Phase II Trial of MEK Inhibitor Selumetinib (AZD6244, ARRY-142886) in Patients with BRAFV600E/K- Mutated Melanoma Federica Catal

Author Manuscript Published OnlineFirst on February 26, 2013; DOI: 10.1158/1078-0432.CCR-12-3476 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Phase II trial of MEK inhibitor selumetinib (AZD6244, ARRY-142886) in patients with BRAFV600E/K- mutated melanoma

Federica Catalanotti6, David B. Solit1,6, Melissa P. Pulitzer4, Michael F. Berger4, Sasinya N. Scott4, Tunc Iyriboz2, Mario E. Lacouture3, Katherine S. Panageas5, Jedd D. Wolchok1,8, Richard D. Carvajal1, Gary K. Schwartz1, Neal Rosen1,7, Paul B. Chapman1

Memorial Sloan-Kettering Cancer Center, Departments of Medicine1, Radiology2, Dermatology3, Pathology4, Epidemiology & Biostatistics5, the Human Oncology and Pathogenesis Program6, Molecular Pharmacology and Chemistry Program7, and the Ludwig Institute for Cancer Research8

Running head: Selumetinib in BRAFV600E/K- mutated melanoma

Key words: phosphorylated AKT, exon-capture, MEK inhibitor, Hedgehog pathway, EGFR mutation.

Funding: This study was funded by the NCI (N01 CM 62206), the American

Recovery and Reinvestment Act (ARRA) of 2009, and the Starr Cancer

Consortium.

Potential conflicts of interest: MEL has consulted for Astra-Zeneca, NR serves on

the Astra Zeneca Scientific Advisory board, DBS receives research funding from

Astra Zeneca for another project.

Corresponding author: Paul B. Chapman, Memorial Sloan-Kettering Cancer

Center, 300 E 66th Street, Rm 1007, New York, New York USA. Fax: 646-888-

4253; email: [email protected].

Word count: 4119 1

Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2013 American Association for Cancer Research. Author Manuscript Published OnlineFirst on February 26, 2013; DOI: 10.1158/1078-0432.CCR-12-3476 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

2 figures; 3 supplementary figures; 3 tables

TRANSLATION RELEVANCE

MEK inhibitors demonstrate potent anti-tumor effects in preclinical models of

BRAF mutant melanoma but induce tumor regression in only a minority of

melanoma patients. Pre-clinical data suggest that BRAF mutant melanomas with

PI3K/AKT pathway activation are less sensitive to MEK inhibition. This study

showed that selumetinib, a selective inhibitor of MEK, induced tumor regression

in 3 of 5 patients with BRAF mutant melanomas that had low expression of

phosphorylated AKT. No responses were seen in the high phosphorylated AKT

group. The results support the hypothesis that activation of AKT is associated

with resistance to MEK inhibition and provide a rationale for co-targeting the MEK

and PI3 kinase/AKT pathways in patients with tumors expressing high levels of

phosphorylated AKT. However, it is likely that additional genetic alterations in

the tumor will also need to be considered for optimal selection of MEK inhibitor

sensitive melanomas.

ABSTRACT

Purpose: Test the hypothesis that in BRAF-mutated melanomas, clinical responses to

selumetinib, a MEK inhibitor, will be restricted to tumors in which the PI3K/AKT pathway

is not activated.

Experimental Design: We conducted a phase II trial in melanoma patients whose

tumors harbored a BRAF mutation. Patients were stratified by phosphorylated-AKT

(pAKT) expression (high vs. low) and treated with selumetinib 75 mg po bid. Pre-

treatment tumors were also analyzed for genetic changes in 230 genes of interest using

an exon-capture approach.

Results: The high pAKT cohort was closed after no responses were seen in the first 10

patients. The incidence of low pAKT melanoma tumors was low (approximately 25% of

melanomas tested) and this cohort was eventually closed because of poor accrual.

However, among the 5 melanoma patients accrued in the low pAKT cohort, there was 1

PR. Two other patients had near PRs before undergoing surgical resection of residual

disease (1 patient) or discontinuation of treatment due to toxicity (1 patient). Among the

2 non-responding, low pAKT melanoma patients, co-mutations in MAP2K1, NF1, and/or

EGFR were detected.

Conclusions: Tumor regression was seen in 3 of 5 patients with BRAF-mutated, low

pAKT melanomas; no responses were seen in the high pAKT cohort. These results

provide rationale for co-targeting MEK and PI3K/AKT in patients with BRAF mutant

melanoma whose tumors express high pAKT. However, the complexity of genetic

changes in melanoma indicates that additional genetic information will be needed for

optimal selection of patients likely to respond to MEK inhibitors.

INTRODUCTION

The mitogen-activated protein kinase (MAPK) pathway transmits

activating signals from the cell surface to the nucleus. In approximately 50% of

melanomas, there is an activating mutation in BRAF, usually BRAFV600E, that

drives cell proliferation(1, 2). Recently, phase II and phase III trials have shown

that approximately 50% of patients with BRAF-mutated melanomas respond to

RAF inhibitors and that RAF inhibitors prolong overall survival(3-5). In 20% of

melanomas, the driver mutation is an activating mutation in NRAS (6). In both

BRAF and NRAS-driven melanomas, the MAPK pathway is constitutively

activated.

Preclinical studies show that BRAFV600E-mutated melanomas are almost

uniformly sensitive to MEK inhibition(7). However, MEK inhibitor treatment of

BRAFV600E-mutated melanomas in which there is co-mutation of PTEN and

activation of the PI3K/AKT pathway results in G1 arrest but not apoptosis(8). On

the other hand, MEK inhibition induces apoptosis in some but not all BRAF-

mutated melanomas in which the PI3K/AKT pathway is not mutationally

activated. Among NRAS-mutated melanoma cells, sensitivity to MEK inhibition is

more variable(7). In contrast, cells in which MEK-ERK signaling is driven by

receptor tyrosine kinases are typically insensitive to MEK inhibition(8). These

observations led us to the hypothesis that BRAF mutant melanomas with low

PI3K/AKT activation would be most sensitive to MEK. This hypothesis is

consistent with recent data from cell lines(9) and consistent with the results of a

recent phase II trial of selumetinib (AZD6244, ARRY-142886), an allosteric

inhibitor of MEK, in unselected melanoma patients. In that trial, 5 of 6

selumetinib responders were found upon retrospective testing to harbor

BRAFV600E mutations(10). The PI3K/AKT status of the tumors was not assessed

in that trial and in fact, the prevalence of PI3K/AKT activation in melanoma

tumors in general is not well-established.

This study, conducted before the availability of BRAF inhibitor therapy,

was designed to test the hypothesis that MEK inhibition will induce clinical

responses in BRAF-mutated melanomas and that such responses are most likely

to be seen in the subset in which the PI3K/AKT pathway is not activated. In this

study, we treated patients with BRAF-mutated melanoma stratified on the basis

of phosphorylated-AKT (pAKT) expression (high vs. low) as a biomarker for

activation of the PI3K/AKT pathway. pAKT expression was used as a marker of

pathway activation since a diversity of molecular events can mediate PI3K/AKT

activation.

MATERIALS AND METHODS

Patient eligibility

This was a single institution, phase II trial in which patients with stage IV,

or unresectable stage III cutaneous melanoma were eligible if the melanoma

harbored a V600E or V600K BRAF mutation. Later in the trial, the protocol was

amended to allow NRAS-mutated melanoma. Two cohorts of patients were

accrued based on the expression of pAKT (high vs. low) as assessed by

immunohistochemistry (see below). If the cohort to which the patient was 6

assigned based on the tumor pAKT expression had been closed to accrual, the

patient was considered ineligible for the study. Other eligibility criteria included:

ECOG performance status of 0 or 1, measurable disease by RECIST 1.0, at least

4 weeks since any prior chemotherapy and 3 months since prior ipilimumab,

adequate hematologic function (WBC >3,000/μL, absolute neutrophil count

≥1,500/μL, platelets >100,000/μL, hemoglobin >9 g/dL not requiring

transfusions), adequate liver function (AST/ALT ≤ 2.5 upper limits of normal,

bilirubin ≤ 1.5 upper limits of normal), and creatinine ≤ 1.5 mg/dL. Patients were

excluded if they had active CNS metastases, uncontrolled serious concomitant

medical conditions including HIV, were pregnant or breast feeding, or were

unable to take oral medication.

Tumor genotyping

Macrodissection on 5μ-thick unstained sections was conducted using

corresponding hematoxylin and eosin–stained sections to ensure greater than

50% tumor nuclei prior to DNA isolation. DNA was extracted using the DNeasy

Tissue kit (QIAGEN) following the manufacturer's recommendations. Extracted

DNA was quantified on the NanoDrop 8000 (Thermo Scientific).

The study initially restricted enrolment to patients whose tumors

harboured a BRAF mutation by Sanger sequencing and/or LNA-

PCR/sequencing. Briefly, standard PCR amplification of a 224-bp fragment

encompassing the entire coding region of exon 15 of the BRAF gene was

performed using the primers BRAF/15F, 5′-TCATAATGCTTGCTCTGATAGG-3′ 7

and BRAF/15R, 5′-GGCCAAAAATTTAATCAGTGG-3′. To increase the sensitivity

of the assay, PCR amplification was also performed using standard primers in

combination with a 21-mer LNA probe, B-RAF LNA-F: 5′-

G+C+T+A+C+A+G+T+G+Aaatctcgatgg/3InvdT/–3′, where the capital letters

preceded by the plus (+) sign designate the locked nucleotides. This probe was

designed to suppress amplification of the wild-type DNA. The PCR products of

both standard and LNA-PCR were purified using Spin Columns (Qiagen) and

sequenced using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied

Biosystems) according to the manufacturer’s protocol on an ABI3730 (48

capillaries) running ABI Prism DNA Sequence Analysis Software.

After October 2010, tumors were genotyped using a Sequenom Mass

ARRAY (Sequenom Inc.) assay. Specifically, samples were tested in duplicate

using a series of multiplexed assays designed to interrogate the most common

BRAF and NRAS mutations. Genomic DNA amplification and single base pair

extension steps were conducted using specific primers designed with the

Sequenom Assay Designer v3.1 software. The allele-specific single base

extension products were then quantitatively analyzed using matrix-assisted laser

desorption/ionization-time of flight/mass spectrometry (MALDI-TOF/MS) on the

Sequenom MassArray Spectrometer. All automated system mutation calls were

confirmed by manual review of the spectra.

Exon-capture sequencing

We profiled genomic alterations in 230 cancer-associated genes using the

IMPACT assay (Integrated Mutation Profiling of Actionable Cancer Targets).

This assay utilizes solution phase hybridization-based exon capture and

massively parallel DNA sequencing to capture all protein-coding exons and

select introns of 230 oncogenes, tumor suppressor genes, and members of

pathways deemed actionable by targeted therapies. Briefly, barcoded sequence

libraries (New England Biolabs, Kapa Biosystems) were subjected to exon

capture by hybridization (Nimblegen SeqCap). 93 to 500 ng of genomic DNA was

used as input for library construction. Libraries were pooled at equimolar

concentrations (100 ng per library) and input to a single exon capture reaction as

previously described(11). To prevent off-target hybridization, a pool of blocker

oligonucleotides complementary to the full sequences of all barcoded adaptors

was spiked in to a final total concentration of 10 µM. DNA was subsequently

sequenced on an Illumina HiSeq 2000 to generate paired-end 75-bp reads.

Sequence data were demultiplexed using CASAVA, and reads were aligned to

the reference human genome (hg19) using the Burrows-Wheeler Alignment

tool(12). Local realignment and quality score recalibration were performed using

the Genome Anlaysis Toolkit (GATK) according to GATK best practices(13). A

mean unique sequence coverage of 553X was achieved.

Sequence data were analyzed to identify three classes of somatic

alterations: single-nucleotide variants, small insertions/deletions (indels), and

copy number alterations. Single-nucleotide variants were identified using muTect

(Cibulskis et al., manuscript in preparation) and retained if the variant allele

frequency in the tumor was >5 times that in the matched normal. For tumors

without matched normal DNA, we filtered out all silent variants and all additional

variants present in dbSNP but not in COSMIC (catalog of somatic mutations in

cancer)(14). Indels were called using the SomaticIndelDetector tool in GATK. All

candidate mutations and indels were reviewed manually using the Integrative

Genomics Viewer(15). Mean sequence coverage was calculated using the

DepthOfCoverage tool in GATK and was used to compute copy number as

described previously(11). Increases and decreases in the coverage ratios

(tumor:normal) were used to infer amplifications and deletions, respectively.

Immunohistochemistry

Eligible patients were assigned to either the high pAKT or low pAKT

cohort depending on the level of pAKT expression as assessed by

immunohistochemistry (IHC) performed on formalin fixed paraffin embedded

(FFPE) tissue sections. Immunostained sections were evaluated in a blinded

fashion by a dermatopathologist (M.P.) with expertise in cutaneous oncology.

Rabbit monoclonal antibody for phosphorylated Akt (Ser473, Cell Signaling

Technology; Beverly, MA, USA catalog #3787) was used at a 1:150 dilution. Five

µ-thick tissue sections were deparaffinized, pre-treated, and treated by standard

methods per manufacturer's instructions.

Biopsies received a numerical score for staining within melanocyte

cytoplasm and nuclei. Intensity of expression was characterized as 0 (no

staining), 1+ (blush), 2+ (intermediate) or 3+ (intense labeling). For the purpose of

this study, cases with 0 or predominantly 1+ staining, with <5% of melanocytes

with 2-3+ staining (usually at the tissue edge) were considered low pAkt

expressors. Cases showing predominant labeling with 2-3+ intensity were

considered to be high pAKT expressors (examples of high pAKT and low pAKT

tumors are shown in Supplementary Figure 1).

Treatment plan

Selumetinib was supplied through the NCI Clinical Therapeutics

Evaluation Program. Patients were treated with selumetinib 75 mg by mouth

twice daily; each cycle was 28 days. All patients signed written informed consent

before participating on this study.

Patients were seen by the treating physician at the end of each cycle and

repeat radiographic evaluations were performed after every other cycle. For

grade III toxicities attributed to selumetinib, drug was held until the toxicity had

resolved to grade I and then treatment was resumed at 50 mg/day. If grade III

toxicity recurred, the patient was removed from study.

The primary endpoint of the trial was response proportion among the two

cohorts as assessed by RECIST 1.0. Since our preclinical data predicted that

some BRAF-mutated tumors would undergo G1 arrest rather than apoptosis, we

also calculated the fraction of patients in each treatment arm who achieved

stable disease lasting at least 4 months. The secondary endpoint was to identify

genetic predictors of response to MEK inhibition through analysis of pre-

treatment tumor tissue. 11

Biostatistics

The plan was to accrue 20 patients into each cohort (low vs. high pAKT

expression). If at least 4 responses were observed in either cohort, selumetinib

would be considered worthy of further testing. If no responses were seen in the

first 10 patients, that cohort would be closed to further accrual. With 20 patients

per cohort, this would provide 90% power to distinguish a true response rate of

30% from a trivial response rate of 5% in each cohort. This design yields a 95%

probability of a negative result if the true response rate is no more than 5%. At

the end of the trial, we planned to report the response proportion for each cohort

along with a 95% confidence interval.

RESULTS

Between March 31, 2009 and July 11, 2011, 190 melanoma patients were

consented and 153 underwent tumor genotyping. Melanomas from 85 patients

(55%) were found to have a BRAF mutation; 7 of these were V600K mutations.

NRAS mutations were identified in 9 patients (tumors were not screened for

NRAS mutations early in the study thus explaining why so few NRAS mutated

melanomas were identified). Of patients with a BRAF or NRAS-mutated

melanoma, 16 signed consent and were registered to be treated although 1

patient withdrew consent prior to receiving any treatment. In sum, 15 patients

were treated on this protocol. All had a BRAFV600E mutation except for 3 patients

in the high pAKT cohort who had either a BRAFV600K mutation (2 patients), and

one patient whose tumor was characterized as BRAF mutant at the time of

screening but on subsequent analysis was shown to harbor a NRASQ61K

mutation. The most common reasons for patients who had BRAF mutant tumors

not being treated were: the patient was currently receiving other therapy (34%),

was found to have high pAKT after that cohort was closed (30%), had brain

metastases discovered on pretreatment evaluation (12%), or the tumor had a

BRAFV600K mutation before the protocol had been amended to allow entry of

patients with V600K mutant tumors (10%). Accrual was rapid when both cohorts

(low and high pAKT) were open. However, as noted below, the high pAKT

cohort was closed at the interim analysis leaving only the low pAKT cohort open

for accrual. Because of the low frequency of low pAKT melanoma tumors,

accrual to this cohort was slow. In October 2012, the trial was amended to

expand the trial eligibility to include melanoma tumors with BRAFV600K or NRAS

mutations. After 5 patients had been accrued to the low pAKT cohort, the trial

was closed because of slow accrual.

Patient characteristics

Table 1 shows the characteristics of the treated patients according to pAKT

expression. Most patients had stage IV, M1c disease (11/15 patients). All but

one patient had received prior systemic therapy, most commonly with

chemotherapy (12/15 patients). Two patients (both in the high pAKT cohort) had

received prior ipilimumab. One patient in each cohort had received prior RAF

inhibitor therapy.

Efficacy

The primary endpoint of this phase II trial was response. Among the 10

patients treated in the high pAKT cohort, there were no anti-tumor responses.

Four patients in the high pAKT cohort had stable disease for at least 16 weeks

but there were no objective responses by formal RECIST criteria. As a result,

the high pAKT cohort was closed at the time of the interim analysis. Five

patients were enrolled into the low pAKT cohort before the trial was closed due to

slow accrual. Three patients demonstrated tumor regressions although only one

fulfilled RECIST criteria for a partial response. Two other patients had near

partial response (27% tumor shrinkage in each case). One of these patients had

residual disease resected after 30 weeks of selumetinib therapy; the other patient

came off study due to toxicity at week 12 requiring discontinuation of selumetinb.

Therefore, in the low pAKT cohort, 3/5 patients had either a partial, or near partial

response. Figure 1 shows a waterfall plot of best overall response for each

patient as a function of treatment arm.

The estimated median progression-free survival was 2.2 months in the

high pAKT cohort and 7.1 months in the low pAKT cohort (Figure 2a). The

estimated median overall survival of the high pAKT cohort was 8 months and 18

months in the low pAKT cohort (Figure 2b). Although there were too few patients

in this study to perform a formal comparison of the two cohorts, the PFS and

overall survival curves suggest a better outcome for the low pAKT cohort.

Adverse events

The most common adverse events included rash, fatigue, and elevated

liver function tests (Table 2). There were few grade III/IV adverse events (rash,

elevated liver function tests, lymphopenia, hypoalbuminemia, dyspnea, cardiac

function). Three patients required dose reduction due to adverse events. One

responding patient in the low pAKT cohort was taken off therapy due to grade III

cardiac toxicity.

Exon capture results

Sufficient tumor-derived DNA was available for next generation

sequencing analysis on all 5 patients in the low pAKT cohort and on 2 patients in

the high pAKT cohort (Table 3). One of the patients in the high pAKT cohort was

found to have a NRASQ61K mutation rather than a BRAF mutation. Mutations

were detected in 40 genes among the 5 low pAKT tumor and 32 genes in the 2

high pAKT tumors. Most of the genes were mutated in only a single tumor but 8

genes were found to be mutated in at least 1 tumor in both cohorts: CDKN2A,

EPHA6, GRIN2A, MAP2K1, NOTCH2, PTPRD, ARID1A, and PTCH1. Six of 7

tumors showed mutations or deletions in CDKN2A. MITF amplifications were

also common (3 patients) (Supplementary Figure 2).

Among the low pAKT cohort, two patients had no response to selumetinib.

One of these patients had a mutation in the MAP2K1 gene that encodes for a

K57N mutation in helix A of MEK1, a primary downstream effector of BRAF

(Supplementary Figure 3). A missense mutation in the amino acid just proximal

(Q56P) has previously shown to be highly activating(16). The other non-

responding patient in the low pAKT cohort had a variety of genetic changes in the

tumor that could have contributed to selumetinib resistance including mutations

in NF1 and EGFR. This patient’s tumor also had a PTCH1 mutation which would

be predicted to cause activation of the Hedgehog pathway.

Discussion

In this phase II trial, we selected patients with a specific tumor genotype and

stratified them based on pAKT expression in the pre-treatment tumor tissue. The

trial design was based upon preclinical data suggesting that mutations that

activate the PI3K/AKT pathway, including alterations in PTEN, are associated

with diminished sensitivity to MEK and BRAF inhibition in BRAF mutant cell

lines(9, 17-19).

On the basis of these preclinical studies, we had predicted that significant

tumor regression would be seen only in tumors with low pAKT expression and

indeed 3/5 patients in the low pAKT cohort had tumor shrinkage of at least 25%,

although only 1 formally met RECIST criteria for a partial response. Melanomas

with high pAKT were far more common than anticipated (approximately 4:1

incidence) but no RECIST responses were seen in this cohort, although 4

patients had prolonged stable disease on selumetinib.

We obtained detailed genetic data on the tumors in the low pAKT arm using

an exon-capture, next generation massively parallel sequencing approach

designed to detect mutations, deletions, and amplifications in 230 genes found to

be commonly altered in human cancer. In this analysis, mutations/deletions in

the MAP2K1, PTEN, CDKN2A, PITCH1, and GRIN2A genes were identified.

Among the two patients with low pAKT, BRAF mutant melanomas that exhibited

de novo resistance to MEK inhibition, one melanoma had a co-mutation of MEK1

(K57N) which by in silico analysis would be predicted to be highly activating.

This finding is notable as a prior report by Emery et al. indicated that mutations in

MEK1 including a Q56P mutation in the helix A induced constitutive activation of

the kinase and MEK inhibitor resistance(16). The other patient in the low pAKT

cohort who did not respond to selumetinib had an alteration upstream of MEK in

the MAPK pathway. In particular, this patient’s tumor harbored a EGFRG735S

mutation, an activating mutation that can transform NIH3T3 cells(20) and has

been found to occur with low frequency in thyroid, lung, and prostate cancers(21-

23). This patient’s tumor also showed a truncating mutation in PTCH1, a tumor

suppressor gene in the Hedgehog pathway. Notably, 2 of the 5 melanomas in

the low pAKT cohort also had a P1315L frameshift mutation in PTCH1(24).

Among the patients who demonstrated tumor regression, two were found to have

MITF amplifications and the third a MEK1 mutation at proline 124 which has

been associated with resistance to vemurafenib (16). It is possible that these

genetic changes mitigated the tumor regressions seen in response to

selumetinib.

Recently, Patel and colleagues reported their experience in 18 unselected

melanoma patients treated with selumetinib(25). They observed 5 clinical

responses among the 9 patients with a BRAF-mutated melanoma. No patient

with wild-type BRAF responded to the MEK inhibitor. They did not report the

AKT activation status of the tumors.

Selumetinib has also been tested clinically in a randomized phase II trial in

melanoma patients who were not genotypically pre-selected as a function of

BRAF status(10). In that trial, 200 patients were randomized to selumetinib or

temozolomide. Six patients randomized to selumetinib had objective PRs; 5 of

whom were found retrospectively to harbor a BRAFV600E mutation. There was no

difference in progression-free survival between the selumetinib and

temozolomide treatment groups, which was the primary endpoint of trial.

Somewhat concerning, however, was the observation that overall survival in the

selumetinib cohort was inferior to overall survival in the temozolomide cohort

despite the fact that cross-over was permitted. These results, along with our

current data, suggest that in carefully selected melanoma patients, selumetinib

can induce tumor regression and prolong overall survival. In contrast, treating

unselected patients will result in a low response rate and may reduce overall

survival.

Recently, a more potent MEK inhibitor, trametinib, has undergone phase

III testing in melanoma in which study entry was restricted to only patients whose

tumors harbored a BRAFV600E/K mutation (26). Trametinib showed a 22%

response rate and superior progression-free and overall survival compared to

dacarbazine or paclitaxel. This indicates that a potent and selective MEK

inhibitor given to a selected (BRAF-mutated) population of melanoma patients

can result in improved survival over chemotherapy. Still, only a minority of

patients responded to trametinib indicating that additional co-mutational or

epigenetic alterations beyond BRAF status also have an impact on MEK inhibitor

sensitivity. Another selective MEK inhibitor, MEK162, was also recently shown to

induce tumor regressions in a subset of both BRAF-mutated and NRAS-mutated

melanomas further highlighting the need to identify additional biomarkers beyond

BRAF and NRAS mutational status that predict for MEK dependence (27).

Our data, although preliminary given the small number of patients treated

and the failure to fully enroll the low pAKT cohort, suggest that selecting

melanoma patients with BRAF-mutated tumors with low expression of pAKT

enriches for those sensitive to MEK inhibition. These results support the

hypothesis that activation of AKT is associated with resistance to MEK inhibition

and provide a rationale for co-targeting the MEK and PI3 kinase/AKT pathways in

patients with tumors expressing high levels of phospho-AKT. Our results also

suggest that additional mutations within the MAPK and Hedgehog pathways may

contribute to resistance to MEK inhibitors. In sum, our data confirm the genetic

complexity of melanoma tumors (28) and suggest that detailed genetic

information will be needed for optimal therapy selection. We believe these

results are especially timely as more potent MEK inhibitors are now available and

physicians will need to understand how to select patients who will respond.

Acknowledgements

We thank Efsevia Vakiani, Gopakumar Iyer, and Irina Linkov for technical assistance,

Cyrus Hedvat for directing the core facility that performed the BRAF and NRAS typing,

and Armando Sanchez and Sherie Mar-Chaim for management of the clinical data.

References

1. Davies H, Bignell G, Cox C, Stephens P, Edkins S, Clegg S, et al. Mutations of the BRAF gene in human cancer. Nature. 2002;417:949 - 54. 2. Curtin JA, Fridlyand J, Kageshita T, Patel HN, Busam KJ, Kutzner H, et al. Distinct sets of genetic alterations in melanoma. N Engl J Med. 2005;353:2135-47. 3. Flaherty KT, Puzanov I, Kim KB, Ribas A, McArthur GA, Sosman JA, et al. Inhibition of mutated, activated BRAF in metastatic melanoma. N Engl J Med. 2010;363:809-19. 4. Chapman PB, Hauschild A, Robert C, Haanen JB, Ascierto P, Larkin J, et al. Improved Survival with Vemurafenib in Melanoma with BRAF V600E Mutation. NEnglJMed. 2011;364:2507-16. 5. Sosman JA, Kim KB, Schuchter L, Gonzalez R, Pavlick AC, Weber JS, et al. Survival in BRAF V600-mutant advanced melanoma treated with vemurafenib. N Engl J Med. 2012;366:707-14. 6. Jakob JA, Bassett RL, Jr., Ng CS, Curry JL, Joseph RW, Alvarado GC, et al. NRAS mutation status is an independent prognostic factor in metastatic melanoma. Cancer. 2011. 7. Joseph EW, Pratilas CA, Poulikakos PI, Tadi M, Wang W, Taylor BS, et al. The RAF inhibitor PLX4032 inhibits ERK signaling and tumor cell proliferation in a V600E BRAF-selective manner. Proc Natl Acad Sci U S A. 2010;107:14903-8. 8. Solit DB, Garraway LA, Pratilas CA, Sawai A, Getz G, Basso A, et al. BRAF mutation predicts sensitivity to MEK inhibition. Nature. 2006;439:358-62. 9. Gopal YN, Deng W, Woodman SE, Komurov K, Ram P, Smith PD, et al. Basal and treatment-induced activation of AKT mediates resistance to cell death by AZD6244 (ARRY-142886) in Braf-mutant human cutaneous melanoma cells. Cancer Res. 2010;70:8736-47. 10. Kirkwood JM, Bastholt L, Robert C, Sosman J, Larkin J, Hersey P, et al. Phase II, open-label, randomized trial of the MEK1/2 inhibitor selumetinib as monotherapy versus temozolomide in patients with advanced melanoma. Clin Cancer Res. 2012;18:555-67. 11. Wagle N, Berger MF, Davis MJ, Blumenstiel B, Defelice M, Pochanard P, et al. High-throughput detection of actionable genomic alterations in clinical tumor samples by targeted, massively parallel sequencing. Cancer discovery. 2012;2:82-93. 12. Li H, Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics. 2009;25:1754-60. 13. Braunschweig AB, Huo F, Mirkin CA. Molecular printing. Nature chemistry. 2009;1:353-8. 14. Forbes SA, Bindal N, Bamford S, Cole C, Kok CY, Beare D, et al. COSMIC: mining complete cancer genomes in the Catalogue of Somatic Mutations in Cancer. Nucleic acids research. 2011;39:D945-50. 15. Robinson JT, Thorvaldsdottir H, Winckler W, Guttman M, Lander ES, Getz G, et al. Integrative genomics viewer. Nature biotechnology. 2011;29:24-6. 16. Emery CM, Vijayendran KG, Zipser MC, Sawyer AM, Niu L, Kim JJ, et al. MEK1 mutations confer resistance to MEK and B-RAF inhibition. Proc Natl Acad Sci U S A. 2009;106:20411-6. 17. Sosman J, Pavlick A, Schuchter L, Lewis KD, Mcarthur GA, Cowey CL, et al. PTEN immunohistochemical expression as part of the correlative analysis in the BRIM-2 trial. ASCO Annual Meeting; 2011; Chicago, IL; 2011.

18. Nathanson KL, Martin AM, Letrero R, D'Andrea K, O'Day S, Infante JR, et al. Tumor genetic analysis of patients with metastatic melanoma treated with the BRAF inhibitor GSK2118436. ASCO Annual Meeting; 2011; Chicago, IL; 2011. 19. Xing F, Persaud Y, Pratilas CA, Taylor BS, Janakiraman M, She QB, et al. Concurrent loss of the PTEN and RB1 tumor suppressors attenuates RAF dependence in melanomas harboring (V600E)BRAF. Oncogene. 2011. 20. Cai CQ, Peng Y, Buckley MT, Wei J, Chen F, Liebes L, et al. Epidermal growth factor receptor activation in prostate cancer by three novel missense mutations. Oncogene. 2008;27:3201-10. 21. Tsao M-S, Sakurada A, Cutz J-C, Zhu C-Q, Kamel-Reid S, Squire J, et al. Erlotinib in Lung Cancer — Molecular and Clinical Predictors of Outcome. NEnglJMed. 2005;353:133-44. 22. Murugan A, Dong J, Xie J, Xing M. Uncommon GNAQ , MMP8 , AKT3 , EGFR , and PIK3R1 Mutations in Thyroid Cancers. Endocrine Pathology. 2011;22:97-102. 23. Douglas DA, Zhong H, Ro JY, Oddoux C, Berger AD, Pincus MR, et al. Novel mutations of epidermal growth factor receptor in localized prostate cancer. Frontiers in bioscience : a journal and virtual library. 2006;11:2518-25. 24. Pastorino L, Cusano R, Nasti S, Faravelli F, Forzano F, Baldo C, et al. Molecular characterization of Italian nevoid basal cell carcinoma syndrome patients. Hum Mutat. 2005;25:322-3. 25. Patel SP, Lazar AJ, Papadopoulos NE, Liu P, Infante JR, Glass MR, et al. Clinical responses to selumetinib (AZD6244; ARRY-142886)-based combination therapy stratified by gene mutations in patients with metastatic melanoma. Cancer. 2012:n/a-n/a. 26. Flaherty KT, Infante JR, Daud A, Gonzalez R, Kefford RF, Sosman J, et al. Combined BRAF and MEK Inhibition in Melanoma with BRAF V600 Mutations. N Engl J Med. 2012. 27. Ascierto PA, Berking C, Agarwala S, Schadendorf D, Van Herpen C, Queirolo P, et al. Efficacy and safety of oral MEK162 in patients with locally advanced and unresectable or metastastic cutaneous melanoma harboring BRAFV600 or NRAS mutations. ASCO Annual Meeting; 2012; Chicago, IL; 2012. 28. Hodis E, Watson IR, Kryukov GV, Arold ST, Imielinski M, Theurillat JP, et al. A landscape of driver mutations in melanoma. Cell. 2012;150:251-63.

Figure Legends:

Figure 1: Waterfall plot showing best overall. Each bar represents an individual

patient. The low pAKT cohort is shown on the left; the high pAKT cohort is

shown on the right. Hatched bars show patients who experienced tumor

shrinkage of at least 25%. All melanomas had a BRAFV600E mutation except for

two patients who had melanomas with a BRAFV600K mutation (indicated by K) and

one patient who had a melanoma with a NRASQ61K mutation (as indicated).

Figure 2: Progression-free survival (A) and overall survival (B) for both the high

pAKT cohort (solid lines) and low pAKT cohort (broken lines). Tick marks

indicate censored patients. For the progression-free survival analysis, 3 patients

are censored who stopped treatment because of toxicity prior to progression.

Table 1: Patient characteristics

High pAKT Low pAKT All patients Number treated 10 5 15 Gender 9 men:1 woman 2 men; 3 women 11 men; 4 women Median age (range) 60 (22-78) 70 (55-74) 68 (22-78) Median ECOG performance 0 (0-1) 1 (0-2) 0 (0-2) status (range) Stage at treatment IIIc 0 1 1 IVA 1 0 1 IVB 2 0 2 IVC 7 4 11

Pre-treatment LDH levels Normal 5 3 8 Elevated 5 2 7

Previous systemic treatment None 1 0 1 Chemotherapy 7 5 12 Immunotherapy 4 1 5 Ipilimumab 2 0 2 BRAF-directed therapy 1 1 2

# of prior therapies/patient 0 1 0 1 1 4 2 6 2 1 2 3 3 1 1 2 >3 3 0 3

Table 2. Adverse events of grade 2 or greater possibly or probably attributable to

selumetininb.

No. of patients Toxicity Grade 2 Grade 3 Grade 4 Acneiform rash 3 2 ↑ AST 2 ↑ ALT 2 1 ↑ Alk phosphatase 2 1 Anemia 2 ↑ glucose 2 Fatigue 2 Diarrhea 2 ↓ lymphocyte count 3 Edema 2 ↓ albumin 1 ↑ bilirubin 2 1 Dyspnea 1 ↓ phosphate 1 LV dysfunction 2 RV dysfunction 1 Vomiting 1 Valvular heart disease 1 Chest wall pain 1 Heart failure 1 ↓ magnesium 1 Pleural effusion 1

Table 3. Exon capture sequencing results of 15 patients treated on study.

Age Gender BORR BRAF NRAS PTEN MEK1 CDKN2a Other

Low pAKT cohort 70 M -27% V600E Deleted NF2 (R187K); RB1 (E79K); PIK3C2G (R1069Q); MITF amp 71¶ F -55% V600E/amp Deleted Deleted MITF amp; PTCH1 (P1315L) 73 F -27% V600E G132C P124S G101W PTPRD (D1521A); KIT amp; GRIN2A (G751W) 75¶ F 3% V600E R80* TP53 (P87S); NF1 (S2701F); EGFR (G796S); ARID1A (1334_1335 insQ); PTCH1 (P1315L) 23 M 54% V600E K57N Deleted NOTCH2 (P6fs); EPHA6 (G277E); TET1 (A896V)

High pAKT cohort 54 M 13% Q61K P124S H83N EPHA6 (P1055L); NOTCH2 (P2335S); GIN2A (E1202K); GNAQ (TD6S)/deleted 69 ¶ M 68% V600K ERB4 (Q707E/N706fs); CDKN2C amp; MITF amp; ARID1A (Q1363*); PTPRD (E905K); PTCH1 (Q628*); GRIN2A (P985L); MYC amp;

DAXX amp 53 F 13% V600E Insufficient tumor available 59 M 11% V600E Insufficient tumor available 75 M 24% V600E Insufficient tumor available 63 M 0% V600E Insufficient tumor available 63 M 13% V600E Insufficient tumor available 79 M -10% V600K Insufficient tumor available 69 M 8% V600E Insufficient tumor available 53 M 14% V600E Insufficient tumor available

Abbreviations: BORR, best overall response as expressed by change in tumor size; amp, amplified. Blank cells indicate wild-type genotypes. *Germline DNA was not available for these patients.

Phase II trial of MEK inhibitor selumetinib (AZD6244) in patients with BRAF V600E/K-mutated melanoma

Federica Catalanotti, David B. Solit, Melissa P. Pulitzer, et al.

Clin Cancer Res Published OnlineFirst February 26, 2013.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-12-3476

Supplementary Access the most recent supplemental material at: Material http://clincancerres.aacrjournals.org/content/suppl/2013/02/26/1078-0432.CCR-12-3476.DC1

Author Author manuscripts have been peer reviewed and accepted for publication but have not yet been Manuscript edited.

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://clincancerres.aacrjournals.org/content/early/2013/02/26/1078-0432.CCR-12-3476. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.