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© 2000 American Society for Clinical Oncology
Detection of Circulating Tumor by Reverse Transcriptase Polymerase Chain Reaction
Institut Gustave Roussy Villejuif Cedex, France To the Editor:I read with interest the article by Sabbatini et al1 in which they reported the detection of circulating carcinoma cells in peripheral blood from patients with nonmetastatic breast cancer by maspin reverse transcriptase polymerase chain reaction (RT-PCR), suggesting that tumor cells are mobilized into the circulation after adjuvant chemotherapy. Results and the conclusion must be interpreted with caution. RT-PCR–based tumor cell detection is an attractive technique because it could be more easily automated than immunocytochemical tumor cell detection. However, the specificity and sensitivity of the analysis can be criticized on several points. First, sensitivity of maspin RT-PCR was determined on serial dilutions of the MCF7 breast cancer cell line. Sensitivity should be tested on several cell lines, because carcinoma cell lines present high heterogeneity in gene expression, and more particularly, maspin gene expression is known to be downregulated in many breast cancer cell lines. Second, a sensitivity of 10 MCF7 cells in 106 peripheral-blood mononuclear cells (PBMCs) was obtained by maspin RT-PCR.2 Immunocytochemical tumor cell detection can reach a much higher sensitivity depending on the amount of cells analyzed, and sensitivity obtained in vitro often overestimates in vivo sensitivity.3 Tumor cell detection techniques should be able to detect less than 10 tumor cells in 106 PBMCs, as the concentration of circulating tumor cells is often lower. Third, RT-PCR was claimed to be highly specific because no maspin transcript could be detected in 17 PBMC and four bone marrow samples from healthy subjects. The very high amplification rate of maspin RT-PCR (two runs of 35 cycles) could amplify minute amounts of maspin transcription. Expression of maspin seems not to be restricted to the breast, as maspin expression has been found in several epithelial tissues and even in lymph nodes from patients with no carcinoma.4 Furthermore, it has been demonstrated that the expression of epithelial-related genes encoding cytokeratin 19 and carcinoembryonic antigen can be induced in PBMC by cytokines and growth factors.5,6 Cytokines and growth factors are found at high concentrations during neutropenia or inflammatory conditions. The evaluation of maspin RT-PCR specificity should include PBMCs from patients receiving chemotherapy for nonepithelial neoplasms and from patients with inflammatory diseases to rule out any induction of maspin transcription. Immunocytochemical tumor cell detection should be performed in parallel to validate the RT-PCR technique. Specificity of high-quality immunocytochemistry is warranted by morphologic recognition of circulating carcinoma cells. A recruitment of tumor cells into peripheral blood cannot be confirmed by maspin RT-PCR alone. Fourth, the authors refer to the study of Brugger et al,7 who reported, using immunocytochemistry, an early increase in the number of tumor cells in peripheral blood after chemotherapy and hematopoietic growth factor support, followed by a second 2-log higher release of tumor cells in patients with bone marrow involvement, coinciding with hematologic recovery. We do not think that the data presented by Sabbatini et al1 confirm the conclusion suggested by Brugger et al.7 REFERENCES
1.
Sabbatini R, Federico M, Morselli M, et al: Detection of circulating tumor cells by reverse transcriptase polymerase chain reaction of maspin in patients with breast cancer undergoing conventional-dose chemotherapy. J Clin Oncol 18: 1914-1920, 2000
2.
Luppi M, Morselli M, Bandieri E, et al: Sensitive detection of circulating breast cancer cells by reverse-transcriptase polymerase chain reaction of maspin gene. Ann Oncol 7: 619-624, 1996 3. Ko Y, Klinz M, Totzke G, et al: Limitations of the reverse transcription-polymerase chain reaction method for the detection of carcinoembryonic antigen-positive tumor cells in peripheral blood. Clin Cancer Res 4: 2141-2146, 1998[Abstract] 4. Merrie AE, Yun K, Gunn J, et al: Analysis of potential markers for detection of submicroscopic lymph node metastases in breast cancer. Br J Cancer 80: 2019-2024, 1999[Medline] 5. Jung R, Krüger W, Hosch S, et al: Specificity of reverse transcriptase polymerase chain reaction assays designed for the detection of circulating cancer cells is influenced by cytokines in vivo and in vitro. Br J Cancer 78: 1194-1198, 1998[Medline] 6. Goeminne JC, Guillaume T, Salmon M, et al: Unreliability of carcinoembryonic antigen (CEA) reverse transcriptase-polymerase chain reaction (RT-PCR) in detecting contaminating breast cancer cells in peripheral blood stem cells due to induction of CEA by growth factors. Bone Marrow Transplant 24: 769-775, 1999[Medline]
7.
Brugger W, Bross KJ, Glatt M, et al: Mobilization of tumor cells and hematopoietic progenitor cells into peripheral blood of patients with solid tumors. Blood 83: 636-640, 1994
ResponseUniversita di Modena Policlinico Modena, Italy In Reply:We appreciate the interest of Drs Goeminne and Guillaume in our recent work,1 although we disagree with most of their criticisms and find it necessary to address all of the issues raised. However, most of these discussions should be, in our opinion, overcome by well-planned and carefully executed quantitative molecular studies. First, we would like to emphasize that we chose to validate the sensitivity of our assay in the MCF7 mammary carcinoma cell line, because this was one of the cell lines with the lowest level of maspin transcription, as reported by Zou et al.2 Downregulation of maspin transcription was so marked in breast carcinoma cell lines, as detected by Northern blot analysis, to allow Zou et al to speculate about possible biologic roles of maspin in breast cancer. More sensitive techniques, such as reverse transcriptase polymerase chain reaction (RT-PCR), have shown that detectable levels of maspin transcript may be documented in breast cancer cell lines in vitro and breast cancer tissues in vivo.3-5 In addition, Lòpez-Guerrero et al6 have confirmed the expression of maspin in the T47D breast cancer cell line, with sensitivity rates comparable to ours in the MCF7 cell line. Second, several reports have emphasized that immunocytochemistry can detect as few as one mammary tumor cell/5 x 105 hematopoietic cells,7,8 so that the finding of a "much higher sensitivity" of immunocytochemical detection of tumor cells compared with RT-PCR is not a rule. Moreover, the risk of overestimation is higher for immunocytochemical than for molecular methods. Finally, it is very difficult to establish the clinical relevance of an extreme sensitivity in tumor cell detection. Third, we propose that RT-PCR for maspin is specific in terms of the detection of "circulating potentially neoplastic mammary cells," which means that maspin is not detectable in healthy subjects. The absence of maspin transcription has been documented in the peripheral blood, bone marrow, and apheresis products from 132 control subjects without breast cancer examined by two other independent groups using our experimental conditions.4,5 We did not propose the RT-PCR for maspin as a marker of tumor cells in tissues other than bone marrow and blood, and, unfortunately, recent data of maspin expression in normal lymph nodes seem to prevent the application of this assay to the search of mammary cells in these sites. It is rather expected that cytokines and growth factors may influence the transcription of maspin, as it occurs for several other epithelial genes. However, Vannucchi et al5 recently showed that maspin transcript was undetectable in the purified CD34+ cell fractions collected from 14 patients with lymphoma and myeloma, suggesting that false-positive results with RT-PCR assay for maspin are a rather uncommon finding, even in patients receiving growth factors and chemotherapy to induce CD34+ cell mobilization. Furthermore, it must be noted that none of our patients received growth factors. In our opinion, the necessary step to validate the clinical use of RT-PCR assay for maspin as well as for other epithelial genes is to perform quantitative RT-PCR tests, which can discriminate between a low level of gene expression that may be detected in non–tumor-related conditions (inflammatory conditions, certain therapies, and so on) and a higher, clinically significant level of gene expression, which may be consistent with the presence of possibly neoplastic cells in a given tissue. Recent articles in the Journal of Clinical Oncology already highlighted the importance of quantitative polymerase chain reaction (PCR) for the detection of micrometastasis in patients with breast cancer, using cytokeratin 19 as the epithelial marker.9,10 We agree with Goeminne and Guillaume that tumor cell detection should be performed in parallel, because it is rather obvious that specificity and sensitivity could be increased by using a combination of RT-PCR assay and immunocytochemistry. It should be noted that in our article, we ourselves introduced notes of caution, emphasizing that "our data raise the intriguing possibility that there is a recruitment of BC [breast cancer] cells into the peripheral blood after the first courses of chemotherapy." It is obvious that immunocytochemistry and, more importantly, clonogenic assays are necessary to unequivocally prove that the maspin-positive cells are neoplastic cells. Regarding the fourth point raised by Goeminne and Guillaume, we never stated that our data confirm those by Brugger et al11 in terms of the kinetic appearance of tumor cells in the peripheral blood. Using a molecular instead of an immunocytochemical approach, we simply provided further evidence in favor of a possible recruitment with chemotherapy of maspin-positive mammary cells, potentially neoplastic, in the peripheral blood of breast cancer patients. Regarding the final remark, we thank Goeminne and Guillaume for discovering the mistake that we made, in the original manuscript submitted to the Editor, in indicating the sensitivity of the RT-PCR assay, although the wrong number was immediately followed by the right reference. We confirm that the sensitivity of the assay is the same as that reported in the original article by Luppi et al.3 The sensitivity of the nested PCR followed by ethidium bromide visualization is, in our hands, not significantly higher than the hybridization of the promoter of the one-step PCR. REFERENCES 1. Sabbatini R, Federico M, Morselli M, et al: Detection of circulating tumor cells by reverse transcriptase polymerase chain reaction of maspin in patients with breast cancer undergoing conventional-dose chemotherapy. J Clin Oncol 18: 1914-1920, 2000
2.
Zou Z, Anisowicz A, Hendrix MJC, et al: Maspin, a serpin with tumor-suppressing activity in human mammary epithelial cells. Science 263: 526-529, 1994 3. Luppi M, Morselli M, Bandieri E, et al: Sensitive detection of circulating breast cancer cells by reverse-transcriptase polymerase chain reaction of maspin gene. Ann Oncol 7: 619-624, 1996 4. Corradini P, Cinque F, Astolfi M, et al: Detection of minimal residual disease in patients with breast cancer: A novel nested-PCR assay using maspin gene. Blood 88:246a, 1996 (abstr) (suppl) 5. Vannucchi AM, Glinz S, Bosi A, et al: Detection of contaminating tumor cells by RT-PCR in breast cancer patients. Bone Marrow Transplant 24: 228-229, 1999[Medline] 6. Lòpez-Guerrero JA, Gilabert BP, Gonzàlez EB, et al: Use of reverse-transcriptase polymerase chain reaction (RT-PCR) for carcinoembryonic antigen, cytokeratin 19, and maspin in the detection of tumor cells in leukapheresis products from patients with breast cancer: Comparison with immunocytochemistry. J Hematother 8: 53-61, 1999[Medline]
7.
Passos-Coelho J, Ross AA, Moss TJ, et al: Absence of breast cancer cells in a single-day peripheral blood progenitor cell collection after priming with cyclophosphamide and granulocyte-macrophage colony-stimulating factor. Blood 85: 1138-1143, 1995 8. Bills AC, Green WF, Tsodikov A, et al: Cytokeratin-positive cells in PBSC collections from normal donors and patients with non-epithelial cell-derived tumors. Cytotherapy 1: 389-399, 1999
9.
Slade MJ, Smith BM, Sinnett HD, et al: Quantitative polymerase chain reaction for the detection of micrometastases in patients with breast cancer. J Clin Oncol 17: 870-879, 1999
10.
Smith BM, Slade MJ, English J, et al: Response of circulating tumor cells to systemic therapy in patients with metastatic breast cancer: Comparison of quantitative polymerase chain reaction and immunocytochemical techniques. J Clin Oncol 18: 1432-1439, 2000 11. Brugger W, Bross KJ, Glatt M, et al: Mobilization of tumor cells and hematopoietic progenitor cells into peripheral blood of patients with solid tumors. Blood 83: 636-640, 1994 Remark: Sensitivity of maspin RT-PCR as published by Luppi et al2 was "10 mammary carcinoma cells in 1 million normal peripheral-blood mononuclear cells" and not "one positive [MCF7] cell in 106 negative [PBMC] cells," as mentioned by Sabbatini.1 Sensitivity was evaluated after hybridization of the first amplification product (35 cycles), whereas the patient samples were submitted to a nested RT-PCR (35 + 35 cycles) without hybridization (as shown in Fig 2 of the article). The sensitivity of this test is not mentioned.
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Copyright © 2000 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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