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© 2003 American Society for Clinical Oncology
Detection and Measurement of Occult Disease for the Prognosis of Solid Tumors
From the Cancer Diagnosis Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute, Rockville, MD; Universitaetsklinikum fuer Frauenheilkunde Leopold-Franzens-Universitaet, Innsbruck, Austria; University of Southern California, Keck School of Medicine, Los Angeles, CA; Universitatsklinikum Hamburg-Eppendorf, Hamburg, Germany; and Memorial Sloan-Kettering Cancer Center, New York, NY. Address reprint requests to Tracy G. Lugo, PhD, Cancer Diagnosis Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute, 6130 Executive Blvd, Room EPN 6035A, Rockville, MD 20892; email: TL82S{at}nih.gov.
DISTANT METASTASIS is the usual cause of death in cancer. The single most important determinant of prognosis and management of cancer is the absence or presence of metastatic dissemination of tumor cells at the time of initial presentation and treatment. However, current measures of disease extent are primitive; standard prognostic indexes, although they provide reliable information about populations of patients, cannot predict which patients will experience disease progression after primary therapy. Better methods for detecting and characterizing subclinical tumor cell deposits in various compartments of the body, primarily the bone marrow and lymph nodes, could enable us to refine our estimates of the risk of recurrence for individual patients and to tailor therapies more effectively. The current routinely applied methods for scanning the body and examining lymph nodes for tumor cells have widely recognized limitations. Numerous studies have reported that it is possible to detect disseminated cancer in patients who are considered cancer-free by standard criteria if the pathologist examines more samples, uses more sensitive detection methods such as immunohistochemistry (IHC) or polymerase chain reaction (PCR), or examines body compartments, such as the bone marrow or peripheral blood, which are not ordinarily examined. Attempts to test the clinical utility of these various detection strategies compared with the standard methods for staging have yielded mixed results, mainly because of the diversity of techniques employed and varying numbers of patient samples examined in each study. Although there is ample evidence to indicate that methods such as IHC and PCR do yield valuable clinical information, there is a need to develop a consensus about how to incorporate these new methods and others as they are developed into the routine evaluation of patients. The Diagnostics Research Branch of the Cancer Diagnosis Program of the National Cancer Institute convened an international workshop in Rockville, MD, on January 7 to 8, 2002, entitled "Detection and Measurement of Occult Disease for the Prognosis of Solid Tumors." The purpose of the workshop was to identify priorities for new clinical studies. Discussion centered on the clinical questions that deserve the most urgent attention and the technical criteria that should determine the choice of an assay technique for a clinical trial. Valerie Rusch, MD, (Memorial Sloan Kettering Cancer Center, New York, NY) served as the chair of the meeting, which was organized by Dr Rusch, Richard Cote, MD (University of Southern California, Los Angeles, CA), Klaus Pantel, MD (University Hospital Eppendorf, Hamburg, Germany), and Tracy Lugo, PhD (National Cancer Institute, Bethesda, MD). The conclusions and recommendations of the workshop are presented.
What Is a Micrometastasis? Staining with hematoxylin and eosin (HE) is the standard method for routine examination of lymph nodes and other specimens for the purpose of staging solid tumors. Current consensus recommendations for reporting metastases in lymph nodes or other tissues set a size threshold of 2 mm or greater in largest diameter for foci of tumor cells that are evident with HE stains. Smaller clusters, isolated cells, or cells detected only by techniques such as IHC or PCR fall into a category described by various terms including micrometastasis, occult metastasis, or minimal residual disease. These single cells or small clusters usually do not exhibit overt metastatic activity such as proliferation, stromal reaction, or lymphatic or vascular invasion.1 A number of tumor-specific molecular characteristics have been identified in cytokeratin-positive epithelial cells in bone marrow, indicating that these cells are indeed tumor cells.2–5 Of course, a critical component has been the morphologic evaluation of the disseminated cells to determine whether they have the cytologic characteristics of malignant cells.6 Because a direct relationship has not been established between the presence of single or small foci of tumor cells outside the primary site and the subsequent development of clinical disease at that site, the term "disseminated tumor cells" has been suggested. Finally, because of the continuing controversy about its clinical significance, occult metastasis detection has not yet become part of the standard work-up for patients with cancer.
The Clinical Context Breast cancer. The most extensive database exists for breast cancer. Approximately 2,500 patients with primary breast carcinomas have been analyzed in five large studies during the last 5 years, and all of these studies show a significant correlation between the presence of immunostained tumor cells in bone marrow and unfavorable clinical outcome.7–11 These studies were all prospectively performed. Stephan Braun, MD (Technical University, Munich, Germany), presented an overview with updated information from two European studies on the value of screening bone marrow for disseminated tumor cells in breast cancer patients. The larger study, originally reported in 2000, enrolled 552 patients with stage I, II, and III breast cancer.9 Bone marrow specimens (2 x 106 cells) were scored for the presence of tumor cells detected by immunohistochemical staining with monoclonal antibody A45-B/B3 directed against a common epitope of cytokeratin proteins. Cytokeratin-positive cells were detected in 36% of the patients. The presence of cytokeratin-positive cells in the bone marrow was an independent predictor of distant metastasis but not locoregional recurrence in this patient population, and it seemed to identify a subset of node-negative patients at high risk for relapse. This study continues to enroll patients, and with accrual now standing at 673 patients, these patterns have held firm. A smaller prospective study of 150 uniformly treated node-negative breast cancer patients (stage I to II) was designed to test the relative significance of tumor cells detected by anticytokeratin immunocytochemistry in lymph nodes versus bone marrow.12 These patients were classified as node-negative on the basis of standard axillary nodal dissection and routine histology. Positive bone marrow aspirates were detected in 44 of 150 (29%) of the patients, and this finding was associated with reduced overall survival even after a relatively short follow-up period. Relatively few patients in this group (13 of 150; 9%) presented with cytokeratin-positive cells in lymph nodes, and this finding was not correlated with poor outcome. However, an insufficient number of patients were studied to address the issue of clinical significance of occult lymph node positivity. Of interest is that the concurrent presence of tumor cells in both sites was quite rare (two of 150 patients). Dr Cote summarized results from studies on 736 patients with stage I to III breast cancer enrolled in the International (Ludwig) Breast Cancer Study Group Trial V. The studies were designed to test the prognostic value of serial sectioning and immunohistochemical evaluation of lymph nodes obtained from standard axillary dissection.13 Immunostaining with anticytokeratin antibodies (AE-1 and CAM 5.2) was more sensitive at detecting occult tumor cells than was HE staining of serial sections of lymph nodes from patients who had been classified as node-negative by standard histology. The presence of occult tumor cells detected by either assay was associated with a prognostically significant reduced overall survival in postmenopausal patients but not in premenopausal patients. These investigators also evaluated bone marrow in a subset of 90 patients and reported that breast cancer cells were rarely found in both the lymph nodes and the bone marrow of the same node-negative patient, supporting the view that tumor cell spread to the regional lymph nodes or to distant organs via the bloodstream represents two distinct biologic processes. These results have set the stage for an ongoing United States trial (American College of Surgeons Oncology Group [ACOSOG] Z0010) that directly compares immunohistochemical and molecular methods for evaluation of the bone marrow aspirates and immunohistochemical evaluation of the sentinel lymph nodes that are negative by routine histologic assessment. The trial will study a large (target accrual: 3,356 patients) prospectively recruited population of breast cancer patients (tumor, node, and metastasis system stages: T1–2, N0, M0). This study will compare the presence or absence of occult metastases with intermediate markers of disease progression (stage and grade) as well as with long-term recurrence and survival rates. In addition, proposals for the next generation of clinical studies, in which staging systems that incorporate the detection of disseminated tumor cells may be used to assign breast cancer patients to treatment protocols, are now being debated. Lung cancer. Dr Rusch summarized the clinical data in non–small-cell lung cancer and the design of the ACOSOG Z0040 trial. Disseminated tumor cells can be detected in pleural fluid, lymph nodes, and bone marrow of lung cancer patients, but estimates of prevalence vary widely. At present, an association between presence of immunostained tumor cells in bone marrow or regional lymph nodes and an unfavorable clinical outcome has been observed in several independent studies.14–17 However, the number of patients analyzed in these studies is relatively small. The ACOSOG Z0040 trial will enroll patients with stage I, II, and IIIa non–small-cell lung cancer for operative pleural lavage, bone marrow aspiration, and lymphadenectomy. The presence of occult metastases in the bone marrow, lymph nodes, and pleural lavage fluid will be compared with intermediate markers of disease progression and long-term disease-free and overall survival rates. Methods for the examination of specimens will include HE staining, anticytokeratin immunocytochemistry, and molecular assays. The objective of this trial is to provide more reliable estimates of prevalence and to make preliminary correlations with disease outcomes. Melanoma. Aspects of the Sunbelt Melanoma Trial were presented by Douglas Reintgen, MD (Lakeland Regional Cancer Center, Lakeland, FL). Although the primary objective of this trial is to test the efficacy of adjuvant interferon alfa in patients with low-volume nodal metastases, it also investigates the usefulness of molecular strategies for the pathologic evaluation of lymph nodes. In this trial sentinel lymph nodes that have been categorized as disease-free by standard histology are tested by PCR for a panel of melanoma-specific markers, and the result is used to assign patients to treatment arms. The standard evaluation includes immunohistochemical staining for S-100 as well as HE staining. Preliminary studies in this patient population (stage I or II melanoma, Breslow thickness > 0.75 mm) have indicated that approximately 52% have evidence of disseminated tumor cells in sentinel nodes by one or more of these assays, of which only 22% are detected by the combination of S-100 and conventional HE staining. In patients who are sentinel node-negative by molecular as well as histologic criteria, surgery alone is curative in more than 95% (D. Reintgen, unpublished data).
The Role of Imaging
The Importance of Study Design First, the answers are not necessarily simple. The clinical significance of disseminated tumor cells can be expected to vary according to the type of cancer, the stage of the disease, and other characteristics of the tumor and the patient. Associations with patient outcomes are affected by the treatments that patients receive and by the selection of clinical end points, such as recurrence or survival. Finally, the choice of detection methods and the selection of probes have a powerful influence on both estimates of prevalence and correlations with outcomes. Second, the large number of published studies provides confusing and sometimes conflicting results. From one study to the next there is enormous variability in the focus of the question asked, the techniques employed, and the manner of presenting and interpreting the data, and thus variability exists in the results and conclusions reported. For example, a MEDLINE search identified 81 articles published before 1994 on micrometastases in lymph nodes in breast cancer patients. Only 32 of these publications reported prevalence of nodal micrometastases in node-negative patients, and the estimates of prevalence fell within a broad range. Twenty of the publications reported associations with outcome, but there was no consistency in the choice of clinical end points (recurrence, disease-free survival, overall survival) or the methods of analysis (number of events, Kaplan-Meier plots). Only 16 of the 20 studies reported estimates that were based on at least 5 years of patient follow-up. What would constitute a definitive study? Dr Groshen emphasized that a definitive study will be a confirmatory study on the basis of a sound scientific rationale and solid preliminary data. It will have a formal design to address one or more highly focused questions. The target population for a definitive study will be well defined by clear eligibility and exclusion criteria. Recruitment and selection of patients must be done according to a method to ensure that the study sample will be representative of the target population. The treatment that patients receive should be standardized. For all of these reasons, prospective study designs are preferred. When patients are enrolled prospectively investigators usually have more control to assess eligibility and to perform all required procedures, treatments, and follow-up, making it more likely that data collection will be complete and accurate. Finally, the studies must have sufficient statistical power to address the issue; that is, enough patients must be enrolled to assure a statistically meaningful result. Most principles of the study design for clinical trials extend to definitive studies to evaluate the clinical implications of occult metastasis. It must be emphasized that fundamental to the design of these studies is the need for laboratory assay methods that are standardized and reproducible, with well-defined sensitivity and specificity. In particular, the design must be specified at the outset. The measure of clinical outcome should be well defined. When end points are overall survival or time to recurrence, investigators should plan to report minimum and median follow-up times and to present the data in the form of survival curves with SEs. Investigators should also decide in advance which standard prognostic variables must be measured and included in the analysis. Finally, as stated above, the sample size must be adequate to provide reasonable (> 85%) power to detect a clinically meaningful result. Several factors may increase the sample size necessary to achieve a desired level of statistical power in studies of disseminated cancer cells. These include the magnitude of the expected clinical effect, the true prevalence of disseminated tumor cells in the patient population under study, the effectiveness of strategies for sampling body tissues, and the rates of laboratory measurement errors.
Methods for the Detection and Measurement of Disseminated Tumor Cells Dr Braun discussed the technical considerations involved in setting up an IHC assay for a large clinical study. Significant variables include the choice of antigen and antibody(ies), the staining technique, criteria for specimen quality, the number of bone marrow sites to be sampled by aspiration, and the number of cells per patient to be examined.18,19 Standardized criteria for each of these variables must be defined and applied throughout the study. Current strategies for breast cancer studies rely on antibodies directed against proteins characteristic of epithelial cells to stain putative breast tumor cells in lymph nodes and bone marrow. Large clinical studies have been conducted using antibodies directed against polymorphic mucins,8 epithelial membrane antigens,7 or epithelial cytokeratins.9,11 Braun cited evidence that the antimucin antibodies react with 2% to 20% of cells in normal bone marrow,20 perhaps accounting for the relatively high prevalence of mucin-positive cells reported in some of these studies. Reliable interpretation of IHC results for a large series of samples requires experienced interpreters and procedures for controlling inter- and intrainterpreter variability. A recent attempt to standardize the subjective readout of immunostained cells has been undertaken by the International Society of Cell Therapy (formerly the International Society for Hematotherapy and Graft Engineering [ISHAGE]).19 New automated devices for the microscopic screening of immunostained slides21–23 may help to assess occult metastases more rapidly. Dave Hoon, DSc (John Wayne Cancer Institute, Santa Monica, CA), discussed the use of reverse transcriptase–PCR–based techniques for detecting occult metastases. These assays are applicable to not only lymph nodes and bone marrow, but also to body tissues such as peripheral blood and CSF. The advantages of PCR include high sensitivity and the ability to perform qualitative and quantitative analysis of multiple markers rapidly.24 Factors that influence specificity include both the selection of and the techniques employed for specimen preparation. To preserve mRNA and to prevent sample contamination, procedures for tissue collection, fixation, and processing must be carefully designed and rigorously controlled. Strategies to improve specificity include the use of primers to detect tissue-specific mRNAs (for example, tyrosinase in melanoma cells), and the use of multiple markers. Large studies are underway to evaluate the clinical utility of PCR-based assays for detection of disseminated tumor cells in sentinel nodes in breast, lung, melanoma, and colon cancer patients. PCR can also reveal the presence of gene mutations, amplifications, and deletions as well as aberrant patterns of gene expression that may have clinical significance. Thomas Moss, MD (IMPATH Laboratories, Los Angeles, CA), emphasized the need to develop more sensitive assay techniques. He reported finding a median of one to five tumor cells per one million bone marrow cells and one tumor cell per three to six million cells in peripheral blood on the basis of the analysis of more than 5,000 specimens from breast cancer patients at IMPATH (a large reference laboratory). A practical method to detect disseminated tumor cells in the peripheral blood would offer advantages, such as the ability to evaluate serial samples to monitor the effectiveness of therapy. Such a method will probably require a strategy for the physical enrichment of tumor cells in a specimen. Currently available strategies for the physical separation of tumor cells from background rely on either density gradients25,26 or the application of magnetic labels.27–29 Jeffrey Chalmers, PhD (Ohio State University, Columbus, OH), reviewed the overall problem of tumor cell enrichment and the specific capabilities of procedures for cell separation on the basis of magnets. Strategies for separation of disseminated tumor cells from peripheral blood usually combine methods for both negative and positive selection of tumor cells, often with heavy reliance on a positive method such as binding to an antibody-coated iron particle. When the performance of alternative methods is compared, several measures of performance must be considered, including purity, recovery, and enrichment. The commercially available systems for tumor cell enrichment via magnets are all batch processes for the binary separation of cells bound to particles of unbound cells. Major differences among the systems include the size of the magnetic particle (bead, colloid, or fluid), as well as the specific ligand employed. The development of flow-through separation systems offers potential advantages,30 especially in situations in which the expression of tumor-specific ligands may not be uniform on all actual target cells.
Research Priorities Panel members first emphasized the primitive state of our understanding of the biologic nature of the putative tumor cells detected by IHC and PCR. In breast and lung cancer patients morphology remains an important criterion for identification6,19 because no molecular markers have yet been consistently associated with most epithelial cancers. Conversely, some immunostained cells present in bone marrow of cancer patients who developed overt metastatic relapse lack the typical morphologic signs of a tumor cell but harbor clear molecular tumor cell characteristics.3 Morphologic analysis alone might therefore not be sufficient to identify isolated tumor cells on cytologic preparations, in particular in view of the fact that most of the samples harbor only one to three stained cells per 2 x 106 bone marrow cells analyzed.9,15 There is also disagreement about the prevalence of benign epithelial cells in the lymph nodes and bone marrow of individuals without cancer that might give rise to false-positive readings. In the largest published cohorts of control patients with nonmalignant conditions, cytokeratin-positive epithelial cells were found in two of 191 and six of 215 bone marrow samples using broad-spectrum and monospecific anticytokeratin antibodies, respectively.9 Whether these are benign cells or malignant cells derived from an unknown primary tumor remains unclear. The characterization of disseminated cytokeratin-positive cells in bone marrow by double labeling and molecular analyses has already provided important information on the heterogeneous phenotype and genotype of disseminated cancer cells.2,3,5 This information needs to be expanded and used to estimate the malignant potential of these cells, and to predict the patient’s prognosis and response to adjuvant therapy. However, panel members and workshop participants also emphasized that even with present techniques, disseminated tumor cells are strongly associated with outcomes in breast cancer and melanoma patients. In the Sunbelt Melanoma Trial, application of IHC and PCR assays for multiple markers to sentinel nodes has identified a subset of patients in which surgery alone is curative in more than 95% of patients, despite the tumor thickness of the primary melanoma. In breast cancer, five large recent studies show a significant correlation between the presence of disseminated tumor cells in bone marrow and unfavorable clinical outcome. Several participants argued strongly that the evidence warrants a clinical trial to test whether different courses of therapy can be prescribed for node-negative (or sentinel node–negative) breast cancer patients in risk categories defined by ultrastaging of both lymph nodes and bone marrow with IHC and PCR. A second panel discussion was convened to discuss the technical requirements for assays to be employed in clinical trials. This discussion was chaired by Dr Cote and the participants were Ronald Ghossein, MD (Memorial Sloan-Kettering Cancer Center, New York, NY), Karen Kaul, MD (Northwestern University, Evanston IL), and Dr Moss of IMPATH, Inc. With the focus on clinical trials that may be organized in the near future, the discussion centered on the methods used to detect occult metastases; in particular, IHC and PCR. Clinical trials that make use of occult metastasis detection for management decisions will require the enrollment of several thousand patients and will require robust, reproducible assays. The panelists emphasized that every aspect of specimen collection, sample processing, and the assay procedure itself has an influence on the final result and must be carefully specified. These aspects include details such as the choice of an anticoagulant and the number of cells to be fed into an enrichment procedure. The use of preservatives to stabilize RNA in tissue for PCR assays usually is not compatible with other assays that call for enrichment of tumor cells or preservation of cell morphology. Several participants noted the lack of a widely accepted model system that can be used for assay optimization and for comparing the technical capabilities of different assay platforms. The development of such model systems, which probably will be based on cell lines, would be useful. There is also need for understanding quality control standards. Before an assay is adopted for use in a clinical trial, as opposed to a preliminary study, the clinical sensitivity and specificity must be defined. This requires careful attention to the definition of normal, for which matched populations should be used. To avoid false-positive findings caused by the high sensitivity of the applied assays, specimens from non–cancer patients should be employed as negative controls. For large-scale testing, even within a single institution, proficiency testing is required. To enhance standardization, procedures should be automated as much as possible. Assays require internal quality controls, especially internal positive controls. To achieve these goals, assay procedures must be built in to clinical trial designs from the beginning.
First, multiple studies show that various solid tumors considered to be early stage and localized by standard clinical evaluation, imaging studies, and pathologic criteria are associated with disseminated tumor cells, either in lymph nodes, bone marrow, or peripheral blood. The presence of disseminated tumor cells seems to be associated with a clinically significant effect on survival. It is now time to perform definitive clinical studies that will establish the prognostic significance of occult metastases. Second, the biologic characteristics of tumor cells in lymph nodes, bone marrow, or peripheral blood that lead to clinically overt systemic disease are as yet undefined, and are an important topic for future research. Such research will likely define new prognostic markers and therapeutic targets. Additional disease-specific markers must be developed. Third, in some studies (primarily of breast cancer) the finding of disseminated tumor cells in lymph nodes is not correlated with the finding of disseminated tumor cells in other body compartments (eg, bone marrow and peripheral blood). There is growing evidence that these events may have distinct biologic and clinical implications. These biologic differences need to be studied at the genomic and protein levels and in relationship to clinical outcomes. Fourth, monitoring the number and phenotype of disseminated tumor cells during adjuvant therapy might provide important information on the efficacy of a particular treatment protocol in an individual patient, and a direct comparison of blood and bone marrow might lead to better understanding of the dynamics of tumor cell spread. These possibilities need to be explored in future clinical trials. Fifth, most studies of disseminated tumor cells have been performed using IHC, but even with this technique there are considerable variations in reported studies. PCR techniques may be more sensitive but less specific than IHC. There are important unresolved issues with respect to standardization of sampling, specimen handling, cell enrichment, and separation. Standardization, automation, and internal controls are needed to make these assays clinically useful. Quality control issues, especially for PCR, require more attention. Consensus should be sought regarding acceptable technical performance criteria (such as false-negative and false-positive rates) for clinical assays, and model systems are needed to permit comparisons between assay platforms. Sixth, the statistical design of some studies correlating clinical outcome with findings of disseminated tumor cells frequently has been inadequate because of poorly defined criteria for eligibility, variability in techniques for specimen acquisition and detection of disseminated tumor cells, unclear end points, and inadequate sample sizes. Prospective study designs with clear, focused questions and high statistical power are required for future definitive trials. Finally, ongoing multicenter studies of disseminated tumor cells will potentially allow stratification of patients by prognostic groups and in selected situations. A few studies are already using the detection of disseminated tumor cells by various means to select patients for treatment. Additional clinical trials should be possible within the next few years. In summary, the existing evidence warrants the consideration of new clinical trials designed to provide definitive information about the prevalence of disseminated tumor cells, the relationship to prognosis, and the clinical utility of these findings in patients with solid tumors. The selection of laboratory assays to detect disseminated tumor cells will be a critical component of the trial design. Trials that evaluate disseminated tumor cells should maximize acquisition and careful archiving of tissue from the largest number of sites (eg, lymph nodes, bone marrow, blood, and primary tumor). Determination of prognosis is clearly a fundamental issue in cancer management. These trials can also incorporate therapeutic monitoring through the evaluation of levels of disseminated tumor cells. The application of occult metastasis detection has the potential of helping us to achieve the goal of patient-specific cancer management.
The views expressed herein do not necessarily represent the views or constitute an endorsement by the National Institutes of Health or the Department of Health and Human Services.
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Copyright © 2003 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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INTRODUCTION
WORKSHOP SUMMARY
