CCR TranslationsCommentary on Taniguchi et al., p. 7808

BEAMing Sheds Light on Drug Resistance

Josh Lauring and Ben Ho Park

Targeted therapies against somatically altered genes are currently used for the treatment of many human

cancers. The nascent technology known as BEAMing has the potential to increase the clinical utility of these

agents because it allows for the detection of cancer mutations in peripheral blood, providing a rapid

assessment of tumor mutation status. Clin Cancer Res; 17(24); 7508–10. �2011 AACR.

In this issue of Clinical Cancer Research, Taniguchi andcolleagues (1) describe the use of a novel technologycalled BEAMing (for Beads, Emulsification, Amplifica-tion, and Magnetics) to query for a common second-siteepidermal growth factor receptor (EGFR) mutation(T790M) that confers resistance to small-molecule EGFRinhibitors in patients with lung cancer, using blood as thesource for mutant DNA detection. As background, somat-ically mutated or altered genes have led to effectivetargeted therapies and improved biomarkers for clinicaldecision-making. As the use of such targeted therapiesbecomes more widespread, there will be a great need forcompanion diagnostic technologies that can reliably andquickly identify patients with cancers that harbor themutation/genetic alteration to be targeted. Particularlychallenging is the fact that the ascertainment of muta-tional status in solid tumors often depends on biopsies ofmetastatic sites, followed by conventional genotypingassays with limited sensitivity. Such biopsies can bedifficult to obtain, even in a research setting, resultingin a great impetus for developing sensitive, noninvasivediagnostic tools to assay for the mutational status of atumor and molecular predictors of response.

BEAMing is one such technology. It relies on single-molecule PCR at a massively parallel scale, similar tonext-generation DNA sequencing technologies (refs. 2and 3; Fig. 1). The mechanics of BEAMing start withamplification of a predetermined locus of interest byconventional PCR. Next, the PCR product is added tohundreds of thousands of oligonucleotide-coupled beadsin oil, and an emulsion is created such that most of thebeads will bind only a single DNA molecule. A secondround of PCR is then performed. After de-emulsificationand magnetic capture are completed, single-base primer

extension or hybridization with mutant-specific probes isperformed with different fluorescently labeled nucleo-tides for either wild-type or mutant sequences. Finally,flow cytometric analysis of hundreds of thousands ofbeads allows for the detection and quantification ofwild-type or mutant alleles. Because BEAMing is a digitalPCR technique that analyzes one allele at a time, it ishighly sensitive for the detection of rare mutant alleleswithin a large population of wild-type alleles. This is theexact molecular environment found in circulating plasmaof cancer patients, i.e., rare circulating tumor DNA(ctDNA) intermixed among a vast pool of noncancerousDNA.

Taniguchi and colleagues (1) applied the BEAMingtechnology for the first time to the analysis of EGFRmutations in non–small cell lung cancer. Activatingmutations in EGFR are found in �10% of Caucasianpatients with non–small cell lung cancer and up to50% of Asian patients, and they are enriched in females,never-smokers, and patients with adenocarcinomas. Suchpatients are now routinely offered therapy with the small-molecule EGFR tyrosine kinase inhibitors (EGFR-TKI)gefitinib and erlotinib. Unfortunately, resistance to theseagents universally occurs. In 50% of cases, resistance isassociated with the emergence of a second-site gatekeepermutation in EGFR: T790M (4, 5). The authors studied twocohorts of patients with stage III or IV non–small cell lungcancer whose diagnostic biopsy specimens revealed acti-vating EGFR mutations. Cohort 1 had progressive diseaseafter treatment with an EGFR-TKI, and cohort 2 had neverreceived an EGFR-TKI. Overall, BEAMing had a sensitivityof 72.7% for detecting the known activating EGFR muta-tion in plasma ctDNA collected at a single time point. Inthe progressive disease cohort, a T790M resistance muta-tion was identified in 43.5% of patients, a value near thepredicted 50%. This is the first use of BEAMing to detect adrug-resistance mutation, although other technologieswere used for that purpose in previous studies (6–9).Because BEAMing is both qualitative and quantitative,Taniguchi and colleagues were able to calculate the ratioof T790M mutations to the original activating EGFRmutations for each patient, with ratios ranging from13.3% to 94%. One would predict that when this ratio

Authors' Affiliation: Sidney Kimmel Comprehensive Cancer Center atJohns Hopkins, Baltimore, Maryland

Corresponding Author: Ben Ho Park, Sidney Kimmel ComprehensiveCancer Center at Johns Hopkins, 1650 Orleans St., Rm. 151, Balti-more, MD 21287. Phone: 410-502-7399; Fax: 410-614-4073; E-mail:bpark2@jhmi.edu

doi: 10.1158/1078-0432.CCR-11-2556

�2011 American Association for Cancer Research.

ClinicalCancer

Research

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reaches 100%, all of the cancer cells with activating EGFRmutations will have also acquired T790M resistancemutations or, alternatively, the T790M-containing cellswill have outgrown or outsurvived the cells withoutT790M mutations. Preclinical data show that T790M cellsare outcompeted for growth by T790T cells in the absenceof selective pressure from EGFR-TKIs (10). Thus, the ratioof resistance to activating mutations may provide a mea-sure of the clonal dominance of the tumor subclones withresistance mutations.Studies such as that of Taniguchi and colleagues are impor-

tant steps in the application of new genotyping techno-logies to clinical cancer care. However, this approach entailssome notable limitations and caveats. As the authors note,theirswas not a prospective study, and they didnot analyzeplasma ctDNA at serial time points. It is not knownwhether any of the patients who had T790M resistancemutations after EGFR-TKI treatment harbored such muta-tions at low levels in their primary tumors before treat-ment, as was previously described (11, 12). To address thisissue in future studies, investigators could also use BEAM-ing on the primary tumor biopsy or surgical specimenbecause its high sensitivity theoretically would enabledetection of this presumably minute drug-resistant pop-ulation. This would give a clearer picture of the potentialpreexisting tumor heterogeneity that may be below thecurrent level of detection with conventional techniques,and it may also give insight into the sensitivity of BEAMingctDNA for detecting microscopic amounts of tumor. If, forexample, a T790M population of cancer cells is present at<1% in the primary tumor at the time of diagnosis/surgery,would thisminority population be detectable by BEAMingof plasma?Other potential applications of BEAMing include nonin-

vasive diagnosis, screening, and detection of micrometa-static disease postoperatively to aid decisions regarding theneed for adjuvant systemic therapies. However, many ques-tions need to be answered in further depth. This will requirenumerous studies involving large populations of patients

across different tumor types treated with a full range oftherapies. In addition, such studies will necessitate theanalysis of BEAMing using serial time points to betterelucidate the factors that influence the generation andstability of ctDNA and to fully understand the sensitivityand specificity of BEAMing as related to a given tumortype and response to specific classes of therapies. Otherquestions will also arise. For example, it is currentlyunclear whether a low level of detectable ctDNA inplasma has clinical meaning or usefulness. In a smallnumber of patients with colon cancer who were studiedretrospectively, detectable mutant DNA predicted even-tual overt disease progression (13); however, it is clearthat more studies are needed. Likewise, in the study byTaniguchi and colleagues, T790M resistance mutationswere found in patients with progressive disease, but we donot know at what quantitative level such mutationspredict clinical failure of EGFR-TKI therapy.

Thus, perhaps the most powerful utility of BEAMing isits ability to quantitate the amount of cancer-specificctDNA. BEAMing has the potential to enable investigatorsto assess tumor burden and tumor heterogeneity withgreat sensitivity and to address important questions withgreater precision. Is a patient with no detectable ctDNAafter surgery cured? Can we rapidly ascertain whether apatient is benefiting from a chosen therapy? Does earlydetection of resistance mutations allow for meaningfultherapeutic interventions? These questions will undoubt-edly be answered as BEAMing and other technologiesmature. Ultimately, the ability to detect microscopicamounts of tumor burden will better inform cliniciansand patients, and allow for a tailored approach for thetreatment of human cancers.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Received October 21, 2011; accepted November 1, 2011; publishedOnlineFirst November 7, 2011.

DNA isolation

Plasma Tumor DNA

Wild-type DNAWater-in-oil emulsion with

magnetic beads

Pre-Amplification Emulsion PCRHybridization

Flow Cytometry Results

Percentage mutant DNA

for example, 0.27%

Q1 Q2

Q4Q3

Mutant

Wild-type

Magnetic beads

coated with primer

Amplified DNA

Figure 1. Principles of BEAMing. Shown are the sequential steps involved with BEAMing. Results are expressed as a ratio of mutant to wild-type DNAmolecules. Flow cytometry provides a quantitative measurement of the mutant DNA present in the plasma. Figure adapted with permission from InosticsGmbH (Hamburg, Germany).

Mutation Detection in Plasma

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References1. Taniguchi K, Uchida J, Nishino K, et al. Quantitative detection of EGFR

mutations in circulating tumor DNA derived from lung adenocarcino-mas. Clin Cancer Res 2011;17:7808–15.

2. Dressman D, Yan H, Traverso G, Kinzler KW, Vogelstein B. Transform-ing single DNA molecules into fluorescent magnetic particles fordetection and enumeration of genetic variations. Proc Natl Acad SciU S A 2003;100:8817–22.

3. Diehl F, Li M, He Y, Kinzler KW, Vogelstein B, Dressman D. BEAMing:single-molecule PCR on microparticles in water-in-oil emulsions. NatMethods 2006;3:551–9.

4. Kobayashi S, Boggon TJ, Dayaram T, J€anne PA, Kocher O, MeyersonM, et al. EGFRmutation and resistanceof non-small-cell lungcancer togefitinib. N Engl J Med 2005;352:786–92.

5. Pao W, Miller VA, Politi KA, Riely GJ, Somwar R, Zakowski MF, et al.Acquired resistance of lung adenocarcinomas to gefitinib or erlotinib isassociated with a second mutation in the EGFR kinase domain. PLoSMed 2005;2:e73.

6. Maheswaran S, Sequist LV, Nagrath S, Ulkus L, Brannigan B, ColluraCV, et al. Detection of mutations in EGFR in circulating lung-cancercells. N Engl J Med 2008;359:366–77.

7. Li J, J€anne PA, Makrigiorgos GM. Biotinylated probe isolation oftargeted gene region improves detection of T790M epidermal growth

factor receptor mutation via peptide nucleic acid-enriched real-timePCR. Clin Chem 2011;57:770–3.

8. Nakamura T, Sueoka-Aragane N, Iwanaga K, Sato A, Komiya K, Abe T,et al. A noninvasive system for monitoring resistance to epidermalgrowth factor receptor tyrosine kinase inhibitors with plasma DNA.J Thorac Oncol 2011;6:1639–48.

9. KuangY,RogersA,YeapBY,WangL,MakrigiorgosM,VetrandK, et al.Noninvasive detection of EGFR T790M in gefitinib or erlotinib resistantnon-small cell lung cancer. Clin Cancer Res 2009;15:2630–6.

10. Chmielecki J, Foo J, Oxnard GR, Hutchinson K, Ohashi K, Somwar R,et al. Optimization of dosing for EGFR-mutant non-small cell lung can-cer with evolutionary cancer modeling. Sci Transl Med 2011;3:90ra59.

11. Bell DW, Gore I, Okimoto RA, Godin-Heymann N, Sordella R, MulloyR, et al. Inherited susceptibility to lung cancer may be associatedwith the T790M drug resistance mutation in EGFR. Nat Genet 2005;37:1315–6.

12. Inukai M, Toyooka S, Ito S, Asano H, Ichihara S, Soh J, et al. Presenceof epidermal growth factor receptor gene T790M mutation as a minorclone in non-small cell lung cancer. Cancer Res 2006;66:7854–8.

13. Diehl F, Schmidt K, Choti MA, Romans K, Goodman S, Li M, et al.Circulating mutant DNA to assess tumor dynamics. Nat Med 2008;14:985–90.

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