BRAF mutation clearly functions as a driver mutation. Selective blockade of the expression of V600E BRAF by RNA interference led to apoptosis of V600E BRAF-mutated colorectal cancer cell lines.

 In addition, V600E BRAF mutation has been shown to transform a “normal” human colon epithelial cell line.

 There is also emerging evidence that V600E BRAF mutations may be an early event in the oncogenesis of serrated colon cancers, as V600E BRAF mutations have been found in precancerous polyps.

 Two groups have generated murine models where there is forced expression of mutated BRAF in the gastrointestinal tract.

 Both of these models exhibit pathological changes resembling serrated colorectal cancer. Rad et al also demonstrated that some of these mice, which expressed BRAF V637E (the murine equivalent of human V600E BRAF mutations), developed carcinomas in the small and large intestine. Interestingly, many of these cancers exhibited microsatellite instability.

	Therapeutic Targeting of BRAF-Mutated Colorectal Cancer

Mutations in KRAS have been clearly demonstrated to predict resistance to EGFR-directed therapy in colorectal cancer.

 However, an area of controversy is the treatment of patients with V600E BRAF-mutated colorectal cancers. Conceptually, BRAF mutations would also seem to suggest resistance to EGFR-directed therapies since both KRAS and BRAF are downstream from EGFR. Consistent with this, several studies have demonstrated a lack of benefit of EGFR-directed therapies in patients with BRAF-mutated colorectal cancer.

 However, controversy exists, because other studies have not reproduced this finding. 

 Despite this controversy, oncologists agree that current therapies are inadequate in treating metastatic BRAF-mutated colorectal cancers and advances are greatly needed.

This review highlights that preclinical data and discusses several ongoing clinical trials that are leveraging this information to explore new therapeutic strategies in targeting BRAF-mutated colorectal cancer.

August 2014 | Contemporary Oncology®

Combination BRAF-MEK Inhibition With Dabrafenib and Trametinib for BRAF-Mutant Melanoma

Immunotherapy Combined With Chemotherapy for Pancreatic Cancer: A Game Changer?

Chimeric Antigen Receptor (CAR) T-Cell Immunotherapy for Leukemia and Beyond

BRAF-Mutated Colorectal Cancer: Early Frustrations and Future Optimism