- Email: [email protected]
Pitfalls in the detection of disseminated non-hematological tumor cells
Annals of Oncology 11: 785-792, 2000. © 2000 Kluwer Academic Publishers. Printed in the Netherlands.
Review Pitfalls in the detection of disseminated non-hematological tumor cells J.-C. Goeminne, T. Guillaume & M. Symann Laboratory of Experimental Oncology and Hematology, Universite Catholique de Louvain, Brussels, Belgium
Summary
There is not yet a consensus on the reliability of the methods that should be used for the detection of rare disseminated tumor cells from non-hematological malignancies. In this review, we will discuss the advantage and drawbacks of the classical approach of immunocytochemistry and the molecular detection by reverse transcriptase polymerase chain reaction
Introduction
Despite the clinical relevance and various methods of detection of solid tumor cells in hematological tissues [1-3], there not yet exists a consensus on the reliability of the methods that should be used for tumor-cell detection. In this review we will discuss the advantages and drawbacks of the classical approach of immunocytochemistry (ICC) as well molecular detection by RT-PCR. The presence of very limited tumor cells in bone marrow or lymph nodes is generally termed micrometastatic disease, although the definition of micrometastasis may vary. It may designate isolated tumor cells or a tumor-cell cluster in the absence of clinical or radiological metastatic lesions [4], or clusters smaller than, 2 mm [5] or 0.5 mm [6]. Detection of rare disseminated tumor cells predict an adverse clinical outcome in most studies [6-8]. However, due to large differences in methodology and discrepancies in findings among various studies, the true clinical correlates still have to be substantiated before it could be used to guide therapeutic decisions, like adjuvant therapy or purging of stem-cell graft products. It is difficult to determine if tumor-cell detection (TCD) is an independent prognostic factor at the time of diagnosis of the primary tumor [9], since TCD is usually related to other factors of poor prognosis, like tumor size [10], lymph node involvement [11], vascular invasion [10,11] and the tumor differentiation grade [7]. In the absence of other factors of poor prognosis, TCD in bone marrow has been found to predict a decreased overall survival and disease free survival in breast cancer [7, 8], gastric [12], colorectal [13], nonsmall-cell lung [14] and prostatic cancer [15], signing an early systemic dissemination of malignancy. Presence of tumor cells in stem-cell graft products is
(RT-PCR). The interpretation of the biological significance of circulating tumor cells and the pitfalls of the detection techniques are the main causes of discrepancy between the conclusions of different tumor-cell detection (TCD) studies. Key words: immunocytochemistry,
RT-PCR, tumor-cell
detection
related to persistence of disease at the time of stem-cell collection and with the exception of neuroblastoma it remains unclear whether the reinjection of contaminating tumor cells contributes to tumor relapse. After and during medical treatment, TCD can be used for monitoring of therapeutic efficiency, persistent minimal residual disease or overt relapse. The discrepancy between the conclusions of different tumor-cell detection (TCD) studies is principally due to the misunderstanding of the biological significance of circulating tumor cells and the pitfalls of the detection techniques. Biological significance of circulating cells TCD does not necessarily reflect metastatic potential. A malignant tumor is continuously shedding tumor cells [16]. This shedding is related to tumor angiogenesis [17, 18], vascular invasion [19], tumor size and proliferation rate [18]. Most of shedded tumor cells are eliminated in the first capillary bed, being the lymph nodes, the lungs and the liver [20] and only a minority will be released progressively and arrested in a secondary capillary bed, the bone marrow being the most easily assessable [21, 22]. Isolated tumor cells found in lymph nodes, blood or bone marrow are the result of this continuous shedding from the primary tumor and most of them will never evolve to macroscopic metastases. This is suggested by finding that tumor cells are detected in the bone marrow of patients with gastrointestinal malignancies [12, 13] at the same frequency as in patients with breast cancer although bone metastases are rarely seen in the former. Furthermore, tumor cells are found in bone marrow from patients who never will relapse and patients can
786 survive several months with a peritoneo-venous shunt for malignant ascites without developing distant metastases, although high amounts of tumor cells are continuously shedded into the venous circulation [23]. Disseminated malignant cells in the bone marrow can remain dormant for many years and escape to the cytotoxic effects of chemotherapy [24]. Consistent with this concept of tumor-cell dormancy, the expression of proliferative markers was found to be low in the tumor cells detected in the bone marrow [25]. Metastases will develop only with the appearance of 'metastatically competent' tumor cell clones, able to survive and proliferate distant from the primary tumor [26]. Reduced expression by tumor cells of HLA I molecules [27], proteins involved in the immune response, tumor-associated proteases [28] may help to identify the capacity of outgrowth of disseminated tumor cells. Tumor-cell detection techniques Immunocytochemistry (ICC) and the reverse transcriptase polymerase chain reaction (RT-PCR) are the two most extensively used techniques of TCD since both methods have been proven much more sensitive than standard microscopic analysis. The sensitivity of detection of both techniques varies between 1 tumor cell in 105 and 107 mononuclear hematological cells. Other techniques such as clonogenic assays or flow cytometry appear less suitable for large scale TCD studies. Common pitfalls of ICC and R T-PCR The random disposition of tumor cells Because tumor cells are not necessarily distributed uniformly in lymph nodes and bone marrow, a single sample or a small volume sample may not reflect the extent of tumor spread. It has been demonstrated that bone marrow taken at several sites rather than only a single one [29] improves sensitivity. Studies have been performed where samples were taken at six [7] or eight different sites [11]. Likewise, the amount of tumor cells in peripheral blood fluctuates. Serial blood samples may therefore give different results [30, 31]. Lack of quantification of tumor burden Most studies on TCD report whether a sample does contain tumor cells or not, without quantifying detected the tumor burden. Quantification of tumor burden could however have an important clinical value: The actual amount of tumor cells detected in bone marrow from patients with breast cancer [32], gastric cancer [12], small-cell lung cancer [33] and prostate cancer [34] seems to have a prognostic significance. In monitoring disease progression during treatment [35] or assessing the efficiency of stem-cell product purging, the detected tumor cell burden should be effectively quantified. The kinetics of circulating cell burden is not well known and needs more quantitative data.
Tumor-cell heterogeneity The principle of TCD by both, ICC and RT-PCR, relies on the recognition of particular antigens or gene transcripts that distinguish tumor cells from surrounding cells, principally hematological cells. The ideal tumorcell marker expressed by all tumor cells and not by other cells has not yet been found. There is great variability in antigenic expression between different cell lines derived from the same cancer histologic type [31, 35] and sometimes even between different cell clones from the very same tumor [36, 37]. Expression of tumor-related antigens on metastatic lesions can be very weak due to downregulation of antigens which are strongly expressed in the primary tumor or in the originating tissue. This has been observed for melanoma markers [38], for cytokeratins [29] and for PSA [39]. In the same fashion, antigen expression can be influenced by treatment. PSA [40], tyrosinase [41] and mucins [42] are all modulated by the prevailing hormonal environment. Immunocytochemistry (ICC) Tumor or tissue specific antigens are targeted by specific antibodies before microscopical analysis. Advantages The specificity of TCD by ICC is greatly improved by simultaneous morphological analysis of the positivestained cells, recognizing the characteristic malignant features characterized by nucleo-cytoplasmic atypia or cell clustering, although some investigators claim to have high specificity without morphological confrontation [8, 29]. Further characterization of immunocytochemical detected tumor cells can be performed by additional ICC staining [25] or by FISH (fluorescent in situ hybridization) for genetic mutations [43]. This can help to confirm malignant nature and to characterize the metastatic features of circulating cells [26]. Further quantification of the detected tumor burden is easily affordable by straightforward microscopic analysis. Problems related with ICC ICC is very labor intensive. In order to obtain high sensitivity a huge cellular volume must be analyzed microscopically. Cellular loss is generally underestimated and can be substantial during the various steps that precede microscopic examination. This includes recovery of mononuclear cells on density gradient, red blood cell lysis, cytospin and the series of washings during immunocytochemical staining. Cellular loss is estimated to be up to 60% [44, 45]. No available antibodies are 100% tumor- or tissuespecific. Due to intra- and inter-tumoral heterogeneity, it is necessary to use a panel of different antibodies recognizing different antigens or to use antibodies that recognize rather common epitopes. The lack of suitable
787 antibodies is mainly seen for instance in neuroblastoma and melanoma. False positive staining have been reported with most extensively used antibodies. Published data on immunocytochemical TCD may as a result be greatly overestimated due to non-specific labeling [46]. There are several particular sources for non-specific labeling: - Some hematological cells, principally in bone marrow [47, 48] and lymph node reticulum cells [49, 50] can express some epithelial antigens. - Macrophages, granulocytes and some lymphocytes have a great propensity to fix antibodies via their Fc receptors in a non-specific manner. - Some plasma cells can react with the APAAP (alkaline phosphatase-anti-alkaline phosphatase) complex, an essential compound for many ICC labeling techniques [51]. - Material phagocytized into hematological cells can be stained immunocyto-chemically in a false positive manner [52, 53]. - Lymph nodes can contain non-malignant epithelial [54] and melanocytic [55] inclusions likewise giving false-positive results. - Very low numbers of epithelial cells have been found in peripheral blood and bone marrow [8] of subjects without malignancy and can be related to benign epithelial proliferative diseases [29, 52] or tissue trauma [12]. For those reasons, the extent of experience of the pathologist is crucial to avoid false positives. To optimize specificity, in some studies microscopical slides are reviewed systematically by a second observer [7, 8]. Future developments Maximization of the volume of cells to be analyzed. - Sensitivity is directly related to the number of cells analyzed. The number is limited by the labour intensity it represents. By increasing the examined cell volume, specificity will also improve, increasing the chance of finding tumor cells with marked morphological characteristics of malignancy. - The detection of tumor cells in bone marrow is directly related to their presence in peripheral blood, but the relative concentration of tumor cells found in blood is much lower than in bone marrow. When sensitivity is increased by analyzing a more substantial amount of cells, peripheral blood analysis possibly could replace bone marrow sampling, which is always a stressful event for the patient. - For quantification of the disseminated tumor burden, high volume samples will give more reliable data by lowering the variability related to the heterogeneous distribution of circulating tumor cells. Some techniques can help in maximizing the analyzed cell volume: - Tumor cells can be tracked by selective enrichment. Most enrichment techniques rely on positive
or negative immunomagnetic selection of target cells [56]. With enrichment methods, blood samples up to 40 ml can be readily analyzed [52], representing about 60 million peripheral blood mononuclear cells, an amount that can not be analyzed by classical microscopical examination. - Slides can be scanned for tumor cells by automated computer-driven image analysis [57]. Promising results are suggested by the solid phase cytometry technique [58]. - Automation of ICC facilitates quantitative analysis, decreases inter-observer variability and allows standardization. Selection of more sensitive and specific antibodies. The use
of anti-cytokeratin antibodies seems much more specific than formerly used anti-epithelial antigens, including epithelial membrane antigen, milk fat globuline, human epithelial antigen-125 [29]. Methodological comparative analysis is required to select the most specific and sensitive antibodies [29, 45, 55]. Semisynthetic antibodies directly conjugated with an enzyme [51] or a fluorochrome can significantly decrease non-specific staining. RT-PCRandPCR TCD by RT-PCR relies on the selective amplification of transcripts (mRNA) of genes that are supposed to be expressed only in tumor cells and not in cells from the surrounding tissue. Most target genes are involved in tissue differentiation (mainly epithelial genes) or in malignant transformation (Table 1). Only chimeric genetranscripts, resulting from a chromosomal translocations are truly tumor-specific. Only in some rare solid tumors common chimeric gene-transcripts have been found such as Ewing's sarcoma. Tumor-related DNA-mutations involving K-ras [59], p53 [59], erbB-2 [60] have been found by PCR in blood and lymph nodes from cancer patients. These findings do not necessarily reflect the presence of circulating tumor cells, as free DNA can be found in serum or lymph nodes [61]. RT-PCR detects the presence of gene-transcripts by amplifying RNA. RNA is very fragile and is preserved as rule only in living cells. Direct PCR on the other hand amplifies the less labile DNA. Advantages of RT-PCR RT-PCR is more rapid and can be more easily automated and standardized than ICC, so it is more suitable for routine laboratory analysis. Theoretically, RT-PCR is more sensitive than ICC, because a very low transcription-level with weak or no translation can be detected by RT-PCR. Problems related to RT-PCR A sample analyzed by ICC can be considered as noncontributory if the quality of an ICC-labeling seems
788 anti-aerosol hoods, and a frequent decontamination of the manipulation area, ... Class Tissue-specificity Gene - mRNA general background. Normal genes can be transcribed at very low rate in many cells of very Cytokeratins 8-18-19-20, CD44 Tissue-related Epithelial different histologic types, a phenomenon known as variants, EGP-2, Apo A-l, markers Squam.C.C.Ag, HER2/neu illegitimate transcription [71]. Some hematopoietic cells appear to have weak expression of epithelial Mucins, EGFR, CEA Adenomatous cell genes [72], as has been demonstrated for cytoOrgan-related Breast Maspin keratins [73], mucin-1 [47] and PSA [66]. For this markers reason the amplification rate cannot be increased Prostate PSA, PSMA, hK2 infinitely [66]. Liver - Induction ofgene expression. We have observed the aFP, albumin induction of CEA-gene transcription in hematoTestis aFP logical cells in the presence of G-CSF [67] resulting Thyroid Thyroglobin, TPO in a loss of specificity of CEA RT-PCR for TCD Neuroblastoma PGP9.5, tyrosine hydroxylase in cancer patients treated with G-CSF or having a Melanoma Tyrosinase, Mucin-18, Melan A, high endogenous expression of G-CSF. Similar MARTI,p97, melanomainduction has been demonstrated for other genes inhibitory activity targeted by RT-PCR for TCD, namely induction by growth factors for CEA and CK19 [74], and by Lung Surfactant, Pgp9.5 hormonal environment for PSA [40, 75], tyrosinase Pancreas Chymotrypsin [76] and mucins [42]. Bladder Uroplakin - Amplification of pseudogenes has been described Parathyroid PTH for CK18 [77] and CK19 [78]. A careful selection of primers with a large intronic jump and systematic TumorMAGE, GAGE related utilization of DNAse can avoid amplification of markers unexpected DNA sequences. Contaminating cells from the surrounding tissues Breast Tumor- and Mammaglobin must be avoided. Several cytokeratins [73], CEA Kidney organ-related MN/CA9 markers [79] and tyrosinase [80] are expressed naturally in (epi)dermis. Ewing (+PNET) TumorFli/EWS, ERG/EWS The risk of false positives can been dramatically specific trans- Papillary thyroid RET/PTC 1 decreased by avoiding contamination by cells from locations with care. the skin, by using a cannula for blood drawing [81] transcripts or by discarding the first milliliters of aspirated Viral-specific EBV, HPV blood [35]. In the operative and postoperative transcripts period there is often an increase of RT-PCR positivity [82, 83]. Non-tumoral cells, shedded into circulation from the operative tissue damage, could unsatisfactory. When analysis is performed by RT-PCR participate in this increase in positivity. the technical quality of the analysis must always be - Non-malignant epithelial or melanocytic lymph optimal, warranting a specificity that approximates node inclusions [54, 55] can not be distinguished 100% and a sensitivity that does not vary, as the quality from malignant tissue by RT-PCR amplifying control of RT-PCR efficiency is rather approximate. tissue-specific gene-expression. Currently specificity and sensitivity may diverge - Recently mRNA for tyrosinase has been amplified importantly between groups. For this reason TCD by from acellular serum samples from melanoma paRT-PCR is much criticized due to the finding of distients [84]. Freely circulating proteolipid complexes, containing RNA could be responsible for this cordant and even contradictory results [3, 62]. observation. If this could be confirmed, the presence of tumor related mRNA should not reflect Loss of specificity. For the most frequently tested genes the presence of circulating tumor cells anymore. used for TCD by RT-PCR, tumor-related gene-expresDue to the high incidence of false positives some sion has been noted in absence of malignant cells, namely CK19 [63, 64], PSA [65, 66], CEA [62, 67], groups have introduced a cut-off for the PCR amplificaprostate-specific membrane antigen [68], mucin-1 [69]. tion rate. A lower sensitivity is accepted to warrant specificity [66, 85]. RT-PCR with low sensitivity can be A number of factors may induce loss of specificity: - Contaminating DNA: To avoid spreading the PCR- a valuable tool for TCD in lymph nodes, in which amplification product and gross contamination infiltrating tumor-cell burden rather than isolated tumor rigorous precautions have to be applied [70]: sepa- cells should have a prognostic value. By RT-PCR a rate rooms for each RT-PCR step, cotton tips, lymph node can be analyzed as a whole, avoiding the Table 1. RT-PCR targeted gene-transcripts for TCD.
789 of the amplification rate. By this manner false positives due to extensive amplification of background geneexpression can be avoided [89]. Traditionally, quantification of the expression rate of a gene-transcript has been Lack ofsensitivity. - The degradation of RNA during the various steps measured by relating it to the expression of a housekeeppreceding the PCR reaction itself accounts greatly ing gene [89] or to an external standard [72]. Recently for the high variability of sensitivity of RT-PCR different models of full automated quantitative PCRresults [86, 87]. Most human tissues, including techniques have been commercialized. In these, the blood and lymph nodes have high levels of amplification rate is monitored at each cycle by an RNAses. The control of RNA integrity by system- internal fluorescent probe [90]. atical amplification of housekeeping genes like - Multimarker-PCR can partially compensate for actin or P2-microglobulin does not exclude partial tumor heterogeneity and possible false positives. degradation of RNA. In several studies, samples Performing PCR for different marker genes simulhad to be treated within two hours after sample taneously increases sensitivity [91], while the recollection [31, 74]. This is unpractical in routine quirement for positivity for more than one gene at clinical practice. the same time improves specificity [92]. - More reliable tumor-related gene-transcripts have - Small technical variations, such as the concentrato be selected. It is possible that rather common tion of some constituents (more particularly MgCl2) of the RT-PCR assay or ageing of equipor inducible genes like cytokeratins, mucin-1 and ment used [74] can seriously affect assay consisCEA will have to be abandoned in favour of genes tency. whose expression seems to be restricted to final organ differentiation like PSA or tyrosinase. - Sensitivity obtained with fresh tumor-contaminated Perhaps new perspectives open with the discovery tissue is often much lower than that obtained with of genes whose expression seems to be limited cultured cells during the setup of a test [88]. Some to organ-specific malignant transformation, like contaminants like heparin, hemoglobin, melanin mammaglobin for breast cancer [93] and MN/ and porphyrins alter PCR efficiency. CA9 for renal cancer [94]. - The gene-expression rate can be very different between different cell-lines of the same malignancy - Cellular enrichment techniques, as described for and even more so between different malignancies ICC, can drastically reduce the risk of false posi[31, 72]. The sensitivity is usually tested on a celltives by decreasing background and contaminants line presenting a high target-gene expression. [95]. Afterwards, this sensitivity is supposed to be found in freshly harvested tumor cells and even extrapolated to other malignancies of different histology. Conclusions This extrapolation is absolutely unjustified. We found poor sensitivity of CEA RT-PCR on three Detection and characterization of disseminated cells different breast cancer cell lines, while the same from non-hematological tumors may provide data of RT-PCR was found to be highly sensitive in gastro- value for tumor staging and for subsequent therapeutic intestinal cell-lines [67]. approach. Currently it cannot be introduced in routine clinical practice due to the contradictory results emerging Non-quantitative. RT-PCR positivity reflects the pres- from different studies. We have discussed the main conence of circulating cells containing target mRNA. It ceptual and technical problems that are responsible for does not, however, correlates with the number of circu- such discordant results. Great effort still remains to be lating cells. The level of gene-specific mRNA level is quite done to increase the reliability of TCD. Solutions can lie variable from one cell line to another. After sampling, in analysis of greater volume samples, further characterthe efficiency of recovery of RNA, the reverse transcrip- ization of circulating tumor cells, quantitative analyses, tion step and the PCR cannot be assessed accurately. automation and standardization of techniques. Currently 'Semi-quantitative' PCR cannot be used to quantify ICC remains more reliable than RT-PCR, and needs to initial tumor-cell burden but can be used to follow the be used for validation of RT-PCR techniques. intensity of expression of a specific gene during theraA consensus about the technical modalities of detecpeutic follow-up. tion methods has to be formulated before the clinical value of TCD can be determined by prospective large scale clinical studies. Future developments RT-PCR protocols are readily automated, including internal sensitivity and specificity controls. Still more RT-PCR kits 'all in one' are being commercialized, References thereby simplifying the technical manipulations and decreasing the risk of contamination and errors. 1. Pantel K., Cote RJ, Fodstad O. Detection and clinical importance Semi-quantitative RT-PCR allows standardization of micrometastatic disease. JNatl Cancer Inst 1999; 91: 1113-24. necessity of time-consuming serial sectioning for microscopical analysis [10].
790 2. Moss TJ. Clinical relevance of minimal residual cancer in patients with solid malignancies. Cancer Metast Rev 1999; 18 (1): 91-100. 3. Lambrechts AC, van 't Veer LJ, Rodenhuis S. The detection of minimal numbers of contaminating epithelial tumor cells in blood or bone marrow: Use, limitations and future of RNA-based methods. Ann Oncol 1998; 92:1269-76. 4. Pelkey TJ, Frierson HF Jr, Bruns DE. Molecular and immunological detection of circulating tumor cells and micrometastases from solid tumors. Clin Chem 1996; 42: 1369-81. 5. Dowlatshahi K, Fan M, Snider HC, Habib FA. Lymph node micrometastases from breast carcinoma: Reviewing the dilemma. Cancer 1997; 80: 1188-97. 6. Natsugoe S, Mueller J, Stein HJ et al. Micrometastasis and tumor cell microinvolvement of lymph nodes from esophageal squamouscell carcinoma: Frequency, associated tumor characteristics, and impact on prognosis. Cancer 1998; 83: 858-66. 7. Diel IJ, Kaufmann M, Costa SD et al. Micrometastatic breast cancer cells in bone marrow at primary surgery: Prognostic value in comparison with nodal status. J Natl Cancer Inst 1996; 88: 1652-8. 8. Braun S, Pantel K, Miiller 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 2000, 342: 525-33. 9. Funke I, Schraut W. Meta-analyses of studies on bone marrow micrometastases: An independent prognostic impact remains to be substantiated. J Clin Oncol 1998; 16: 557-66. 10. International (Ludwig) Breast Cancer Study Group. Prognostic importance of occult axillary lymph node micrometastases from breast cancers. Lancet 1990; 335 (8705): 1565-8. 11. Berger U, Bettelheim R, Mansi JL et al. The relationship between micrometastases in the bone marrow, histopathologic features of the primary tumor in breast cancer and prognosis. Am J Clin Pathol 1988; 90: 1-6. 12. Jauch KW, Heiss MM, Gruetzner U et al. Prognostic significance of bone marrow micrometastases in patients with gastric cancer. J Clin Oncol 1996; 14: 1810-7. 13. Lindemann F, Schlimok G, Dirschedl P et al. Prognostic significance of micrometastatic tumour cells in bone marrow of colorectal cancer patients. Lancet. 1992; 340: 685-9. 14. Pantel K, Izbicki J, Passlick B et al. Frequency and prognostic significance of isolated tumour cells in bone marrow of patients with non-small-cell lung cancer without overt metastases. Lancet 1996 9; 347: 649-53. 15. Wood DP Jr, Banerjee M. Presence of circulating prostate cells in the bone marrow of patients undergoing radical prostatectomy is predictive of disease-free survival. J Clin Oncol 1997; 15: 3451-7. 16. Liotta LA, Saidel MG, Kleinerman J. The significance of hematogenous tumor-cell clumps in the metastatic process. Cancer Res 1976; 36: 889-94. 17. McCulloch P, Choy A, Martin L. Association between tumour angiogenesis and tumour cell shedding into effluent venous blood during breast cancer surgery. Lancet 1995; 346: 1334-5. 18. Fox SB, Leek RD, Bliss J et al. Association of tumor angiogenesis with bone marrow micrometastases in breast cancer patients. J Natl Cancer Inst 1997; 89: 1044-9. 19. Choy A, McCulloch P. Induction of tumour cell shedding into effluent venous blood breast cancer surgery. Br J Cancer 1996; 73: 79-82. 20. Weiss L, Dimitrov DS. Afluidmechanical analysis of the velocity, adhesion, and destruction of cancer cells in capillaries during metastasis. Cell Biophys 1984; 6 (1): 9-22. 21. Garcia-Olmo D, Ontanon J, Garcia-Olmo DC et al. Detection of genomically-tagged cancer cells in different tissues at different stages of tumor development: Lack of correlation with the formation of metastasis. Cancer Lett 1999; 140: 11-20. 22. Koike C, Watanabe M, Oku N et al. Tumor cells with organspecific metastatic ability show distinctive trafficking in vivo: Analyses by positron emission tomography and bioimaging. Cancer Res 1997; 57: 3612-9. 23. Tarin D, Price JE, Kettlewell MG et al. Mechanisms of human
24.
25. 26.
27.
28.
29. 30.
31.
32. 33. 34.
35. 36.
37.
38.
39. 40.
41. 42.
tumor metastasis studied in patients with peritoneovenous shunts. Cancer Res 1984; 44: 3584-92. Braun S, Kentenich C, Janni W et al. Lack of effect of adjuvant chemotherapy on the elimination of single dormant tumor cells in bone marrow of high-risk breast cancer patients. J Clin Oncol 2000; 18: 80-6. Pantel K, Schlimok G, Braun S et al. Differential expression of proliferation-associated molecules in individual micrometastatic carcinoma cells. J Natl Cancer Inst 1993; 85: 1419-24. Putz E, Witter K, Offner S et al. Phenotypic characteristics of cell lines derived from disseminated cancer cells in bone marrow of patients with solid epithelial tumors: Establishment of working models for human micrometastases. Cancer Res 1999; 59: 241-8. Pantel K, Schlimok G, (Cutter D et al. Frequency of downregulation of major histocompatibility class I antigen expression on individual micrometastatic carcinoma cells. Cancer Res 1991; 51: 4715. Heiss MM, Allgayer H, Gruetzner K.U et al. Individual development and uPA-receptor expression of disseminated tumour cells in bone marrow: A reference to early systemic disease in solid cancer. Nature Med 1995; 1: 1035-9. Pantel K, Schlimok G, Angstwurm M et al. Methodological analysis of immunocytochemical screening for disseminated epithelial tumor cells in bone marrow. J Hematother 1994; 3: 165-73. Jung R, Ahmad-Nejad P, Wimmer M et al. Quality management and influential factors for the detection of single metastatic cancer cells by reverse transcriptase polymerase chain reaction. Eur J Clin Chem Clin Biochem 1997; 35 (1): 3-10. Jonas S, Windeatt S, O-Boateng A et al. Identification of carcinoembryonic antigen-producing cells circulating in the blood of patients with colorectal carcinoma by reverse transcriptase polymerase chain reaction. Gut 1996; 39 (5): 717-21. Cote RJ, Rosen PP, Lesser ML et al. Prediction of early relapse in patients with operable breast cancer by detection of occult bone marrow micrometastases. J Clin Oncol 1991; 9 (10): 1749-56. Pasini F, Pelosi G, Verlato G et al. Positive immunostaining with MLuCl of bone marrow aspirate predicts poor outcome in patients with small-cell lung cancer. Ann Oncol 1998; 9 (2): 181-5. Weckermann D, Wawroschek F, Krawczak G et al. Does the immunocytochemical detection of epithelial cells in bone marrow (micrometastasis) influence the time to biochemical relapse after radical prostatectomy? Urol Res 1999; 27 (4): 285-90. Peck K, Sher YP, Shih JY et al. Detection and quantitation of circulating cancer cells in the peripheral blood of lung cancer patients. Cancer Res 1998; 58: 2761-5. Braun S, Hepp F, Sommer HL, Pantel K. Tumor-antigen heterogeneity of disseminated breast cancer cells: Implications for immunotherapy of minimal residual disease. Int J Cancer 1999; 84(1): 1-5. Canon JL, Humblet Y, Lebacq-Verheyden AM et al. Immunodetection of small-cell lung cancer metastases in bone marrow using three monoclonal antibodies. M Eur J Cancer Clin Oncol 1988; 24(2): 147-50. Marincola FM, Hijazi YM, Fetsch Pet al. Analysis of expression of the melanoma-associated antigens MART-1 and gplOO in metastatic melanoma cell lines and in situ lesions. J Immunother 1996; 19: 192-205. Qiu SD, Young CY. Bilhartz DL et al. In situ hybridization of prostate-specific antigen mRNA in human prostate. J Urol 1990; 144 (6): 1550-6. Young CY, Montgomery BT, Andrews PE et al. Hormonal regulation of prostate-specific antigen messenger RNA in human prostatic adenocarcinoma cell line LNCaP. Cancer Res 1991; 51: 3748-52. Fuller BB, Niekrasz I, Hoganson GE. Down-regulation of tyrosinase mRNA levels in melanoma cells by tumor promoters and by insulin. Mol Cell Endocrinol 1990; 72 (2): 81-7. Gollub EG, Waksman H, Goswami S, Marom Z. Mucin genes are regulated by estrogen and dexamethasone. Biochem Biophys Res Commun 1995; 217: 1006-14.
791 43. Forus A, Hoifodt HK, Overli GETet al. Sensitive fluorescent in situ hybridisation method for the characterisation of breast cancer cells in bone marrow aspirates. J Clin Path Mol Pathol 1999; 52: 68-74. 44. Kobayashi TK, Ueda M, Yamaki T, Yakushiji M. Evaluation of cytocentrifuge apparatus with special reference to the cellular recovery rate. Diagn Cytopathol 1992; 8: 420-3. 45. Franklin WA, Shpall EJ, Archer P et al. Immunocytochemical detection of breast cancer cells in marrow and peripheral blood of patients undergoing high-dose chemotherapy with autologous stem-cell support. Breast Cancer Res Treat 1996; 41: 1-13. 46. Lagrange M, Ferrero JM, Lagrange JL et al. Non-speciflcally labelled cells that simulate bone marrow metastases in patients with non-metastatic breast cancer. J Clin Pathol 1997; 50: 206-11. 47. BruggerW, Buhring HJ, Grunebach Fet al. Expression of MUC1 epitopes on normal bone marrow: Implications for the detection of micrometastatic tumor cells. J Clin Oncol 1999; 17 (5): 1535-44. 48. Delsol G, Gatter KC, Stein H et al. Human lymphoid cells express epithelial membrane antigen. Implications for diagnosis of human neoplasms. Lancet 1984; 2: 1124-9. 49. Doglioni C, DelFOrto P, Zanetti G et al. Cytokeratin-immunoreactive cells of human lymph nodes and spleen in normal and pathological conditions. An immunocytochemical study. Virch Arch A Pathol Anat Histopathol 1990; 416: 479-90. 50. Domagala W, Bedner E, Chosia M et al. Keratin-positive reticulum cells in fine needle aspirates and touch imprints of hyperplastic lymph nodes. A possible pitfall in the immunocytochemical diagnosis of metastatic carcinoma. Acta Cytol 1992; 36: 241-5. 51. Borgen E, Beiske K, Trachsel S et al. Immunocytochemical detection of isolated epithelial cells in bone marrow: Non-specific staining and contribution by plasma cells directly reactive to alkaline phosphatase. J Pathol 1998; 185: 427-34. 52 Brandt B, Junker R, Griwatz C et al. Isolation of prostate-derived single cells and cell clusters from human peripheral blood. Cancer Res 1996; 56: 4556-61. 53. Nicholson AG, Graham AN, Pezzella F et al. Does the use of immunohistochemistry to identify micrometastases provide useful information in the staging of node-negative non-small-cell lung carcinomas? Lung Cancer 1997; 18: 231-40. 54. Fisher CJ, Hill S, Millis RR. Benign lymph node inclusions mimicking metastatic carcinoma. J Clin Pathol 1994; 47: 245-7. 55. Yu LL, Flotte TJ, Tanabe KK et al. Detection of microscopic melanoma metastases in sentinel lymph nodes. Cancer 1999; 86: 617-27. 56. Naume B, Borgen E, Beiske K et al. Immunomagnetic techniques for the enrichment and detection of isolated breast carcinoma cells in bone marrow and peripheral blood. J Hematother 1997; 6: 103-14. 57. Mesker WE, v d Burg JM, Oud PS et al. Detection of immunocytochemically stained rare events using image analysis. Cytometry 1994; 17: 209-15. 58. Mignon-Godefroy K, Guillet JG, Butor C. Solid phase cytometry for detection of rare events. Cytometry 1997; 27: 336-44. 59. Hibi K, Robinson CR, Booker S et al. Molecular detection of genetic alterations in the serum of colorectal cancer patients. Cancer Res 1998; 58: 1405-7. 60. Chiang PW, Beer DG, Wei WL et al. Detection of erbB-2 amplifications in tumors and sera from esophageal carcinoma patients. Clin Cancer Res 1999; 5: 1381-6. 61. Yamamoto N, Kato Y, Yanagisawa A et al. Predictive value of genetic diagnosis for cancer micrometastasis: Histologic and experimental appraisal. Cancer 1997; 80: 1393-8. 62. Zippelius A, Kufer P, Honold G et al. Limitations of reversetranscriptase polymerase chain reaction analyses for detection of micrometastatic epithelial cancer cells in bone marrow. J Clin Oncol 1997; 15: 2701-8. 63. Yun K, Gunn J, Merrie AE et al. Keratin 19 mRNA is detectable by RT-PCR in lymph nodes of patients without breast cancer. Br J Cancer 1997; 76 (8): 1112-3.
64. Krismann M, Todt B, Schroder J et al. Low specificity of cytokeratin 19 reverse transcriptase-polymerase chain reaction analyses for detection of hematogenous lung cancer dissemination. J Clin Oncol 1995; 13: 2769-75. 65. Thiounn N, Saporta F, Flam TA et al. Positive prostate-specific antigen circulating cells detected by reverse transcriptasepolymerase chain reaction does not imply the presence of prostatic micrometastases. Urology 1997; 50: 245-50. 66. Gala JL, Heuslerspreute M, Loric S et al. Expression of prostatespecific antigen and prostate-specific membrane antigen transcripts in blood cells: Implications for the detection of hematogenous prostate cells and standardization. Clin Chem 1998; 44: 472-81. 67. 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 1999; 24: 76975. 68. Lintula S, Stenman UH. The expression of prostate-specific membrane antigen in peripheral blood leukocytes. J Urol 1997; 157: 1969-72. 69. Dent GA, Civalier CJ, Brecher ME, Bentley SA. MUC1 expression in hematopoietic tissues. Am J Clin Pathol 1999; 111: 741-7. 70. Kwok S, Higuchi R. Avoiding false positives with PCR. Nature 1989; 339: 237-8. 71. Chelly J, Concordet JP, Kaplan JC, Kahn A. Illegitimate transcription: Transcription of any gene in any cell type. Proc Natl Acad Sci USA 1989; 86: 2617-21. 72. De Graaf H, Maelandsmo GM, Ruud P et al. Ectopic expression of target genes may represent an inherent limitation of RT-PCR assays used for micrometastasis detection: Studies on the epithelial glycoprotein gene EGP-2. Int J Cancer 1997; 72: 191-6. 73. Traweek ST, Liu J, Battifora H. Keratin gene expression in nonepithelial tissues. Detection with polymerase chain reaction. Am J Pathol 1993; 142(4): 1111-8. 74. Jung R, Kriiger 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 1998; 78: 1194-8. 75. Yu H, Diamandis EP, Zarghami N, Grass L. Induction of prostate specific antigen production by steroids and tamoxifen in breast cancer cell lines. Breast Cancer Res Treat 1994; 32 (3): 291-300. 76. Kippenberger S, Loitsch S, Solano F et al. Quantification of tyrosinase, TRP-1, and TRP-2 transcripts in human melanocytes by reverse transcriptase-competitive multiplex PCR-regulation by steroid hormones. J Invest Dermatol 1998; 110: 364-7. 77. Neumaier M, Gerhard M, Wagener C. Diagnosis of micrometastases by the amplification of tissue-specific genes. Gene 1995; 159 (1): 43-7. 78. Ruud P, Fodstad O, Hovig E. Identification of a novel cytokeratin 19 pseudogene that may interfere with reverse transcriptasepolymerase chain reaction assays used to detect micrometastatic tumor cells. Int J Cancer 1999; 80 (1): 119-25. 79. Metze D, Bhardwaj R, Amann U et al. Glycoproteins of the carcinoembryonic antigen (CEA) family are expressed in sweat and sebaceous glands of human fetal and adult skin. J Invest Dermatol 1996; 106 (1): 64-9. 80. Battyani Z, Xerri L, Hassoun J et al. Tyrosinase gene expression in human tissues. Pigment Cell Res 1993; 6 (6): 400-5. 81. Jonas SK, Klokouzas A, Wharton RQ, AIlen-Mersch TG. Identification of circulating tumour cells by RT-PCR in patients with colorectal cancer: Detection prior to evidence of metastasis. Proc Am Assoc Cancer Res 1997; 38: 19 (Abstr O12). 82. Eschwege P, Dumas F, Blanchet P et al. Haematogenous dissemination of prostatic epithelial cells during radical prostatectomy. Lancet 1995; 346 (8989): 1528-30. 83. Weitz J, Kienle P, Lacroix J et al. Dissemination of tumor cells in patients undergoing surgery for colorectal cancer. Clin Cancer Res 1998; 4 (2): 343-8.
792 84. Kopreski MS, Benko FA, Kwak LW, Gocke CD. Detection of tumor messenger RNA in the serum of patients with malignant melanoma. Clin Cancer Res 1999; 5: 1961-5. 85. Schoenfeld A, Luqmani Y, Smith D et al. Detection of breast cancer micrometastases in axillary lymph nodes by using polymerase chain reaction. Cancer Res 1994; 54: 2986-90. 86. Keilholz U, Willhauck M, Rimoldi D et al. Reliability of reverse transcription-polymerase chain reaction (RT-PCR)-based assays for the detection of circulating tumour cells: A quality-assurance initiative of the EORTC Melanoma Cooperative Group. Eur J Cancer 1998; 34: 750-3. 87. Schittek B, Blaheta HJ, Florchinger G et al. Increased sensitivity for the detection of malignant melanoma cells in peripheral blood using an improved protocol for reverse transcription-polymerase chain reaction. Br J Dermatol 1999; 141: 37-43. 88. 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 1998; 4: 2141-6. 89. 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 1999; 17: 870-9. 90. Orlando C, Pinzani P, Pazzagli M. Developments in quantitative PCR. Clin Chem Lab Med 1998; 36: 255-69. 91. Cheung IY, Barber D, Cheung NK. Detection of microscopic neuroblastoma in marrow by histology, immunocytology, and reverse transcription-PCR of multiple molecular markers. Clin Cancer Res 1998; 4: 2801-5.
92. Palmien G, Strazzullo M, Ascierto P et al. Polymerase chain reaction-based detection of circulating melanoma cells as an effective marker of tumor progression. J Clin Oncol 1999; 17: 304-11. 93. Min CJ, Tafra L, Verbanac KM. Identification of superior markers for polymerase chain reaction detection of breast cancer metastases in sentinel lymph nodes. Cancer Res 1998; 58: 4581^1. 94. McKJernan JM, Buttyan R, Bander NH et al. The detection of renal carcinoma cells in the peripheral blood with an enhanced reverse transcriptase-polymerase chain reaction assay for MN/CA9. Cancer 1999; 86: 492-7. 95. Makarovskiy AN, Ackerley W, Wojcik L et al. Application of immunomagnetic beads in combination with RT-PCR for the detection of circulating prostate cancer cells. J Clin Lab Anal 1997; 11:346-50. Received 5 January 2000; accepted 21 March 2000.
Correspondence to: J.-C. Goeminne, MD Service d'Oncologie Medicale Institut Gustave Roussy 39, rue Camille Desmoulins 94805 Villejuif France E-mail: [email protected]
Review Pitfalls in the detection of disseminated non-hematological tumor cells J.-C. Goeminne, T. Guillaume & M. Symann Laboratory of Experimental Oncology and Hematology, Universite Catholique de Louvain, Brussels, Belgium
Summary
There is not yet a consensus on the reliability of the methods that should be used for the detection of rare disseminated tumor cells from non-hematological malignancies. In this review, we will discuss the advantage and drawbacks of the classical approach of immunocytochemistry and the molecular detection by reverse transcriptase polymerase chain reaction
Introduction
Despite the clinical relevance and various methods of detection of solid tumor cells in hematological tissues [1-3], there not yet exists a consensus on the reliability of the methods that should be used for tumor-cell detection. In this review we will discuss the advantages and drawbacks of the classical approach of immunocytochemistry (ICC) as well molecular detection by RT-PCR. The presence of very limited tumor cells in bone marrow or lymph nodes is generally termed micrometastatic disease, although the definition of micrometastasis may vary. It may designate isolated tumor cells or a tumor-cell cluster in the absence of clinical or radiological metastatic lesions [4], or clusters smaller than, 2 mm [5] or 0.5 mm [6]. Detection of rare disseminated tumor cells predict an adverse clinical outcome in most studies [6-8]. However, due to large differences in methodology and discrepancies in findings among various studies, the true clinical correlates still have to be substantiated before it could be used to guide therapeutic decisions, like adjuvant therapy or purging of stem-cell graft products. It is difficult to determine if tumor-cell detection (TCD) is an independent prognostic factor at the time of diagnosis of the primary tumor [9], since TCD is usually related to other factors of poor prognosis, like tumor size [10], lymph node involvement [11], vascular invasion [10,11] and the tumor differentiation grade [7]. In the absence of other factors of poor prognosis, TCD in bone marrow has been found to predict a decreased overall survival and disease free survival in breast cancer [7, 8], gastric [12], colorectal [13], nonsmall-cell lung [14] and prostatic cancer [15], signing an early systemic dissemination of malignancy. Presence of tumor cells in stem-cell graft products is
(RT-PCR). The interpretation of the biological significance of circulating tumor cells and the pitfalls of the detection techniques are the main causes of discrepancy between the conclusions of different tumor-cell detection (TCD) studies. Key words: immunocytochemistry,
RT-PCR, tumor-cell
detection
related to persistence of disease at the time of stem-cell collection and with the exception of neuroblastoma it remains unclear whether the reinjection of contaminating tumor cells contributes to tumor relapse. After and during medical treatment, TCD can be used for monitoring of therapeutic efficiency, persistent minimal residual disease or overt relapse. The discrepancy between the conclusions of different tumor-cell detection (TCD) studies is principally due to the misunderstanding of the biological significance of circulating tumor cells and the pitfalls of the detection techniques. Biological significance of circulating cells TCD does not necessarily reflect metastatic potential. A malignant tumor is continuously shedding tumor cells [16]. This shedding is related to tumor angiogenesis [17, 18], vascular invasion [19], tumor size and proliferation rate [18]. Most of shedded tumor cells are eliminated in the first capillary bed, being the lymph nodes, the lungs and the liver [20] and only a minority will be released progressively and arrested in a secondary capillary bed, the bone marrow being the most easily assessable [21, 22]. Isolated tumor cells found in lymph nodes, blood or bone marrow are the result of this continuous shedding from the primary tumor and most of them will never evolve to macroscopic metastases. This is suggested by finding that tumor cells are detected in the bone marrow of patients with gastrointestinal malignancies [12, 13] at the same frequency as in patients with breast cancer although bone metastases are rarely seen in the former. Furthermore, tumor cells are found in bone marrow from patients who never will relapse and patients can
786 survive several months with a peritoneo-venous shunt for malignant ascites without developing distant metastases, although high amounts of tumor cells are continuously shedded into the venous circulation [23]. Disseminated malignant cells in the bone marrow can remain dormant for many years and escape to the cytotoxic effects of chemotherapy [24]. Consistent with this concept of tumor-cell dormancy, the expression of proliferative markers was found to be low in the tumor cells detected in the bone marrow [25]. Metastases will develop only with the appearance of 'metastatically competent' tumor cell clones, able to survive and proliferate distant from the primary tumor [26]. Reduced expression by tumor cells of HLA I molecules [27], proteins involved in the immune response, tumor-associated proteases [28] may help to identify the capacity of outgrowth of disseminated tumor cells. Tumor-cell detection techniques Immunocytochemistry (ICC) and the reverse transcriptase polymerase chain reaction (RT-PCR) are the two most extensively used techniques of TCD since both methods have been proven much more sensitive than standard microscopic analysis. The sensitivity of detection of both techniques varies between 1 tumor cell in 105 and 107 mononuclear hematological cells. Other techniques such as clonogenic assays or flow cytometry appear less suitable for large scale TCD studies. Common pitfalls of ICC and R T-PCR The random disposition of tumor cells Because tumor cells are not necessarily distributed uniformly in lymph nodes and bone marrow, a single sample or a small volume sample may not reflect the extent of tumor spread. It has been demonstrated that bone marrow taken at several sites rather than only a single one [29] improves sensitivity. Studies have been performed where samples were taken at six [7] or eight different sites [11]. Likewise, the amount of tumor cells in peripheral blood fluctuates. Serial blood samples may therefore give different results [30, 31]. Lack of quantification of tumor burden Most studies on TCD report whether a sample does contain tumor cells or not, without quantifying detected the tumor burden. Quantification of tumor burden could however have an important clinical value: The actual amount of tumor cells detected in bone marrow from patients with breast cancer [32], gastric cancer [12], small-cell lung cancer [33] and prostate cancer [34] seems to have a prognostic significance. In monitoring disease progression during treatment [35] or assessing the efficiency of stem-cell product purging, the detected tumor cell burden should be effectively quantified. The kinetics of circulating cell burden is not well known and needs more quantitative data.
Tumor-cell heterogeneity The principle of TCD by both, ICC and RT-PCR, relies on the recognition of particular antigens or gene transcripts that distinguish tumor cells from surrounding cells, principally hematological cells. The ideal tumorcell marker expressed by all tumor cells and not by other cells has not yet been found. There is great variability in antigenic expression between different cell lines derived from the same cancer histologic type [31, 35] and sometimes even between different cell clones from the very same tumor [36, 37]. Expression of tumor-related antigens on metastatic lesions can be very weak due to downregulation of antigens which are strongly expressed in the primary tumor or in the originating tissue. This has been observed for melanoma markers [38], for cytokeratins [29] and for PSA [39]. In the same fashion, antigen expression can be influenced by treatment. PSA [40], tyrosinase [41] and mucins [42] are all modulated by the prevailing hormonal environment. Immunocytochemistry (ICC) Tumor or tissue specific antigens are targeted by specific antibodies before microscopical analysis. Advantages The specificity of TCD by ICC is greatly improved by simultaneous morphological analysis of the positivestained cells, recognizing the characteristic malignant features characterized by nucleo-cytoplasmic atypia or cell clustering, although some investigators claim to have high specificity without morphological confrontation [8, 29]. Further characterization of immunocytochemical detected tumor cells can be performed by additional ICC staining [25] or by FISH (fluorescent in situ hybridization) for genetic mutations [43]. This can help to confirm malignant nature and to characterize the metastatic features of circulating cells [26]. Further quantification of the detected tumor burden is easily affordable by straightforward microscopic analysis. Problems related with ICC ICC is very labor intensive. In order to obtain high sensitivity a huge cellular volume must be analyzed microscopically. Cellular loss is generally underestimated and can be substantial during the various steps that precede microscopic examination. This includes recovery of mononuclear cells on density gradient, red blood cell lysis, cytospin and the series of washings during immunocytochemical staining. Cellular loss is estimated to be up to 60% [44, 45]. No available antibodies are 100% tumor- or tissuespecific. Due to intra- and inter-tumoral heterogeneity, it is necessary to use a panel of different antibodies recognizing different antigens or to use antibodies that recognize rather common epitopes. The lack of suitable
787 antibodies is mainly seen for instance in neuroblastoma and melanoma. False positive staining have been reported with most extensively used antibodies. Published data on immunocytochemical TCD may as a result be greatly overestimated due to non-specific labeling [46]. There are several particular sources for non-specific labeling: - Some hematological cells, principally in bone marrow [47, 48] and lymph node reticulum cells [49, 50] can express some epithelial antigens. - Macrophages, granulocytes and some lymphocytes have a great propensity to fix antibodies via their Fc receptors in a non-specific manner. - Some plasma cells can react with the APAAP (alkaline phosphatase-anti-alkaline phosphatase) complex, an essential compound for many ICC labeling techniques [51]. - Material phagocytized into hematological cells can be stained immunocyto-chemically in a false positive manner [52, 53]. - Lymph nodes can contain non-malignant epithelial [54] and melanocytic [55] inclusions likewise giving false-positive results. - Very low numbers of epithelial cells have been found in peripheral blood and bone marrow [8] of subjects without malignancy and can be related to benign epithelial proliferative diseases [29, 52] or tissue trauma [12]. For those reasons, the extent of experience of the pathologist is crucial to avoid false positives. To optimize specificity, in some studies microscopical slides are reviewed systematically by a second observer [7, 8]. Future developments Maximization of the volume of cells to be analyzed. - Sensitivity is directly related to the number of cells analyzed. The number is limited by the labour intensity it represents. By increasing the examined cell volume, specificity will also improve, increasing the chance of finding tumor cells with marked morphological characteristics of malignancy. - The detection of tumor cells in bone marrow is directly related to their presence in peripheral blood, but the relative concentration of tumor cells found in blood is much lower than in bone marrow. When sensitivity is increased by analyzing a more substantial amount of cells, peripheral blood analysis possibly could replace bone marrow sampling, which is always a stressful event for the patient. - For quantification of the disseminated tumor burden, high volume samples will give more reliable data by lowering the variability related to the heterogeneous distribution of circulating tumor cells. Some techniques can help in maximizing the analyzed cell volume: - Tumor cells can be tracked by selective enrichment. Most enrichment techniques rely on positive
or negative immunomagnetic selection of target cells [56]. With enrichment methods, blood samples up to 40 ml can be readily analyzed [52], representing about 60 million peripheral blood mononuclear cells, an amount that can not be analyzed by classical microscopical examination. - Slides can be scanned for tumor cells by automated computer-driven image analysis [57]. Promising results are suggested by the solid phase cytometry technique [58]. - Automation of ICC facilitates quantitative analysis, decreases inter-observer variability and allows standardization. Selection of more sensitive and specific antibodies. The use
of anti-cytokeratin antibodies seems much more specific than formerly used anti-epithelial antigens, including epithelial membrane antigen, milk fat globuline, human epithelial antigen-125 [29]. Methodological comparative analysis is required to select the most specific and sensitive antibodies [29, 45, 55]. Semisynthetic antibodies directly conjugated with an enzyme [51] or a fluorochrome can significantly decrease non-specific staining. RT-PCRandPCR TCD by RT-PCR relies on the selective amplification of transcripts (mRNA) of genes that are supposed to be expressed only in tumor cells and not in cells from the surrounding tissue. Most target genes are involved in tissue differentiation (mainly epithelial genes) or in malignant transformation (Table 1). Only chimeric genetranscripts, resulting from a chromosomal translocations are truly tumor-specific. Only in some rare solid tumors common chimeric gene-transcripts have been found such as Ewing's sarcoma. Tumor-related DNA-mutations involving K-ras [59], p53 [59], erbB-2 [60] have been found by PCR in blood and lymph nodes from cancer patients. These findings do not necessarily reflect the presence of circulating tumor cells, as free DNA can be found in serum or lymph nodes [61]. RT-PCR detects the presence of gene-transcripts by amplifying RNA. RNA is very fragile and is preserved as rule only in living cells. Direct PCR on the other hand amplifies the less labile DNA. Advantages of RT-PCR RT-PCR is more rapid and can be more easily automated and standardized than ICC, so it is more suitable for routine laboratory analysis. Theoretically, RT-PCR is more sensitive than ICC, because a very low transcription-level with weak or no translation can be detected by RT-PCR. Problems related to RT-PCR A sample analyzed by ICC can be considered as noncontributory if the quality of an ICC-labeling seems
788 anti-aerosol hoods, and a frequent decontamination of the manipulation area, ... Class Tissue-specificity Gene - mRNA general background. Normal genes can be transcribed at very low rate in many cells of very Cytokeratins 8-18-19-20, CD44 Tissue-related Epithelial different histologic types, a phenomenon known as variants, EGP-2, Apo A-l, markers Squam.C.C.Ag, HER2/neu illegitimate transcription [71]. Some hematopoietic cells appear to have weak expression of epithelial Mucins, EGFR, CEA Adenomatous cell genes [72], as has been demonstrated for cytoOrgan-related Breast Maspin keratins [73], mucin-1 [47] and PSA [66]. For this markers reason the amplification rate cannot be increased Prostate PSA, PSMA, hK2 infinitely [66]. Liver - Induction ofgene expression. We have observed the aFP, albumin induction of CEA-gene transcription in hematoTestis aFP logical cells in the presence of G-CSF [67] resulting Thyroid Thyroglobin, TPO in a loss of specificity of CEA RT-PCR for TCD Neuroblastoma PGP9.5, tyrosine hydroxylase in cancer patients treated with G-CSF or having a Melanoma Tyrosinase, Mucin-18, Melan A, high endogenous expression of G-CSF. Similar MARTI,p97, melanomainduction has been demonstrated for other genes inhibitory activity targeted by RT-PCR for TCD, namely induction by growth factors for CEA and CK19 [74], and by Lung Surfactant, Pgp9.5 hormonal environment for PSA [40, 75], tyrosinase Pancreas Chymotrypsin [76] and mucins [42]. Bladder Uroplakin - Amplification of pseudogenes has been described Parathyroid PTH for CK18 [77] and CK19 [78]. A careful selection of primers with a large intronic jump and systematic TumorMAGE, GAGE related utilization of DNAse can avoid amplification of markers unexpected DNA sequences. Contaminating cells from the surrounding tissues Breast Tumor- and Mammaglobin must be avoided. Several cytokeratins [73], CEA Kidney organ-related MN/CA9 markers [79] and tyrosinase [80] are expressed naturally in (epi)dermis. Ewing (+PNET) TumorFli/EWS, ERG/EWS The risk of false positives can been dramatically specific trans- Papillary thyroid RET/PTC 1 decreased by avoiding contamination by cells from locations with care. the skin, by using a cannula for blood drawing [81] transcripts or by discarding the first milliliters of aspirated Viral-specific EBV, HPV blood [35]. In the operative and postoperative transcripts period there is often an increase of RT-PCR positivity [82, 83]. Non-tumoral cells, shedded into circulation from the operative tissue damage, could unsatisfactory. When analysis is performed by RT-PCR participate in this increase in positivity. the technical quality of the analysis must always be - Non-malignant epithelial or melanocytic lymph optimal, warranting a specificity that approximates node inclusions [54, 55] can not be distinguished 100% and a sensitivity that does not vary, as the quality from malignant tissue by RT-PCR amplifying control of RT-PCR efficiency is rather approximate. tissue-specific gene-expression. Currently specificity and sensitivity may diverge - Recently mRNA for tyrosinase has been amplified importantly between groups. For this reason TCD by from acellular serum samples from melanoma paRT-PCR is much criticized due to the finding of distients [84]. Freely circulating proteolipid complexes, containing RNA could be responsible for this cordant and even contradictory results [3, 62]. observation. If this could be confirmed, the presence of tumor related mRNA should not reflect Loss of specificity. For the most frequently tested genes the presence of circulating tumor cells anymore. used for TCD by RT-PCR, tumor-related gene-expresDue to the high incidence of false positives some sion has been noted in absence of malignant cells, namely CK19 [63, 64], PSA [65, 66], CEA [62, 67], groups have introduced a cut-off for the PCR amplificaprostate-specific membrane antigen [68], mucin-1 [69]. tion rate. A lower sensitivity is accepted to warrant specificity [66, 85]. RT-PCR with low sensitivity can be A number of factors may induce loss of specificity: - Contaminating DNA: To avoid spreading the PCR- a valuable tool for TCD in lymph nodes, in which amplification product and gross contamination infiltrating tumor-cell burden rather than isolated tumor rigorous precautions have to be applied [70]: sepa- cells should have a prognostic value. By RT-PCR a rate rooms for each RT-PCR step, cotton tips, lymph node can be analyzed as a whole, avoiding the Table 1. RT-PCR targeted gene-transcripts for TCD.
789 of the amplification rate. By this manner false positives due to extensive amplification of background geneexpression can be avoided [89]. Traditionally, quantification of the expression rate of a gene-transcript has been Lack ofsensitivity. - The degradation of RNA during the various steps measured by relating it to the expression of a housekeeppreceding the PCR reaction itself accounts greatly ing gene [89] or to an external standard [72]. Recently for the high variability of sensitivity of RT-PCR different models of full automated quantitative PCRresults [86, 87]. Most human tissues, including techniques have been commercialized. In these, the blood and lymph nodes have high levels of amplification rate is monitored at each cycle by an RNAses. The control of RNA integrity by system- internal fluorescent probe [90]. atical amplification of housekeeping genes like - Multimarker-PCR can partially compensate for actin or P2-microglobulin does not exclude partial tumor heterogeneity and possible false positives. degradation of RNA. In several studies, samples Performing PCR for different marker genes simulhad to be treated within two hours after sample taneously increases sensitivity [91], while the recollection [31, 74]. This is unpractical in routine quirement for positivity for more than one gene at clinical practice. the same time improves specificity [92]. - More reliable tumor-related gene-transcripts have - Small technical variations, such as the concentrato be selected. It is possible that rather common tion of some constituents (more particularly MgCl2) of the RT-PCR assay or ageing of equipor inducible genes like cytokeratins, mucin-1 and ment used [74] can seriously affect assay consisCEA will have to be abandoned in favour of genes tency. whose expression seems to be restricted to final organ differentiation like PSA or tyrosinase. - Sensitivity obtained with fresh tumor-contaminated Perhaps new perspectives open with the discovery tissue is often much lower than that obtained with of genes whose expression seems to be limited cultured cells during the setup of a test [88]. Some to organ-specific malignant transformation, like contaminants like heparin, hemoglobin, melanin mammaglobin for breast cancer [93] and MN/ and porphyrins alter PCR efficiency. CA9 for renal cancer [94]. - The gene-expression rate can be very different between different cell-lines of the same malignancy - Cellular enrichment techniques, as described for and even more so between different malignancies ICC, can drastically reduce the risk of false posi[31, 72]. The sensitivity is usually tested on a celltives by decreasing background and contaminants line presenting a high target-gene expression. [95]. Afterwards, this sensitivity is supposed to be found in freshly harvested tumor cells and even extrapolated to other malignancies of different histology. Conclusions This extrapolation is absolutely unjustified. We found poor sensitivity of CEA RT-PCR on three Detection and characterization of disseminated cells different breast cancer cell lines, while the same from non-hematological tumors may provide data of RT-PCR was found to be highly sensitive in gastro- value for tumor staging and for subsequent therapeutic intestinal cell-lines [67]. approach. Currently it cannot be introduced in routine clinical practice due to the contradictory results emerging Non-quantitative. RT-PCR positivity reflects the pres- from different studies. We have discussed the main conence of circulating cells containing target mRNA. It ceptual and technical problems that are responsible for does not, however, correlates with the number of circu- such discordant results. Great effort still remains to be lating cells. The level of gene-specific mRNA level is quite done to increase the reliability of TCD. Solutions can lie variable from one cell line to another. After sampling, in analysis of greater volume samples, further characterthe efficiency of recovery of RNA, the reverse transcrip- ization of circulating tumor cells, quantitative analyses, tion step and the PCR cannot be assessed accurately. automation and standardization of techniques. Currently 'Semi-quantitative' PCR cannot be used to quantify ICC remains more reliable than RT-PCR, and needs to initial tumor-cell burden but can be used to follow the be used for validation of RT-PCR techniques. intensity of expression of a specific gene during theraA consensus about the technical modalities of detecpeutic follow-up. tion methods has to be formulated before the clinical value of TCD can be determined by prospective large scale clinical studies. Future developments RT-PCR protocols are readily automated, including internal sensitivity and specificity controls. Still more RT-PCR kits 'all in one' are being commercialized, References thereby simplifying the technical manipulations and decreasing the risk of contamination and errors. 1. Pantel K., Cote RJ, Fodstad O. Detection and clinical importance Semi-quantitative RT-PCR allows standardization of micrometastatic disease. JNatl Cancer Inst 1999; 91: 1113-24. necessity of time-consuming serial sectioning for microscopical analysis [10].
790 2. Moss TJ. Clinical relevance of minimal residual cancer in patients with solid malignancies. Cancer Metast Rev 1999; 18 (1): 91-100. 3. Lambrechts AC, van 't Veer LJ, Rodenhuis S. The detection of minimal numbers of contaminating epithelial tumor cells in blood or bone marrow: Use, limitations and future of RNA-based methods. Ann Oncol 1998; 92:1269-76. 4. Pelkey TJ, Frierson HF Jr, Bruns DE. Molecular and immunological detection of circulating tumor cells and micrometastases from solid tumors. Clin Chem 1996; 42: 1369-81. 5. Dowlatshahi K, Fan M, Snider HC, Habib FA. Lymph node micrometastases from breast carcinoma: Reviewing the dilemma. Cancer 1997; 80: 1188-97. 6. Natsugoe S, Mueller J, Stein HJ et al. Micrometastasis and tumor cell microinvolvement of lymph nodes from esophageal squamouscell carcinoma: Frequency, associated tumor characteristics, and impact on prognosis. Cancer 1998; 83: 858-66. 7. Diel IJ, Kaufmann M, Costa SD et al. Micrometastatic breast cancer cells in bone marrow at primary surgery: Prognostic value in comparison with nodal status. J Natl Cancer Inst 1996; 88: 1652-8. 8. Braun S, Pantel K, Miiller 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 2000, 342: 525-33. 9. Funke I, Schraut W. Meta-analyses of studies on bone marrow micrometastases: An independent prognostic impact remains to be substantiated. J Clin Oncol 1998; 16: 557-66. 10. International (Ludwig) Breast Cancer Study Group. Prognostic importance of occult axillary lymph node micrometastases from breast cancers. Lancet 1990; 335 (8705): 1565-8. 11. Berger U, Bettelheim R, Mansi JL et al. The relationship between micrometastases in the bone marrow, histopathologic features of the primary tumor in breast cancer and prognosis. Am J Clin Pathol 1988; 90: 1-6. 12. Jauch KW, Heiss MM, Gruetzner U et al. Prognostic significance of bone marrow micrometastases in patients with gastric cancer. J Clin Oncol 1996; 14: 1810-7. 13. Lindemann F, Schlimok G, Dirschedl P et al. Prognostic significance of micrometastatic tumour cells in bone marrow of colorectal cancer patients. Lancet. 1992; 340: 685-9. 14. Pantel K, Izbicki J, Passlick B et al. Frequency and prognostic significance of isolated tumour cells in bone marrow of patients with non-small-cell lung cancer without overt metastases. Lancet 1996 9; 347: 649-53. 15. Wood DP Jr, Banerjee M. Presence of circulating prostate cells in the bone marrow of patients undergoing radical prostatectomy is predictive of disease-free survival. J Clin Oncol 1997; 15: 3451-7. 16. Liotta LA, Saidel MG, Kleinerman J. The significance of hematogenous tumor-cell clumps in the metastatic process. Cancer Res 1976; 36: 889-94. 17. McCulloch P, Choy A, Martin L. Association between tumour angiogenesis and tumour cell shedding into effluent venous blood during breast cancer surgery. Lancet 1995; 346: 1334-5. 18. Fox SB, Leek RD, Bliss J et al. Association of tumor angiogenesis with bone marrow micrometastases in breast cancer patients. J Natl Cancer Inst 1997; 89: 1044-9. 19. Choy A, McCulloch P. Induction of tumour cell shedding into effluent venous blood breast cancer surgery. Br J Cancer 1996; 73: 79-82. 20. Weiss L, Dimitrov DS. Afluidmechanical analysis of the velocity, adhesion, and destruction of cancer cells in capillaries during metastasis. Cell Biophys 1984; 6 (1): 9-22. 21. Garcia-Olmo D, Ontanon J, Garcia-Olmo DC et al. Detection of genomically-tagged cancer cells in different tissues at different stages of tumor development: Lack of correlation with the formation of metastasis. Cancer Lett 1999; 140: 11-20. 22. Koike C, Watanabe M, Oku N et al. Tumor cells with organspecific metastatic ability show distinctive trafficking in vivo: Analyses by positron emission tomography and bioimaging. Cancer Res 1997; 57: 3612-9. 23. Tarin D, Price JE, Kettlewell MG et al. Mechanisms of human
24.
25. 26.
27.
28.
29. 30.
31.
32. 33. 34.
35. 36.
37.
38.
39. 40.
41. 42.
tumor metastasis studied in patients with peritoneovenous shunts. Cancer Res 1984; 44: 3584-92. Braun S, Kentenich C, Janni W et al. Lack of effect of adjuvant chemotherapy on the elimination of single dormant tumor cells in bone marrow of high-risk breast cancer patients. J Clin Oncol 2000; 18: 80-6. Pantel K, Schlimok G, Braun S et al. Differential expression of proliferation-associated molecules in individual micrometastatic carcinoma cells. J Natl Cancer Inst 1993; 85: 1419-24. Putz E, Witter K, Offner S et al. Phenotypic characteristics of cell lines derived from disseminated cancer cells in bone marrow of patients with solid epithelial tumors: Establishment of working models for human micrometastases. Cancer Res 1999; 59: 241-8. Pantel K, Schlimok G, (Cutter D et al. Frequency of downregulation of major histocompatibility class I antigen expression on individual micrometastatic carcinoma cells. Cancer Res 1991; 51: 4715. Heiss MM, Allgayer H, Gruetzner K.U et al. Individual development and uPA-receptor expression of disseminated tumour cells in bone marrow: A reference to early systemic disease in solid cancer. Nature Med 1995; 1: 1035-9. Pantel K, Schlimok G, Angstwurm M et al. Methodological analysis of immunocytochemical screening for disseminated epithelial tumor cells in bone marrow. J Hematother 1994; 3: 165-73. Jung R, Ahmad-Nejad P, Wimmer M et al. Quality management and influential factors for the detection of single metastatic cancer cells by reverse transcriptase polymerase chain reaction. Eur J Clin Chem Clin Biochem 1997; 35 (1): 3-10. Jonas S, Windeatt S, O-Boateng A et al. Identification of carcinoembryonic antigen-producing cells circulating in the blood of patients with colorectal carcinoma by reverse transcriptase polymerase chain reaction. Gut 1996; 39 (5): 717-21. Cote RJ, Rosen PP, Lesser ML et al. Prediction of early relapse in patients with operable breast cancer by detection of occult bone marrow micrometastases. J Clin Oncol 1991; 9 (10): 1749-56. Pasini F, Pelosi G, Verlato G et al. Positive immunostaining with MLuCl of bone marrow aspirate predicts poor outcome in patients with small-cell lung cancer. Ann Oncol 1998; 9 (2): 181-5. Weckermann D, Wawroschek F, Krawczak G et al. Does the immunocytochemical detection of epithelial cells in bone marrow (micrometastasis) influence the time to biochemical relapse after radical prostatectomy? Urol Res 1999; 27 (4): 285-90. Peck K, Sher YP, Shih JY et al. Detection and quantitation of circulating cancer cells in the peripheral blood of lung cancer patients. Cancer Res 1998; 58: 2761-5. Braun S, Hepp F, Sommer HL, Pantel K. Tumor-antigen heterogeneity of disseminated breast cancer cells: Implications for immunotherapy of minimal residual disease. Int J Cancer 1999; 84(1): 1-5. Canon JL, Humblet Y, Lebacq-Verheyden AM et al. Immunodetection of small-cell lung cancer metastases in bone marrow using three monoclonal antibodies. M Eur J Cancer Clin Oncol 1988; 24(2): 147-50. Marincola FM, Hijazi YM, Fetsch Pet al. Analysis of expression of the melanoma-associated antigens MART-1 and gplOO in metastatic melanoma cell lines and in situ lesions. J Immunother 1996; 19: 192-205. Qiu SD, Young CY. Bilhartz DL et al. In situ hybridization of prostate-specific antigen mRNA in human prostate. J Urol 1990; 144 (6): 1550-6. Young CY, Montgomery BT, Andrews PE et al. Hormonal regulation of prostate-specific antigen messenger RNA in human prostatic adenocarcinoma cell line LNCaP. Cancer Res 1991; 51: 3748-52. Fuller BB, Niekrasz I, Hoganson GE. Down-regulation of tyrosinase mRNA levels in melanoma cells by tumor promoters and by insulin. Mol Cell Endocrinol 1990; 72 (2): 81-7. Gollub EG, Waksman H, Goswami S, Marom Z. Mucin genes are regulated by estrogen and dexamethasone. Biochem Biophys Res Commun 1995; 217: 1006-14.
791 43. Forus A, Hoifodt HK, Overli GETet al. Sensitive fluorescent in situ hybridisation method for the characterisation of breast cancer cells in bone marrow aspirates. J Clin Path Mol Pathol 1999; 52: 68-74. 44. Kobayashi TK, Ueda M, Yamaki T, Yakushiji M. Evaluation of cytocentrifuge apparatus with special reference to the cellular recovery rate. Diagn Cytopathol 1992; 8: 420-3. 45. Franklin WA, Shpall EJ, Archer P et al. Immunocytochemical detection of breast cancer cells in marrow and peripheral blood of patients undergoing high-dose chemotherapy with autologous stem-cell support. Breast Cancer Res Treat 1996; 41: 1-13. 46. Lagrange M, Ferrero JM, Lagrange JL et al. Non-speciflcally labelled cells that simulate bone marrow metastases in patients with non-metastatic breast cancer. J Clin Pathol 1997; 50: 206-11. 47. BruggerW, Buhring HJ, Grunebach Fet al. Expression of MUC1 epitopes on normal bone marrow: Implications for the detection of micrometastatic tumor cells. J Clin Oncol 1999; 17 (5): 1535-44. 48. Delsol G, Gatter KC, Stein H et al. Human lymphoid cells express epithelial membrane antigen. Implications for diagnosis of human neoplasms. Lancet 1984; 2: 1124-9. 49. Doglioni C, DelFOrto P, Zanetti G et al. Cytokeratin-immunoreactive cells of human lymph nodes and spleen in normal and pathological conditions. An immunocytochemical study. Virch Arch A Pathol Anat Histopathol 1990; 416: 479-90. 50. Domagala W, Bedner E, Chosia M et al. Keratin-positive reticulum cells in fine needle aspirates and touch imprints of hyperplastic lymph nodes. A possible pitfall in the immunocytochemical diagnosis of metastatic carcinoma. Acta Cytol 1992; 36: 241-5. 51. Borgen E, Beiske K, Trachsel S et al. Immunocytochemical detection of isolated epithelial cells in bone marrow: Non-specific staining and contribution by plasma cells directly reactive to alkaline phosphatase. J Pathol 1998; 185: 427-34. 52 Brandt B, Junker R, Griwatz C et al. Isolation of prostate-derived single cells and cell clusters from human peripheral blood. Cancer Res 1996; 56: 4556-61. 53. Nicholson AG, Graham AN, Pezzella F et al. Does the use of immunohistochemistry to identify micrometastases provide useful information in the staging of node-negative non-small-cell lung carcinomas? Lung Cancer 1997; 18: 231-40. 54. Fisher CJ, Hill S, Millis RR. Benign lymph node inclusions mimicking metastatic carcinoma. J Clin Pathol 1994; 47: 245-7. 55. Yu LL, Flotte TJ, Tanabe KK et al. Detection of microscopic melanoma metastases in sentinel lymph nodes. Cancer 1999; 86: 617-27. 56. Naume B, Borgen E, Beiske K et al. Immunomagnetic techniques for the enrichment and detection of isolated breast carcinoma cells in bone marrow and peripheral blood. J Hematother 1997; 6: 103-14. 57. Mesker WE, v d Burg JM, Oud PS et al. Detection of immunocytochemically stained rare events using image analysis. Cytometry 1994; 17: 209-15. 58. Mignon-Godefroy K, Guillet JG, Butor C. Solid phase cytometry for detection of rare events. Cytometry 1997; 27: 336-44. 59. Hibi K, Robinson CR, Booker S et al. Molecular detection of genetic alterations in the serum of colorectal cancer patients. Cancer Res 1998; 58: 1405-7. 60. Chiang PW, Beer DG, Wei WL et al. Detection of erbB-2 amplifications in tumors and sera from esophageal carcinoma patients. Clin Cancer Res 1999; 5: 1381-6. 61. Yamamoto N, Kato Y, Yanagisawa A et al. Predictive value of genetic diagnosis for cancer micrometastasis: Histologic and experimental appraisal. Cancer 1997; 80: 1393-8. 62. Zippelius A, Kufer P, Honold G et al. Limitations of reversetranscriptase polymerase chain reaction analyses for detection of micrometastatic epithelial cancer cells in bone marrow. J Clin Oncol 1997; 15: 2701-8. 63. Yun K, Gunn J, Merrie AE et al. Keratin 19 mRNA is detectable by RT-PCR in lymph nodes of patients without breast cancer. Br J Cancer 1997; 76 (8): 1112-3.
64. Krismann M, Todt B, Schroder J et al. Low specificity of cytokeratin 19 reverse transcriptase-polymerase chain reaction analyses for detection of hematogenous lung cancer dissemination. J Clin Oncol 1995; 13: 2769-75. 65. Thiounn N, Saporta F, Flam TA et al. Positive prostate-specific antigen circulating cells detected by reverse transcriptasepolymerase chain reaction does not imply the presence of prostatic micrometastases. Urology 1997; 50: 245-50. 66. Gala JL, Heuslerspreute M, Loric S et al. Expression of prostatespecific antigen and prostate-specific membrane antigen transcripts in blood cells: Implications for the detection of hematogenous prostate cells and standardization. Clin Chem 1998; 44: 472-81. 67. 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 1999; 24: 76975. 68. Lintula S, Stenman UH. The expression of prostate-specific membrane antigen in peripheral blood leukocytes. J Urol 1997; 157: 1969-72. 69. Dent GA, Civalier CJ, Brecher ME, Bentley SA. MUC1 expression in hematopoietic tissues. Am J Clin Pathol 1999; 111: 741-7. 70. Kwok S, Higuchi R. Avoiding false positives with PCR. Nature 1989; 339: 237-8. 71. Chelly J, Concordet JP, Kaplan JC, Kahn A. Illegitimate transcription: Transcription of any gene in any cell type. Proc Natl Acad Sci USA 1989; 86: 2617-21. 72. De Graaf H, Maelandsmo GM, Ruud P et al. Ectopic expression of target genes may represent an inherent limitation of RT-PCR assays used for micrometastasis detection: Studies on the epithelial glycoprotein gene EGP-2. Int J Cancer 1997; 72: 191-6. 73. Traweek ST, Liu J, Battifora H. Keratin gene expression in nonepithelial tissues. Detection with polymerase chain reaction. Am J Pathol 1993; 142(4): 1111-8. 74. Jung R, Kriiger 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 1998; 78: 1194-8. 75. Yu H, Diamandis EP, Zarghami N, Grass L. Induction of prostate specific antigen production by steroids and tamoxifen in breast cancer cell lines. Breast Cancer Res Treat 1994; 32 (3): 291-300. 76. Kippenberger S, Loitsch S, Solano F et al. Quantification of tyrosinase, TRP-1, and TRP-2 transcripts in human melanocytes by reverse transcriptase-competitive multiplex PCR-regulation by steroid hormones. J Invest Dermatol 1998; 110: 364-7. 77. Neumaier M, Gerhard M, Wagener C. Diagnosis of micrometastases by the amplification of tissue-specific genes. Gene 1995; 159 (1): 43-7. 78. Ruud P, Fodstad O, Hovig E. Identification of a novel cytokeratin 19 pseudogene that may interfere with reverse transcriptasepolymerase chain reaction assays used to detect micrometastatic tumor cells. Int J Cancer 1999; 80 (1): 119-25. 79. Metze D, Bhardwaj R, Amann U et al. Glycoproteins of the carcinoembryonic antigen (CEA) family are expressed in sweat and sebaceous glands of human fetal and adult skin. J Invest Dermatol 1996; 106 (1): 64-9. 80. Battyani Z, Xerri L, Hassoun J et al. Tyrosinase gene expression in human tissues. Pigment Cell Res 1993; 6 (6): 400-5. 81. Jonas SK, Klokouzas A, Wharton RQ, AIlen-Mersch TG. Identification of circulating tumour cells by RT-PCR in patients with colorectal cancer: Detection prior to evidence of metastasis. Proc Am Assoc Cancer Res 1997; 38: 19 (Abstr O12). 82. Eschwege P, Dumas F, Blanchet P et al. Haematogenous dissemination of prostatic epithelial cells during radical prostatectomy. Lancet 1995; 346 (8989): 1528-30. 83. Weitz J, Kienle P, Lacroix J et al. Dissemination of tumor cells in patients undergoing surgery for colorectal cancer. Clin Cancer Res 1998; 4 (2): 343-8.
792 84. Kopreski MS, Benko FA, Kwak LW, Gocke CD. Detection of tumor messenger RNA in the serum of patients with malignant melanoma. Clin Cancer Res 1999; 5: 1961-5. 85. Schoenfeld A, Luqmani Y, Smith D et al. Detection of breast cancer micrometastases in axillary lymph nodes by using polymerase chain reaction. Cancer Res 1994; 54: 2986-90. 86. Keilholz U, Willhauck M, Rimoldi D et al. Reliability of reverse transcription-polymerase chain reaction (RT-PCR)-based assays for the detection of circulating tumour cells: A quality-assurance initiative of the EORTC Melanoma Cooperative Group. Eur J Cancer 1998; 34: 750-3. 87. Schittek B, Blaheta HJ, Florchinger G et al. Increased sensitivity for the detection of malignant melanoma cells in peripheral blood using an improved protocol for reverse transcription-polymerase chain reaction. Br J Dermatol 1999; 141: 37-43. 88. 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 1998; 4: 2141-6. 89. 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 1999; 17: 870-9. 90. Orlando C, Pinzani P, Pazzagli M. Developments in quantitative PCR. Clin Chem Lab Med 1998; 36: 255-69. 91. Cheung IY, Barber D, Cheung NK. Detection of microscopic neuroblastoma in marrow by histology, immunocytology, and reverse transcription-PCR of multiple molecular markers. Clin Cancer Res 1998; 4: 2801-5.
92. Palmien G, Strazzullo M, Ascierto P et al. Polymerase chain reaction-based detection of circulating melanoma cells as an effective marker of tumor progression. J Clin Oncol 1999; 17: 304-11. 93. Min CJ, Tafra L, Verbanac KM. Identification of superior markers for polymerase chain reaction detection of breast cancer metastases in sentinel lymph nodes. Cancer Res 1998; 58: 4581^1. 94. McKJernan JM, Buttyan R, Bander NH et al. The detection of renal carcinoma cells in the peripheral blood with an enhanced reverse transcriptase-polymerase chain reaction assay for MN/CA9. Cancer 1999; 86: 492-7. 95. Makarovskiy AN, Ackerley W, Wojcik L et al. Application of immunomagnetic beads in combination with RT-PCR for the detection of circulating prostate cancer cells. J Clin Lab Anal 1997; 11:346-50. Received 5 January 2000; accepted 21 March 2000.
Correspondence to: J.-C. Goeminne, MD Service d'Oncologie Medicale Institut Gustave Roussy 39, rue Camille Desmoulins 94805 Villejuif France E-mail: [email protected]