循环肿瘤细胞的检测

Detection of Androgen Receptor Mutations

in Circulating Tumor Cells:

Highlights of the Long Road to Clinical Qualification

Hans Lilja 1,2,3*and Howard I.Scher 3

There is evidence that androgen receptor (AR )4gene function not only is necessary for the growth and dif-ferentiation of the healthy prostate gland but also may contribute critically to treatment failure in progressive stages of castration-resistant prostate cancer (CRPC)5(1–3).The mechanisms include AR overexpression,AR gene amplification,an increase in the levels of the androgen synthetic machinery leading to increased in-tratumoral androgens,ligand-independent activation,and gain-of-function mutations in the gene itself (2).The clinical importance of these findings has been val-idated in trials of the novel androgen receptor antago-nist MDV3100,which was developed in a cell-based screen for activity in cells with overexpressed AR,and of abiraterone acetate,a 17,20lyase inhibitor that blocks androgen synthesis in the testis,the adrenals,and the tumor (3,4).

The development and validation of reliable,repro-ducible protocols to accurately assess functional status,frequency,and functional relevance of mutations in AR could then be critical for the successful development and clinical implementation of novel targeted strate-gies for patients with advanced stages of prostate can-cer,particularly those with progressive CRPC.Al-though a recent study on the integrative genomic profiling of human prostate cancer was limited by the interrogation of only a modest number of genes and patient samples,the reported data strongly suggested that the overall rate of mutations may be low in pros-tate cancer (5).In particular,mutations were infre-quent among common,broadly mutated oncogenes,such as PIK3CA (phosphoinositide-3-kinase,catalytic,

alpha polypeptide),KRAS (v-Ki-ras2Kirsten rat sar-coma viral oncogene homolog),and BRAF (v-raf mu-rine sarcoma viral oncogene homolog B1).Impor-tantly,however,the study reported a higher frequency of AR mutations.Alterations in AR ,including muta-tions,gene amplification,and overexpression,were common in the metastatic tumor samples analyzed but were notably absent in tumor samples representing un-treated localized disease (5).Assessing the frequency of specific molecular changes in CRPC,however,is lim-ited by the general unavailability of metastatic tumor samples for profiling,as well as by a lack of analytically valid assays for measurement.

Circulating tumor cells (CTCs)isolated from blood have been hypothesized to be capable of fulfilling the unmet need for tumor tissue for molecular profil-ing for biomarkers that predict sensitivity to treat-ment.Biomarker qualification requires analytically valid assays that can then be studied in prospective clinical trials designed for a specific context of use.A variety of CTC assays are available and under study,but at present only 1,CellSearch ?(Veridex/Johnson &Johnson),has undergone full analytical validation for enumeration (6)and been shown in prospective trials to provide prognostic information at baseline and after treatment in patients with breast,colorectal,or pros-tate cancer (7–9).The US Food and Drug Administra-tion (FDA)clearance is as an “aid to monitoring”dis-ease in conjunction with other measures.The assay is not cleared for the assessment of predictive markers.

A limitation of CellSearch is that CTCs are not detected in patients with clinically localized disease or in patients who are in the clinical state of increasing prostate-specific antigen (10,11).To address this is-sue,we measured the mRNA copy number in 2AR -regulated prostate-specific genes [KLK3(kallikrein-related peptidase 3)and KLK2(kallikrein-related peptidase 2)](11)in blood prepared with the PAXgene Blood RNA System (PreAnalytiX).A direct compari-son of the 2assays revealed highly concordant results that were significantly associated with the presence of skeletal metastases and with overall bin-ing these measures,however,did not seem to contrib-ute importantly to enhancing the predictive accuracy

1

Departments of Clinical Laboratories,2Surgery (Urology),and 3Medicine (Genito-Urinary Oncology),Memorial Sloan-Kettering Cancer Center,New York,NY.

*Address correspondence to this author at:Memorial Sloan-Kettering Cancer Center,1275York Ave.,Box 213,New York,NY 10021.Fax 646-422-2379;e-mail liljah@.

Received July 1,2010;accepted July 6,2010.

Previously published online at DOI:10.1373/clinchem.2010.1508964

Human genes:AR ,androgen receptor;PIK3CA ,phosphoinositide-3-kinase,cat-alytic,alpha polypeptide;KRAS ,v-Ki-ras2Kirsten rat sarcoma viral oncogene homolog;BRAF ,v-raf murine sarcoma viral oncogene homolog B1;KLK3,kallikrein-related peptidase 3;KLK2,kallikrein-related peptidase 2.5

Nonstandard abbreviations:CRPC,castration-resistant prostate cancer;CTC,circulating tumor cell;FDA,US Food and Drug Administration.

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above that of the CTC counts obtained with the FDA-cleared CellSearch assay(10,11).

Marketed along with the CellSearch Circulating Tumor Cell Kit for CTC enumeration is the CellSearch Tumor Cell Kit for molecular analysis(although the FDA has not cleared the latter).In this issue of the Journal,Jiang et al.(12)used the Circulating Tumor Cell and Profile kits with the AllPrep DNA/RNA Micro Kit(Qiagen)to amplify AR exons3–8by the PCR.The amplicons were subsequently subjected to digestion with the SURVEYOR endonuclease and fractionation by WAVE?denaturing HPLC(Transgenomic).Assays for mutations were postulated to be more sensitive and spe-cific than assays for the presence of nonmutated tissue-specific genes.The authors used KRAS mutations as the target and obtained results with direct resequencing that suggested the possibility of detecting mutations in the tar-get gene in mixtures containing as little as2.5%of the target in the enriched CTC sample(12).They subse-quently detected27AR mutations(19missense muta-tions,2silent mutations,5deletions,and1insertion)in CTC samples from20(57%)of35informative patients with CRPC.Whereas other investigators had previously reported many alterations as somatic mutations in tissue samples from metastatic lesions,Jiang et al.also discov-ered novel AR mutations of putative functional signifi-cance(12).The estimated frequency of AR mutations among metastatic prostate cancer patients reported by Jiang et al.is strikingly similar to that of Taylor et al.(5), who used a very different approach to address this issue.

To confirm these findings prospectively first re-quires a focus on the analytical techniques used and then documentation of the patient population studied. The first concern is the large number of cycles used in the nested PCR protocol,which in theory could detect a single copy of the gene target.The second concern is that the limits of detection for AR mutations were not studied directly,but their determination will ulti-mately be required for analytical validation of this ap-proach.Additional concerns include the detection of AR mutations in patient samples with low or0CTC counts,as well as the low correlation between the CTC count and the frequency of mutated AR genes in each sample.The latter concern may be explained by the difference in the biomarker reported,because the CellSearch Profile Kit measures the total number of epithelial cell adhesion molecule–positive cells cap-tured by the immunomagnetic bead separation,not the fraction of cells that meet the strict FDA-cleared defi-nition of a CTC,which only includes epithelial cell ad-hesion molecule–positive cells that are intact with a 4?,6-diamidino-2-phenylindole–positive nucleus sur-rounded by cytoplasm,cytokeratin positive,and CD45 negative.

To determine whether an alteration in the AR gene represents a somatic change requires concurrent anal-ysis of DNA from nonpathologic lymphocytes,as re-ported by Taylor et al.(5).Jiang et al.did not perform this assessment in their study(12),nor did they evalu-ate age-matched,healthy,nonsymptomatic male vol-unteers as potential negative controls.Recognizing that the alterations present in late-stage CRPC reflect both the intrinsic biology of the tumor and the specific ther-apies the tumor has been exposed to and ultimately progressed from will require the evaluation of a suffi-cient number of patients in representative discrete clin-ical cohorts before any results can be considered for evaluation in prospective trials.For the latter issue,an understanding of the functional significance of specific AR mutations and the association of the presence of a lesion with a clinical outcome will be required to better enable the design of a prospective trial that assesses their role as predictive biomarkers.

Author Contributions:All authors confirmed they have contributed to the intellectual content of this paper and have met the following3re-quirements:(a)significant contributions to the conception and design, acquisition of data,or analysis and interpretation of data;(b)drafting or revising the article for intellectual content;and(c)final approval of the published article.

Authors’Disclosures of Potential Conflicts of Interest:Upon manuscript submission,all authors completed the Disclosures of Poten-tial Conflict of Interest form.Potential conflicts of interest: Employment or Leadership:None declared.

Consultant or Advisory Role:None declared.

Stock Ownership:H.Lilja,Arctic Partners.

Honoraria:None declared.

Research Funding:None declared.

Expert Testimony:None declared.

Other:H.Lilja holds patents with Arctic Partners for free prostate-specific antigen(PSA),intact PSA,and human kallikrein2assays. Role of Sponsor:The funding organizations played no role in the design of study,choice of enrolled patients,review and interpretation of data,or preparation or approval of manuscript.

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