Sigrid Eckardt

-mutant metastatic melanoma, expanding the number of effective treatment options that can improve quality and length of life for many patients.

Although single-agent therapy with targeted inhibitors of the BRAF kinase yields initial high response rates and a proven survival benefit, approximately half of all patients relapse after 6 to 7 months due to acquired resistance. The underlying escape mechanisms are complex and dynamic and involve reactivation of the mitogen-activated protein kinase (MAPK) pathway through genetic changes affecting BRAF, RAS, and MEK kinase activity, epigenetic changes, and immune evasion.

Concomitant inhibition of both BRAF and MEK kinases with targeted inhibitors has become, &ldquo;The recommend approach for molecularly targeted therapy,&rdquo; according to Keith T. Flaherty, MD, with Massachusetts General Hospital Cancer Center, in Boston, Massachusetts.

When BRAF kinase is constitutively activated due to a mutation (eg, V600E), treatment with BRAF inhibitors results in blockade of downstream MAPK pathway.

In cells with wild-type BRAF, treatment with BRAF inhibitors results in transactivation of CRAF and promotion of RAF dimerization, causing paradoxical activation of downstream MAPK pathway in the presence of upstream oncogenic changes, such as RAS mutations.

The rationale for combining BRAF and MEK inhibition is to delay resistance associated with the reactivation of MEKERK signalling after single-agent BRAF inhibition. Upfront inhibition of both BRAF and MEK kinases can delay emergence of treatment resistance and decrease the incidence of cutaneous hyperproliferative lesions associated with the paradoxical activation of the MAPK pathway following BRAF inhibitor monotherapy.

&ldquo;There are two bases for adding a MEK inhibitor to a BRAF inhibitor,&rdquo; said Flaherty. &ldquo;One related to overcoming resistance and the other to countering a mechanism of toxicity.&rdquo;

Combined BRAF-MEK Inhibition: Dabrafenib/ Trametinib

Based on data from two phase III studies demonstrating overall survival (OS) prolongation, the dabrafenib/trametinib combination regimen received full FDA approval for the treatment of patients with unresectable or metastatic

The most recent analysis from the COMBI-v study presented at the 2015 European Cancer Congress (ECC) reported a 7.6-month prolongation of OS with dabrafenib and trametinib compared with single-agent vemurafenib (25.6 vs 18.0 months; HR, 0.66; 95% CI, 0.53-0.81;

The estimated 2-year OS rate was 51% with dabrafenib and trametinib compared with 38% with vemurafenib monotherapy.

COMBI-v was a two-arm, open-label phase III study comparing dabrafenib/trametinib (150 mg twice daily/2 mg once daily) or vemurafenib (960 mg twice daily) in patients with

V600E/K mutation-positive unresectable or metastatic melanoma (n = 704). The study was stopped early in July 2014 following the demonstration of benefit at an interim analysis (

COMBI-v was the second study to demonstrate superiority for the BRAF/MEK inhibitor combination therapy over monotherapy in advanced melanoma. Final OS data from the COMBI-d trial, which had been initiated concomitantly and randomized 423 patients with

V600E/K-mutant melanoma to receive dabrafenib with trametinib (n = 211) or placebo (n = 212), reported a median OS of 25.1 months with the combination compared with 18.7 months with dabrafenib alone (HR, 0.71; 95% CI, 0.55-0.92;

= .011). The 2-year OS rate with the combination was 51% versus 42% with single-agent dabrafenib.

According to Caroline Robert, MD, PhD, lead author of the COMBI-v study, &ldquo;Dabrafenib plus trametinib has demonstrated superiority over BRAF inhibitor monotherapy now in two phase III trials, supporting its use as a standard-of-care therapy in patients with

Updated analyses from both trials also demonstrate increasing OS advantages of combination therapy over time. In the COMBI-v study, the absolute OS difference increased from 9% at 1 year, to 10% at 18 months, and to 13% at 2 years,4 and in the COMBI-d study from 6% at 1 year to 9% at 2 years.

In both studies, the addition of MEK inhibition significantly increased the likelihood of having an objective response (ORR; COMBI-d 69% vs 53% for BRAF inhibitor monotherapy,

= .0008), and more patients achieved complete responses (CR; COMBI-d, 16% vs 13%; COMBI-v, 17% vs 10%). Combination therapies also resulted in longer median duration of response (DoR; COMBI-d, 12.9 vs 10.6 months; COMBI-v, 13.8 vs 8.5 months).

Updated analyses of progression-free survival (PFS), the primary endpoint of the COMBI-v study, showed a significant prolongation with the combination (12.6 vs 7.3 months; HR, 0.61; 95% CI, 0.51-0.73,

<.001), which according to Robert &ldquo;is the longest achieved in a randomized study for patients with the

The dabrafenib/trametinib combination was associated with fewer adverse events (AE) compared with dabrafenib monotherapy.

Importantly, both studies confirmed that combination therapy reduced the incidence of oncogenic toxic effects related to MAPK pathway activation following BRAF inhibitor monotherapy.

Cutaneous toxicities including hyperkeratosis (6% vs 33%) and cutaneous squamous carcinoma (3% vs 9%) were less common in the dabrafenib/trametinib group than in the dabrafenib monotherapy group.6 Similarly, in COMBI-v, cutaneous squamous-cell carcinoma and keratoacanthoma were observed in 1% of patients in the combination-therapy group but in 18% in the vemurafenib group.

The most common AE associated with BRAF/MEK inhibition in both studies was pyrexia (52% and 53% in COMBI-v and COMBI-d, respectively).

Patients of the COMBI-v dabrafenib and trametinib treatment arm showed statistically significant and clinically meaningful improvements in health-related quality of life compared with patients on vemurafenib monotherapy, with improvements in overall health, physical and social functioning, and specific symptoms such as pain, insomnia, and loss of appetite.

BRAF/MEK inhibitor combinations have emerged as &ldquo;the new standard of care in terms of targeted agents for melanoma,&rdquo; said Rene Gonzalez, MD, with the University of Colorado Melanoma Research Clinics. &ldquo;All three of those studies were positive in the sense that it without a doubt showed that combination therapy is better than single-agent BRAF inhibitor. The overall survival is in the range of two years. The combination progression-free survival is 11 to 12 months, which is better than a single-agent BRAF inhibitor. I can barely think of a role for when a patient would receive just BRAF or MEK inhibitor alone.&rdquo;

Combined BRAF-MEK Inhibition: Vemurafenib andCobimetinib

The combination of vemurafenib and cobimetinib received FDA approval in November 2015, based on significant prolongation of PFS shown in the phase III coBRIM trial for previously untreated patients (n = 495) with

V600 mutation-positive unresectable locally advanced or metastatic melanoma.

Updated analyses confirmed previously reported clinical benefits from adding cobimetinib (60 mg once daily on days 1-21 of a 28-day cycle) to continuous vemurafenib (960 mg twice daily), which resulted in significant prolongation of PFS (median, 12.3 vs 7.2 months; HR, 0.56; 95% CI, 0.45-0.70;

<.001) and higher probability of response (ORR; 69.6% vs 50%;

<.001), including CR (16% vs 11%) compared with vemurafenib monotherapy.

The median DoR was 12.98 versus 9.23 months, with cobimetinib and placebo, respectively.

Overall survival data after a median of 18.5 months were presented at the 2015 Society for Melanoma Research Congress and revealed OS prolongation of 4.9 months with the combination (median OS, 23.3 vs 17.4 months, combination and vemurafenib alone, respectively; HR, 0.70; 95% CI, 0.55-0.90;

The OS rates at 17 months were 65% versus 50% and after 2 years the rates were 48% and 38%, for the combination and single-agent, respectively.

The presenting author Victoria Atkinson, MD, of the Princess Alexandra Hospital in Queensland, Australia, reported: &ldquo;The benefit of the combination was seen in all subgroups assessed, including ECOG performance status, LDH level, and presence of V600E or V600K mutations.&rdquo;

Unique AEs of the vemurafenib/cobimetinib regimen include phototoxicity, including photosensitivity and central serous retinopathy, which were observed in 28% and 20% of patients.10 Other AE more commonly observed with the combination were diarrhea (57% vs 28%), nausea or vomiting (39% vs 24% and 21% vs 12%, respectively), elevated liver enzyme levels (ALT, 24% vs 18%; AST, 22% vs 13%), and an increased creatine kinase level (30% vs 3%).

Similar to observations with dabrafenib and trametinib, combined BRAF/MEK inhibition was associated with fewer cutaneous toxicities than BRAF inhibitor monotherapy, including hyperkeratosis (10% vs 29%), cutaneous squamous cell carcinomas (3% vs 11%), and keratoacanthomas (<1% vs 8%). Also less frequent were joint pain (33% vs 40%) and hair loss (14% vs 29%).

Combination Immunotherapy: Nivolumab/Ipilimumab

Dual checkpoint blockade with a combination regimen of the CTLA-4 inhibitor ipilimumab and anti-PD-1 agent nivolumab continues to demonstrate efficacy in advanced melanoma. The combination regimen received accelerated approval for the frontline treatment of patients with

wild-type unresectable or metastatic melanoma in October 2015, based on improved PFS and ORR shown in the phase II CheckMate-069 and phase III CheckMate-067 studies, with expansion of the indication in January 2016 to include patients with

Recently reported exploratory data from the 2-year follow-up of the phase II CheckMate-069 trial indicate an OS benefit with the combination.

The 2-year OS rate with first-line combination ipilimumab plus nivolumab therapy (3mg/kg and 1mg/kg, respectively) was 64% versus 54% with nivolumab alone.

Across the entire study population (n = 142), which included patients with

mutant melanoma (n = 33), median OS had not been reached in either treatment group.

Patients with BRAF wild-type melanoma receiving the combination (n = 72) had a 42% OS improvement compared with ipilimumab monotherapy. Median OS had not been reached and was 24.8 months with ipilimumab monotherapy (HR, 0.58; 95% CI, 0.31-1.08). Two-year OS rates were 69% versus 53% with the combination and monotherapy, respectively.

According to lead author Michael Postow, MD, with Memorial Sloan Kettering Cancer Center, New York, high survival rates in the ipilimumab monotherapy arm were related to a large amount of crossover to other agents, including PD-1 agents, at progression.

OS results from the double-blind phase III CheckMate-067 study are pending.

Combination immunotherapy with ipilimumab/nivolumab capitalizes on non-redundant mechanisms of action of CTLA-4 and PD-1 inhibition that may result in synergistic effects. Specifically, ipilimumab blockade of CTLA-4 augments T-cell activation and proliferation, whereas nivolumab-mediated suppression of PD-1 activity restores the active T-cell response within the tumor microenvironment.

Outcomes from CheckMate-067 trial (n = 945 patients with both

V600 mutant and wild-type advanced melanoma) have shown that first-line nivolumab alone or in combination with ipilimumab doubled the median time to disease progression compared with ipilimumab monotherapy (median PFS, 11.5 months for the combination, 6.9 months for nivolumab monotherapy, and 2.9 months for ipilimumab monotherapy).12 Compared with ipilimumab monotherapy, the combination regimen and nivolumab monotherapy were associated with 58% and 43% reduction in the risk of disease progression, respectively (HR, 0.42;

Two-year assessment data from the phase II CheckMate-069 study showed similar findings. Among all randomized patients, median PFS had also not been reached in the combination treatment arm and was 3.0 months with ipilimumab monotherapy (HR, 0.36;

<.0001), which according to Postow &ldquo;reflects a 64% reduction in the hazard of progression or death in this all-randomized patient population, which is very impressive and statistically significant.&rdquo;

Ipilimumab/nivolumab combination therapy has produced ORRs of up to 61% among patients with

wild-type tumors in the CheckMate-069 study,

and 50% in the phase III CheckMate-067 study,

including 22% and 11.5% complete responses, respectively, all of which were significantly higher than those with ipilimumab monotherapy (ORR, 11% and 14%, respectively; CR, 0% and 7%, respectively).

In both studies, the combination regimen delivered rapid and durable responses, with time to response of 2.9 to 2.7 months.12,13 At the 2-year follow-up of the CheckMate-067 study, the median DoR was not reached in either arm, and 80% of responses to the combination (45 of 56) and ipilimumab monotherapy (4 of 5) were ongoing.

Median tumor burden by RECIST v1.1 decreased by 70% and 51% in patients on dual inhibitor therapy in CheckMate-069 and -67, respectively, but increased by 5% and 5.9% among patients receiving ipilimumab only (

TABLE 1. Efficacy of Frontline Combination Regimens in

Articles

A number of new combination approaches have gained approval in the past year for the treatment of patients with BRAF-mutant metastatic melanoma, expanding the number of effective treatment options that can improve quality and length of life for many patients.

Combination Regimens for Metastatic Melanoma

mutation status, tumor growth rate, and the presence or absence of cancer-related symptoms.

Immunotherapies with agents that block CTLA-4 or PD-1/PD-L1 checkpoints have been associated with durable responses in a subset of patients, and are often considered for patients with low-volume, asymptomatic metastatic melanoma. Targeted therapies, on the other side, are preferred for patients with

-mutant tumors who have symptomatic disease and benefit from the rapid response associated with these agents.

A main concern with targeted therapies is tumor resistance that ultimately develops in most patients within 6-12 months. However, as Jason J. Luke, MD, with the University of Chicago Medicine states, &ldquo;A sizable fraction of patients do very, very well for a long time on targeted therapy.&rdquo;

A long-term analysis has shown that approximately one fifth of patients with

V600-mutant melanoma receiving combined dabrafenib/trametinib remained progression-free after 3 years, suggesting a plateau of the survival curve.

According to lead author Georgina Long, MD, with the Melanoma Institute Sydney, Australia, and co-authors,

this defines, &ldquo;a need to understand the characteristics of patients who derive the greatest benefit from each mode of therapy.&rdquo; Specifically, &ldquo;future selection of patients for combined BRAF and MEK inhibition, and the rational design of clinical trials for those who fail it, may be assisted by an analysis of the clinicopathological features of those experiencing long-term benefit and those who progress.&rdquo;

In their study, Long and colleagues found that good prognostic features such as normal lactate dehydrogenase (LDH) levels, earlier-stage melanoma, and fewer metastatic sites were consistently associated with durable response and prolonged overall survival (OS) with BRAF/MEK inhibitor therapy.

Patients who survived longest on treatment included those with a complete response (CR), according to RECIST, and patients with a normal baseline LDH (63% and 62%, respectively). Data were from 2 cohorts of BRAF-inhibitor na&iuml;ve patients (n = 78) who had been treated with dabrafenib (150 mg twice daily) plus trametinib (2 mg daily).

Similarly, a study evaluating long-term outcomes of patients who received either BRAF or combined BRAF and MEK inhibitor regimens in phase I to III trials with single agent dabrafenib or vemurafenib, or combined dabrafenib/trametinib found that female sex and normal pretreatment LDH levels were the only factors associated with longer progression-free survival (PFS) and OS.

LDH below the upper limit of normal (ULN) was significantly associated with prolonged survival of patients receiving BRAF/MEK inhibitor combination therapy with dabrafenib/trametinib.2 Patients with a normal LDH at baseline had a median OS of 45.5 months (95% CI, 45.5 to not reached) compared with 16.6 months (95% CI, 11.1-22.6) for patients with an elevated LDH.2 Overall survival rates of patients with normal versus elevated LDH levels at baseline, respectively, were 88% versus 68% at 1 year, 75% versus 18% at 2 years, and 62% versus 5% at 3 years.

Recent data from phase III COMBI-v trial have also linked baseline LDH serum levels to survival benefits with the dabrafenib/trametinib combination.

According to lead author Caroline Robert, MD, of Institut Gustave Roussy in Paris, &ldquo;The longest benefit was in patients with an LDH less than or equal to the upper limit of normal, who had a 2-year overall survival rate of 66% and a median progression-free survival of 17.5 months.&rdquo;

In this patient group, median OS was not yet reached with dabrafenib/trametinib and was 21.5 months with vemurafenib (HR, 0.56), translating to a 44% OS benefit with the combination. Patients with an LDH above the ULN had less benefit with a median OS of 10.6 months with the combination and 8.9 months with vemurafenib monotherapy (HR, 0.81).

Similar observations are emerging from the phase III evaluation of cobimetinib/vemurafenib. OS benefits of the combination were observed in all patient subgroups.

For patients with both LDH baseline levels, median OS was not reached with the combination and was 23.3 months with vemurafenib. Patients with elevated LDH levels had an OS of 14.8 months with the combination and 11.2 months with vemurafenib alone. According to lead author Victoria Atkinson, MD, of the Princess Alexandra Hospital in Queensland, Australia, &ldquo;This is consistent with previous combination studies showing that patients with poor prognostic factors do worse.&rdquo;

Longer-term analyses of patients who had received both BRAF monotherapy and combined BRAF/MEK therapy have also reported a significant association of normal LDH levels with prolonged survival. Patients with normal LDH levels had a median OS of 23.5 months compared with 7.3 months in patients with elevated LDH levels.

Across all studies, normal serum LDH levels correlated with continued long-term response without progression (per Cox proportional hazards regression analysis;

= .024,2 or per univariate and multivariate analyses).

In COMBI-v, in patients with low LDH levels, median PFS was 17.5 and 9.2 months with the combination and monotherapy, respectively (HR, 0.55) and 5.5 versus 4.0 months in patients with elevated LDH levels (HR, 0.70).

According to Alexander Menzies, MBBS, with the Melanoma Institute Sydney, Australia, and co-authors,3 this evidence &ldquo;suggests that LDH is not simply prognostic. The biology of metastatic tumors that produce different levels of LDH may be distinct; for example, it has been shown that a high serum LDH level reflects an anaerobic oxidative metabolic state. As such, the best treatments for metastatic patients with normal and elevated LDH levels may be different.&rdquo;

According to Robert, patients with &ldquo;low LDH respond extremely well to targeted treatment. Patients with high LDH, whatever treatment we use, remain a big medical need. They derive a small benefit each time we evaluate a new, effective therapy. The benefit is always less in this population.&rdquo;

Tumor Burden and Timing of BRAF MEK Inhibitor Therapy

Consistent features associated with better outcomes with combined BRAF/MEK inhibitor in long-term analyses also included earlier-stage melanoma and fewer metastatic sites. Furthermore, according to Long and co-authors, &ldquo;good prognostic features at progression seemed to prolong survival from the time of progression but were also associated with receiving subsequent systemic therapy.&rdquo;

TABLE 1. Survival by Baseline Characteristics With Dabrafenib/Trametinib

Abbreviations: LDH, lactate dehydrogenase; ULN, upper limit of normal.

The presence of less than 3 organ disease sites was a baseline feature that met stringent criteria for association with longer OS (Cox proportional hazard regression analysis).

The median OS in patients with less than 3 metastatic sites was 45.5 versus 17.5 months in patients with &ge;3 metastatic sites (HR, 0.36; 95% CI, 0.18-0.69).

The corresponding OS rates were 92% versus 68% at 1 year, 73% versus 30% at 2 years, and 58% versus 19% at 3 years.

In patients with earlier stage disease (IIIC/M1a/M1b vs M1c), the median OS had not been reached and was 21.9 months in patients with M1c stage disease (HR, 0.36; 95% CI, 0.18-0.72). Other baseline features evaluated, including age, sex, ECOG performance status, and prior immunotherapy, did not correlate with added benefit from combination therapy

TABLE 2. Survival Comparison by Baseline Characteristics With Dabrafenib/Trametinib

Several indicators of a good prognosis, including the presence of fewer than three metastatic organ sites, lower median sum of diameters, lower melanoma stage, lower ECOG performance status, and no history of brain metastases were baseline factors that were also more frequent among patients who had a continued long-term response without progression.

Consistent with these observations, patients who progressed and did not receive further systemic chemotherapy had higher ECOG performance status, higher LDH, larger sum of diameters of RECIST target lesions at progression, greater absolute change in the RECIST sum of diameters from nadir, progression in the brain, and female sex

Findings that low tumor burden predicts superior long-term outcomes imply that BRAF/MEK inhibitor therapy should also be considered earlier in the disease course, in healthier patients with lower disease burden, in whom immunotherapy is often preferred. Long and co-authors noted that, &ldquo;on the basis of longer-term survival data from the current study and other studies, it would not be possible to use baseline features to select one therapy over another. Specifically, this study showed that long-term survival and durable responses with dabrafenib plus trametinib are associated with good prognostic features at baseline, including factors associated with low-volume diseaseclassically considered a hallmark for frontline immunotherapy.&rdquo;

Observations from a phase Ib trial have also identified tumor burden as a predictor of clinical outcome with BRAF/MEK inhibition with vemurafenib and cobimetinib.

This study involved 35 BRAF inhibitor-na&iuml;ve patients with

V600-mutant melanoma and found that early and progressive metabolic response of tumors to dual inhibitor therapy was associated with improved OS. Lead author Grant A. McArthur, MBBS, PhD, with the Peter MacCallum Cancer Centre, Melbourne, Australia, and colleagues evaluated several 18-F-fluorodeoxyglucose (FDG) uptake measures on positron emission tomography (PET) as indicators of tumor burden and glycolytic metabolism in tumors.6

Patients with low values of tumor burden measures at baseline compared with the median (percentage injected dose <0.36% or metabolic tumor volume < 47.5 ml) had a longer OS than patients with tumor burden measures above the median (HR, 0.19,

The ORR to treatment was 87% by RECIST and median PFS was 13.7 months. Complete metabolic responses (CMR) were obtained by 51% of patients during the second treatment cycle, and patients achieving CMR during the first treatment cycle (14%) had a longer PFS than patients achieving a partial MR (>30% decrease in standard maximum uptake volume; 86%). Tumor burden at baseline was not correlated with metabolic response.

The authors concluded that, &ldquo;Marked, early and progressive metabolic response on FDG-PET [was] consistent with successful inhibition of ERK-signaling.&rdquo;

According to lead author McArthur, these findings fit in with hypotheses that suggest that, &ldquo;Enhanced inhibition of the RAF/MEK/ERK pathway may lead to more cell death or even a change in tumor microenvironment that is less compatible with long-term cell survival or the reacquisition of a proliferative state [&hellip;] and that more effective or complete inhibition of RAF/MEK/ERK signaling may indeed produce biological responses that improve overall survival.&rdquo;

On-Treatment Features and Biomarkers of Response

Both degree of response and response category have been linked with benefit for targeted therapies. Patients with a complete response (CR; per RECIST) to the combination of dabrafenib/trametinib had numerically higher OS rates than patients with partial response (PR) or stable disease (SD). OS rates at 1, 2, and 3 years were 100%, 88%, and 63% for patients with CR versus 79%, 48%, and 33% for patients with PR, and 69%, 35%, and 35% for patients with SD.

Among patients receiving either BRAF inhibitor monotherapy or BRAF/MEK inhibitor combination regimens, the RECIST response correlated with PFS and OS (both

<.001) as a continuous variable, and patients with CR had longer PFS and OS.

According to the authors of this study, the association of potential biomarkers that may associate with response and clinical outcome is complex, because &ldquo;Mixed responses to treatment often occur with BRAF inhibitors, in which some metastases shrink while others remain stable or grow. Such responses are probably a consequence of molecular heterogeneity, which further affects clinical outcome and confounds biomarker studies.&rdquo;

Candidate biomarkers of response identified in small studies include markers of MAPK pathway activation and TORC1 activity early during treatment, PTEN status, cell cycle gene aberrations (CCND1 and CDKN2A copy number), and stromal growth factor expression.

Recent findings from the phase III coBRIM trial of vemurafenib/cobimetinib versus vemurafenib indicate that the presence of co-existing oncogenic mutations in addition to

V600 does not impact response to combination treatment.

, or receptor tyrosine kinases (RAS/RAF/RTK) were detected in 11% (n = 46) of the study population, and resulted in higher levels of ERK and MEK activation in tumors as detected by protein analysis. However, no correlation with PFS or ORR was identified.

The analysis of biomarkers that may correlate with response to treatment and treatment toxicity is the focus of an ongoing randomized phase III study (ClinicalTrials.gov identifier NCT02314143). The 3-arm study has been designed to assess sequential effects of BRAF and MEK inhibition on skin, blood and tumor biomarkers, and will compare continuous dabrafenib/trametinib combination therapy with upfront monotherapy with dabrafenib or trametinib followed by combination therapy.

National Comprehensive Cancer Network (NCCN). NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines&reg;): Melanoma.

Long GV, Weber JS, Infante JR, et al. Overall Survival and Durable Responses in Patients With BRAF V600-Mutant Metastatic Melanoma Receiving Dabrafenib Combined With Trametinib.

Menzies AM, Wilmott JS, Drummond M, et al. Clinicopathologic features associated with efficacy and long-term survival in metastatic melanoma patients treated with BRAF or combined BRAF and MEK inhibitors.

Robert C, Karaszewska B, Schachter J, et al. Two year estimate of overall survival in COMBI-v, a randomized, open-label, phase III study comparing the combination of dabrafenib (D) and trametinib (T) with vemurafenib (Vem) as first-line therapy in patients (pts) with unresectable or metastatic BRAF V600E/K mutation-positive cutaneous melanoma. Presented at: 2015 European Cancer Congress; September 25-29; Vienna, Austria Abstract 3301.

Atkinson V, Larkin J, McArthur GA, et al. Improved overall survival with cobimetinib (COBI) and vemurafenib (VEM) in advanced BRAFV600-mutated melanoma and biomarker correlates of efficacy. Presented at: 12th International Congress of the Society for Melanoma Research in San Francisco, California, 21 November 2015.

With the development of novel targeted and immunotherapeutic agents that are more efficacious than traditional chemotherapy, treatment paradigms in melanoma have undergone major changes.

Distinct Features Associated With Benefit for BRAF/MEK Inhibition

About half of all melanomas contain somatic mutations that result in constitutive activation of the BRAF kinase, leading to hyperactive signaling of the growth-promoting mitogen-activated protein kinase (MAPK) pathway. Systemic therapies with selective small-molecule inhibitors of the BRAF kinase, both in monotherapy and in combination with inhibitors of the MEK kinase, can extend survival and reduce recurrence risk in patients with

mutation testing has therefore become an essential tool in the diagnostic workup and management of patients with advanced melanoma.

Activity of Selective BRAF Inhibitors Beyond

According to Keith T. Flaherty, MD, with the Massachusetts General Hospital Cancer Center in Boston, &ldquo;At the moment, the only molecular feature in melanoma that is critical to treatment decision making is the presence or absence of a V600

mutation.&rdquo;  Activating mutations of

are present in 40% to 60% of all melanoma cases, with more than 90% of mutations found at codon 600 in exon 15. The activating V600E amino acid substitution is by far the most common V600 mutation and accounts for 74% to 95% of these mutations, followed by V600K in 5% to 30%, V600R in 5% to 7%, and V600M in about 4%.

More rare mutations include V600D and mutations in other codons with activating, impairing, or unknown effects on kinase activity.

Clinical evidence for the activity selective BRAF inhibitors derives from trials that were largely restricted to patients with V600E- and V600K-mutant tumors, as concurrently developed

mutation testing assays were optimized for the detection of these two most common mutations.

Prospective testing of the BRAF inhibitors vemurafenib and dabrafenib as single agents in phase III trials was conducted among patients with V600E-positive metastatic melanoma, demonstrating response rates in the range of 50%, clinical benefit rates close to 90%, and prolongation of progression-free survival (PFS) and overall survival (OS) compared with dacarbazine.

Similarly, phase III studies with combination regimens of dabrafenib and trametinib or vemurafenib with cobimetinib revealed significant PFS and OS benefits with dual BRAF/MEK inhibition over single agent BRAF inhibitors. Patients with V600E- or V600K-mutant melanoma represented the majority of the study population.

Indications for these agents based on phase III trial data therefore incorporate the presence of specific mutations: Combination dabrafenib/trametinib and vemurafenib/cobimetinib therapies and single agent trametinib are indicated for the treatment of patients with V600E or V600K mutations, whereas approved use of single-agent vemurafenib or dabrafenib is limited to the treatment of patients with V600E mutant melanoma.

This rationale is supported by findings that

mutation type can affect response to treatment with selective BRAF inhibitors. Phase II study findings have shown that although dabrafenib is active in patients with V600K-mutant tumors, ORR and PFS in this subgroup were lower than in patients with V600E-mutant tumors.

However, evidence also exists that patients with less common BRAF mutations not represented in phase III trials can benefit from BRAF inhibitor therapy.

In a cohort study including patients with

V600R mutations (n = 9), objective responses to monotherapy with selective BRAF inhibitors (dabrafenib or vemurafenib) were seen in 5 of 6 patients evaluable for response assessment. The V600R mutation is common in males and those with ulcerated primary melanomas. Similar to V600K melanoma, V600R is more frequently observed in older individuals.

According to study author Oliver Klein, MD, with Westmead Hospital, Sydney, Australia, and co-authors, these partial responses occurred rapidly and included &ldquo;a reduction in the size of lung metastases, liver metastases, stabilization of brain metastases, and a marked reduction in subcutaneous metastases to the scalp. The rapid clinical responses of patients with V600R

mutation-positive melanoma mirror those observed in patients with V600E and V600K mutations, indicating that selective BRAF inhibitors are active against melanoma with this genotype.&rdquo;

Other reports have shown responses to selective BRAF inhibitors in patients with V600R alterations, including a case report of a patient who responded to vemurafenib treatment

and a phase II study of patients with discrepant genotyping results for

V600E and V600R, who had a confirmed partial response to trametinib with a PFS of at least 57 weeks.

Testing for BRAF Status-Specimen Considerations

mutation status in patients with melanoma is essential for the selection of first-line therapy. Adequate genetic testing relies on a sequence of processes that include selection of tissue source, biopsy approach, specimen preparation and evaluation of tumor content, and choice of test assay.

According to current recommendations, routine

testing of the primary cutaneous melanoma should not be performed in patients without metastatic disease. In patients with metastatic disease, biopsy of the metastatic lesion, if feasible, is considered the preferred method to obtain sufficient tissue for genetic testing, if systemic therapy is planned, but archival material can also be used.

According to Flaherty, these recommendations are based on observations that

mutations arise early during melanocytic proliferation and that up to 80% of benign nevi also harbor V600 mutations, rendering mutation testing of primary tumor tissue non-informative.

&ldquo;The best available evidence indicates that

mutations are stably present in all tumor cells in lymph node and visceral metastases, whereas there can be heterogeneity in the primary melanoma. [This supports] the recommended practice of performing

mutation testing in metastatic tumor sites, with previously resected regional lymph nodes (for patients who had stage III disease prior to developing stage IV disease) being a reliable source of tumor material for this testing,&rdquo; he said.

Tumor molecular profiling is usually conducted on DNA extracted from formalin-fixed paraffin-embedded (FFPE) tissue specimens after pathological examination has confirmed the presence of tumor cells and determined tumor content. Factors that may affect testing results include tissue integrity, presence of high levels of pigmentation, or lymphocyte infiltration.

According to Monika Jurkowska, PhD, with Genomed Health Center in Warsaw, Poland, and co-authors, key steps for reliable testing involves the selection of &ldquo;block surface with sufficient tumor cellularity (>5-20% depending of the analytical sensitivity of the applied method and feasibility of subsequent dissection) but also without extensive necrosis, blood or melanin pigments content.&rdquo;

Status-Scope of Current Diagnostic Strategies

Validated approaches for the detection of

mutations in FFPE tumor material tissue include polymerase chain reaction (PCR)-based assays, sequencing technologies (Sanger, pyrosequencing, next generation sequencing [NGS]) or immunohistochemistry, among others. Methods differ in sensitivity, specificity, and cost. Depending on scope, detection methods can be broadly grouped into targeted assays that only identify a genetic alteration of interest, such as allele-specific PCR tests, and screening assays that can simultaneously identify all mutations in a larger genomic region, such as bidirectional direct Sanger sequencing.

Direct sequencing is highly specific but has a relatively low sensitivity (80%-93.4%), requiring a relatively high proportion of mutant tumor cells within the sample.

High Resolution Melt analysis (HRM) and Sanger sequencing technologies have proven highly specific and relatively sensitive (100% specific and up to a 6.6% allele frequency sensitivity), whereas pyrosequencing is sensitive (5% allele frequency) but less specific (90%).

PCR-based tests have a higher sensitivity than Sanger sequencing (97.5%) and require only small amounts of DNA. The FDA approved Cobas 4800 BRAF V600 and THxID BRAF tests were developed concurrently with vemurafenib and dabrafenib, respectively, and were optimized to detect the two most common V600 mutations, V600E and V600K, with differences in specificity.

The Cobas 4800 BRAF V600 mutation test received approval along with vemurafenib as a companion diagnostic.

This targeted assay combines allele-specific real-time PCR and TaqMelt technology to determine

V600 mutation status in DNA isolated from FFPE tumor tissue and has an approximate sensitivity of 5% mutation-bearing cells in a mixed sample.

The assay is highly specific for the V600E mutation (98%) but also has cross-reactivity with V600K (approximately 50%), and with V600D and V600E2 mutations.

The test only identifies the presence of a mutation without differentiation between mutations and does not reliably detect V600 mutations other than V600E.

The THxID BRAF test was developed to identify melanoma patients with

V600E or V600K mutations for treatment with dabrafenib and trametinib.

This PCR-based assay incorporates an internal control to validate adequate DNA integrity and relies on primers that are specific for each of the two mutations. The assay is largely non-cross reactive with other V600 mutants and identifies V600E and V600K mutations with high concordance with high-resolution melt (HRM) analysis and Sanger sequencing results (95.9%).

Approaches to Optimize BRAF Mutation Testing

Based on studies comparing PCR-based assays with sequencing approaches, the exclusive use of PCR-based assays for the detection of

mutations will fail to identify patients with more rare mutations, such as V600R and D, who may also benefit from targeted therapy.

Comparison of PCR/Sanger sequencing assays with the Cobas 4800 BRAF V600 test in archival material (236 FFPE samples, cutaneous melanoma lymph node metastases) found that both methods detected

mutations at a similar rate (60.9% vs. 61.0%), with a concordance of 95.2%.

Sanger sequencing failed to produce results in 2.5% of samples but was highly specific and reproducible in identifying V600E, K, and D mutations, whereas the Cobas assay produced results for all samples but false negative results in 2.5% of samples that contained V600E, K, and D mutations.

mutation detection methods in 295 melanoma FFPE samples found that relative to Sanger sequencing, the Cobas 4800 BRAF V600 test had only 80.5% sensitivity (95% CI, 72.4%-86.6%; 118 vs 96 of 275 samples).

False negative samples per Cobas assay included tumors in which sequencing identified V600E, K, and R mutations.

Kevin Qu, with Quest Diagnostics Nichols Institute, and coauthors explained that the presence of more than one mutant nucleotide (dinucleotide) could affect test outcomes: &ldquo;The cobas test is a single-oligonucleotide probe-based test that has greater analytical sensitivity than Sanger sequencing. However, as our findings suggest, its specificity for a single-nucleotide mutation means that it may miss relevant dinucleotide mutations in some cases. Sanger sequencing can detect single-nucleotide and dinucleotide mutations in the same region, which accounts for its higher detection rate in this study.&rdquo;

To address this, an ideal testing strategy may call for samples that test negative according to PCR assays to be additionally validated by a second sequencing approach that reliably identifies non-V600E mutations, or confirm their absence. This may represent a practical and comprehensive strategy to identify patients with

mutant melanoma. Kevin Qu and coauthors conclude: &ldquo;Use of Sanger sequencing as a second-line test for samples that are negative on the cobas assay could be a rational approach to maximizing the potential benefit of vemurafenib.&rdquo;

BRAF mutation testing has become an essential tool in the diagnostic workup and management of patients with advanced melanoma.

Sigrid Eckardt | Authors

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