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Annals of Surgical Oncology 9:71-76 (2002)
© 2002 Society of Surgical Oncology

ORIGINAL ARTICLES

Persistence of Tumor DNA in Plasma of Breast Cancer Patients After Mastectomy

Jose M. Silva, PhD, Jose M. Garcia, Gemma Dominguez, PhD, Javier Silva, Celia Miralles, PhD, Blanca Cantos, Santiago Coca, PhD, Mariano Provencio, PhD, Pilar España, PhD and Felix Bonilla, PhD

From the Department of Medical Oncology, Clinica Puerta de Hierro (JMS, JMG, GD, JS, CM, BC, MP, PE, FB); and the Department of Pathology, Hospital Militar del Aire (SC), Madrid, Spain.

Correspondence: Address correspondence and reprint requests to: Felix Bonilla, Department of Medical Oncology, Molecular Genetics Unit, Clinica Puerta de Hierro, C/San Martin de Porres, 4, 28035-Madrid, Spain; Fax: 34-91-373 7667; E-mail: felixbv{at}stnet.es


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: We investigated tumor DNA changes before and after mastectomy in the plasma of breast cancer patients with no disseminated disease and eventually investigated these changes’ relationship to specific pathological parameters of the tumors.

Methods: We studied 41 patients. DNA extracted from tumor and normal breast tissues, mononuclear blood cells, and plasma was used for molecular studies. Alterations in the microsatellite markers D17S855, D17S654, D16S421, TH2, D10S197, and D9S161, as well as point mutations in the p53 gene and aberrant methylation of p16INK4a, were used to identify and characterize tumor and plasma DNA. A number of tumor clinicopathological parameters were analyzed in each patient.

Results: We found that 18 (44%) of the 27 patients with alterations in tumor DNA presented the same plasma DNA alteration before mastectomy, and persistence of the same molecular features was detected in plasma DNA 4 to 6 weeks postmastectomy in 8 (19.5%) patients. Patients with vascular invasion, more than three lymph node metastases, and higher histological grade at diagnosis displayed plasma DNA after mastectomy with a significant difference.

Conclusions: Persistence of plasma DNA with features of tumor DNA may be present after mastectomy in breast cancer patients, and its relation to bad-prognosis histological parameters may suggest undetectable micrometastatic disease.

Key Words: Postmastectomy • Mammary malignancies • Circulating DNA • Poor prognosis • Genetic markers


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In most developed and many developing countries, breast cancer is the most frequent cancer and the leading cause of cancer death in women.1 Although the mortality rate for breast cancer has not increased to the same degree as its prevalence, the development of strategies capable of detecting metastatic or recurrent disease in preclinical or presymptomatic phases of the disease is desirable.

Malignant tumor development is a multistep process that involves genetic changes, such as the activation of oncogenes and inactivation of tumor suppressor genes.2 In breast cancer, numerous chromosomal regions have been identified as harboring structural and epigenetic alterations, but the majority have not demonstrated a specific clinical marker role.3–10 However, the molecular genetic alterations detected in tumor cells may be indirectly used to specifically define the characteristics of tumor DNA found outside the primary tumor. In this way, the presence of tumor cells or specific tumor DNA in diverse samples, such as sputum,11 urine,12 pancreatic juice,13 stool,14 and nipple discharge,15 can provide a useful tool in early diagnosis and prognosis.

In recent years it has been possible to identify free circulating DNA in several tumor types, including those of higher prevalence, such as lung cancer,16,17 breast carcinomas,18–21 and colon cancer.22,23 The influence of this molecular alteration in patient survival has not yet been determined, and only one study in pancreatic carcinoma has offered evidence on its role as a prognostic factor.24

Two decades ago some studies reported variations in total free plasma DNA in cancer patients after radiotherapy, but these studies were unable to determine whether these variations corresponded to plasma DNA with tumor DNA characteristics.25 This study was designed to investigate the changes in this plasma DNA in breast cancer patients with no metastatic disease, before and after mastectomy, and eventually to investigate the relationship of these changes to specific pathological parameters of the primary tumors.


   PATIENTS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Tissue Sampling, DNA Extraction, and Tumor Characteristics
Between January 1, 1999, and December 31, 2000, tumor tissue and corresponding normal tissue samples were obtained sequentially at surgery from 41 patients undergoing mastectomy. Informed consent was obtained from all participants after the nature of the study, which was approved by the Research Ethics Board of our hospital, was explained. The samples were snap-frozen in liquid nitrogen until processing. No evidence of metastatic dissemination was revealed during pathological diagnosis or clinical evaluation in any patient at the time of mastectomy. Only patients undergoing mastectomy on the basis of either tumor size or clinical conditions were included, in an attempt to avoid the presence of residual tumor. Two blood samples were collected from each patient, the first sample being taken on the day of surgery before mastectomy and the second between 4 and 6 weeks postmastectomy. None of the patients showed recurrent clinical disease at this time. No patient received chemotherapy or radiotherapy before the extraction of the second blood sample. Blood samples were also obtained from 13 healthy controls, and plasma and mononuclear cell DNA were extracted.

DNA extraction from tumor and normal tissue samples and peripheral blood mononuclear cells was performed by a nonorganic method (S-4520 Kit; Oncor Inc., Gaithersburg, MD). Plasma DNA was purified on Qiagen columns (Qiamp Blood Kit; Qiagen Inc., Hilden, Germany) according to the blood and body fluids protocol, with the following modifications: Between 7.5 and 12 ml of plasma was heated to 99°C for 5 minutes on a heat block. The heated sample was then centrifuged at 14,000 rpm for 30 minutes, after which the clear supernatant, approximately 1 ml, was collected. Proteinase K, 20 mg/ml (Boehringer Mannheim, Mannheim, Germany), and buffer AL (Qiagen Inc.) were added in a 1:10 proportion with respect to the supernatant collected and were incubated overnight at 55°C. One column was used repeatedly until the complete sample had been processed. The DNA extracted was quantified spectrophotometrically.

Dates of birth and diagnosis, family history of the disease, and menopausal status data were obtained from the medical record of each patient, together with the following pathological characteristics of the tumors: tumor size, lymph node metastases, presence of steroid receptors (estrogen and progesterone), histological type, vascular invasion, histological grade, pathological stage, and proliferative index.

Microsatellite Analysis and Polymerase Chain Reaction Conditions
The characterization of tumor and plasma DNA was studied by investigating the presence of alterations in several loci of these DNA samples with six microsatellite markers. Polymorphic markers D17S855,26 D17S654,27 D16S421,28 TH2,8 D10S197,5 and D9S16129 were used because they have been reported to show a high rate of change in breast carcinomas.18 Microsatellite analysis was fulfilled by the polymerase chain reaction (PCR) method performed in 10-µl volumes with 50 ng of template DNA, .3 U of AmpliTaq Gold DNA polymerase (Perkin-Elmer, Roche Molecular Systems, Inc., Branchburg, NJ), 1 µl of 10x PCR buffer, 200 µM of deoxynucleotide triphosphate, .6 µM of each primer, and different concentrations of MgCl2, depending on the polymorphic marker. A 40-cycle amplification was performed in a thermal cycler (Perkin-Elmer, Cetus, Foster City, CA). The alleles were separated by mixing 10 µl of the PCR products with a 3-µl volume of loading buffer (total volume, 13 µl), .02% xylene cyanol, and .02% bromophenol blue. Electrophoresis was run on nondenaturing 8% to 12% polyacrylamide gels for 12 to 15 hours at 500 V. After gel electrophoresis, the allelic band intensity was detected by a nonradioisotopic technique by use of a commercially available silver staining method.30 Allelic intensities were analyzed by densitometry. The gel image was captured by a GS-690 Imaging Densitometer (Bio-Rad Laboratories, Hercules, CA), digitized at 400 dpi, and analyzed with Multi-Analyst/PC (Bio-Rad) (Fig. 1). In this study, loss of heterozygosity was considered to exist when the signal of the allele was reduced by more than 75%, and microsatellite instability was considered to exist when one or more novel bands appeared in tumor or plasma DNA.



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  		FIG. 1. Representative photograph of several gels from different patients; these gels were taken under normal light after staining with a (NO3)Ag method showing loss of heterozygosity in marker 9S191 in tumor (T) and plasma DNA samples before mastectomy (Pb) and after mastectomy (Pa); extra bands in exon 7 of the p53 gene in tumor tissue and plasma samples before mastectomy, but not in plasma sample after surgery; and methylation of p16INK4a in tumor tissue and plasma DNA before mastectomy. N, normal breast tissue; L, lymphocyte; M, methylated DNA; UM, unmethylated DNA.

 
Mutational Study of the p53 Gene
Tumor and plasma DNA were also analyzed for the presence of TP53 mutations at conserved exons by use of a PCR–single-strand conformation polymorphism (SSCP) method according to a modification reported by Orita et al.31 Exons 5, 6, 7, and 8 of the p53 gene were amplified by use of the primer set and PCR conditions as previously described.18,32 The allelic band intensity on the gels was detected by a nonradioisotopic method by use of a commercially available silver staining method.30 Specimens that showed a differential band at SSCP were amplified to obtain templates for DNA sequencing. These amplifications were independent of those used for SSCP analysis. Amplified DNA fragments were purified from .9% agarose gels by using the Geneclean Kit (Bio-101, Inc., La Jolla, CA) and were used for direct DNA sequencing by the deoxynucleotide triphosphate method with the Sequenase Kit (United States Biochemical Corp., Cleveland, OH).

Methylation Study of p16INK4a
DNA methylation patterns in the CpG island of the p16INK4a gene were determined by methylation-specific PCR, as previously described.33 Briefly, methylation-specific PCR distinguishes unmethylated from methylated alleles in a given gene on the basis of sequence changes produced after bisulfite treatment of DNA, which converts unmethylated (but not methylated) cytosine to uracil, and subsequent PCR with primers designed for either methylated or unmethylated DNA. In this way, DNA from tumor, normal tissue, plasma, and lymphocytes was denatured by NaOH and modified by sodium bisulfite. DNA samples were then purified with Wizard DNA purification resin (Promega, Madison, WI), again treated with NaOH, precipitated with ethanol, and resuspended in water. PCR reaction was directly loaded onto nondenaturing 8% polyacrylamide gels and stained with a nonradioisotopic specific method.30

Statistical Analysis
The variables analyzed were contrasted by means of the {chi}2 test, the {chi}2 test with the Yates correction, or Fisher’s exact test when any of the expected frequencies were <5. Two-tailed P values of <.05 were considered to be significant. Statistical analyses were performed with the EPI-INFO package, version 6.04 (CDC, Atlanta, GA) .


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
We analyzed 41 breast cancer patients and used the six aforementioned microsatellites, mutations of the Tp53 gene, and the methylation status of p16INK4a, as molecular markers. These molecular alterations have been reported to be present in a significant number of breast carcinomas. Plasma DNA was found in all 41 cases at concentrations ranging from 30 to 165 ng/ml (mean concentration, 122 ng/ml).

Tumor and Plasma DNA Alterations Before Mastectomy
LOH at a minimum of one locus of the six studied was observed in 23 (56%) breast carcinomas from the group of informative cases. The same analysis of the plasma DNA extracted from blood before mastectomy revealed 14 (34%) patients with the same microsatellite changes observed in tumors and 9 (26%) patients with no alterations. No MI was detected in tumor DNA. In two cases it was also observed that plasma DNA showed allelic loss not present in the tumor at marker D17S654.

Tumor mutations in the p53 gene were detected in seven (17%) carcinomas; the same mutations were identified in plasma DNA in two (5%) of these tumors. One of these two did not display concomitant LOH at any marker tested in either tumor or plasma DNA (Table 1). With respect to the methylation status of p16INK4a, the analysis revealed a considerable proportion of methylated tumor DNA; nine (22%) individuals, among them four (10%) patients, exhibited aberrant methylation in plasma DNA, and three of these did not present LOH at any marker studied in plasma DNA (Table 1 and Fig. 1). Molecular analysis included the same reactions (DNA of normal blood cells, normal breast tissue, tumor tissue, and plasma) in all cases, and no differences between normal blood cell and normal breast tissue DNA were observed in any of the reactions. Overall, with the markers used, molecular alterations were detected in 27 (66%) breast carcinomas, and 18 (44%) patients displayed a similar alteration in plasma DNA (Table 1).


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  		TABLE 1. Molecular alterations detected in tumor and plasma DNA before and after mastectomy in patients with breast cancer, revealed by analysis of microsatellite, study of p53 gene mutations, and aberrant methylation status of p16INK4a
 
Plasma DNA Alterations After Mastectomy
When the microsatellites, TP53 mutations, and methylation of p16INK4a in plasma DNA extracted from the blood samples taken between 4 and 6 weeks after mastectomy were analyzed, 6 (15%) patients of the 14 who had shown microsatellite alterations before mastectomy were found to retain the same molecular changes observed in tumor DNA and in premastectomy plasma DNA (Table 1). No TP53 mutations were observed in the two patients who displayed mutations in plasma DNA before mastectomy, and two (5%) of the four patients with aberrant methylation maintained this change in plasma DNA after mastectomy.

Altogether, 8 (19.5%) of the 18 (44%) patients with molecular alterations in premastectomy plasma DNA displayed plasma DNA that retained the same molecular alteration 4 to 6 weeks postmastectomy (Table 1 and Fig. 1).

In 10 of the 13 healthy controls, the amount of plasma DNA extracted allowed amplification with the same molecular markers as in the patients. In each case, the markers displayed the same patterns in both plasma and normal blood cell DNA.

Tumor Characteristics and Molecular Changes
In the search for an explanation for the persistence of this plasma DNA with tumor DNA characteristics in blood samples taken after mastectomy, a study was made of the correlation between the presence or absence of plasma DNA with these features postmastectomy and the previously mentioned clinicopathological characteristics of the tumors at diagnosis. It was found that tumors concomitantly exhibiting three characteristics classically associated with high-grade malignancy (more than three lymph nodes affected, vascular invasion, and histological grade III) exhibited a statistically significant difference (P = .04) (Table 2), suggesting that in their follow-up these patients could be more prone to metastatic disease. The same correlation study between patients with abnormal plasma DNA and patients without tumor plasma DNA before mastectomy, with respect to the same clinicopathological parameters of the tumors, displayed only a tendency to significance (P = .07) in the subgroup of patients with the three mentioned characteristics concomitantly as related to a bad prognosis (Table 3).


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  		TABLE 2. Correlation between patients with molecular alterations in plasma DNA after mastectomy and high-grade malignancy characteristics
 

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  		TABLE 3. Correlation between patients with molecular alterations in plasma DNA before mastectomy and high-grade malignancy characteristics
 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study was designed to investigate the persistence of plasma DNA after mastectomy, providing evidence of the continued presence of circulating DNA with characteristics of tumor DNA postmastectomy. In this subset of breast cancer patients it was found that 47% of patients presented alterations in plasma DNA similar to those existing in tumor DNA premastectomy; this result is equivalent to other previous reports on breast carcinoma.18–21 However, this report describes an original datum: the persistence of plasma DNA after mastectomy in breast cancer patients.

The variations of free plasma DNA in cancer patients, regarding a therapeutical approach such as radiotherapy, were analyzed two decades ago. Diverse decreasing or increasing proportions during and after treatment were observed, but several issues remained to be resolved in this report25: (1) radiotherapy is not a radical treatment and sometimes serves only as palliative therapy; (2) no information about the posttreatment disease status was reported; and (3) the specific characterization of this plasma DNA was inaccurate. Recently, Lo et al.34 reported a quantitative analysis to evaluate cell-free Epstein-Barr virus DNA in plasma from patients with nasopharyngeal carcinoma; they observed fluctuations after radiotherapy and persistence of the disease in patients with no regression of the molecular alteration. This report investigated the persistence of tumor DNA in plasma of breast cancer patients after radical treatments, such as mastectomy, without clinical recurrence or macroscopic residual tumor at the time the blood samples were taken.

Some factors that may influence this finding could be related to the clearance of tumor DNA from plasma. This phenomenon has yet to be studied in patients with tumors, but under other circumstances, such as the clearance of fetal DNA from maternal plasma, it occurs very rapidly.35 However, the possible incorporation of tumor DNA or tumor cells from micrometastatic sources should also be considered. In this case, this molecular alteration may be an indicator of clinically silent microdisseminated disease.

We considered it important to determine whether the persistence of plasma DNA after mastectomy was significantly associated with any of the pathological characteristics of the primary tumors. No parameters were found, among those evaluated, that differed significantly among patients without tumor plasma DNA, patients with abnormal plasma DNA before surgery only, and patients with circulating DNA after mastectomy. However, when we analyzed a parameter comprising three characteristics associated with a bad prognosis, such as involvement of more than three lymph nodes, histological grade III, and vascular invasion, a significant concentration of this parameter was observed in the patient subgroup with aberrant plasma DNA after mastectomy.

These results suggest that persistence of plasma DNA after mastectomy may identify a group of patients whose disease has more aggressive features that could also have facilitated micrometastatic dissemination, the possible origin of the circulating DNA detected at this time. Its validity as a predictive marker of recurrent breast carcinoma will have to be evaluated in prospective follow-up studies.


   Acknowledgments
 
Supported by grants from Fundacion Caja Madrid; Fondo de Investigacion Sanitaria n° 01/0505; the Spanish Association Against Cancer; and Bristol-Myers, S.A. We are indebted to all the women who participated in the study and to Martha Messman and Martin Hadley-Adams for the revision and preparation of this manuscript.

Received for publication June 22, 2001. Accepted for publication August 29, 2001.


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 ABSTRACT
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 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
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