Is Immunohistochemical Analysis an Appropriate Diagnostic Technique for Bone Marrow Micrometastases?
Authors: K. C. Redmond, J. H. Wang, K. K. Austin, N. Relihan, G. McGreal, W. O. Kirwan, D. Richardson, J. Kelly, H. P. Redmond, Stephan Braun, Nadia Harbeck, Klaus Pantel
Journal: Journal of Clinical Oncology (2001)

Article Tools SPECIAL DEPARTMENTS Article Tools OPTIONS & TOOLS Export Citation Track Citation Add To Favorites Rights & Permissions COMPANION ARTICLES No companion articles ARTICLE CITATION DOI: 10.1200/JCO.2001.19.15.3589 Journal of Clinical Oncology - published online before print September 21, 2016 PMID: 11481372 Is Immunohistochemical Analysis an Appropriate Diagnostic Technique for Bone Marrow Micrometastases? K. C. RedmondxK. C. RedmondSearch for articles by this author , J. H. WangxJ. H. WangSearch for articles by this author , K. K. AustinxK. K. AustinSearch for articles by this author , N. RelihanxN. RelihanSearch for articles by this author , G. McGrealxG. McGrealSearch for articles by this author , W. O. KirwanxW. O. KirwanSearch for articles by this author , D. RichardsonxD. RichardsonSearch for articles by this author , J. KellyxJ. KellySearch for articles by this author , H. P. RedmondxH. P. RedmondSearch for articles by this author Stephan BraunxStephan BraunSearch for articles by this author , Nadia HarbeckxNadia HarbeckSearch for articles by this author , Klaus PantelxKlaus PantelSearch for articles by this author Show More Cork University, South Infirmary Hospitals, Cork, IrelandTechnical University, Munich, GermanyUniversity Hospital Eppendorf, Hamburg, Germany https://doi.org/10.1200/JCO.2001.19.15.3589 First Page Full Text PDF Figures and Tables © 2001 by American Society of Clinical OncologyjcoJ Clin OncolJournal of Clinical OncologyJCO0732-183X1527-7755American Society of Clinical OncologyResponse01082001In Reply:Established prognostic markers do not enable identification of patients with occult tumor cell dissemination among primary breast cancer patients who have been treated by complete tumor resection (stage R0). Even among so-called low-risk patients, such as node-negative breast cancer patients, 30% or even more will experience recurrence of incurable metastatic disease within 5 years after diagnosis. This present diagnostic dilemma motivated clinical evaluation of several approaches to identify patients with early tumor-cell dissemination, including immunocytochemical screening for occult metastatic cells in bone marrow and extended pathologic examination of lymph nodes using immunochemistry.In a passionate response to our study,1 Redmond et al raised several questions about methodologic details that could confer problems in interpreting immunocytochemical and immunohistochemical data. We agree with Redmond et al that immunocytochemistry and immunohistochemistry are time consuming and thus limited to analysis of a certain amount of cells, unless automated imaging analysis is used as it is now available.2 We further agree that any diagnostic approach that intends to draw clinical conclusions from the finding of as little as a single tumor cell against the background of 2 × 106 autochthonous bone marrow cells requires extreme precautions and care with regard to sensitivity, specificity, reliability, and validity as well as interpretation. Over the past 10 years, our careful investigations of these confounding variables showed that the antibody A45-B/B3 directed against the cytokeratin (CK) heterodimers CK8/18 and CK8/19 as well as a common CK epitope is a specific and sensitive tool to identify such micrometastatic tumor cell spread.3,4 In view of the well-known clinical evidence that approximately one third of node-negative breast cancer patients relapse at distant sites within 5 years after diagnosis, we believe that a micrometastasis prevalence of 29% in our series cannot be called too “low.” The outlined immunoassay yielded a similar prevalence of tumor cells in pre- and postoperative bone marrow samples, thus indicating a high reproducibility of results.4 All studies published by our co-workers were based on data resulting from the use of an appropriate assay system, which included appropriate positive and negative control stainings, as stated explicitly in each published report so far.Redmond et al further questioned whether analysis of 2 × 106 bone marrow cells per patient as well as of few (in our series, four) adjacent lymph node sections is sufficient to eliminate concerns about a sampling error. Of course, our approach still carries the danger of a certain sampling error. Yet, there are convincing arguments in favor of the clinical relevance of our analytical approach, since the analysis of 2 × 106 bone marrow cells is obviously enough to provide clinically relevant data on prognosis of early-stage breast cancer patients.1,3 Furthermore, clinical routine diagnosis with its limited capacities also limits itself to analyzing only two histologic sections per axillary lymph node.Even though the sensitivity of reverse transcriptase polymerase chain reaction (RT-PCR) techniques for detection of single tumor cells is thought to considerably surpass that of immunocytochemistry, the RT-PCR sensitivities reported so far have been unexpectedly low due to inhomogeneity or even lack of expression of the respective tumor-associated mRNA used.5 In addition, epithelial tissue–specific mRNAs, such as CK 18, CK 19, and epithelial cell adhesion molecule, as well as tumor-associated mRNAs, such as HER2, carcinoembryonic antigen, and MUC-1, were found to be expressed in mesenchymal tissues, such as blood, bone marrow, and lymph nodes.6Given observer expertise, which is an obvious prerequisite for every cytologic approach that is intended for clinical use, only a few staining results remain ultimately undeterminable,3,4 as is the case for every analytical approach that involves the human eye and expertise for evaluation, including Papanicolaou smears of the cervix or histopathologic grading.The robustness of the immunocytochemical approach to identifying hematogenous tumor cell spread is endorsed by the unanimous prognostic relevance shown by those recent studies that used validated immunoassays with the anti-CK antibodies. Because the overall number of patients analyzed for occult metastatic cells is still relatively small, a formal meta-analysis is not possible at present. Thus, we acknowledge the need for a multicenter prospective trial in order to obtain level I evidence for the prognostic value. (At present, there are at least three studies underway in the United States and Europe to undertake this task. Because of the limited data available so far, we added a caveat as to overinterpretation of our data in all of our reports.)Another concern raised by Redmond et al is the prognostic impact of cells that are detected but nonviable as opposed to those viable cells that are indeed capable of initiating metastases. This concern involves questions about tumor-biologic features, such as phenotype and genotype of CK-positive cells. Phenotyping and genotyping of single tumor cells at the single-cell level is not trivial from a technical point of view. So far, only limited—but nevertheless quite intriguing—data are available. CK-positive cells in bone marrow carry characteristics exclusively known for tumor cells, such as HER2 amplification and protein overexpression,7,8 as well as genomic changes that are identical to those of the respective primary tumor.9 We recently showed for the first time that HER2 expression on bone marrow micrometastases is associated with poor patient prognosis in breast cancer, independent of established prognostic factors.7 Hosch et al10 were able to demonstrate the tumorigenicity and metastatic potential of a cell line established from an immunohistochemically positive lymph node in severe combined immunodeficiency mice.In our opinion, it is not the limited information on biologic characteristics of bone marrow micrometastases that needs to be debated but the lack of specific therapeutic opportunities that exist for patients with early-stage breast cancer and micrometastatic disease. This lack of a specifically targeted therapy is true not only for the presence of bone marrow micrometastases but also for almost all other prognostic factors in primary breast cancer, except for expression of estrogen receptor and HER2 oncoprotein. In contrast to all available prognostic markers, bone marrow micrometastases can be utilized for patient monitoring during adjuvant treatment, both for conventional systemic therapies as well as for novel tumor-biologic therapies.11 Phenotyping of bone marrow micrometastases for potential therapeutic targets may even offer the unique opportunity for a more direct detection of the actual adjuvant therapy targets than analysis of primary tumor tissue. Thus, immunocytochemical analysis of bone marrow aspirates can be used to stratify patients in adjuvant therapy trials, to evaluate specifically targeted treatment strategies (eg, trastuzumab), as well as to monitor therapeutic efficacy and relieve both patients and clinicians from waiting for 5 years or more before the efficacy of adjuvant therapy can be determined.In view of the evidence discussed above, it is our conviction that immunocytochemical identification of bone marrow micrometastases offers promising opportunities for the future management of patients with breast cancer and other solid tumors.1. Braun S, Cevatli BS, Assemi C, et al: Comparative analysis of micrometastasis to the bone marrow and lymph nodes of node-negative breast cancer patients receiving no adjuvant therapy. J Clin Oncol 19:: 1468,2001-1475, Link, Google Scholar2. Bauer KD, de la Torre-Bueno J, Diel IJ, et al: Reliable and sensitive analysis of occult bone marrow metastases using automated cellular imaging. Clin Cancer Res 6:: 3552,2000-3559, Medline, Google Scholar3. Braun S, Pantel K, Müller P, et al: Cytokeratin-positive cells in the bone marrow and survival of patients with stage I, II, or III breast cancer. N Engl J Med 342:: 525,2000-533, Crossref, Medline, Google Scholar4. Pantel K, Schlimok G, Angstwurm M, et al: Methodological analysis of immunocytochemical screening for disseminated epithelial tumor cells in bone marrow. J Hematother 3:: 165,1994-173, Crossref, Medline, Google Scholar5. Mapara MY, Körner IJ, Hildebrandt M, et al: Monitoring of tumor cell purging after highly efficient immunomagnetic selection of CD34 cells from leukapheresis products in breast cancer patients: Comparison of immunocytochemical tumor cell staining and reverse transcriptase-polymerase chain reaction. Blood 89:: 337,1997-344, Crossref, Medline, Google Scholar6. Zippelius A, Kufer P, Honold G, et al: Limitations of reverse transcriptase-polymerase chain reaction for detection of micrometastatic epithelial cancer cells in bone marrow. J Clin Oncol 15:: 2701,1997-2708, Link, Google Scholar7. Braun S, Jeumos I, Schlimok G, et al: ErbB2 over-expression on occult metastatic cells in bone marrow predicts poor clinical outcome of stage I-III breast cancer patients. Cancer Res 61:: 1890,2001-1895, Medline, Google Scholar8. Pantel K, Schlimok G, Braun S, et al: Differential expression of proliferation-associated molecules in individual micrometastatic carcinoma cells. J Natl Cancer Inst 85:: 1419,1993-1424, Crossref, Medline, Google Scholar9. Klein CA, Schmidt-Kittler O, Schardt JA, et al: Comparative genomic hybridization, loss of heterozygosity, and DNA sequence analysis of single cells. Proc Natl Acad Sci U S A 96:: 4494,1999-4499, Crossref, Medline, Google Scholar10. Hosch S, Kraus J, Scheunemann P, et al: Malignant potential and cytogenetic characteristics of occult disseminated tumor cells in esophageal cancer. Cancer Res 60:: 6836,2000-6840, Medline, Google Scholar11. Braun S, Pantel K: Biological characteristics of micrometastatic cancer cells in bone marrow. Cancer Metastasis Rev 18:: 75,1999-90, Crossref, Medline, Google Scholar