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Increased Risk of Systemic Relapses Associated With Bone Marrow Micrometastasis and Circulating Tumor Cells in Localized Ewing Tumor

Gudrun Schleiermacher, Martine Peter, Odile Oberlin, Thierry Philip, Hervé Rubie, Françoise Mechinaud, Danièle Sommelet-Olive, Judith Landman-Parker, Danièle Bours, Jean Michon, Olivier Delattre for the Société Française d’Oncologie Pédiatrique

From the Laboratoire de Pathologie Moléculaire des Cancers, INSERM U 509, Section de Recherche, and Laboratoire de Transfert, and Unité de Biostatistique, and Département d’Oncologie Pédiatrique, Institut Curie, and Service d’Hémato-Oncologie Pédiatrique, Hôpital Trousseau, Paris; Département d’Oncologie Pédiatrique, Institut Gustave Roussy, Villejuif; Service de Pédiatrie, Centre Léon Bérard, Lyon; Service d’Hématologie Oncologie, Hôpital des Enfants, Toulouse; Service d’Oncologie Pédiatrique, CHU de Nantes, Nantes; and Service de Médecine Infantile, Hôpital des Enfants, Vanduvre, France.

Address reprint requests to Olivier Delattre, MD, PhD, Laboratoire de Pathologie Moléculaire des Cancers, L’Institut National de la Santé et de la Recherche Médicale, U 509, 26 rue d’Ulm, 75248 Paris Cedex 05, France; email: delattre{at}curie.fr.

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Purpose: The presence of metastasis is a major prognostic factor in Ewing tumor (ET). The relapse pattern of patients with localized tumors has long indicated that cases with disseminated ET cells escape detection at diagnosis. ET cells are characterized by specific gene fusions that can be detected with high sensitivity and specificity by reverse transcriptase polymerase chain reaction (RT-PCR).

Patients and Methods: RT-PCR targeting EWS-FLI-1 or EWS-ERG transcripts was used to search for occult tumor cells in peripheral blood (PB) and bone marrow (BM) at diagnosis in 172 patients with ET, and the prognostic significance of this parameter was assessed.

Results: As we suggested previously in a smaller series of patients, RT-PCR positivity of the BM was correlated with a high risk of adverse outcome in the overall study population (P = .007). More interestingly, among patients with otherwise localized tumors, BM micrometastasis also predicted significantly poorer disease-free survival rates (P = .043). The presence of circulating tumor cells (CTC) was more frequently observed in patients with large tumors (P = .006). CTC were associated with a poor outcome among patients with clinically localized disease (P = .045). Patients with clinically localized disease and peripheral occult tumor cells as evidenced by BM and/or PB RT-PCR positivity had axial or proximal tumors and experienced relapses at a systemic rather than at a local level.

Conclusion: Patients with localized ET and BM micrometastasis or CTC are comparable to patients with metastases in terms of the localization of the primary tumor, outcome, and relapse pattern.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
TUMORS OF the Ewing family are undifferentiated malignancies of childhood and early adulthood, the prognosis of which is highly dependent on the presence of dissemination at diagnosis. Indeed, although overall survival of patients with localized Ewing tumors (ETs) can be estimated at 60% to 80% after multimodal therapy, outcome for patients with evidence of metastasis at diagnosis remains much poorer.¹

At diagnosis, 20% to 30% of patients have evidence of metastasis that involves mainly the lung, bone, and bone marrow through a hematogenous spread. These patients have a high risk of metastatic relapse, resulting frequently in fatal outcomes. Such relapses can also occur in clinically localized forms of ET, despite a good local control, and so far, they cannot be efficiently predicted by any prognostic factors. This observation has led to the hypothesis that micrometastases, present at diagnosis but undetectable by conventional methods, may not be eradicated by current treatment approaches and, thus, lead to distant recurrences.

ET cells are characterized by a specific tumor cell marker consisting, in most cases, of a chromosomal translocation between chromosome 22 and 11 or, less frequently, between chromosome 22 and chromosome 21, 7, 17, or 2, leading to the fusion of the EWS gene to members of the ETS oncogene family.^2–10 These translocations are observed in 95% to 98% of ET cases.¹¹ The resulting fusion genes are transcribed in tumor cells, and the fusion transcripts can readily be detected using the reverse transcriptase polymerase chain reaction (RT-PCR) technique. Indeed, RT-PCR has been used to search for tumor cells in bone marrow (BM) and peripheral blood (PB) with a much higher sensitivity than conventional cytological analysis, as one tumor cell per one million background cells can be detected by this approach.^12–18 The studies using this technique have shown that 20% to 30% of ET patients have PB or BM tumor cells at diagnosis.^12–16,18 In a series of 62 patients, we previously observed that RT-PCR positivity in the BM was significantly associated with the presence of clinical metastasis and was a prognostic factor in univariate analysis, an observation that indicated that RT-PCR evaluation of BM might constitute an important criterion for staging.¹⁸ However, until recently, the prognostic significance of tumor cells detected in BM or PB at diagnosis in patients with otherwise localized ET could not be investigated, given the low numbers of patients included in the reported series.

We have now extended RT-PCR analysis of PB and BM to 172 patients with ET, and we report on the follow-up of these patients with regard to the extent of the disease and the presence or absence of tumor cells in PB and BM.

	PATIENTS AND METHODS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Patients
The study population consisted of patients treated for ET at French centers between 1993 and 1999. Only patients with an unequivocal histologic diagnosis of ET were included. Local extension was evaluated by standard radiography and magnetic resonance imaging. The volume of the tumor was calculated on computed tomographic scans or magnetic resonance imaging using previously described methods.¹⁹ The search for metastasis included pulmonary computed tomographic scans, bone scans with technetium-99, BM aspirates (obtained from up to 10 different sites), and trephine biopsies. The BM was evaluated only at sites different from that of the primary tumor.

Altogether, 172 patients, including 64 previously described cases, were studied.¹⁸ Median age of this population was 12 years (range, 1 to 56 years). There were 100 males and 72 females. Follow-up ranged from 7 to 97 months, with a median of 27 months. Forty-seven patients had evidence of metastasis at diagnosis: 20 had only lung metastasis, 13 had bone and/or BM involvement, and 14 had a combination of lung and either bone or BM metastases.

A total of 140 patients were included in the protocols of the French Society of Pediatric Oncology (SFOP EW 93 and EW 97, the latter being identical to the former but renamed after its approval by the local ethics committee). Informed consent was obtained for all patients included in the definitive version. The number of patients reported in this study corresponds to approximately 30% of all patients included in these protocols. For localized disease, these protocols included induction treatment using doxorubicin and cyclophosphamide. Histopathologic response was evaluated at surgery through determination of the percentage of viable tumor cells on the surgical specimen. Good responders, defined by a percentage of viable tumor cells lower than 10%, received a maintenance treatment with vincristine and dactinomycin in addition to the aforementioned drugs. In cases of metastatic disease, treatment included induction therapy with cyclophosphamide, doxorubicin, ifosfamide, and etoposide followed by a myeloablative chemotherapy using busulfan/melphalan with autologous stem cell or BM reinjection. The same intensive regimen was applied for localized tumors with a poor histologic response to induction therapy. Indication of radiotherapy was also stratified according to histologic response.

Sixteen young adult patients were treated according to the aforementioned EW 93 and EW 97 protocols without being included in the study. Finally, another 16 patients were treated with adult protocols that used the same drugs but with slightly different schedules.

The search for tumor cells in PB or BM samples was performed as a retrospective study of blood and BM samples used for the initial staging of patients and required no additional sampling. In no instance did the results influence therapeutic decisions. According to French law at the time of the study, no informed consent was required for the retrospective analysis of archived biologic material.

Molecular Analysis
The search for specific fusion transcripts between the EWS and FLI-1 or ERG genes in the primary tumor was performed for the 117 cases for which adequate tissue was available, according to techniques published previously.¹¹ Cases with fusions involving the ETV1, E1AF, or FEV genes were not included in this study. Detection of Ewing cells in 144 PB or 131 BM samples, obtained at diagnosis, was performed by RT-PCR targeting EWS-FLI-1- or EWS-ERG-specific transcripts as described previously.^12,18 In brief, PB or pooled BM samples from different sites were shipped to the laboratory at room temperature, and nucleated cells were isolated within 24 hours. Isolation of RNA, evaluation of its quality, reverse transcription, and nested PCR in a single tube were performed according to previously described procedures.^12,18 When known from the analysis of the primary tumor, the relevant fusion transcript was specifically targeted in peripheral sites. Otherwise, both EWS-FLI-1 and EWS-ERG transcripts were sought.

Statistical Analysis
Associations between clinical and molecular data were searched for using the ² method or Fisher’s exact test when necessary. Overall survival (OS) and disease-free survival (DFS) rates were computed from the date that treatment commenced. The survival curves were calculated using the Kaplan-Meier method, and the difference between survival curves was analyzed using the log-rank test. All tests were two-sided, and a P value of less than 0.05 was considered to indicate statistical significance. When multiple tests were performed, a correction taking into account the number of tests was applied to maintain an overall type I error rate of 0.05, thus changing the threshold that allows conclusion of statistical significance (the Bonferroni correction).²⁰ A stratified log-rank test was used to adjust for the molecular biology results on the disease status (metastatic or localized) of the patients.

	RESULTS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
In this study, an RT-PCR assay previously shown to enable reproducibly the detection of one Ewing cell per million leukocytes was used to detect tumor cells in PB and BM at diagnosis in 172 patients with ET. For this population, the 2-year DFS and OS rates (± 2 SE) were 71% (± 7.4%) and 74% (± 7.2%), respectively. For patients with metastatic and localized disease, 2-year DFS was 54% (± 16.2%) and 77% (± 8%), respectively, which did not differ significantly from the DFS observed for all patients included in the EW 93 protocol, indicating the absence of bias in the selection of patients entered in the molecular study.

Analysis of BM by RT-PCR revealed 36 of 131 patients (27%) who scored positive (Table 1). Positive results were obtained in 18 of 39 metastatic patients (46%) versus 18 of 92 nonmetastatic patients (19%), indicating that RT-PCR positivity in the BM was strongly associated with the presence of clinical metastases (P = .0018, ² test; Tables 1 and 2). As expected, the 12 patients with cytologic evidence of BM metastasis who could be analyzed by molecular methods also scored positive by RT-PCR in the BM. Among metastatic patients without cytologic evidence of BM involvement, RT-PCR was positive in the BM of three of eight patients with bone and three of 18 patients with lung metastasis, indicating that in patients without cytologic evidence of BM involvement, the frequency of BM RT-PCR positivity is similar to that observed in patients with clinically localized disease. RT-PCR positivity of BM was not associated with the volume of the primary tumor nor with the histologic response to primary chemotherapy (Table 2).

Univariate analysis of the whole study population showed that RT-PCR positivity in BM predicted a poorer DFS rate (P = .0003, log-rank test). Because this prognostic significance might only reflect the poor outcome of patients with metastatic tumors, who frequently have RT-PCR-positive BM, we performed a stratified log-rank test to adjust for the metastatic status of patients (P = .007, stratified log-rank; Table 2, Fig 1A) and further studied the follow-up of the 92 patients with clinically localized tumors whose BM was analyzed. Within this group, the 18 patients scoring positive by RT-PCR in BM are described in Table 3 and will be referred to as patients with BM micrometastasis. Patients with clinically localized disease and BM RT-PCR negativity are considered strictly localized cases.

View larger version (14K): [in this window] [in a new window]   Fig 1. Prognostic significance of bone marrow (BM) micrometastasis and circulating tumor cells in Ewing tumors (ET). Disease-free survival (DFS) of all patients (A) and of patients with clinically localized ET (B) according to RT-PCR positivity/negativity in BM. (C) DFS of patients with clinically localized ET according to RT-PCR positivity/negativity in peripheral blood.

Univariate analysis revealed that, in the overall population, the presence of CTC was not associated with a poorer DFS, as previously observed (P = .1, log-rank test; Table 2). However, of particular interest were the 107 patients with clinically localized disease whose PB was analyzed by RT-PCR. The 21 patients with clinically localized disease and PB RT-PCR positivity are described in detail in Table 4 and will be referred to as localized cases with CTC. Among localized cases with CTC, 12 of 21 have relapsed, versus 23 of 86 localized cases without CTC. The 2-year DFS (± 2 SE) was 59% (± 22%) for the PB RT-PCR-positive versus 79% (± 9.2%) for the PB RT-PCR-negative cases, with no significant difference between median follow-up for the two groups of patients (29 months for the CTC-positive group and 27 months for the localized group; P = .4). Thus, among the patients with clinically localized disease, the presence of CTC was associated with an increased risk of relapse (P = .045, log-rank-test; Fig 1C).

Concerning the localization of the primary tumor, 23% of the patients from the strictly localized group had distal tumors, compared with none of the patients from the BM micrometastatic or CTC group and 6% from the metastatic group. This indicates that the localization of the primary tumor to the distal limbs is extremely rare in patients with peripheral occult tumor cells, compared with the strictly localized group (P = .008, ² test; Table 5).

Patients with peripheral occult tumor cells thus share characteristics of patients with metastatic disease with regard to tumor localization and relapse pattern as well as outcome.

	DISCUSSION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
In this study, tumor cells were detected at diagnosis in BM and PB of patients with ET using an RT-PCR assay targeting the ET-specific EWS-FLI or EWS-ERG fusion transcripts, and their significance was investigated. These transcripts could be identified in the tumors of 117 patients for whom the primary site could be analyzed. In 55 patients, no material was available for the study of this site. Both EWS-FLI-1 and EWS-ERG transcripts were sought in the PB and/or BM of these patients. We cannot exclude the possibility of false-negative results caused by the absence of EWS-FLI-1 and EWS-ERG transcripts in the tumor cells of these patients. However, these cases are expected to be rare for the following reasons: EWS-FLI-1 or EWS-ERG transcripts have been shown to be present in more than 95% of ETs with typical histology,¹¹ and only cases with such a characteristic histology were included in this study; a recently published study of a series of 215 patients with ET indicated that only three cases (1.5%) exhibited variant transcripts different from the EWS-FLI-1 or EWS-ERG transcripts targeted in the present study;²¹ and finally, a similar proportion of positive PB or BM was observed in the group of 55 patients without analysis of the primary site compared with the 117 patients for whom primary tumor tissue was available (18 positive PB or BM samples out of 88 analyzed samples in the first group [20%] compared with 47 of 187 [25%] in the second group [P = .6, ² test]).

The frequency of BM RT-PCR-positive cases in the overall population (25%) and in clinically nonmetastatic patients (17%) was similar to that described previously.^14,16 As reported, BM RT-PCR positivity was of strong prognostic significance in the overall population.¹⁸ In this study, patients with clinically localized disease and with BM micrometastasis also experienced a poorer outcome. The observation that seven of the nine BM micrometastatic patients who relapsed had good local response to treatment strongly indicates that the adverse evolution of BM-micrometastatic patients is linked to progression of micrometastatic sites rather than to poor local control.

The overall frequency of CTC was 19% and was thus comparable to that previously reported in several studies.^13,16 The observation that CTC are more frequently observed in large tumors supports the hypothesis that the presence of CTC might reflect the overall tumor burden, in itself an indicator of poor prognosis. The prognostic significance of CTC among patients with clinically localized disease may possibly be explained by their association with large tumor volumes, but the numbers in this series of patients were too low to enable reliable multivariate analysis.

In this study, it could be shown that patients with localized disease and peripheral occult tumor cells as evidenced by BM micrometastasis or CTC share several important characteristics with metastatic patients. First, the localization of the primary tumor is most frequently axial or proximal and almost never distal. Second, these patients exhibit a poor outcome linked to a higher frequency of relapses, as evidenced by a poorer DFS. Third, these relapses occur predominantly at a systemic level. These findings are consistent with a previously published study,¹⁴ in which, among seven patients with clinically localized disease and with BM micrometastasis, relapses have been observed for three cases, two of which occurred at systemic levels. However, the follow-up of our study does not enable determination of whether CTC or BM micrometastasis will also influence the frequency of late recurrences, which are commonly observed in ET.²²

These observations strongly support the idea that peripheral occult tumor cells, present at the time of diagnosis, may not be eradicated by current treatment approaches and lead to tumor recurrences. The search for occult tumor cells should thus be included in the staging of patients with ET at diagnosis.

The observation that relapses at a systemic level were also observed in a subset of patients with localized ET without evidence of tumor cell dissemination and good local response to chemotherapy indicates that some disseminated tumor cells still escape detection by the present technique, the sensitivity of which should be increased. Indeed, false-negative results may be linked to the presence of a number of tumor cells below the limit of sensitivity of this technique, to focal BM invasions not included in the sampled sites, or to transitory shedding of tumor cells into the peripheral circulation. Optimization could therefore rely on an increase of the template RNA used for RT-PCR, possibly using polyadenylated RNA instead of total RNA, on an increase in the number of BM and PB samples tested, or on the use of real-time PCR.^21,23

At present, there is considerable interest in the analysis of minimal metastatic disease in solid tumors based on a variety of technical approaches, including immunohistochemistry and molecular methods using RT-PCR.^24,25 Although preliminary reports indicate that BM micrometastasis might be a prognostic marker in a number of malignancies such as breast cancer, colorectal cancer, prostatic cancer, or malignant melanoma^26–37 and that CTC have been suggested to be of prognostic significance in some cancer types such as malignant melanoma or prostatic cancer,^31,38,39 a precise clinical evaluation of this parameter is hampered, in most cancers, by the lack of tumor cell–specific markers. In this respect, some pediatric cancers, including ET and alveolar rhabdomyosarcoma, constitute interesting models inasmuch as they are characterized by specific chromosome translocations that can be detected with a high sensitivity and specificity by RT-PCR.⁴⁰ The present observation that peripheral occult tumor cells in patients with ET with clinically localized disease harbor a pejorative prognostic significance should therefore stimulate research on occult tumor cells in other solid tumors.

	ACKNOWLEDGMENTS

 
We thank D. Amsallem, A. Babin-Boilletot, B. Bui N’guyen, P. Boutard, L. Brugières, P. Chastagner, V. Combaret, C. Coze, F. Demeocq, A. Deville, G. Leverger, G. Marguerite, O. Minckes, F. Millot, J. Otten, Y. Perel, D. Plantaz, E. Plouvier, P. Pouillard, D. Ranchere-Vince, C. Schmitt, J.L. Stephan, M.D. Tabone, and J.P. Vannier for providing samples used in this study.

We thank V. Mosseri and B. Asselain for their help in the statistical analysis of data, and E. Gilbert for technical assistance.

	NOTES

 
Supported in part by grants from the Programme Hospitalier de Recherche Clinique, the Plateforme de Soutien au Transfert et à la Recherche Clinique, the Association pour la Recherche contre le Cancer, the Caisse Nationale d’Assurance Maladie des Travailleurs Salariés, the Institut Curie, and the Institut National de la Santé et de la Recherche Médicale, Paris, France.

	REFERENCES

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
1. Horowitz ME, Malawer MM, Woo SY, et al: Ewing’s sarcoma family of tumors: Ewing’s sarcoma of bone and soft tissue and the peripheral primitive neuroectodermal tumors, in Pizzo PA, Poplack DG (eds): Principles and Practice of Pediatric Oncology (ed 3). Philadelphia, PA, Lippincott-Raven, 1997, pp 831–864

2. Aurias A, Rimbaut A, Buffe D, et al: Chromosomal translocation in Ewing’s sarcoma. N Engl J Med 309:496–497, 1983[Medline]

3. Turc-Carel C, Philip I, Berger MP, et al: Chromosomal translocations in Ewing’s sarcoma. N Engl J Med 309:497–498, 1983

4. Delattre O, Zucman J, Plougastel B, et al: Gene fusion with an ETS DNA-binding domain caused by chromosome translocation in human tumours. Nature 359:162–165, 1992[CrossRef][Medline]

5. Zucman J, Melot T, Desmaze C, et al: Combinatorial generation of variable fusion proteins in the Ewing family of tumours. EMBO J 12:4481–4487, 1993[Medline]

6. Sorensen PH, Lessnick SL, Lopez-Terrada D, et al: A second Ewing’s sarcoma translocation, t(21;22), fuses the EWS gene to another ETS-family transcription factor, ERG. Nat Genet 6:146–151, 1994[CrossRef][Medline]

7. Jeon IS, Davis JN, Braun BS, et al: A variant Ewing’s sarcoma translocation (7;22) fuses the EWS gene to the ETS gene ETV1. Oncogene 10:1229–1234, 1995[Medline]

8. Kaneko Y, Yoshida K, Handa M, et al: Fusion of an ETS-family gene, E1AF, to EWS by t(17;22)(q12;q12) chromosome translocation in an undifferentiated sarcoma of infancy. Genes Chromosom Cancer 15:115–121, 1996[CrossRef][Medline]

9. Peter M, Couturier J, Pacquement H, et al: A new member of the Ets family fused to EWS in Ewing tumors. Oncogene 14:115–121, 1997[CrossRef][Medline]

10. Kovar H: Ewing’s sarcoma and peripheral primitive neuroectodermal tumors after their genetic union. Curr Opin Oncol 10:334–342, 1998[Medline]

11. Delattre O, Zucman J, Melot T, et al: The Ewing family of tumors—a subgroup of small round-cell tumors defined by specific chimeric transcripts. N Engl J Med 331:294–299, 1994[Abstract/Free Full Text]

12. Peter M, Magdelenat H, Michon J, et al: Sensitive detection of occult Ewing’s cells by the reverse-transcriptase polymerase chain reaction. Br J Cancer 72:96–100, 1995[Medline]

13. Pfleiderer C, Zoubek A, Gruber B, et al: Detection of tumor cells in peripheral blood and bone marrow from Ewing tumor patients by RT-PCR. Int J Cancer 64:135–139, 1995[Medline]

14. Zoubek A, Ladenstein R, Windhager R, et al: Predictive potential of testing for bone marrow involvement in Ewing tumor patients by RT-PCR: A preliminary evaluation. Int J Cancer 79:56–60, 1998[CrossRef][Medline]

15. Toretsky JA, Neckers L, Wexler LH: Detection of (11;22)(q24;q12) translocation-bearing cells in peripheral blood progenitor cells of patients with Ewing’s sarcoma family of tumors. J Natl Cancer Inst 87:385–386, 1995[Free Full Text]

16. West DC, Grier HE, Swallow MM, et al: Detection of circulating tumor cells in patients with Ewing’s sarcoma and peripheral primitive neuroectodermal tumor. J Clin Oncol 15:583–588, 1997[Abstract/Free Full Text]

17. De Alava E, Lozano MD, Patino A, et al: Ewing family tumors: Potential prognostic value of reverse-transcriptase polymerase chain reaction detection of minimal residual disease in peripheral blood samples. Diagn Mol Pathol 7:52–157, 1998

18. Fagnou C, Michon J, Peter M, et al: Presence of tumor cells in bone marrow but not in blood is associated with adverse prognosis in patients with Ewing’s tumor. J Clin Oncol 16:1707–1711, 1998[Abstract]

19. Gobel V, Jurgens H, Etspuler G, et al: Prognostic significance of tumor volume in localized Ewing’s sarcoma of bone in children and adolescents. J Cancer Res Clin Oncol 113:187–191, 1987[CrossRef][Medline]

20. Hoschberg Y: A sharper Bonferroni procedure for multiple tests of significance. Biometrika 75:800–802, 1988[Abstract/Free Full Text]

21. Peter M, Gilbert E, Delattre O: A multiplex real time PCR assay for the detection of gene fusions observed in solid tumors. Lab Invest 81:905–912, 2001[Medline]

22. Horowitz ME, Kinsella TJ, Wexler LH, et al: Total-body irradiation and autologous bone marrow transplant in the treatment of high-risk Ewing’s sarcoma and rhabdomyosarcoma. J Clin Oncol 11:1911–1918, 1993[Abstract/Free Full Text]

23. Merino ME, Navid F, Christensen BL, et al: Immunomagnetic purging of Ewing’s sarcoma from blood and bone marrow: Quantitation by real-time polymerase chain reaction. J Clin Oncol 19:3649–3659, 2001[Abstract/Free Full Text]

24. Pantel K, Cote RJ, Fodstad O: Detection and clinical importance of micrometastatic disease. J Natl Cancer Inst 91:1113–1124, 1999[Abstract/Free Full Text]

25. Ghossein RA, Bhattacharya S: Molecular detection and characterisation of circulating tumor cells and micrometastasis in solid tumors. Eur J Cancer 36:1681–1694, 2000[CrossRef][Medline]

26. Braun S, Pantel K, Müller P, et al: Cytokeratin-positive cells in the bone marrow and survival of patients with stage I, II or III breast cancer. N Engl J Med 342:525–533, 2000[Abstract/Free Full Text]

27. Corey E, Corey MJ: Detection of disseminated prostate cells by reverse transcription-polymerase chain reaction (RT-PCR): Technical and clinical aspects. Int J Cancer 77:655–673, 1998[CrossRef][Medline]

28. Deguchi T, Yang M, Ehara H, et al: Detection of micrometastatic prostate cancer cells in the bone marrow of patients with prostate cancer. Br J Cancer 75:634–638, 1997[Medline]

29. Diel IJ, Kaufmann M, Goerner R, et al: Detection of tumor cells in bone marrow of patients with primary breast cancer: A prognostic factor for distant metastasis. J Clin Oncol 10:1534–1539, 1992[Abstract/Free Full Text]

30. Fields KK, Elfenbein GJ, Trudeau WL, et al: Clinical significance of bone marrow metastases as detected using the polymerase chain reaction in patients with breast cancer undergoing high-dose chemotherapy and autologous bone marrow transplantation. J Clin Oncol 14:1868–1876, 1996[Abstract/Free Full Text]

31. Ghossein RA, Coit D, Brennan M, et al: Prognostic significance of peripheral blood and bone marrow tyrosinase messenger RNA in malignant melanoma. Clin Cancer Res 4:419–428, 1998[Abstract/Free Full Text]

32. Lindemann F, Schlimok G, Dirschedl P, et al: Prognostic significance of micrometastatic tumour cells in bone marrow of colorectal cancer patients. Lancet 340:685–689, 1992[CrossRef][Medline]

33. Okegawa T, Nutahara K, Higashihara E: Preoperative nested reverse transcriptase polymerase chain reaction for prostatic specific membrane antigen predicts biochemical recurrence after radical prostatectomy. Br J Urol 84:112–117, 1999

34. Schoenfeld A, Kruger KH, Gomm J, et al: The detection of micrometastasis in the peripheral blood and bone marrow of patients with breast cancer using immunohistochemistry and reverse transcriptase polymerase chain reaction for keratin 19. Eur J Cancer 33:854–861, 1997[CrossRef][Medline]

35. Soeth E, Röder C, Juhl H, et al: The detection of disseminated tumor cells in bone marrow from colorectal-cancer patients by a cytokeratin-20-specific nested reverse transcriptase polymerase chain reaction is related to the stage of disease. Int J Cancer 69:278–282, 1996[CrossRef][Medline]

36. Weitz J, Kienle P, Magener A, et al: Detection of disseminated colorectal cancer cells in lymph nodes, blood and bone marrow. Clin Cancer Res 5:1830–1836, 1999[Abstract/Free Full Text]

37. Wood DP Jr, Banks ER, Humphreys S, et al: Identification of bone marrow micrometastases in patients with prostate cancer. Cancer 74:2533–2540, 1994[CrossRef][Medline]

38. Mellado B, Gutierrea L, Castel T, et al: Prognostic significance of the detection of circulating malignant cells by reverse transcriptase polymerase chain reaction in long-term clinically disease-free melanoma patients. Clin Cancer Res 5:1843–1848, 1999[Abstract/Free Full Text]

39. Mejean A, Vona G, Nalpas B, et al: Detection of circulating prostate derived cells in patients with prostate adenocarcinoma is an independent risk for tumor recurrence. J Urol 163:2022–2029, 2000[CrossRef][Medline]

40. Kelly KM, Womer RB, Barr FG: Minimal disease detection in patients with alveolar rhabdomyosarcoma using a reverse transcriptase polymerase chain reaction method. Cancer 78:1320–1327, 1996[CrossRef][Medline]

Submitted March 2, 2001; accepted August 28, 2002.

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				A. Ohali, S. Avigad, I.J. Cohen, I. Meller, Y. Kollender, J. Issakov, I. Gelernter, Y. Goshen, I. Yaniv, and R. Zaizov Association Between Telomerase Activity and Outcome in Patients With Nonmetastatic Ewing Family of Tumors J. Clin. Oncol., October 15, 2003; 21(20): 3836 - 3843. [Abstract] [Full Text] [PDF]

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