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Tumor Biology |
Department of Medical Oncology, Clinica Puerta de Hierro [J. M. S., G. D., J. M. G., R. G., M. J. V., F. N., M. P., P. E., F. B.], and Department of Gynecology, Hospital Santa Cristina [S. S. M.], 28035 Madrid, Spain
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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It is known that malignant cell transformation is accompanied by well-defined molecular genetic changes within the original cell. Today, it is possible to detect many of them not only in research studies but also in clinical diagnostics in tumor DNA of tissue samples obtained from diverse sources reached by the tumor cells, including sputum (4) , urine (5) , pancreatic juice (6) , and stool (7) . In breast cancer, a case has been reported with c-erbB-2 gene amplification found in nipple discharge (8) .
Previous studies have found that free DNA is circulating in both healthy and ill individuals. In control subjects, the mean concentration of soluble DNA in plasma was 14 ng/ml (9) , and in patients with different types of neoplasias, the mean concentration rose to 180 ng/ml (10) . A mean concentration of 118 ng/ml was also detected in benign gastrointestinal processes (9) , and mean concentrations above those registered in controls have been reported in patients with autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, or other inflammatory conditions (11) . An active process explaining the enrichment of plasma DNA in cancer patients has also been suggested (12) . More recently, qualitative and quantitative studies have demonstrated the presence of fetal DNA in maternal serum, with implications for noninvasive prenatal diagnosis (13, 14, 15) . Likewise, the examination of graft and host interactions has revealed the presence of donor-specific DNA in the plasma of kidney and liver transplant recipients (16) .
In recent years, using molecular techniques such as PCR for amplification of small amounts of DNA, it has been possible to identify the same alterations observed in tumor DNA in the plasma DNA of patients bearing diverse types of tumors. The alterations found include K-ras, N-ras, and p53 gene mutations, aberrant promoter hypermethylation of tumor suppressor genes, and changes in microsatellites detected by polymorphic markers in the following cancers: pancreatic cancer (17 , 18) ; colon cancer (19, 20, 21) ; myelodysplastic syndromes (22) ; small cell lung cancer (23 , 24) ; non-small cell lung cancer (25 , 26) ; head and neck carcinomas (27) ; clear cell renal carcinoma (28) ; and breast (24) and liver cancer (29) .
Breast cancer is associated with different types of molecular genetic aberrations such as somatic mutations of oncogenes (30, 31, 32) and tumor suppressor genes (33, 34, 35, 36) as well as allelic loss and MI3 in several chromosomal regions (37, 38, 39, 40, 41, 42) . The determination of these anomalies can be used as a specific tool in the histological diagnosis, even early diagnosis, and can possibly be used as a prognostic factor. As observed in other tumors, the specific molecular alterations shown by breast carcinomas may also be found in the plasma DNA of patients harboring a breast cancer tumor.
Based on these facts, we designed the present study with two goals: (a) to determine the presence at diagnosis of tumor DNA in the plasma of patients with breast cancer, characterized by alterations in microsatellites and in tumor suppressor genes; and (b) to analyze the distribution of 13 clinicopathological parameters in patients with and without specific plasma DNA.
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PATIENTS AND METHODS |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Analysis of Clinicopathological Parameters.
The following parameters were obtained from the medical records of the 62 patients studied: (a) birth and diagnosis dates; (b) family history of the disease; (c) menopausal status; (d) tumor size; (e) LNMs; (f) presence of steroid receptors (estrogen and progesterone); (g) histological type; (h) peritumoral vessel invasion; and (i) histological grade. Pathological stage was assessed using the tumor-node-metastasis (TNM) classification. All tumors were graded with regard to two parameters: (a) nuclear polymorphism (uniform and regular size, 1; moderate pleomorphism, 2; highly pleomorphic with giant nucleus, 3); and (b) mitotic index (<1 mitosis, 1; 2, 2; >3, 3). The final grade was determined by adding the two scores: grade 1, 1–2; grade 2, 3–4; grade 3, 5–6. Presence of peritumoral vessel invasion was analyzed, and the steroid receptor content (estrogen and progesterone) was determined by an immunohistochemical procedure, the results of which were considered to be positive when 25% or more of the cells stained positively. The proliferative index of the tumors was demonstrated by Ki-67 antigen (Immunotech, Westbrook, ME) in an immunohistochemical analysis; the Ki-67 labeling index was considered high when 15% or more of the cells stained positively.
Microsatellite Analysis and PCR Conditions.
PCR was performed in 25-µl volumes using 100 ng of template DNA, 0.75 unit of Ampli Taq Gold DNA polymerase (Perkin-Elmer, Roche Molecular Systems, Inc., Branchburg, NJ), 2.5-µl of 10x PCR buffer, 200 µM dNTP, 0.6 µM of each primer, and different concentrations of MgCl2, depending on the polymorphic marker. A 30-cycle amplification was done in a thermal cycler (Perkin-Elmer, Foster City, CA). Six microsatellite markers were used to determine LOH on the following chromosomes: (a) on chromosome 17, D17S855 (43)
and D17S654 (44)
; (b) on chromosome 16, D16S421 (45)
; (c) on chromosome 11, TH2 (42
; (d) on chromosome 10, D10S197 (37)
; and (e) on chromosome 9, D9S161 (46)
. These markers were chosen because they have been reported to show a high rate of alterations in breast carcinomas. The alleles were separated by mixing 25 µl of the PCR products with a 10-µl volume of loading buffer (total volume, 35 µl), 0.02% xylene cyanol, and 0.02% bromphenol blue. Electrophoresis was run on nondenaturing 8–12% polyacrylamide gels for 12–15 h at 500 V. After gel electrophoresis, the allelic band intensity was detected by a nonradioisotopic technique using a commercially available silver staining method (47)
. We analyzed the allelic intensities by densitometry. The gel image was captured by a GS-690 Imaging Densitometer (Bio-Rad Laboratories, Hercules, CA), digitized at 400 dpi, and analyzed using Multi-Analyst/PC (Bio-Rad Laboratories). An allele should be considered lost when its signal is reduced by 100% with respect to that observed in the normal counterpart DNA. However, it is accepted that possible variations in the amplification process and, more frequently, the presence of small amounts of normal cells among the tumor tissue or, in our study, of normal blood cells DNA in plasma DNA can result in the expression of different band intensities in the total absence of one allele. In this study, we considered LOH to exist when the signal of the allele was reduced by more than 75% and considered MI to exist when one or more novel bands appeared in tumor or plasma DNA. Despite the fact that the latter alteration is rare in breast carcinoma (37
, 38)
, we checked for it among the results of the microsatellite study.
Mutational Study of the p53 Gene.
To establish the presence of point mutations in the conserved exons of TP53, PCR-SSCP analysis was performed according to a modification of the method reported by Orita et al. (48)
. We amplified exons 5, 6, 7, and 8 of the p53 gene, and the primers used were: exon 5, 5'-TCCTTCCTCTTCCTACAG and 5'-ACCCTGGGCAACCAGCCCTGT; exon 6, 5'-ACAGGGCTGGTTGCCCAGGGT and 5'-AGTTGCAAACCAGACCTCAGGCG; exon 7, 5'-TCCTAGGTTGGCTCTGACTGT and 5'-AGTGGCCCTGACCTGGAGTCT; and exon 8, 5'-GGGACAGGTAGGACCTGATTTCCTT and 5'-ATCTGAAGGCATAACTGCACCCTTGG. The annealing temperatures were 65°C, 67°C, 62°C and 68°C, respectively. PCR was performed under standard conditions in 25-µl that contained 2-µl (100 ng) of DNA template (tumor, normal or plasma DNA); 2.5-µl of 10 x PCR buffer and 0.75 U of Ampli Taq Gold (Perkin-Elmer, Roche Molecular Systems Inc., Branchburg, NJ); 200 µM dNTP mix; 0.6 µM of each primer; and different concentrations of magnesium chloride depending on the primer, and distilled H2O needed to reach the total volume. For PCR amplification, the samples underwent 40 cycles of 94°C for 1 min, subjected to different annealing temperatures depending on the primer, and 70°C for 1 min. The amplified products were denatured by mixing with 15 µl of denaturing stop solution that contained 98% formamide, 10 mml/L edathamil (pH 8.0), 0.02% xylene cyanol and 0.02% bromphenol blue, heated to 95°C for 5 min and rapidly cooled on ice. Electrophoresis was run on nondenaturing 8%–12% polyacrylamide gels for 12–15 h at 250 v. The allelic band intensity on the gels was detected by a nonradioisotopic method using a commercially available silver staining method (47)
. The specimens that showed a differential band at SSCP were amplified to obtain templates for DNA sequencing. These amplifications were independent from those used for SSCP analysis. Amplified DNA fragments were purified from 0.9% agarose gels using the Geneclean Kit (Bio-101, Inc. La Jolla, CA), and used for direct DNA sequencing by the dNTP method with the Sequenase Kit (United States Biochemical Corp. Cleveland, OH).
Methylation Study of the First Exon of p16INK4a.
We also used the methylation status of the first exon of p16INK4a as a molecular alteration to identify patients with tumor DNA in plasma. The study was performed in the 43 cases in which plasma DNA was available after microsatellite and p53 gene mutational analysis. A PCR-based methylation assay was performed, based on the inability of some restriction enzymes to cut methylated sequences (49)
. The PstI and SacII restriction enzyme sites were examined. Analysis of DNA digests was performed according to the manufacturer’s instructions (Promega, Madison, WI), and the digested DNA was amplified with primers flanking the restriction sites. Amplification of ß-globin was used as internal control of the reaction in a multiplex PCR. (There are no restriction sites in the ß-globin sequence selected for PstI and SacII.) The primer set used for methylation analysis of exon 1 of p16INK4a was 5'-GGGAGCAGCATGGAGCCG and 5'-AGTCGCCCGCCATCCCCT, and the primer set for ß-globin was 5'-CAACTTCATCCACGTTCACC and 5'-GAAGAGCCAAGGACAGGTAC. The conditions used were as follows: 2.5 µl of 10x buffer II (Perkin-Elmer, Roche Molecular Systems Inc.); 200 µM dNTPs; 2.5 mM MgCl2; 0.6 µM exon 1 primers; 0.6 µM ß-globin primers; 100 ng of the digested DNA as template; and 25 µl of distilled H2O. This reaction mix was amplified with Ampli Taq Gold (Perkin-Elmer, Roche Molecular Systems Inc.) at 94°C for 12 min with 35 cycles of 94°C for 30 s, 61°C for 30 s, and 72°C for 30 s, followed by incubation at 72°C for 11 min. PCR products were resolved in 6% acrylamide and stained with a nonisotopic silver nitrate method (47)
. PCR-based methylation analysis using restriction enzymes may be subject to variability if the DNA digestions are not complete. To rule out the possibility of incomplete restriction, all samples were digested overnight in two independent experiments. PCR amplifications from each of the duplicate digests were repeated twice to ensure reproducibility of the results.
Statistical Analysis.
A descriptive statistical study was performed in which the categorical variables were tabulated according to their absolute value proportion and 95% CI. The continuous variables are expressed in terms of the mean and 95% CI. The categorical variables were contrasted by means of the 
2 test with the Yates correction (50)
or Fisher’s exact test when any of the expected frequencies was less than 5. We considered P < 0.05 to be significant Statistical analyses were performed using the EPI-INFO package, version 6.04.
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RESULTS |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Plasma DNA was found in all 62 patients at concentrations ranging from 24–170 ng/ml (mean concentration, 115 ng/ml).
Microsatellite Analysis.
Although the microsatellites used were selected on the basis of their high rate of LOH in breast carcinomas, about 40% of our cases were found to be uninformative. Fifty-one breast carcinomas (82%) showed allelic loss at at least one locus. The same analysis of plasma DNA disclosed 38 patients (61%) with the same microsatellite alterations (Fig. 1)
. The highest individual rate of LOH in tumor DNA corresponded to markers D17S654 and D16S421, with a 53% and 50% LOH, respectively (Table 1)
. All markers studied showed allelic loss in plasma DNA, but with variable percentages with respect to tumor DNA; TH2 displayed the highest coincidence rate, with 100% of cases (Table 1)
. No MI was detected in tumor DNA. However, we observed that in five cases, plasma DNA demonstrated other additional molecular alterations not present in tumor DNA: two MI and one LOH for marker D17S654; and two LOH for D9S161.
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and 3
); in four of these tumors, LOH was also found in regions other than 17p13, and, in two tumors, LOH was not observed in any of the microsatellites studied. In three cases (5%), the same mutation was identified in plasma DNA, including one patient without concomitant LOH at any marker tested (Table 1)
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, two of them corresponded to cases with neither LOH nor p53 gene mutations (Table 1)
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In all, we identified 56 cases (90%) in which there was at least one molecular event in tumor DNA and 41 cases (66%) with a similar alteration in plasma DNA. Of the 15 patients that showed molecular changes in tumor DNA but not in plasma DNA, 9 patients displayed the same molecular pattern in normal tissue, normal blood cells, and plasma DNA; in 6 cases, no amplification of the plasma DNA samples was obtained. The six cases in which there were no molecular alterations in the primary tumor had a mean plasma DNA concentration of 70 ng/ml, but the molecular study revealed no differences among normal, normal blood cells, tumor, and plasma DNA.
Correlations between Clinicopathological Parameters and Molecular Changes.
To establish a clinical meaning for the presence of plasma DNA with the characteristics of tumor DNA, we studied the correlation between the presence or absence of plasma DNA of tumor origin and 13 clinicopathological parameters at diagnosis (Table 2)
. Statistical analysis revealed statistically significant differences in the following independent variables: (a) involvement of three or more lymph nodes (P = 0.005); (b) IDC (P = 0.05); and (c) a high proliferative index (P = 0.02). When we considered the subgroup of patients whose tumors concomitantly exhibited three characteristics classically associated with high-grade malignancy (LNMs, histological grade III, and PVI) and analyzed them with respect to the presence or absence of plasma DNA, a significant difference (P = 0.009) was also observed. The rest of the variables analyzed displayed no statistically significant differences (Table 2)
. During the 1-year study period reported here, there were no cases of relapse or death, regardless of the presence or absence of plasma DNA.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Another aim of this study was to determine whether the presence of plasma DNA at diagnosis was significantly associated with some of the clinicopathological characteristics of the patients. Among the 13 parameters evaluated, we found 4 that showed statistically significant differences between patients with and without plasma DNA with tumor characteristics: (a) three (high proliferating index, three or more affected lymph nodes, and IDC) as independent variables; and (b) one comprised of PVI, axillary involvement, and high histological grade, which are three classical pathological parameters of poor prognosis (Table 2)
. Our patients showed no significant differences in other independent variables such as tumor size, stage, histological grade, steroid receptors, or PVI that are also indicative of tumor extension and aggressiveness and, secondarily, of tumor cell turnover and capacity for metastatic spread. To date, six studies have analyzed the presence of tumor DNA in plasma and disease stage, and the results have not been uniform. Two studies in head and neck carcinoma (27)
and small cell lung cancer (23)
reported a large number of patients with advanced disease and the presence of tumor DNA in plasma, and four studies also associated this molecular event with the early stages of non-small cell lung cancer (25)
, colon cancer (19
, 20) , and clear cell renal carcinoma (28)
. Taken together, the data available concerning the extension of tumor disease and the appearance of plasma DNA with tumor characteristics do not offer a conclusive explanation, suggesting that other unexplored biological characteristics of the tumor cells could be related to this phenomenon. Thus, the results of our study may indicate that the presence of plasma DNA of tumor origin in breast cancer patients is significantly associated with some histological features of highly malignant lesions.
In addition to the possible role of free plasma DNA as a prognostic factor in breast cancer, other applications can be also considered. The early diagnosis of breast cancer is currently one of the best strategies for improving the survival of these patients. If the results of this procedure were found to be positive in disease stage I or with tumors measuring less than 1 cm, it would be a good implementation of the current radiographic procedures. Moreover, the identification of premalignant disease with defined molecular alterations (54) would permit the investigation of these molecular events in patients with preneoplastic lesions, hypothetically changing the management of these processes.
Previous observations have suggested that plasma DNA can undergo quantitative changes in cancer patients after radiation therapy (10) ; however, its utility in monitoring chemotherapy and radiotherapy has yet to be fully defined.
The identification of recurrent breast cancer at a preclinical stage is an essential strategy in the fight against this disease, but no reliable serological predictive markers are currently available (55) . Considering the prevalence and incidence of this disease, the discovery of a valid marker could have a strong impact on its management and probably on survival. We consider that the utilization of a simpler methodology that, as in our case, does not involve radioisotopic techniques may help to introduce plasma DNA analysis into the study and management of breast cancer. In the coming years, prospective studies, such as that recently reported in pancreatic carcinomas (56) , will provide us with definitive information on the value of this research tool as a prognostic factor in cancer patients.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by grants from the Fundación Caja Madrid and FIS number 98/0847. 
2 To whom requests for reprints should be addressed, at Molecular Genetics Unit, Clinica del Trabajo, Avenida de la Reina Victoria 21, 28003-Madrid, Spain. Phone: 34-1-533-8255; Fax: 34-1-554-9398; E-mail: felixbv{at}stnet.es
3 The abbreviations used are: MI, microsatellite instability; dNTP, deoxynucleotide triphosphate; LOH, loss of heterozygosity; CI, confidence interval; SSCP, single-strand conformational polymorphism; PVI, peritumoral vessel involvement; LNM, lymph node metastasis; IDC, invasive ductal carcinoma.
Received 1/20/99. Accepted 5/ 3/99.
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G. Dominguez, J. Carballido, J. Silva, J. M. Silva, J. M. Garcia, J. Menendez, M. Provencio, P. Espana, and F. Bonilla p14ARF Promoter Hypermethylation in Plasma DNA as an Indicator of Disease Recurrence in Bladder Cancer Patients Clin. Cancer Res., April 1, 2002; 8(4): 980 - 985. [Abstract] [Full Text] [PDF]
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H.-Z. Zou, B.-M. Yu, Z.-W. Wang, J.-Y. Sun, H. Cang, F. Gao, D. H. Li, R. Zhao, G.-G. Feng, and J. Yi Detection of Aberrant p16 Methylation in the Serum of Colorectal Cancer Patients Clin. Cancer Res., January 1, 2002; 8(1): 188 - 191. [Abstract] [Full Text] [PDF]
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J. M. Silva, F. Bonilla, H. Blons, F. Coulet, and P. Laurent-Puig Correspondence re: F. Coulet et al., Detection of Plasma Tumor DNA in Head and Neck Squamous Cell Carcinoma by Microsatellite Typing and p53 Mutation Analysis. Cancer Res., 60: 707-709, 2000. Cancer Res., December 1, 2001; 61(23): 8595 - 8596. [Full Text] [PDF]
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P. Cairns, M. Esteller, J. G. Herman, M. Schoenberg, C. Jeronimo, M. Sanchez-Cespedes, N.-H. Chow, M. Grasso, L. Wu, W. B. Westra, et al. Molecular Detection of Prostate Cancer in Urine by GSTP1 Hypermethylation Clin. Cancer Res., September 1, 2001; 7(9): 2727 - 2730. [Abstract] [Full Text] [PDF]
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J. M. Silva, G. Dominguez, J. Silva, J. M. Garcia, A. Sanchez, O. Rodriguez, M. Provencio, P. Espana, and F. Bonilla Detection of Epithelial Messenger RNA in the Plasma of Breast Cancer Patients Is Associated with Poor Prognosis Tumor Characteristics Clin. Cancer Res., September 1, 2001; 7(9): 2821 - 2825. [Abstract] [Full Text] [PDF]
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G. Sozzi, D. Conte, L. Mariani, S. Lo Vullo, L. Roz, C. Lombardo, M. A. Pierotti, and L. Tavecchio Analysis of Circulating Tumor DNA in Plasma at Diagnosis and during Follow-Up of Lung Cancer Patients Cancer Res., June 1, 2001; 61(12): 4675 - 4678. [Abstract] [Full Text] [PDF]
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S. L. B. Rosas, W. Koch, M. d. G. d. C. Carvalho, L. Wu, J. Califano, W. Westra, J. Jen, and D. Sidransky Promoter Hypermethylation Patterns of p16, O6-Methylguanine-DNA-methyltransferase, and Death-associated Protein Kinase in Tumors and Saliva of Head and Neck Cancer Patients Cancer Res., February 1, 2001; 61(3): 939 - 942. [Abstract] [Full Text]
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X. q. Chen, H. Bonnefoi, M.-F. Pelte, J. Lyautey, C. Lederrey, S. Movarekhi, P. Schaeffer, H. E. Mulcahy, P. Meyer, M. Stroun, et al. Telomerase RNA as a Detection Marker in the Serum of Breast Cancer Patients Clin. Cancer Res., October 1, 2000; 6(10): 3823 - 3826. [Abstract] [Full Text]
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J. A. Shaw, B. M. Smith, T. Walsh, S. Johnson, L. Primrose, M. J. Slade, R. A. Walker, and R. C. Coombes Microsatellite Alterations in Plasma DNA of Primary Breast Cancer Patients Clin. Cancer Res., March 1, 2000; 6(3): 1119 - 1124. [Abstract] [Full Text]
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F. Coulet, H. Blons, A. Cabelguenne, T. Lecomte, O. Laccourreye, D. Brasnu, P. Beaune, J. Zucman, and P. Laurent-Puig Detection of Plasma Tumor DNA in Head and Neck Squamous Cell Carcinoma by Microsatellite Typing and p53 Mutation Analysis Cancer Res., February 1, 2000; 60(3): 707 - 711. [Abstract] [Full Text]
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M. Sanchez-Cespedes, M. Esteller, L. Wu, H. Nawroz-Danish, G. H. Yoo, W. M. Koch, J. Jen, J. G. Herman, and D. Sidransky Gene Promoter Hypermethylation in Tumors and Serum of Head and Neck Cancer Patients Cancer Res., February 1, 2000; 60(4): 892 - 895. [Abstract] [Full Text]
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