- Email: [email protected]
Detection of circulating testicular cancer cells in peripheral blood
Cancer Letters 143 (1999) 57±62
Detection of circulating testicular cancer cells in peripheral blood Takeshi Yuasa a, Tatsuhiro Yoshiki a,*, Tsutomu Tanaka a, Takahiro Isono b, Yusaku Okada a b
a Department of Urology, Shiga University of Medical Science, Otsu, Japan Central Research Laboratory, Shiga University of Medical Science, Seta, Otsu 520-2192 , Japan
Received 17 February 1999; received in revised form 6 May 1999; accepted 6 May 1999
Abstract Patients who receive peripheral blood stem cell transplants are at risk of developing cancer recurrence due to the presence of malignant cells in the transplants. We investigated a sensitive method to detect malignant cells in the peripheral blood and peripheral blood stem cells of patients with testicular cancer using nested, reverse transcription-polymerase chain reaction (RTPCR) to measure alpha-fetoprotein gene expression. Using this technique, a single cancer cell could be detected in 10 6 peripheral blood mononuclear cells. This is the ®rst report of an attempt to detect circulating malignant cells in the peripheral blood of patients with testicular cancer by nested RT-PCR. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Peripheral blood stem cell; Alpha fetoprotein; Polymerase chain reaction; Testicular cancer
1. Introduction Testicular cancer is the most common cancer in young men. It is curable in many cases through cisplatinum-based chemotherapy in combination with radiotherapy and/or surgical management. According to an Indiana University trial, patients with minimally or moderately disseminated testicular cancer did well with standard chemotherapy, whereas patients with advanced disease had only a 53% therapeutic response [1]. Therefore, a more aggressive chemotherapeutic regimen is needed for patients with advanced stages of this disease. Peripheral blood stem cell transplantation (PBSCT) is a major
* Corresponding author. Tel.: 1 81-77-548-2273; fax: 1 81-77548-2400. E-mail address: [email protected] (T. Yoshiki)
component of aggressive chemotherapy for testicular cancer and hematologic malignancies [2±4]. Multidrug anticancer chemotherapy regimens using PBSCT have improved the survival of patients with advanced disease. However, there have been several reports of recurrences after PBSCT [5±8]. Previous reports have demonstrated that the malignant cells in peripheral blood stem cell (PBSC) harvests can proliferate after reinfusion into the patient's body [5]. Therefore, we believe that PBSC harvests should be evaluated for tumor contamination prior to reinfusion. Although the risk of cancer recurrence due to reinfusion of contaminated PBSC harvests is unclear, we believe that contaminated PBSC harvests should not be transplanted until further studies con®rm the safety of these transplants. Recently, polymerase chain reaction (PCR)-based methods have been reported to be highly sensitive in detecting circulating tumor cells in blood [5±9].
0304-3835/99/$ - see front matter q 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(99)00194-9
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T. Yuasa et al. / Cancer Letters 143 (1999) 57±62
In this paper, we established a method using nested reverse transcription-PCR (RT-PCR) to detect the expression of alpha fetoprotein (AFP) in the peripheral blood and PBSC harvests of patients with advanced-stage testicular cancer and elevated serum AFP. 2. Materials and methods 2.1. Samples Samples were obtained from ®ve patients with non-seminomatous germ cell tumor (NSGCT). Specimens, 5 mm in diameter, were cut in half. One sample was frozen immediately and stored at 2708C until RNA extraction. The other was ®xed with neutralized, buffered formalin for routine histopathologic examination. Peripheral blood samples and PBSC harvests were obtained before and after ®rst-line chemo-therapy, respectively. Histologic diagnoses and clinical states were determined by the World Health Organization and Tumor-NodeMetastasis classi®cation, respectively [10,11]. The clinicopathologic features of the testicular cancers are summarized in Table 1. 2.2. Analysis of AFP gene expression by nested RTPCR Prior to total RNA extraction, blood samples were treated with isotonic ammonium chloride±Tris solution (pH 7.4) for 5 min at 378C to lyse the erythrocytes. These samples were then washed once in phosphate-buffered saline (PBS). Total cellular RNA from tissue or blood samples was extracted by the
acid-guanidinium thiocyanate-phenol-chloroform method [12]. RNA samples were treated with DNase 1 (RNase-free) (Pharmacia Biotech., Uppsala, Sweden) as recommended by the manufacturer. Single-strand cDNA was synthesized from 5 mg of total RNA using 20 units of RAV-2 reverse transcriptase (Takara, Otsu, Japan) and random primers (Takara). AFP gene-speci®c primers (#1, 5 0 -CAGTGAGGACAAACTATTGGC-3 0 , bases 1392±1413 of human AFP; #2, 5 0 -CTCTTCACCAAAGCAGACTTC-3 0 , bases 1765±l786 of human AFP; #MI, 5 0 GCTGACATTATTATCGGACAC-3 0 , bases 1429± l450 of human AFP; #M2, 5 0 -AGCCTCAAGTTGTTCCTCTGT-3 0 , bases 1690±1711 of human AFP) were synthesized according to the nucleotide sequence of the human AFP gene [9,13]. Portions (1 ml) of the cDNA were ampli®ed by PCR using primers #1 and #2. The reaction mixture (50 ml) consisted of 25 mM Tris±HCl (pH 9.0), containing 50 mM KCl, 2 mM MgCl2, 1 mM dithiothreitol, 200 mM of dNTPs, 1.25 units of Ampli-Taq DNA polymerase (Takara) and 200 ng of each primer. Ampli®cation was performed using 30 cycles of denaturation (948C, 1 min), annealing (558C, 1 min) and extension (728C, 1 min). Aliquots (1 ml) of the ampli®ed RTPCR product were ampli®ed again using the #M1 and #M2 primers in 50 ml reaction mixture. The human bactin gene-speci®c primer pair (5 0 -GTGGGGCGCCCCAGGCACCA-3 0 , bases 144±163 of human b-actin and 5 0 -CTCCTTAATGTCACGCACGATTTC-3 0 bases 660±683 of human b-actin) was used as a control primer pair as described previously [14,15]. Ampli®cation of the human b-actin gene was performed for 25 cycles using the same temperature pro®le described above. After ampli®cation,
Table 1 Clinicopatholologic summary of the patients with NSGCT Case
Age
Histology a
Stage
pTNM b
AFP (ng/ml)
b-HCG (ng/ml)
1 2 3 4 5 6
27 39 35 21 40 46
EC 1 SL LC 1 IT 1 SE EC IT EC 1 SL EC 1 SL 1 YST
III C I I I III A III B2
pT1N3M1 pT1N0M0 pT3N0M0 pT1N0M0 pT1N3M0 pT3N0M1
12885 218 8992 95 281 2827
2 4.63 2 2 8.92 2
a b
EC, embryonal carcinoma; IT, immature teratoma; SE, seminoma; YST, yolk sac tumor. TNM, Tumor-Node-Metastasis classi®cations.
T. Yuasa et al. / Cancer Letters 143 (1999) 57±62
10 ml of the RT-PCR products were subjected to electrophoretic analysis on 2% agarose gel with ethidium bromide. 2.3. Determination of detection sensitivity The hepatocellular carcinoma cell line, HepG2 [16], was used to determine the sensitivity of the assay. HepG2 cells were serially diluted from 10 3 cells to one cell by mixing with 10 6 peripheral blood mononuclear cells (PBMC). The total RNA isolated from the diluents was subjected to nested RT-PCR to determine our ability to detect AFP gene-expressing malignant cells in peripheral blood. 2.4. Nucleotide sequencing of the PCR products Nested RT-PCR products were blunted by Pfu DNA polymerase (Stratagene, Los Angeles, CA) and ligated with a cloning vector pZErOTM4-2.1 (Invitrogen Corporation, San Diego, CA) digested by EcoRV (Takara). Products were then transformed to Escherichia coli, XL2-Blue MRF 0 (Stratagene). The recombinant clones were grown overnight in a LB medium containing 50 mg/ml kanamycin. Plasmid DNA was isolated from recombinant clones using the FlexiPrep kit (Amersham Pharmacia Biotech, Uppsala, Sweden) and sequenced by the dideoxy chain-termination method [17] using Thermo Sequenasee (Amersham Pharmacia Biotech, Buckingham, UK).
59
3. Results 3.1. Expression of the AFP gene in NSGCT cells We analyzed the expression of the AFP gene in NSGCT cells by RT-PCR. The clinicopathologic characteristics of the ®ve patients (cases 1±5) with testicular cancer are summarized in Table 1. All of the patients with NSGCT had elevated levels of serum AFP. Two patients (cases 2 and 5) had slightly elevated serum levels of human chorionic gonadotropin-beta (HCG-b). Ampli®ed bands corresponding in size to the AFP gene (395 bp) were found in all ®ve NSGCT samples and the HepG2 hepatic cancer cell line (Fig. 1). Ampli®ed AFP products were not found in the ten seminoma and three normal testis samples (data not shown). These results indicate that AFP is expressed in NSGCT cells of patients with NSGCT and elevated serum AFP. These results also suggest that RT-PCR can be used to detect NSGCT cells by measuring AFP expression. 3.2. Assessment of a system to detect AFP gene expression by nested RT-PCR We determined the sensitivity of nested RT-PCR for detecting cancer cells by using the HepG2 hepatic cancer cell line. HepG2 cells were diluted and mixed with mononuclear cells. AFP gene expression could be detected by RT-PCR in samples containing one HepG2 malignant cell in 10 6 mononuclear cells. However, ampli®ed products were not obtained from the three peripheral blood samples of healthy
Fig. 1. RT-PCR analysis for the expression of the AFP gene in NSGCT. Five primary NSGCTs with associated elevated serum AFP were applied to the analysis. Normal testicular tissue and seminoma tissue were used as negative controls, and HepG2, a hepatoma cell line was used as a positive control. The expression of b-actin was used as an internal control. Numbers are equivalent to Table 1. Te, normal testis; Se, seminoma.
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T. Yuasa et al. / Cancer Letters 143 (1999) 57±62
Fig. 2. Nested RT-PCR analysis for expression of the AFP gene in serial dilutions of HepG2 cells in 10 6 peripheral blood mononuclear cells (PBMC). Samples containing HepG2 cells (l0 3, 10 2, 10 and 1 per 10 6 cells) were analyzed by nested RT-PCR. Peripheral blood samples from three healthy volunteers were used as negative controls, and HepG2 was used as a positive control. Expression of b-actin was used as an internal control.
volunteers (Fig. 2). These experiments were performed in triplicate, and the results indicate that a single cancer cell can be detected in 10 6 PBMC by the nested RT-PCR assay. 3.3. Detection of the expression of AFP in patients with testicular cancer AFP gene expression was examined in the primary tumors of ®ve patients and in peripheral blood, obtained before initial chemotherapy, from these patients. Following chemotherapy, AFP expression was determined in the PBSC harvests from two patients (cases 5 and 6) (Fig. 3). The expression of
AFP was detected in the primary tumors of all ®ve patients and in the peripheral blood sample of case 6. The expression of AFP was not detected in the peripheral blood of case 5 or in the PBSC harvests of case 5 and 6. Sequencing analysis of the nested RT-PCR products revealed that the sequence data was identical to that previously reported for AFP [13]. 4. Discussion PBSCT is being used more frequently in patients with advanced testicular cancer for hematopoietic reconstitution after high-dose chemotherapy. How-
Fig. 3. Nested RT-PCR analysis for expression of the AFP gene in two elevated AFP and testicular cancer patients (cases 5 and 6). Expression of b-actin was used as an internal control. Tumor, primary tumor sample; PBMC, peripheral blood mononuclear cells before ®rst-line chemotherapy; PBSC, peripheral blood stem cell harvest.
T. Yuasa et al. / Cancer Letters 143 (1999) 57±62
ever, there is an increased risk of recurrence in patients who receive PBSC harvests that are contaminated with malignant cells. Although circulating cancer cells have not yet been reported in patients with testicular cancer, they have been identi®ed in patients with various malignancies such as neuroblastoma [18], prostatic cancer [19] and transitional cell carcinoma [20]. Brenner et al. have reported that patients who received PBSC harvests that were contaminated with malignant cells reacted poorly [5] They also showed that reinfusion of contaminated PBSC harvests could lead to cancer recurrence. These reports suggest that PBSC harvests should be checked for contamination prior to reinfusion. Therefore, we developed a sensitive system to detect cancer cells in PBSC harvests. Since most advanced NSCCTs contain AFP-expressing cancer cells, AFP was thought to be a good marker for detecting testicular cancer cells in PBSC harvests. Before testing the peripheral blood and PBSC harvests of patients with testicular cancer, we evaluated this detection system using tissue samples of embryonal carcinomas, which contain AFP mRNA (Fig. 1). Using this system, we detected AFP expression by nested RT-PCR in the peripheral blood of one patient with testicular cancer and an elevated serum AFP (case 6) (Fig. 3). The nucleotide sequence of the PCR product was found to be identical to the reported sequence of the AFP gene [13], con®rming the existence of AFP gene expression in the peripheral blood. AFP gene expression was not found in the PBSC harvests of either patient (Fig. 3). They are both alive and cancer-free more than 1 year after treatment. PBSC harvests have not been routinely examined for contamination by testicular cancer cells at most urological centers. This report suggests that this detection system should be used to prevent iatrogenic recurrence after PBSCT, although we did not detect AFP expression in the PBSC harvests of the two patients we studied. In summary, this may be the ®rst report of an attempt to detect circulating malignant cells in the peripheral blood of patients with testicular cancer by nested RTPCR. The detection of AFP expression in PBSC harvests by nested RT-PCR may provide useful information for the treatment of testicular cancer patients. In addition to AFP, we have been studying sperm-speci®c antigens [21±27], which may also be useful for the
61
detection of small numbers of cancer cells in the peripheral blood of patients with testicular cancer. Acknowledgements This work was partly supported by a Grant-in-Aid for Scienti®c Research from the Ministry of Education, Science, Sports and Culture of Japan. We thank Mr. Masafumi Suzaki, Dr. Mutsuki Mishina and Dr. Mitsuhiro Narita for technical assistance and providing samples. References [1] R. Birch, S. Williams, A. Cone, L. Finhorn, P. Roark, S. Turner, F.A. Greco, Prognostic factors for a favorable outcome in disseminated germ cell tumors, J. Clin. Oncol. 4 (1986) 400±407. [2] W. Bensinger, J. Singer, F. Appelbaum, K. Lilleby, K. Longin, S. Rowley, F. Clarke, K. Clift, J. Hansen, T. Shields, K. Storb, C. Weaver, P. Weider, C.D. Buckner, Autologous transplantation with peripheral blood mononuclear cells collected after administration of recombinant granulocyte stimulating factor, Blood 81 (1993) 3158±3163. [3] T.R. Klumpp, K.F. Mangan, S.L. Goldberg, L.S. Pearlman, G.R. MacDonald, Colony-stimulating factor accelerates neutrophil engraftment following peripheral-blood stem-cell transplantation: a prospective, randomized trial, J. Clin. Oncol. 13 (1995) 1323±1327. [4] B.K. Margolin, J.H. Doroshow, C. Ahn, V. Hamasaki, L. Leong, R. Morgan, J. Raschko, S. Shibata, G. Somlo, M.J. Tetef, Treatment of germ cell cancer with two cycles of high-dose ifosfamide, carboplatin, and etoposide with autologous stem-cell support, J. Clin. Oncol. 14 (1996) 2631± 2637. [5] M.K. Brenner, D.R. Rill, K.C. Moen, K.A. Krance, J. Mirro Jr, W.A. Anderson, J.N. Ihle, Gene-marking to trace origin of relapse after autologous bone marrow transplantation, Lancet 341 (1993) 85±86. [6] J.E. Hardingham, D. Kotasek, R.E. Sage, L.T. Gooley, J.X. Mi, A. Dobrovic, J.B. Norman, A.E. Bolton, B.M. Dale, Signi®cance of molecular marker-positive cells after autologous peripheral-blood stem-cell transplantation for non-Hodgkin's lymphoma, J. Clin. Oncol. 13 (1995) 1073±1079. [7] J.G. Sharp, A. Kessinger, S. Mann, D.A. Crouse, J.O. Armitage, P. Biermaii, D.D. Weisenburger, Outcome of high-dose therapy and autologous transplantation in non-Hodgkin's lymphoma based on the presence of tumor in the marrow or infused hematopoietic harvest, J. Clin. Oncol. 14 (1996) 214± 219. [8] P. Corradini, M. Astol®, C. Cherasco, M. Ladetto, C. Voena, D. Caraciolo, A. Pileri, C. Tarella, Molecular monitoring of minimal residual disease in follicular and mantle cell non-
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T. Yuasa et al. / Cancer Letters 143 (1999) 57±62 Hodgkin's lymphomas treated with high-dose chemotherapy and peripheral blood progenitor cell autografting, Blood 89 (1997) 724±731. M. Matsumura, Y. Niwa, Y. Hikida, K. Okano, N. Kato, S. Shima, Y. Shiratori, M. Omata, Sensitive assay for detection of hepatocellular carcinoma associated gene transcription (alpha fetoprotein mRNA) in blood, Biochem. Biophys. Res. Commun. 207 (1995) 813±818. International Union Against Cancer (UICC), TNM Classi®cation of Malignant Tumours, 3rd edition, Springer-Verlag, Berlin, 1989. F. Most®, L.H. Sobin, Histological Typing of Testis Tumours Internaunnal Histological Classi®cation of Tumours, 16, WHO, Geneva, 1977. P. Chomczynski, N. Sacchi, Single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction, Anal. Biochem. 162 (1987) 156±159. T. Morinaga, M. Sakai, T.G. Wegmann, T. Tamaoki, Primary structures of human alpha-fetoprotein and its mRNA, Proc. Natl. Acad. Sci. USA 80 (1983) 4604±4608. P. Ponte, S.Y. Ng, J. Engel, P. Gunning, L. Kedes, Evolutionary conservation in the untranslated regions of actin mRNAs: DNA sequence of a human beta-actin cDNA, Nucleic Acids Res. 12 (1984) 1687±l696. O. Tachibana, H. Nakazawa, J. Lampe, K. Watanabe, P. Kleihues, H. Ohgaki, Expression of Fas/APO-1 during the progression of astrocytomas, Cancer Res. 55 (1995) 5528±5530. D.P. Aden, A. Fogel, S. Plotkin, I. Damjanov, B.B. Knowles, Controlled synthesis of HBsAg in a differentiated human liver carcinoma-derived cell line, Nature 282 (1979) 615±616. F. Sanger, S. Nicklens, A.R. Coulson, sequencing with chain terminating inhibitors, Proc. Natl. Acad. Sci. USA 74 (1977) 5463±5467. T.J. Moss, D.G. Sanders, L.C. Lasky, B. Bostrom, Contaminanon of peripheral blood stem cell harvests by circulating neuroblastoma cell, Blood 76 (1990) 1879±1883.
[19] M.V. Seiden, P.W. Kantoff, K. Krithivas, K. Propert, M. Bryant, E. Haltom, L. Gaynes, I. Kaplan, G. Bubley, W.J. De Wolf, Detection of circulating tumor cells in men with localized prostate cancer, J. Clin. Oncol. 12 (1994) 2634± 2639. [20] T. Yuasa, T. Yoshiki, T. Tanaka, C. Kim, T. Isono, Y. Okada, Expression of uroplakin Ib and uroplakin III genes in tissues and peripheral blood of patients with transitional cell carcinoma, Jpn. J. Cancer Res. 89 (1998) 879±882. [21] Y.Y. Yang, T. Yoshiki, Y. Lee, C.Y. Lee, Anti-trophoblast monoclonal antibodies for prenatal genetic diagnosis, Ann. NY Acad. Sci. 731 (1994) 178±180. [22] T. Yoshiki, Y.Y. Yang, Y. Lee, C.Y. Lee, Generation and characterization of monoclonal antibodies speci®c to surface antigens of human trophoblast cells, Am. J. Reprod. Immunol. 34 (1995) 148±155. [23] T. Yoshiki, J.C. Herr, C.Y.G. Lee, Puri®cation and characterization of a sperm antigen recognized by HSA-5 monoclonal antibody, J. Reprod. Immunol. 29 (1995) 209±222. [24] C.Y.G. Lee, T. Yoshiki, Chui, P. McChesney, J.C. Herr, E.S. Hwang, E.S. Huang, Epitope analysis of a sperm acrosomal antigen de®ned by HSA-5 monoclonal antibody, J. Reprod. Immunol. 29 (1995) 223±238. [25] J.D. Ambrose, K. Kajamahendran, T. Yoshiki, C.Y. Lee, Antibull sperm monoclonal antibodies I: identi®ca¯on of major antienic domains of bull sperm and manifestation of interspecies cross-reactivity, J. Androl. 17 (1996) 567±578. [26] T. Yoshiki, B.C. Lai, S. Dorjee, C.Y. Lee, Molecular characterizations of an intraacrosomal antigen de®ned by HS-33 monoclonal antibody, J. Androl. 17 (1996) 666±673. [27] T. Yoshiki, K. Johnin, C.H. Kun, M.C. Chou, B. Lai, C.Y. Lee, Molecular nature of a sperm acrosomal antigen recognized by HS-13 monoclonal antibody, J. Reprod. Immunol. 36 (l997) 61±75.
Detection of circulating testicular cancer cells in peripheral blood Takeshi Yuasa a, Tatsuhiro Yoshiki a,*, Tsutomu Tanaka a, Takahiro Isono b, Yusaku Okada a b
a Department of Urology, Shiga University of Medical Science, Otsu, Japan Central Research Laboratory, Shiga University of Medical Science, Seta, Otsu 520-2192 , Japan
Received 17 February 1999; received in revised form 6 May 1999; accepted 6 May 1999
Abstract Patients who receive peripheral blood stem cell transplants are at risk of developing cancer recurrence due to the presence of malignant cells in the transplants. We investigated a sensitive method to detect malignant cells in the peripheral blood and peripheral blood stem cells of patients with testicular cancer using nested, reverse transcription-polymerase chain reaction (RTPCR) to measure alpha-fetoprotein gene expression. Using this technique, a single cancer cell could be detected in 10 6 peripheral blood mononuclear cells. This is the ®rst report of an attempt to detect circulating malignant cells in the peripheral blood of patients with testicular cancer by nested RT-PCR. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Peripheral blood stem cell; Alpha fetoprotein; Polymerase chain reaction; Testicular cancer
1. Introduction Testicular cancer is the most common cancer in young men. It is curable in many cases through cisplatinum-based chemotherapy in combination with radiotherapy and/or surgical management. According to an Indiana University trial, patients with minimally or moderately disseminated testicular cancer did well with standard chemotherapy, whereas patients with advanced disease had only a 53% therapeutic response [1]. Therefore, a more aggressive chemotherapeutic regimen is needed for patients with advanced stages of this disease. Peripheral blood stem cell transplantation (PBSCT) is a major
* Corresponding author. Tel.: 1 81-77-548-2273; fax: 1 81-77548-2400. E-mail address: [email protected] (T. Yoshiki)
component of aggressive chemotherapy for testicular cancer and hematologic malignancies [2±4]. Multidrug anticancer chemotherapy regimens using PBSCT have improved the survival of patients with advanced disease. However, there have been several reports of recurrences after PBSCT [5±8]. Previous reports have demonstrated that the malignant cells in peripheral blood stem cell (PBSC) harvests can proliferate after reinfusion into the patient's body [5]. Therefore, we believe that PBSC harvests should be evaluated for tumor contamination prior to reinfusion. Although the risk of cancer recurrence due to reinfusion of contaminated PBSC harvests is unclear, we believe that contaminated PBSC harvests should not be transplanted until further studies con®rm the safety of these transplants. Recently, polymerase chain reaction (PCR)-based methods have been reported to be highly sensitive in detecting circulating tumor cells in blood [5±9].
0304-3835/99/$ - see front matter q 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(99)00194-9
58
T. Yuasa et al. / Cancer Letters 143 (1999) 57±62
In this paper, we established a method using nested reverse transcription-PCR (RT-PCR) to detect the expression of alpha fetoprotein (AFP) in the peripheral blood and PBSC harvests of patients with advanced-stage testicular cancer and elevated serum AFP. 2. Materials and methods 2.1. Samples Samples were obtained from ®ve patients with non-seminomatous germ cell tumor (NSGCT). Specimens, 5 mm in diameter, were cut in half. One sample was frozen immediately and stored at 2708C until RNA extraction. The other was ®xed with neutralized, buffered formalin for routine histopathologic examination. Peripheral blood samples and PBSC harvests were obtained before and after ®rst-line chemo-therapy, respectively. Histologic diagnoses and clinical states were determined by the World Health Organization and Tumor-NodeMetastasis classi®cation, respectively [10,11]. The clinicopathologic features of the testicular cancers are summarized in Table 1. 2.2. Analysis of AFP gene expression by nested RTPCR Prior to total RNA extraction, blood samples were treated with isotonic ammonium chloride±Tris solution (pH 7.4) for 5 min at 378C to lyse the erythrocytes. These samples were then washed once in phosphate-buffered saline (PBS). Total cellular RNA from tissue or blood samples was extracted by the
acid-guanidinium thiocyanate-phenol-chloroform method [12]. RNA samples were treated with DNase 1 (RNase-free) (Pharmacia Biotech., Uppsala, Sweden) as recommended by the manufacturer. Single-strand cDNA was synthesized from 5 mg of total RNA using 20 units of RAV-2 reverse transcriptase (Takara, Otsu, Japan) and random primers (Takara). AFP gene-speci®c primers (#1, 5 0 -CAGTGAGGACAAACTATTGGC-3 0 , bases 1392±1413 of human AFP; #2, 5 0 -CTCTTCACCAAAGCAGACTTC-3 0 , bases 1765±l786 of human AFP; #MI, 5 0 GCTGACATTATTATCGGACAC-3 0 , bases 1429± l450 of human AFP; #M2, 5 0 -AGCCTCAAGTTGTTCCTCTGT-3 0 , bases 1690±1711 of human AFP) were synthesized according to the nucleotide sequence of the human AFP gene [9,13]. Portions (1 ml) of the cDNA were ampli®ed by PCR using primers #1 and #2. The reaction mixture (50 ml) consisted of 25 mM Tris±HCl (pH 9.0), containing 50 mM KCl, 2 mM MgCl2, 1 mM dithiothreitol, 200 mM of dNTPs, 1.25 units of Ampli-Taq DNA polymerase (Takara) and 200 ng of each primer. Ampli®cation was performed using 30 cycles of denaturation (948C, 1 min), annealing (558C, 1 min) and extension (728C, 1 min). Aliquots (1 ml) of the ampli®ed RTPCR product were ampli®ed again using the #M1 and #M2 primers in 50 ml reaction mixture. The human bactin gene-speci®c primer pair (5 0 -GTGGGGCGCCCCAGGCACCA-3 0 , bases 144±163 of human b-actin and 5 0 -CTCCTTAATGTCACGCACGATTTC-3 0 bases 660±683 of human b-actin) was used as a control primer pair as described previously [14,15]. Ampli®cation of the human b-actin gene was performed for 25 cycles using the same temperature pro®le described above. After ampli®cation,
Table 1 Clinicopatholologic summary of the patients with NSGCT Case
Age
Histology a
Stage
pTNM b
AFP (ng/ml)
b-HCG (ng/ml)
1 2 3 4 5 6
27 39 35 21 40 46
EC 1 SL LC 1 IT 1 SE EC IT EC 1 SL EC 1 SL 1 YST
III C I I I III A III B2
pT1N3M1 pT1N0M0 pT3N0M0 pT1N0M0 pT1N3M0 pT3N0M1
12885 218 8992 95 281 2827
2 4.63 2 2 8.92 2
a b
EC, embryonal carcinoma; IT, immature teratoma; SE, seminoma; YST, yolk sac tumor. TNM, Tumor-Node-Metastasis classi®cations.
T. Yuasa et al. / Cancer Letters 143 (1999) 57±62
10 ml of the RT-PCR products were subjected to electrophoretic analysis on 2% agarose gel with ethidium bromide. 2.3. Determination of detection sensitivity The hepatocellular carcinoma cell line, HepG2 [16], was used to determine the sensitivity of the assay. HepG2 cells were serially diluted from 10 3 cells to one cell by mixing with 10 6 peripheral blood mononuclear cells (PBMC). The total RNA isolated from the diluents was subjected to nested RT-PCR to determine our ability to detect AFP gene-expressing malignant cells in peripheral blood. 2.4. Nucleotide sequencing of the PCR products Nested RT-PCR products were blunted by Pfu DNA polymerase (Stratagene, Los Angeles, CA) and ligated with a cloning vector pZErOTM4-2.1 (Invitrogen Corporation, San Diego, CA) digested by EcoRV (Takara). Products were then transformed to Escherichia coli, XL2-Blue MRF 0 (Stratagene). The recombinant clones were grown overnight in a LB medium containing 50 mg/ml kanamycin. Plasmid DNA was isolated from recombinant clones using the FlexiPrep kit (Amersham Pharmacia Biotech, Uppsala, Sweden) and sequenced by the dideoxy chain-termination method [17] using Thermo Sequenasee (Amersham Pharmacia Biotech, Buckingham, UK).
59
3. Results 3.1. Expression of the AFP gene in NSGCT cells We analyzed the expression of the AFP gene in NSGCT cells by RT-PCR. The clinicopathologic characteristics of the ®ve patients (cases 1±5) with testicular cancer are summarized in Table 1. All of the patients with NSGCT had elevated levels of serum AFP. Two patients (cases 2 and 5) had slightly elevated serum levels of human chorionic gonadotropin-beta (HCG-b). Ampli®ed bands corresponding in size to the AFP gene (395 bp) were found in all ®ve NSGCT samples and the HepG2 hepatic cancer cell line (Fig. 1). Ampli®ed AFP products were not found in the ten seminoma and three normal testis samples (data not shown). These results indicate that AFP is expressed in NSGCT cells of patients with NSGCT and elevated serum AFP. These results also suggest that RT-PCR can be used to detect NSGCT cells by measuring AFP expression. 3.2. Assessment of a system to detect AFP gene expression by nested RT-PCR We determined the sensitivity of nested RT-PCR for detecting cancer cells by using the HepG2 hepatic cancer cell line. HepG2 cells were diluted and mixed with mononuclear cells. AFP gene expression could be detected by RT-PCR in samples containing one HepG2 malignant cell in 10 6 mononuclear cells. However, ampli®ed products were not obtained from the three peripheral blood samples of healthy
Fig. 1. RT-PCR analysis for the expression of the AFP gene in NSGCT. Five primary NSGCTs with associated elevated serum AFP were applied to the analysis. Normal testicular tissue and seminoma tissue were used as negative controls, and HepG2, a hepatoma cell line was used as a positive control. The expression of b-actin was used as an internal control. Numbers are equivalent to Table 1. Te, normal testis; Se, seminoma.
60
T. Yuasa et al. / Cancer Letters 143 (1999) 57±62
Fig. 2. Nested RT-PCR analysis for expression of the AFP gene in serial dilutions of HepG2 cells in 10 6 peripheral blood mononuclear cells (PBMC). Samples containing HepG2 cells (l0 3, 10 2, 10 and 1 per 10 6 cells) were analyzed by nested RT-PCR. Peripheral blood samples from three healthy volunteers were used as negative controls, and HepG2 was used as a positive control. Expression of b-actin was used as an internal control.
volunteers (Fig. 2). These experiments were performed in triplicate, and the results indicate that a single cancer cell can be detected in 10 6 PBMC by the nested RT-PCR assay. 3.3. Detection of the expression of AFP in patients with testicular cancer AFP gene expression was examined in the primary tumors of ®ve patients and in peripheral blood, obtained before initial chemotherapy, from these patients. Following chemotherapy, AFP expression was determined in the PBSC harvests from two patients (cases 5 and 6) (Fig. 3). The expression of
AFP was detected in the primary tumors of all ®ve patients and in the peripheral blood sample of case 6. The expression of AFP was not detected in the peripheral blood of case 5 or in the PBSC harvests of case 5 and 6. Sequencing analysis of the nested RT-PCR products revealed that the sequence data was identical to that previously reported for AFP [13]. 4. Discussion PBSCT is being used more frequently in patients with advanced testicular cancer for hematopoietic reconstitution after high-dose chemotherapy. How-
Fig. 3. Nested RT-PCR analysis for expression of the AFP gene in two elevated AFP and testicular cancer patients (cases 5 and 6). Expression of b-actin was used as an internal control. Tumor, primary tumor sample; PBMC, peripheral blood mononuclear cells before ®rst-line chemotherapy; PBSC, peripheral blood stem cell harvest.
T. Yuasa et al. / Cancer Letters 143 (1999) 57±62
ever, there is an increased risk of recurrence in patients who receive PBSC harvests that are contaminated with malignant cells. Although circulating cancer cells have not yet been reported in patients with testicular cancer, they have been identi®ed in patients with various malignancies such as neuroblastoma [18], prostatic cancer [19] and transitional cell carcinoma [20]. Brenner et al. have reported that patients who received PBSC harvests that were contaminated with malignant cells reacted poorly [5] They also showed that reinfusion of contaminated PBSC harvests could lead to cancer recurrence. These reports suggest that PBSC harvests should be checked for contamination prior to reinfusion. Therefore, we developed a sensitive system to detect cancer cells in PBSC harvests. Since most advanced NSCCTs contain AFP-expressing cancer cells, AFP was thought to be a good marker for detecting testicular cancer cells in PBSC harvests. Before testing the peripheral blood and PBSC harvests of patients with testicular cancer, we evaluated this detection system using tissue samples of embryonal carcinomas, which contain AFP mRNA (Fig. 1). Using this system, we detected AFP expression by nested RT-PCR in the peripheral blood of one patient with testicular cancer and an elevated serum AFP (case 6) (Fig. 3). The nucleotide sequence of the PCR product was found to be identical to the reported sequence of the AFP gene [13], con®rming the existence of AFP gene expression in the peripheral blood. AFP gene expression was not found in the PBSC harvests of either patient (Fig. 3). They are both alive and cancer-free more than 1 year after treatment. PBSC harvests have not been routinely examined for contamination by testicular cancer cells at most urological centers. This report suggests that this detection system should be used to prevent iatrogenic recurrence after PBSCT, although we did not detect AFP expression in the PBSC harvests of the two patients we studied. In summary, this may be the ®rst report of an attempt to detect circulating malignant cells in the peripheral blood of patients with testicular cancer by nested RTPCR. The detection of AFP expression in PBSC harvests by nested RT-PCR may provide useful information for the treatment of testicular cancer patients. In addition to AFP, we have been studying sperm-speci®c antigens [21±27], which may also be useful for the
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detection of small numbers of cancer cells in the peripheral blood of patients with testicular cancer. Acknowledgements This work was partly supported by a Grant-in-Aid for Scienti®c Research from the Ministry of Education, Science, Sports and Culture of Japan. We thank Mr. Masafumi Suzaki, Dr. Mutsuki Mishina and Dr. Mitsuhiro Narita for technical assistance and providing samples. References [1] R. Birch, S. Williams, A. Cone, L. Finhorn, P. Roark, S. Turner, F.A. Greco, Prognostic factors for a favorable outcome in disseminated germ cell tumors, J. Clin. Oncol. 4 (1986) 400±407. [2] W. Bensinger, J. Singer, F. Appelbaum, K. Lilleby, K. Longin, S. Rowley, F. Clarke, K. Clift, J. Hansen, T. Shields, K. Storb, C. Weaver, P. Weider, C.D. Buckner, Autologous transplantation with peripheral blood mononuclear cells collected after administration of recombinant granulocyte stimulating factor, Blood 81 (1993) 3158±3163. [3] T.R. Klumpp, K.F. Mangan, S.L. Goldberg, L.S. Pearlman, G.R. MacDonald, Colony-stimulating factor accelerates neutrophil engraftment following peripheral-blood stem-cell transplantation: a prospective, randomized trial, J. Clin. Oncol. 13 (1995) 1323±1327. [4] B.K. Margolin, J.H. Doroshow, C. Ahn, V. Hamasaki, L. Leong, R. Morgan, J. Raschko, S. Shibata, G. Somlo, M.J. Tetef, Treatment of germ cell cancer with two cycles of high-dose ifosfamide, carboplatin, and etoposide with autologous stem-cell support, J. Clin. Oncol. 14 (1996) 2631± 2637. [5] M.K. Brenner, D.R. Rill, K.C. Moen, K.A. Krance, J. Mirro Jr, W.A. Anderson, J.N. Ihle, Gene-marking to trace origin of relapse after autologous bone marrow transplantation, Lancet 341 (1993) 85±86. [6] J.E. Hardingham, D. Kotasek, R.E. Sage, L.T. Gooley, J.X. Mi, A. Dobrovic, J.B. Norman, A.E. Bolton, B.M. Dale, Signi®cance of molecular marker-positive cells after autologous peripheral-blood stem-cell transplantation for non-Hodgkin's lymphoma, J. Clin. Oncol. 13 (1995) 1073±1079. [7] J.G. Sharp, A. Kessinger, S. Mann, D.A. Crouse, J.O. Armitage, P. Biermaii, D.D. Weisenburger, Outcome of high-dose therapy and autologous transplantation in non-Hodgkin's lymphoma based on the presence of tumor in the marrow or infused hematopoietic harvest, J. Clin. Oncol. 14 (1996) 214± 219. [8] P. Corradini, M. Astol®, C. Cherasco, M. Ladetto, C. Voena, D. Caraciolo, A. Pileri, C. Tarella, Molecular monitoring of minimal residual disease in follicular and mantle cell non-
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