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Originally published as JCO Early Release 10.1200/JCO.2008.21.2092 on February 23 2009 © 2009 American Society of Clinical Oncology.
Bone Marrow Aspiration for Disseminated Tumor Cell Detection: A Must-Have Test or Is the Jury Still Out?Genitourinary Oncology Service, Department of Medicine, Sidney Kimmel Center for Prostate and Urologic Cancers, Sloan-Kettering Cancer Center; and the Department of Medicine, Joan and Sanford E. Weill College of Medicine of Cornell University, New York, NY
Institute of Tumour Biology, Center of Experimental Medicine, University Medical Center Hamburg Eppendorf, Hamburg, Germany It has long been recognized that metastatic foci develop from malignant epithelial cells that are derived from the primary tumor. For bone metastases, this means hematogenous shedding of the cells through the bloodstream, followed by attachment and proliferation in the bone marrow. A major controversy in the metastasis field is the time when shedding actually occurs relative to the steps in the development of a tumor from the time of malignant transformation, the in situ stage, or after the invasive phenotype was effectively established. The report by Weckermann et al1 is an observational study of the clinical outcomes of patients with localized or regionally metastatic prostate cancer to lymph nodes treated by radical prostatectomy who had bone marrow aspirates analyzed for the presence of cytokeratin- positive cells pre- and postoperatively. Previous work has shown that cells with this phenotype represent malignant epithelial cells or disseminated tumor cells (DTCs) that have spread from the primary tumor.2 The patients were variably observed per hospital routine with blood and imaging tests and monitored for clinical outcomes, including biochemical recurrence, local recurrence, or the development of overt metastatic disease. The results showed that patients in whom DTCs were detected preoperatively were more likely to relapse within the first 2 years after surgery. The detection of DTCs in the postoperative setting was not associated with biochemical recurrence or the development of metastatic disease. The results contrast with what has been observed in breast cancer patients studied in a similar manner, in which the detection of DTCs, both at the time of surgery3 as well as postoperatively,4–6 was predictive of clinical relapse. The authors suggest that DTCs can serve as a biomarker of prognosis that can be used in medical decision making and in clinical trials. In addition, preliminary data are presented of a comparative genomic hybridization (CGH) analysis of the cytokeratin-positive cells isolated pre- and postoperatively from the bone marrow and from the primary tumor. Overall, the number of significant copy number alterations was slightly higher in the primary tumors compared to DTCs from M0 patients isolated before or after surgery or during biochemical relapse, whereas DTCs isolated from patients with manifest metastases (stage M1) showed significantly more chromosomal aberrations. Surprisingly, the M1-specific changes were rarely detected in areas microdissected from the primary tumors. The authors suggest that the more aggressive phenotype of DTCs identified in the bone marrow samples of M1 patients reflects activation of the malignant process by factors extrinsic to the tumor itself. The Oncology Biomarker Qualification Initiative (OBQI) is an agreement between the US Food and Drug Administration, Centers for Medicare and Medicaid Services (CMS), and the National Cancer Institute (NCI) that outlines a road map for the development and qualification of biomarkers for a specific intended use in clinical practice.7 There are two key components of this initiative: the availability of analytically valid assays that provide reproducible measurements and that can be conducted in CLIA-certified laboratories, and evidence from prospective trials studying the relationship of the biomarker to specific clinical outcomes. The authors of the current report conclude that the detection of DTCs in the bone marrow of patients with localized or locoregional prostate cancer is a biomarker of prognosis, and that patients in whom cells are detected should be considered for treatment with antiproliferative therapies in the perioperative period to prevent recurrent disease. They also conclude that the detection of DTCs postoperatively does not predict for a lethal disease phenotype, and that the reason for this peculiar finding might be a dormant (ie, nonactivated) state of these cells. So, as practicing physicians and clinical researchers, we asked ourselves the following—is the test ready for prime time? Must or should we perform this test on our patients, and if so, should we ask patients to pay for it when an insurance carrier says no? If it is not ready, is further study warranted, and if so, what are the questions? A critical examination shows that at this time the evidence is at best preliminary to qualify DTC detection and CGH profiling for each of these intended uses, but that further study is indeed warranted. Consider first the disease. The clinical spectrum of prostate cancers that are detected range from innocuous to significant, curable to incurable, with a ratio of incidence to mortality in the United States that is approximately 7:1. To address this continuum, we consider the disease as a series of states representing milestones easily recognized in practice to guide patient management.8 These states include localized disease, noncastrate- or castrate-rising prostate-specific antigen, and noncastrate or castrate clinical metastases. States are distinct from stages in that the latter are only applicable to the newly diagnosed, untreated patient (clinical stage) or one who has undergone definitive treatment of a primary tumor (pathologic stage). As the prognosis of patients within each state is highly variable and may span 10 years or more, the central question is how to identify lethal cancers early so that effective therapies can be administered. For prostate cancer, this is considered to be the development of detectable disease on an imaging study, a point where the mortality from prostate cancer exceeds that of other causes. Second, consider the assay. Immunocytochemical methods to detect cytokeratin positivity are difficult to interpret, and various antibodies and staining techniques have been used over the past decades.9,10 Recent efforts to standardize cytokeratin-based assays,10 which are supported by a European consortium focused on DTC detection and characterization,11 have helped to address this problem by establishing consensus protocols for cytokeratin-based DTC detection. This platform may also be used to establish a consensus method for the whole genome analysis of single DTCs to provide consistent results in different laboratories, which is necessary before such a method can be considered for use in clinical trials or in clinical practice. INTENDED USE 1 One intended use for DTCs would be as a biomarker of prognosis that predicts for disease recurrence after surgery. The prostate cancer literature is replete with studies of prognostic models to predict recurrent disease after surgery.12 Most were designed to predict a biochemical recurrence or PSA relapse, and include a variety of clinical, biologic, and more recently, molecular determinants. The factors are typically combined into nomograms that allow parameters to be considered in a continuous manner as appropriate in contrast to discrete cutoffs, and may include the outcomes of thousands of men treated. Ignoring the issues surrounding the use of these nomograms in practice, any new biomarker, such as DTC detection, would need to be considered in relation to the established norms and not against the individual parameters outlined here. The current analysis on the prognostic value of DTC measurements in preoperative bone marrow samples confirms the recent study of Köllermann et al.13 The present investigation includes patients with localized disease only and those with localized disease and regional metastases with inherently different prognoses. The high rate of margin positivity is also of concern, as this factor alone is predictive for recurrence. A more difficult hurdle, but a more important question, is whether DTC detection could predict for metastatic disease or death from prostate cancer. To do so would require a prospective trial of equivalently staged patients who underwent the same procedure, and who were observed at fixed intervals with a predefined and fixed schedule of visits, clinical tests, and imaging. As a first step, a trial restricted to patients with a high risk of recurrence could be studied, to see if the ability to detect DTCs adds incremental value. With this preliminary data, a definitive trial of appropriate size and power could be designed to address the question. Since repeated bone marrow sampling is more time consuming and less acceptable to the patient than blood sampling, the detection of circulating tumor cells in the blood of cancer patients has recently received considerable attention.8 Although a variety of isolation techniques are being evaluated, only one, CellSearch (Veridex, Raritan, NJ), is both analytically valid for cirulating tumor cell (CTC) enumeration and US Food and Drug Administration–cleared to monitor prognosis and assess the response to therapy in patients with breast, colorectal, and prostate cancer.14 The ability to obtain repeated samples easily by phlebotomy and with minimal patient discomfort offers the opportunity for real-time monitoring.15,16 A comparative analysis of DTCs and CTCs in breast cancer patients using a different CTC capture method, however, showed that the two assays provide complementary information.16,17 Additional studies are needed. INTENDED USE 2 A second intended use of DTCs would be to identify patients in need of antiproliferative therapy in the perioperative or postoperative period. In addition to the uncertain predictive power of assays for recurrence, there was no evidence provided that the DTCs identified in the current study have a high proliferative index (eg, based on Ki-67 staining). In breast cancer, for example, most DTCs (and CTCs) are Ki-67–negative and therefore in G0 or early G1 phase of the cell cycle.16,18,19 The suggestion that cytokine release during surgery stimulates the DTCs to proliferate is therefore speculative at best. Recognizing that the trials were not enriched for patients with DTC-positive bone marrow, one could argue that this experiment has already been done in randomized trials exploring neoadjuvant androgen-depletion therapy prior to radial prostatectomy, which showed no difference on PSA relapse-free survival. At this point, the data are insufficient to justify an intervention trial to address this question. INTENDED USE 3 The postoperative detection of DTCs does not predict for a lethal disease phenotype. This finding contrasts with similar analyses in breast cancer. The authors propose that DTCs in the bone marrow of prostate cancer patients remain in a state of dormancy for extended periods. The concept of dormancy has gained considerable attention, particularly in the field of breast cancer,9,20 which implies that tumor cells can escape their dormant state and resume proliferating through as yet uncharacterized signals and as a consequence, form overt metastases.21 Under this scenario, the postoperative presence of DTCs should pose a risk for subsequent metastatic relapse, as is the case in breast cancer.4,5 Unclear, however, is why most DTCs in prostate cancer patients are put to sleep so efficiently when those cells should have been present also at surgery and have received the postulated activation signal. Besides these biologic aspects, the observed discrepancies might also be explained by technical issues. First, considering the low number of DTCs, it is not clear how many tests must be positive to be considered positive and how many tests considered negative to be true negative. Moreover, the preoperative samples were taken under general anesthesia, but the postoperative bone marrow samples were obtained under local anesthesia. Not stated is whether the postoperative bone marrow samples were also bilateral or whether they were unilateral to reduce patient discomfort. The latter would introduce bias.22 Finally, the postoperative bone marrow samples were taken at various time points. In a breast cancer study, it was shown that the timing of the postoperative sampling itself was prognostic.6 Additional questions arise because of the overlap between the pre- and postoperative patient cohorts, since the authors analyzed changes in DTC status (eg, from positive to negative) in 131 patients. A subset analysis of these patients alone would be of interest. It is also stated that DTCs were consistently present postoperatively, but no information was provided on the time course of DTC detection, and DTCs were found repeatedly in only 26 patients. Since only one to two DTCs were detected per sample, there is a real possibility that some DTCs were missed due to a Poisson distribution of rare events. Also uncertain is how stable the DTC measurements are in individual patients, and what change in DTC counts per sample is called a change. INTENDED USE 4 Another intended use would be to identify patients with tumors that have transitioned to a lethal disease phenotype. This observation is perhaps the most interesting and the one that can be studied more definitively in the short term. In this study, the authors fail to show that the consecutive gain of genomic aberrations in DTCs of individual patients monitored between surgery and metastatic relapse contributes to the escape from dormancy and the formation of metastases. The interpretation of the genomic DTC data from M1 patients is limited by the fact that these DTCs could have been derived from either the original primary tumor or the metastases themselves. A comparative analysis of primary tumors, DTCs isolated at different postoperative time points, and overt metastases was not performed; such an analysis would be very difficult, given that on average only one to two DTCs were detected in each bone marrow sample. This poses an additional problem—how representative is the genome of one or two DTCs of the total burden of disseminated tumor cells in the patient? Considering an equal distribution of DTCs throughout the bone marrow, we previously estimated that the total number of DTCs in the patient might be approximately 1 million19; clearly, reliance on the profile of the one or two DTCs detected for medical decision making could be misleading. Despite the issues raised, the authors must be commended for the foresight to design and complete a provocative study that will form the basis for many prospective trials to test the hypotheses that have been generated. Biomarker development and credentialing is difficult, and data on the genomic make-up of DTCs in prostate cancer patients are rare.23,24 To change clinical practice and impact medical decision making requires more than just a P value or a hazard ratio based on a single analysis. It requires multiple prospective trials focused on specific intended uses that are clinically relevant. Is the significance of DTCs in the bone marrow of prostate cancer patients different from that in breast cancer? Can these cells provide a snapshot into the biologic make-up of an individual patient's cancer to guide treatment selection? Is the performance of serial bone marrow aspirations a must-have test? Only time will tell. AUTHORS' DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST The author(s) indicated no potential conflicts of interest. AUTHOR CONTRIBUTIONS Manuscript writing: Howard I. Scher, Klaus Pantel Final approval of manuscript: Howard I. Scher, Klaus Pantel Acknowledgment Supported by Grant No. P50 CA92629 from the NCI SPORE in Prostate Cancer; Grant No. PA 341/15-2 from Deutsche Forschungsgemeinschaft; Grant No. 10-2181-Pa from Deutsche Krebshilfe, Bonn, Germany; the European Commission (DISMAL project, contract no. LSHC-CT-2005-018911); the Prostate Cancer Foundation; and the Sidney Kimmel Center for Prostate and Urologic Cancers. REFERENCES
1. Weckermann D, Polzer B, Ragg T, et al: Perioperative activation of disseminated tumor cells in bone marrow of patients with prostate cancer. J Clin Oncol 27:1549–1556, 2009. 2. Cristofanilli M, Mendelsohn J: Circulating tumor cells in breast cancer: Advanced tools for "tailored" therapy? Proc Natl Acad Sci U S A 103:17073–17074, 2006. 3. Braun S, Vogl FD, Naume B, et al: A pooled analysis of bone marrow micrometastasis in breast cancer. N Engl J Med 353:793–802, 2005. 4. Wiedswang G, Borgen E, Karesen R, et al: Isolated tumor cells in bone marrow three years after diagnosis in disease-free breast cancer patients predict unfavorable clinical outcome. Clin Cancer Res 10:5342–5348, 2004. 5. Janni W, Rack B, Schindlbeck C, et al: The persistence of isolated tumor cells in bone marrow from patients with breast carcinoma predicts an increased risk for recurrence. Cancer 103:884–891, 2005.[CrossRef][Medline] 6. Naume B, Fehm T, Wiedswang G, et al. Persistence of isolated tumor cells in the bone marrow of breast cancer patients predicts increased risk for relapse - a European pooled analysis San Antonio Breast Cancer Symposium, 2008. 7. US Food and Drug Administration. Memorandum of understanding between Food and Drug Administration (FDA) and the National Cancer Institute (NCI) and Centers for Medicare and Medicaid services. http://www.fda.gov/oc/mous/domestic/FDA-NCI-CMS.html. 8. Scher HI, Heller G: Clinical states in prostate cancer: Towards a dynamic model of disease progression. Urology 55:323–327, 2000.[CrossRef][Medline] 9. Pantel K, Brakenhoff RH, Brandt B: Detection, clinical relevance and specific biological properties of disseminating tumour cells. Nat Rev Cancer 8:329–340, 2008.[CrossRef][Medline] 10. Fehm T, Braun S, Muller V, et al: A concept for the standardized detection of disseminated tumor cells in bone marrow from patients with primary breast cancer and its clinical implementation. Cancer 107:885–892, 2006.[CrossRef][Medline] 11. DISMAL Project. http://www.dismal-project.eu/. 12. Shariat SF, Walz J, Roehrborn CG, et al: External validation of a biomarker-based preoperative nomogram predicts biochemical recurrence after radical prostatectomy. J Clin Oncol 26:1526–1531, 2008. 13. Kollermann J, Weikert S, Schostak M, et al: Prognostic significance of disseminated tumor cells in the bone marrow of prostate cancer patients treated with neoadjuvant hormone treatment. J Clin Oncol 26:4928–4933, 2008. 14. Veridex. 501(k) Summary. http://www.fda.gov/cdrh/pdf7/K073338.pdf. 15. de Bono JS, Scher HI, Montgomery B, et al: Circulating tumor cells predict overall survival following treatment in metastatic prostate cancer. Clinical Cancer Res 14:6302–6309, 2008. 16. Muller V, Stahmann N, Riethdorf S, et al: Circulating tumor cells in breast cancer: Correlation to bone marrow micrometastases, heterogeneous response to systemic therapy and low proliferative activity. Clin Cancer Res 11:3678–3685, 2005. 17. Pierga JY, Bonneton C, Vincent-Salomon A, et al: Clinical significance of immunocytochemical detection of tumor cells using digital microscopy in peripheral blood and bone marrow of breast cancer patients. Clin Cancer Res 10:1392–1400, 2004. 18. Pantel K, Schlimok G, Braun S, et al: Differential expression of proliferation-associated molecules in individual micrometastatic carcinoma cells. J Natl Cancer Inst 85:1419–1424, 1993. 19. Pantel K, Brakenhoff RH: Dissecting the metastatic cascade. Nat Rev Cancer 4:448–456, 2004.[CrossRef][Medline] 20. Aguirre-Ghiso JA: Models, mechanisms and clinical evidence for cancer dormancy. Nat Rev Cancer 7:834–846, 2007.[CrossRef][Medline] 21. Pantel K, Alix-Panabieres C, Riethdorf S: Cancer micrometastases. Nature Clin Pract Oncol in press. 22. Pantel K, Schlimok G, Angstwurn M, et al: Methodological analysis of immunocytochemical screening for disseminated epithelial tumor cells in bone marrow. J Hematother 3:165–173, 1994.[Medline] 23. Holcomb IN, Grove DI, Kinnunen M, et al: Genomic alterations indicate tumor origin and varied metastatic potential of disseminated cells from prostate cancer patients. Cancer Res 68:5599–5608, 2008. 24. Schmidt H, DeAngelis G, Eltze E, et al: Asynchronous growth of prostate cancer is reflected by circulating tumor cells delivered from distinct, even small foci, harboring loss of heterozygosity of the PTEN gene. Cancer Res 66:8959–8965, 2006.
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Copyright © 2009 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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