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Molecular Cancer Therapeutics
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Spotlight on Clinical Response

Intratumoral Molecular Heterogeneity in a BRAF-Mutant, BRAF Inhibitor-Resistant Melanoma: A Case Illustrating the Challenges for Personalized Medicine

James S. Wilmott, Varsha Tembe, Julie R. Howle, Raghwa Sharma, John F. Thompson, Helen Rizos, Roger S. Lo, Richard F. Kefford, Richard A. Scolyer and Georgina V. Long
James S. Wilmott
1Melanoma Institute Australia; 2The University of Sydney; 3Westmead Institute for Cancer Research; 4Westmead Millennium Institute; 5Westmead Hospital; 6Royal Prince Alfred Hospital; Sydney, New South Wales, Australia; 7Divisions of Dermatology/Department of Medicine; 8Jonsson Comprehensive Cancer Center; and 9Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, California
1Melanoma Institute Australia; 2The University of Sydney; 3Westmead Institute for Cancer Research; 4Westmead Millennium Institute; 5Westmead Hospital; 6Royal Prince Alfred Hospital; Sydney, New South Wales, Australia; 7Divisions of Dermatology/Department of Medicine; 8Jonsson Comprehensive Cancer Center; and 9Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, California
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Varsha Tembe
1Melanoma Institute Australia; 2The University of Sydney; 3Westmead Institute for Cancer Research; 4Westmead Millennium Institute; 5Westmead Hospital; 6Royal Prince Alfred Hospital; Sydney, New South Wales, Australia; 7Divisions of Dermatology/Department of Medicine; 8Jonsson Comprehensive Cancer Center; and 9Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, California
1Melanoma Institute Australia; 2The University of Sydney; 3Westmead Institute for Cancer Research; 4Westmead Millennium Institute; 5Westmead Hospital; 6Royal Prince Alfred Hospital; Sydney, New South Wales, Australia; 7Divisions of Dermatology/Department of Medicine; 8Jonsson Comprehensive Cancer Center; and 9Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, California
1Melanoma Institute Australia; 2The University of Sydney; 3Westmead Institute for Cancer Research; 4Westmead Millennium Institute; 5Westmead Hospital; 6Royal Prince Alfred Hospital; Sydney, New South Wales, Australia; 7Divisions of Dermatology/Department of Medicine; 8Jonsson Comprehensive Cancer Center; and 9Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, California
1Melanoma Institute Australia; 2The University of Sydney; 3Westmead Institute for Cancer Research; 4Westmead Millennium Institute; 5Westmead Hospital; 6Royal Prince Alfred Hospital; Sydney, New South Wales, Australia; 7Divisions of Dermatology/Department of Medicine; 8Jonsson Comprehensive Cancer Center; and 9Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, California
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Julie R. Howle
1Melanoma Institute Australia; 2The University of Sydney; 3Westmead Institute for Cancer Research; 4Westmead Millennium Institute; 5Westmead Hospital; 6Royal Prince Alfred Hospital; Sydney, New South Wales, Australia; 7Divisions of Dermatology/Department of Medicine; 8Jonsson Comprehensive Cancer Center; and 9Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, California
1Melanoma Institute Australia; 2The University of Sydney; 3Westmead Institute for Cancer Research; 4Westmead Millennium Institute; 5Westmead Hospital; 6Royal Prince Alfred Hospital; Sydney, New South Wales, Australia; 7Divisions of Dermatology/Department of Medicine; 8Jonsson Comprehensive Cancer Center; and 9Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, California
1Melanoma Institute Australia; 2The University of Sydney; 3Westmead Institute for Cancer Research; 4Westmead Millennium Institute; 5Westmead Hospital; 6Royal Prince Alfred Hospital; Sydney, New South Wales, Australia; 7Divisions of Dermatology/Department of Medicine; 8Jonsson Comprehensive Cancer Center; and 9Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, California
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Raghwa Sharma
1Melanoma Institute Australia; 2The University of Sydney; 3Westmead Institute for Cancer Research; 4Westmead Millennium Institute; 5Westmead Hospital; 6Royal Prince Alfred Hospital; Sydney, New South Wales, Australia; 7Divisions of Dermatology/Department of Medicine; 8Jonsson Comprehensive Cancer Center; and 9Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, California
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John F. Thompson
1Melanoma Institute Australia; 2The University of Sydney; 3Westmead Institute for Cancer Research; 4Westmead Millennium Institute; 5Westmead Hospital; 6Royal Prince Alfred Hospital; Sydney, New South Wales, Australia; 7Divisions of Dermatology/Department of Medicine; 8Jonsson Comprehensive Cancer Center; and 9Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, California
1Melanoma Institute Australia; 2The University of Sydney; 3Westmead Institute for Cancer Research; 4Westmead Millennium Institute; 5Westmead Hospital; 6Royal Prince Alfred Hospital; Sydney, New South Wales, Australia; 7Divisions of Dermatology/Department of Medicine; 8Jonsson Comprehensive Cancer Center; and 9Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, California
1Melanoma Institute Australia; 2The University of Sydney; 3Westmead Institute for Cancer Research; 4Westmead Millennium Institute; 5Westmead Hospital; 6Royal Prince Alfred Hospital; Sydney, New South Wales, Australia; 7Divisions of Dermatology/Department of Medicine; 8Jonsson Comprehensive Cancer Center; and 9Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, California
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Helen Rizos
1Melanoma Institute Australia; 2The University of Sydney; 3Westmead Institute for Cancer Research; 4Westmead Millennium Institute; 5Westmead Hospital; 6Royal Prince Alfred Hospital; Sydney, New South Wales, Australia; 7Divisions of Dermatology/Department of Medicine; 8Jonsson Comprehensive Cancer Center; and 9Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, California
1Melanoma Institute Australia; 2The University of Sydney; 3Westmead Institute for Cancer Research; 4Westmead Millennium Institute; 5Westmead Hospital; 6Royal Prince Alfred Hospital; Sydney, New South Wales, Australia; 7Divisions of Dermatology/Department of Medicine; 8Jonsson Comprehensive Cancer Center; and 9Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, California
1Melanoma Institute Australia; 2The University of Sydney; 3Westmead Institute for Cancer Research; 4Westmead Millennium Institute; 5Westmead Hospital; 6Royal Prince Alfred Hospital; Sydney, New South Wales, Australia; 7Divisions of Dermatology/Department of Medicine; 8Jonsson Comprehensive Cancer Center; and 9Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, California
1Melanoma Institute Australia; 2The University of Sydney; 3Westmead Institute for Cancer Research; 4Westmead Millennium Institute; 5Westmead Hospital; 6Royal Prince Alfred Hospital; Sydney, New South Wales, Australia; 7Divisions of Dermatology/Department of Medicine; 8Jonsson Comprehensive Cancer Center; and 9Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, California
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Roger S. Lo
1Melanoma Institute Australia; 2The University of Sydney; 3Westmead Institute for Cancer Research; 4Westmead Millennium Institute; 5Westmead Hospital; 6Royal Prince Alfred Hospital; Sydney, New South Wales, Australia; 7Divisions of Dermatology/Department of Medicine; 8Jonsson Comprehensive Cancer Center; and 9Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, California
1Melanoma Institute Australia; 2The University of Sydney; 3Westmead Institute for Cancer Research; 4Westmead Millennium Institute; 5Westmead Hospital; 6Royal Prince Alfred Hospital; Sydney, New South Wales, Australia; 7Divisions of Dermatology/Department of Medicine; 8Jonsson Comprehensive Cancer Center; and 9Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, California
1Melanoma Institute Australia; 2The University of Sydney; 3Westmead Institute for Cancer Research; 4Westmead Millennium Institute; 5Westmead Hospital; 6Royal Prince Alfred Hospital; Sydney, New South Wales, Australia; 7Divisions of Dermatology/Department of Medicine; 8Jonsson Comprehensive Cancer Center; and 9Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, California
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Richard F. Kefford
1Melanoma Institute Australia; 2The University of Sydney; 3Westmead Institute for Cancer Research; 4Westmead Millennium Institute; 5Westmead Hospital; 6Royal Prince Alfred Hospital; Sydney, New South Wales, Australia; 7Divisions of Dermatology/Department of Medicine; 8Jonsson Comprehensive Cancer Center; and 9Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, California
1Melanoma Institute Australia; 2The University of Sydney; 3Westmead Institute for Cancer Research; 4Westmead Millennium Institute; 5Westmead Hospital; 6Royal Prince Alfred Hospital; Sydney, New South Wales, Australia; 7Divisions of Dermatology/Department of Medicine; 8Jonsson Comprehensive Cancer Center; and 9Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, California
1Melanoma Institute Australia; 2The University of Sydney; 3Westmead Institute for Cancer Research; 4Westmead Millennium Institute; 5Westmead Hospital; 6Royal Prince Alfred Hospital; Sydney, New South Wales, Australia; 7Divisions of Dermatology/Department of Medicine; 8Jonsson Comprehensive Cancer Center; and 9Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, California
1Melanoma Institute Australia; 2The University of Sydney; 3Westmead Institute for Cancer Research; 4Westmead Millennium Institute; 5Westmead Hospital; 6Royal Prince Alfred Hospital; Sydney, New South Wales, Australia; 7Divisions of Dermatology/Department of Medicine; 8Jonsson Comprehensive Cancer Center; and 9Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, California
1Melanoma Institute Australia; 2The University of Sydney; 3Westmead Institute for Cancer Research; 4Westmead Millennium Institute; 5Westmead Hospital; 6Royal Prince Alfred Hospital; Sydney, New South Wales, Australia; 7Divisions of Dermatology/Department of Medicine; 8Jonsson Comprehensive Cancer Center; and 9Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, California
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Richard A. Scolyer
1Melanoma Institute Australia; 2The University of Sydney; 3Westmead Institute for Cancer Research; 4Westmead Millennium Institute; 5Westmead Hospital; 6Royal Prince Alfred Hospital; Sydney, New South Wales, Australia; 7Divisions of Dermatology/Department of Medicine; 8Jonsson Comprehensive Cancer Center; and 9Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, California
1Melanoma Institute Australia; 2The University of Sydney; 3Westmead Institute for Cancer Research; 4Westmead Millennium Institute; 5Westmead Hospital; 6Royal Prince Alfred Hospital; Sydney, New South Wales, Australia; 7Divisions of Dermatology/Department of Medicine; 8Jonsson Comprehensive Cancer Center; and 9Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, California
1Melanoma Institute Australia; 2The University of Sydney; 3Westmead Institute for Cancer Research; 4Westmead Millennium Institute; 5Westmead Hospital; 6Royal Prince Alfred Hospital; Sydney, New South Wales, Australia; 7Divisions of Dermatology/Department of Medicine; 8Jonsson Comprehensive Cancer Center; and 9Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, California
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Georgina V. Long
1Melanoma Institute Australia; 2The University of Sydney; 3Westmead Institute for Cancer Research; 4Westmead Millennium Institute; 5Westmead Hospital; 6Royal Prince Alfred Hospital; Sydney, New South Wales, Australia; 7Divisions of Dermatology/Department of Medicine; 8Jonsson Comprehensive Cancer Center; and 9Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, California
1Melanoma Institute Australia; 2The University of Sydney; 3Westmead Institute for Cancer Research; 4Westmead Millennium Institute; 5Westmead Hospital; 6Royal Prince Alfred Hospital; Sydney, New South Wales, Australia; 7Divisions of Dermatology/Department of Medicine; 8Jonsson Comprehensive Cancer Center; and 9Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, California
1Melanoma Institute Australia; 2The University of Sydney; 3Westmead Institute for Cancer Research; 4Westmead Millennium Institute; 5Westmead Hospital; 6Royal Prince Alfred Hospital; Sydney, New South Wales, Australia; 7Divisions of Dermatology/Department of Medicine; 8Jonsson Comprehensive Cancer Center; and 9Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, California
1Melanoma Institute Australia; 2The University of Sydney; 3Westmead Institute for Cancer Research; 4Westmead Millennium Institute; 5Westmead Hospital; 6Royal Prince Alfred Hospital; Sydney, New South Wales, Australia; 7Divisions of Dermatology/Department of Medicine; 8Jonsson Comprehensive Cancer Center; and 9Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, California
1Melanoma Institute Australia; 2The University of Sydney; 3Westmead Institute for Cancer Research; 4Westmead Millennium Institute; 5Westmead Hospital; 6Royal Prince Alfred Hospital; Sydney, New South Wales, Australia; 7Divisions of Dermatology/Department of Medicine; 8Jonsson Comprehensive Cancer Center; and 9Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, California
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DOI: 10.1158/1535-7163.MCT-12-0530 Published December 2012
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Abstract

Targeted therapies are increasingly being used to treat a variety of cancers. Their efficacy depends upon the accurate detection and targeting of a specific mutation or aberration in the tumor. All cancers, such as melanoma, are molecularly heterogeneous, with drug-resistant subclones present before the treatment or emerging as a result of targeted therapies. Here, we show intralesional molecular heterogeneity in a progressing V600E BRAF-mutant melanoma metastasis from a patient treated for 7 months with the BRAF inhibitor vemurafenib. In the single metastasis, two distinct subclones were observed, both V600E BRAF-mutant and only one with an additional G13R NRAS mutation. Molecular heterogeneity even at the intralesional level shows that personalizing or adjusting therapies based on genotyping of a portion of a single lesion may not accurately depict the molecular profile or drivers of oncogenesis across the entire patient's melanoma. Mol Cancer Ther; 11(12); 2704–8. ©2012 AACR.

Introduction

New tumor mutation–specific targeted therapies are revolutionizing the treatment of many solid tumors (1) including melanoma (2, 3). However, tumor molecular heterogeneity is rapidly emerging as a complicating factor in the efficacy of targeted therapies, the durability of responses, and the development of drug resistance. The selective pressure induced by targeted therapies can result in the dominance or acquisition of additional driver mutations or molecular aberrations in tumor subclones.

Constitutive activating mutations of the B-raf (BRAF) gene occur in approximately half of all patients with metastatic melanoma (3). BRAF inhibitors have proven highly active (3) and improve overall survival compared with DTIC in patients with BRAF mutant metastatic melanoma (2). Eventually, most patients develop resistance to BRAF inhibition and relapse. Multiple mechanisms of resistance have been elucidated (4), including the reactivation of the MAPK pathway through the development of an NRAS mutation (5). Minority NRAS mutant subclones may preexist BRAF inhibitor treatment or the BRAF mutant population may acquire a secondary NRAS mutation after BRAF inhibitor exposure. This study shows tumor heterogeneity exists within a BRAF inhibitor–resistant lesion, which has implications for biopsy driven personalized medicine.

Case report

A 71-year-old man presented in February 2009 with a paraumbilical subcutaneous melanoma metastasis. He had a past history of a primary cutaneous melanoma of the right lower leg diagnosed in April 2005 and a right groin recurrence in May 2007. By May 2009, he had multiple distant melanoma metastases involving the subcutis, lung, left axilla, and adrenal glands and an isolated 6 mm brain metastasis. The patient's paraumbilical metastasis was tested for genomic mutations within exon 15 of the BRAF gene using high resolution melt and Sanger sequencing, revealing the presence of a BRAFV600E point mutation. He was treated with stereotactic radiosurgery to the brain metastasis, progressed after 3 months of stable disease on temozolomide +/− ABT-888/placebo (Trial registration ID: NCT00804908), and was then enrolled on the phase II study of vemurafenib, BRIM2 (6). He commenced vemurafenib (960 mg twice daily) on December 2, 2009 and achieved a RECIST (response evaluation criteria in solid tumors) defined partial response (7), but progressed on June 29, 2010, both clinically and radiographically on computed tomography scanning (Fig. 1). Excision biopsy of subcutaneous metastases was conducted one day before the commencement of vemurafenib, on day 14 of treatment, and again on disease progression on July 20, 2010 as part of the Treat Excise Analyze for Melanoma Study at the Melanoma Institute Australia (Sydney, Australia) as approved by the Royal Prince Alfred Hospital Research Ethics Committee Protocol Nos. X10-0305 and HREC/10/RPAH. His subsequent systemic therapies included single-agent trametinib (previously GSK1120212, an MEK inhibitor; ref. 8), the combination of dabrafenib (previously GSK2118436, a BRAF inhibitor) and trametinib (9) and E7080 (a multikinase inhibitor with specific activity against VEGF), and finally he was rechallenged with vemurafenib (Fig. 1A). For each systemic therapy, excision biopsy of a melanoma metastasis was conducted on days 4 to 7 following therapy commencement and on RECIST progression, providing multiple independent tumor samples for analysis and comparison.

Figure 1.
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Figure 1.

A, patient's treatment timeline with biopsy points in yellow boxes and treatments in blue. B–D, photographs and MRI images taken of the biopsy excised on vemurafenib progression at different treatment stages (yellow arrows mark the biopsied lesion), with the Sanger sequencing result showing the detection of the NRAS G13R mutation.

Results and Discussion

We sought to determine whether the patient's initial vemurafenib resistance developed through the reactivation of the MAPK pathway due to additional mutations to the NRAS or BRAF genes. Sanger sequencing was conducted on the patient's vemurafenib-resistant metastasis to detect somatic mutations in exon 1 and exon 2 of the NRAS gene and in exon 11 and exon 15 of the BRAF gene. This revealed a G13R NRAS mutation in addition to the original BRAF V600E mutation within the single vemurafenib-resistant metastasis (Fig. 1B–D). We used immunocytochemistry to analyze the expression levels of downstream signaling proteins within the progression biopsy to identify any subclones of tumor cells within the lesion. Immunohistochemistry was conducted on the progressed metastasis using antibodies against Ki-67, p-ERK1/2, p-AKT, cyclin D1, MITF, p27Kip1, p16INK4a, p53, BCL-2, Mcl-1, PTEN, MAP3K8 (COT), IGF1Rβ, and PDGFRβ. The staining for p-ERK1/2 expression clearly identified 2 areas of tumor with differential staining (Fig. 2A). The percentage of immunopositive tumor cells was assessed for each subclone using a Dako ACIS III Automated Image Analysis System. One subclone had high p-ERK1/2 expression (95% tumor cells positive, labeled subclone A; Fig. 2D), and was adjacent to melanoma cells with low expression of p-ERK1/2 (3% tumor cells positive, labeled subclone B; Fig. 2E). The proliferative rate represented by Ki-67 expression was higher in the subclone A (10% tumor cells positive) than in subclone B (3% tumor cells positive; Fig. 2F and G). Conversely, the percentage of cyclin D-positive tumor cells was lower in subclone A than in subclone B (46% and 74%, respectively; Fig. 2H and I). The expression of the remaining proteins did not seem to differ between the subclones.

Figure 2.
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Figure 2.

A, expression of p-ERK1/2, which is high in subclone A and relatively negative in subclone B (scale bar, 1 mm). B and C, allele-specific RT-PCR showing the presence of NRAS wild-type in both subclones A and B, but NRAS G13R in only subclone B. D, high magnification image of p-ERK1/2 expression in subclone A (scale bar, 100 μm). E, high magnification image of p-ERK1/2 expression in subclone B (scale bar, 100 μm). F, Ki-67 expression in subclone A (scale bar, 100 μm). G, Ki-67 expression in subclone B (scale bar, 100 μm). H, cyclin D1 expression in subclone A (scale bar, 100 μm). I, cyclin D1 expression in subclone B (scale bar, 100 μm).

We then carried out mutation testing separately on each of the subclones A and B, which were identified by their p-ERK1/2 expression. Each subclone was macrodissected and genomic DNA was extracted from each individual subclone. BRAF and NRAS mutation status of the subclones was determined by allele-specific specific real-time PCR (RT-PCR). In addition, subclone genomic DNA was analyzed by Mass Spectrometric SNP genotyping with OncoCarta v0.1 and the MelaCarta panel v0.1 to detect any additional somatic mutations. The genomic DNA mutational status of subclones A and B was different; subclone A was NRAS wild-type and BRAFV600E mutant (Fig. 2B), whereas subclone B was NRAS G13R mutant and BRAFV600E mutant (Fig. 2C). The sensitivity of this allele-specific RT-PCR allowed the detection of as little as 2% mutant NRAS and BRAF allele (data not shown; ref. 10). For this reason, it is unlikely that the differential mutational status of the subclones is due to the assay sensitivity. The OncoCarta and MelaCarta did not reveal any additional mutations.

Mutational analysis using mutant-specific RT-PCR of the antecedent primary melanoma and lymph node metastases that occurred 5 and 3 years before the diagnosis of widely disseminated metastatic disease, revealed the presence of V600E BRAF mutation, but not the NRAS G13R mutation (Fig. 1A). Similarly, analysis of 7 metastatic tumors resected after cessation of treatment with vemurafenib and during treatment with single-agent trametinib or the combination of trametinib and dabrafenib detected a BRAF V600E mutation without a NRAS G13R mutation in all biopsies (Fig. 1A).

Here, we show intratumoral molecular heterogeneity in a progressing V600E BRAF-mutant melanoma metastasis from a patient treated for 7 months with the BRAF inhibitor vemurafenib. Within the same individual progressing and vemurafenib-resistant metastasis, 2 distinct subpopulation were observed, both V600E BRAF-mutant, and only one G13R NRAS-mutant. The acquisition of an additional NRAS mutation causing MAPK pathway reactivation has been proposed as a mechanism of BRAF inhibitor resistance in BRAF mutant melanoma patients (5). In this study, the NRAS G13R mutation may be one but not the sole driver of disease progression. This issue of tumor heterogeneity is further complicated by incomplete tumor shrinkage, at the macroscopic and radiologic levels, induced by BRAF inhibitors in the majority of tumors and patients. Thus, a biopsy of tumor progression on any BRAF inhibitor may frequently yield a mixture of tumor cells with distinct kinetic growth properties. That an NRAS mutation was not detected in the subsequent tumor biopsies raises tantalizing questions as to how the MEK inhibitor treatment alone (first sequential therapy after failure on vemurafenib alone) might have influenced the selective pressure, for or against, the double BRAF/NRAS mutant melanoma population. Our tissue biopsy series here illustrate the dynamic nature of tumor evolution on multiple distinct targeted regimens.

In this patient, we have shown that intralesional genetic heterogeneity is present within BRAF inhibitor-resistant metastatic melanoma and highlighted the fact that sampling error is a potential pitfall of fine-needle aspiration or punch biopsy techniques. Advances in genome analysis methods such as deep sequencing (11–13) and sequencing of circulating tumor cells (14) may provide superior information regarding the heterogeneity of a patient's tumors. These methods remain exploratory due in part to the complex analyses required. Knowledge of heterogeneity may help predict and improve a patient's response to combination therapies based on the level and signature of mutational heterogeneity. These technologies may also help confront another likely issue arising from the tumor heterogeneity: multiple mechanisms of acquired drug resistance in the same progressing tumor or multiple progressing tumors in the same patient.

However, the current method of assessing heterogeneity in a subset of sampled lesions is unlikely to adequately predict tumor heterogeneity in vivo, nor reflect the ongoing genetic changes that occur during treatment (13, 15). Thus, the strategy of biopsying metastatic disease to decide the next systemic therapy after progression on a targeted therapy will be complicated by heterogeneity. We need to anticipate multiple mechanisms of resistance, predict the critical downstream effector pathways, and treat with the most effective broad-ranging combination of therapies at first diagnosis of metastatic disease. These therapies may include the targeting of oncogenes along with immunotherapy or chemotherapy (16–18).

Disclosure of Potential Conflicts of Interest

R.F. Kefford is a consultant/advisory board member and has a honoraria and travel support from GlaxoSmithKline. G.V. Long is a consultant/advisory board member in Roche, Bristol-Myers Squibb, and GlaxoSmithKline and has a honoraria from Roche and MERCK and travel support from GlaxoSmithKline and Roche. G.V. Long also has a commercial research support from Roche. R.A. Scolyer is a consultant/advisory board member of Roche and GlaxoSmithKline and has a honoraria from Abbott Molecular and Roche. J.F. Thompson has a honoraria and is a consultant/advisory board member of Roche and GlaxoSmithKline. G. V. Long and R. A. Scolyer are funded by the Cancer Institute New South Wales Fellowship program. H. Rizos is a recipient of a Cancer Institute New South Wales Research Fellowship and an NHMRC Senior Research Fellowship. The funding body had no role in the design or conduct of the study. No potential conflicts of interest were disclosed by the other authors.

Authors' Contributions

Conception and design: J.S. Wilmott, J.R. Howle, R. Sharma, H. Rizos, R.S. Lo, R.F. Kefford, R.A. Scolyer, G.V. Long

Development of methodology: J.S. Wilmott, V. Tembe, H. Rizos, R.F. Kefford, R.A. Scolyer, G.V. Long

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): J.S. Wilmott, J.R. Howle, R. Sharma, J.F. Thompson, R.F. Kefford, R.A. Scolyer, G.V. Long

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): J.S. Wilmott, V. Tembe, H. Rizos, R.S. Lo, R.F. Kefford, R.A. Scolyer, G.V. Long

Writing, review, and/or revision of the manuscript: J.S. Wilmott, J.R. Howle, J.F. Thompson, H. Rizos, R.S. Lo, R.F. Kefford, R.A. Scolyer, G.V. Long

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): H. Rizos, R.A. Scolyer, G.V. Long

Study supervision: R.F. Kefford, R.A. Scolyer, G.V. Long

Grant Support

This work is supported by the Program Grant 402761 of the National Health and Medical Research Council of Australia (NHMRC), Translational Research Program Grant 05/TPG/1-01 of the Cancer Institute New South Wales (NSW), and an infrastructure grant to Westmead Millennium Institute by the Health Department of NSW through Sydney West Area Health Service. Westmead Institute for Cancer Research is the recipient of capital grant funding from the Australian Cancer Research Foundation.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

  • Received May 28, 2012.
  • Revision received August 26, 2012.
  • Accepted August 31, 2012.
  • ©2012 American Association for Cancer Research.

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Molecular Cancer Therapeutics: 11 (12)
December 2012
Volume 11, Issue 12
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Intratumoral Molecular Heterogeneity in a BRAF-Mutant, BRAF Inhibitor-Resistant Melanoma: A Case Illustrating the Challenges for Personalized Medicine
James S. Wilmott, Varsha Tembe, Julie R. Howle, Raghwa Sharma, John F. Thompson, Helen Rizos, Roger S. Lo, Richard F. Kefford, Richard A. Scolyer and Georgina V. Long
Mol Cancer Ther December 1 2012 (11) (12) 2704-2708; DOI: 10.1158/1535-7163.MCT-12-0530

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Intratumoral Molecular Heterogeneity in a BRAF-Mutant, BRAF Inhibitor-Resistant Melanoma: A Case Illustrating the Challenges for Personalized Medicine
James S. Wilmott, Varsha Tembe, Julie R. Howle, Raghwa Sharma, John F. Thompson, Helen Rizos, Roger S. Lo, Richard F. Kefford, Richard A. Scolyer and Georgina V. Long
Mol Cancer Ther December 1 2012 (11) (12) 2704-2708; DOI: 10.1158/1535-7163.MCT-12-0530
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