Originally published as Ann Surg Oncol Early Release 10.1245/ASO.2004.03.049 on May 18, 2004
Annals of Surgical Oncology 11:560-567 (2004)
© 2004 Society of Surgical Oncology
DNA Fingerprints Provide a Patient-Specific Breast Cancer Marker
SuEllen Toth-Fejel, PhD,
Patrick Muller, BS,
Bruce Ham, MD,
Kevin Esvelt,
Nicole Dumas,
Kristine Calhoun, MD and
Rodney Pommier, MD
From the Division of Surgical Oncology, Department of General Surgery, Oregon Health & Science University, Portland, Oregon.
Correspondence: Address correspondence and reprint requests to: Rodney Pommier, MD, Division of Surgical Oncology, 3181 S. W. Sam Jackson Park Road, Mail Code L223A, Portland, Oregon; Fax: 503-494-7573; e-mail: pommierr{at}ohsu.edu
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ABSTRACT
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Background: Detection of systemic breast cancer recurrence is limited by lack of universally expressed tumor cell markers. We hypothesized that a test that detects genetic alterations specific to breast cancer cells of an individual patient would provide a superior cancer marker.
Methods: DNA was extracted from blood, primary tumor, and axillary lymph nodes of 33 breast cancer patients and normal breast tissue of 12 control patients. A patient’s genome was scanned by PCR amplification between Alu sequences. A DNA fingerprint of approximately 17–40 bands was produced for comparison between normal blood and sampled tissues.
Results: There were 7 stage I, 18 stage II, 7 stage III, and 1 stage IV breast cancer cases; 33 of 33 cancer cases showed DNA fingerprint differences between blood and primary tumor (P < .0001).This test predicted 100% of positive nodes. No false-negatives occurred, and in two cases malignancy was detected in histologically negative nodes. Three of the 12 controls showed a single similar band change.
Conclusions: DNA fingerprinting is a method for detecting and characterizing genetic alterations specific to an individual patient’s primary tumor in 100% of cases tested. These specific changes were also identified in 100% of positive nodes, proving the capacity of the test to detect metastases.
Key Words: Alu-PCR • Breast cancer • DNA fingerprinting • Tumor markers
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INTRODUCTION
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Breast cancer is associated with multiple molecular genetic findings, including loss of heterozygosity (LOH),1 amplification and deletions of genes, defects in DNA-mismatch repair genes, and BRCA1 and BRCA2 mutations in hereditary breast cancer.2 As tumor progression continues, early malignant molecular changes cascade into large genomic instability reflected as chromosome gains and losses.3 Although no single rearrangement is diagnostic for breast cancer, several rearrangements occur at higher-than-expected frequencies. These include rearrangements leading to gain and losses of genetic material from chromosomes 1, 3, 6, and 8. The most common numerical aberration (and occasionally the sole one) is an extra copy of chromosome 7, 8, 18, or 20.4
The genetic variability in breast cancer has been a barrier to finding universal markers for this cancer. Marker tests rely on the expression of specific genes in breast cancer cells to serve as the target of detection.5–8 However, it appears that there is no single tumor marker that is consistently and specifically expressed by all primary breast cancers. In addition, marker expression may differ between the primary tumor and its metastases.5
This study was initiated to determine the feasibility of developing a molecular test that would not rely on any specific gene expression but rather would use the unique DNA changes in each patient’s tumor to provide a marker for the breast cancer. In order to detect a wide variety of genetic changes, which could occur throughout the genome, we chose to use a polymerase chain reaction (PCR) method that would provide a large-scale scan of the entire genome. We hypothesized that utilizing Alu repeat sequences as the primer sites for PCR would enable us to achieve this goal.
Alu repeat elements are short sequences of DNA of about 100–300 base pairs (bp) in length. They occur semirandomly throughout the genome and are located within and between genes.9 When DNA is amplified between Alu sequences by a PCR method, multiple regions of the genome are simultaneously amplified. These regions vary in length. When the DNA that is produced by this process is separated by length through a gel matrix, a DNA fingerprint is produced that resembles a bar code.10,11 Because the spacing of Alu sequences varies between individuals, the bar code pattern reflects the unique qualities of a person’s DNA, and we have thus chosen to refer to the pattern obtained as a "fingerprint."12,13
DNA abnormalities that occur in breast cancer will shift the genomic positions of Alu sequences and alter the DNA fingerprint pattern. When the DNA fingerprint from normal tissue is defined as the normal standard in a given patient, it can then be compared with a DNA fingerprint made from breast cancer DNA. Any differences between the two DNA fingerprints highlight the abnormal changes associated with the patient’s own breast cancer, and thus a breast cancer fingerprint for that patient is established. It has been reported that the insertion of an Alu I sequence into a BRCA2 gene resulted in disruption of the gene and in breast cancer.14
It was the goal of this study to evaluate the reliability and sensitivity of this PCR-based molecular test for detecting malignancy in all breast cancer patients, regardless of the genetic etiology of their disease.
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MATERIALS AND METHODS
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Study Population and Samples
Patients with breast cancer who had not received any prior treatments were eligible for this study. In addition, women undergoing breast-reduction operations were enrolled as normal controls. Women with breast cancer had blood samples drawn to be used as a source of their normal DNA fingerprint. During their breast operations, a 1.5-cm3 portion of their primary tumor and axillary nodes were obtained and used to produce DNA fingerprints of their primary tumor and lymph nodes. Two to four lymph nodes were obtained from patients undergoing axillary dissection. These nodes were bivalved for distribution to surgical pathology and this study. When patients developed recurrent disease, tissue from the sites of recurrence or metastases was obtained and processed similarly to the primary tumor samples. A new blood sample was also obtained. Blood samples were drawn and breast tissue was obtained from normal control patients to be used as sources of control DNA fingerprints. The study was approved by the Oregon Health & Science University Institutional Review Board.
Molecular Testing
DNA was extracted from breast cancer, lymph nodes, and blood with a spin column protocol (Qiagen). PCR amplification of DNA from each of the tissue samples was performed for each patient with use of up to three different Alu I primers. It is important to note that the Alu I repeat was not the region that was amplified; rather, the DNA between Alu repeats was amplified.13
The Alu-specific primers (used one at a time) were R12A/267: AGCGAGACTCCG; R14B/264: CAGAGCGAGACTCT; and A/8: TGAGCCACCGCG. The 50-µl reactions were performed in 10mM tris-HCl (pH, 8.8), 50 mM KCl, 1.5 mM MgCl2, 100 µM dNTPs each, 1 µM primer, 1 unit DNA polymerase (AccuprimeTaq; Invitrogen), and 250 ng DNA template. The PCR reaction consisted of denaturation at 94°C for 7 minutes, 27 amplification cycles (30 seconds at 94°C, 45 seconds at 50°C, and 120 seconds at 72°C), and an extension at 72°C for 7 minutes. Samples were amplified in duplicate or until a DNA fingerprint was firmly established.
Following amplification, PCR products were run in parallel in adjacent lanes during electrophoresis to separate DNA fragments according to length. The resulting DNA fragments were visualized as a series of bands (a DNA fingerprint) on a nondenaturing 3.5% polyacrylamide gel with ethidium bromide nucleic acid staining. Genetic changes between a patient’s normal blood DNA and other samples were identifiable as the presence or absence of a band or a staining intensity difference of a specific band (Fig. 1). Kodak 1D Image Analysis Software (Kodak) was used to quantify differences between the patient’s tumor DNA fingerprint and the normal DNA fingerprint.
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FIG. 1. Schematic diagram of the Alu PCR technique, demonstrating band differences between a normal fingerprint, fingerprint with band-intensity changes, and fingerprint with band changes.
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Statistical Analysis
For the purpose of this test, an abnormal DNA fingerprint was defined as one having any band change or any band-intensity change that was at least three times normal in comparison with the normal blood fingerprint. A normal DNA fingerprint was defined as one that matched the normal blood DNA fingerprint with no band-intensity changes greater than or equal to three times normal.
Positive and negative predictive value (PPV and NPV) and sensitivity and specificity were calculated with the following formulas, where TP indicates true-positive, TN indicates true-negative, FP indicates false-positive, and FN indicates false-negative results: sensitivity = TP/(TP + FN); specificity = TN/(TN + FP); PPV = TP/(TP + FP); NPV = TN/(TN + FN). Statistical significance of the ability of the test to discriminate between normal breast tissue and breast cancer or between normal lymph nodes and lymph nodes containing metastases was determined by Fisher exact test.
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RESULTS
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Tissue and blood samples were obtained from 45 women. This included 33 women with breast cancer, 11 normal control patients undergoing breast reduction operations, and 1 patient for whom the malignancy status of her breast mass was uncertain at the time of surgery. One patient listed as stage IV was documented as stage III at presentation but was restaged as stage IV when CT scans were completed (Table 1).
Amplification of DNA from tumor, blood, and breast tissue of all patients and normal controls produced DNA fragments with lengths ranging between 400 base pairs and 2000 base pairs. Depending on which primer was used, the number of fragments (i.e., bands) per DNA fingerprint ranged between 17 and 40.
Among the normal controls, nine of 12 showed no band changes between their normal blood DNA and breast tissue DNA fingerprints (Fig. 2). When DNA fingerprint bands were compared for band intensity differences between normal blood DNA and normal breast DNA, no magnitude change was observed over 1.9. In other words, the genetic material from one source was never twice greater than the other source. Three patients showed a difference of a single band. In all three cases the extra band was approximately 1300 base pairs in length. Band intensity variation observed in these three samples was similar to the other normal sample. The single patient for whom the breast mass pathology was unknown at the time of operation had no band alterations between her normal blood and breast mass DNA fingerprints. A diagnosis of benign breast tissue was confirmed by histology. This patient was included among the normal controls for the purposes of this study.
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FIG. 2. Examples of DNA fingerprints obtained from blood (26B, 44B) and normal breast tissue (26M, 44M) from two normal control patients.
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In all 33 patients with breast cancer, a DNA fingerprint was produced from their breast tumor DNA that differed from that obtained from their normal DNA (Fig. 3). The changes in number of bands and band intensities per breast cancer DNA fingerprint ranged between 1 and 13 in total and were reliable and reproducible (Fig. 4). Both losses and gains of genetic material were observed in tumors, with the majority of fingerprint differences occurring between 600 and 2000 base pairs. The number of band changes did not correlate significantly with tumor size or stage. All tumor samples were confirmed by histopathology as malignant. The sensitivity of the DNA fingerprint test was 100% (P < .0001). The specificity to identify malignancy was 75%. The PPV was 92% and the NPV was 100%.
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FIG. 3. Examples of DNA fingerprints obtained from blood (8B, 56B) and breast cancer tissue (8T, 56T) from two patients with breast cancer. Arrows point to the regions of band discrepancies at 1600–1700 base pairs in patient 8 and 1300 base pairs in patient 56.
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FIG. 4. Bar graph indicating the total number of band changes and band-intensity changes observed in tumor DNA fingerprints of breast cancer patients.
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DNA from 39 lymph node samples from breast cancer patients was examined. Twenty-five lymph nodes produced DNA fingerprints that matched the normal blood DNA fingerprint (Fig. 5). These nodes were also diagnosed as negative for metastases by surgical pathology. Nine nodes with DNA fingerprints matching that of the primary tumor were diagnosed by surgical pathology as positive for metastases (Fig. 6). Two nodes that were diagnosed as normal by surgical pathology were malignant by our test, with a DNA fingerprint that matched that of the primary tumor. Follow-up of these two patients will be necessary to determine the significance of these findings.
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FIG. 5. Examples of DNA fingerprints obtained from blood (60B), breast cancer tissue (60T), and two normal axillary lymph nodes (60NA, 60NB) from a patient with breast cancer. Arrows point to the regions of band discrepancies at 1400 base pairs and an abnormal band intensity change at 1600 base pairs. No such abnormal band changes are seen in the two axillary lymph fingerprints.
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FIG. 6. Examples of DNA fingerprints obtained from normal breast tissue from the contralateral breast (36M), blood (36B), breast cancer tissue (36T), and two abnormal axillary lymph nodes (36NA, 35NB) from a patient with breast cancer. Arrows point to a 1550-base-pair band with abnormal intensity changes in tumor and two axillary lymph nodes.
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There were three anomalous nodes examined in the study. One was found within the tail of the breast of a woman undergoing total mastectomy for breast cancer, as she refused axillary dissection. The lymph node was diagnosed as positive for metastases by surgical pathology, but the DNA fingerprint matched neither the normal blood nor the primary tumor fingerprints. She refused all adjuvant therapies. At the time of an axillary dissection for a massive axillary recurrence, two additional nodes underwent DNA fingerprinting and histological examination. Both nodes were diagnosed as positive for metastases by surgical pathology. Both nodes were shown to have a DNA fingerprint that again did not match the primary tumor but was identical to the fingerprint of the node removed during mastectomy.
With respect to the detection of metastases in lymph nodes, the sensitivity of the DNA fingerprint test was 100% (P < .0001). The specificity was 93%. The PPV was 86% and the NPV was 100%.
One patient had a recurrence with distant metastases during this study. MRI following a seizure revealed two brain lesions. Both lesions were surgically excised, with half of each specimen going to surgical pathology and for this study, but pathologists were unable to establish the origin of these tumors by histology and immunohistochemical staining. DNA fingerprinting, however, showed that both tumors had fingerprints identical to that of the primary breast tumor, suggesting that the origin was her breast tumor. In addition, blood obtained at the time of resection of the brain lesions differed from that of her initial blood sample in that it showed evidence of circulating tumor cells (Fig. 7).
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FIG. 7. DNA fingerprints from a patient with breast cancer metastatic to the brain. Fingerprints from blood, tumor, and axillary lymph node obtained at time of initial diagnosis (1-6B, 1-6T, and 1-6N), blood (62B2), and two metastatic tumors obtained from the brain (62C1 and 62C2). Arrows indicate the loss of bands at 1600 and 1800 base-pair bands in the tumor. Samples C1 and C2 also show loss of band 1600. The blood sample 62B2 is different from the normal 1-6B sample and similar to the primary (1-6T ) and metastatic tumor (C1 and C2 ) fingerprints.
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The results of serial DNA fingerprinting during the treatment course of one patient with stage IIIB breast cancer are shown in Fig. 8. This patient presented 2 months after a normal mammogram with extensive inflammatory breast cancer. DNA fingerprinting of her blood and her breast tumor revealed no band changes. Following neoadjuvant chemotherapy with Cytoxan, doxorubicin, and taxotere, her blood and DNA fingerprints changed. A band at approximately 1700 base pairs was no longer present in either fingerprint. However, 70 days later and 4 weeks after completion of neoadjuvant radiation therapy, the 1700-base-pair band was once again present in the DNA fingerprints of both blood and tumor obtained from the mastectomy specimen. A blood sample obtained 27 days after mastectomy, which was 25 days before death from fulminate pulmonary metastases, revealed increased amplification of this band.
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FIG. 8. DNA fingerprints from a patient with inflammatory breast cancer. Fingerprints from blood and tumor obtained at time of initial diagnosis (34B and 34T), blood and tumor obtained during neoadjuvant chemotherapy (51B2 and 51T2), contralateral breast tissue and tumor from mastectomy (57M and 57T3), and blood (61B3 ) 23 days prior to death. Arrow indicates the 1700-base-pair band that is present or lost at different time periods during her disease course. Please note that the patient had extensive lymphatic invasion of pectoralis major muscle at mastectomy, and this may explain the presence of the 1700-base-pair band in the contralateral breast tissue.
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DISCUSSION
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Several research groups have sought to develop breast cancer markers by employing tests that detect the expression of specific genes. However, to date there are no consistent breast cancer–specific molecular markers universally found among breast cancer patients. Markers such as CK18, CK19, CK20, MUC-1, and mammoglobin that have been associated with breast cancer are sometimes found in normal cells too.6,8 CK-19, c-Met, MUC-1, and beta-GalNAc-T are expressed in normal lymph nodes.5 In an attempt to increase marker sensitivity and specificity, a multimarker approach that searches for panels of mRNA markers in tumors, lymph nodes, and circulating blood has been developed.6 One such study panel that included beta-hCG, c-Met, beta-GalNAc-T, and MAGE-A3 showed that although primary tumors were found to have at least one marker in all but 1 of 25 tumors, only 4 of 25 expressed all four markers. At least one tumor marker was detected in only 69% of the blood samples. Mammoglobin and CEA mRNAs have been detected in 75% and 95% of primary tumors, respectively, showing 97% and 79% sensitivity in detecting malignant lymph nodes.5 CA27.29 appears to be superior to CA15.3 for detecting relapse in stage II patients (80% versus 10%–29%, respectively),7 but neither have been shown to be of clinical benefit.15
Given the broad genetic variation that occurs among breast cancers and the fact that modifications in gene expression are often a direct result of genomic alterations, the lack of consistent tumor markers is not surprising. The approach taken in this study was to develop an individually tailored molecular test that could serve as a marker for breast cancer that did not rely on gene expression or any specific genetic abnormality. Rather, it was based on an understanding of the genetic complexity of the disease and exploited the fact that the genetic changes for each patient’s breast cancer are, if not unique, certainly different from their normal DNA. By using Alu-PCR DNA fingerprinting, we were able to take advantage of the genomic rearrangements characteristic of breast cancer to provide a method for detecting the presence of malignancy in all cases. The findings of this study showed that a breast cancer DNA fingerprint, which is a reflection of genomic rearrangements, is distinct from that obtained from normal tissue and can identify the same malignancy in other tissues and organs.
This method of testing detected changes specific to the breast cancer cells of an individual patient and thereby provided a breast cancer DNA fingerprint that was a patient-specific cancer marker. This method was 100% sensitive among the 33 patients with breast cancer tested, which is superior to the sensitivity reported for other breast cancer markers. It also has an NPV of 100%. The specificity and PPV of 75% and 92%, respectively, are lower because of the single band change found in three normal women undergoing breast reduction, but they are comparable to those reported for other markers. The significance of this band change, and whether it would be found in women who don’t have breast hypertrophy, is unknown and warrants further study.
Our study showed that tumor DNA fingerprints were maintained in malignant lymph nodes in all but one case. The single case in which the lymph node fingerprints differed from the primary tumor fingerprint may represent further progression of disease or, alternatively, represent the detection of an occult second primary. DNA fingerprint results from the other 36 lymph nodes and the correlation between the initial lymph node fingerprint and that of the massive axillary recurrence suggest the latter possibility. With respect to lymph nodes that are diagnosed as positive for metastases by surgical pathology, our test had a sensitivity and NPV of 100%. The specificity and PPV of 93% and 86%, respectively, are lower because of the two lymph nodes that the test called positive but were negative by surgical pathology. However, it is has been reported that both serial sectioning of lymph nodes and PCR techniques are more sensitive than standard histopathologic examinations at detecting the presence of metastases in lymph nodes.16,17
A future goal for this technique will be to use it as a simple blood test to monitor patients’ responses to treatments, detect residual disease after treatments, and provide early detection of recurrence. We anticipated that this will require considerable refinements and increases in the sensitivity of the test before this will be practical. We were pleased to find that, at this early level of development, our technique is already demonstrating some of this capability. The DNA fingerprints of our patient with very aggressive inflammatory breast cancer suggest that malignant cells were circulating in her blood prior to treatment and that only after treatment were they reduced. They again appeared in the blood prior to the appearance of pulmonary metastases and her death. Similarly, when our other patient developed brain metastases, her blood DNA fingerprint showed evidence of circulating cancer cells. It is also apparent from these cases that blood does not provide a good source of normal DNA in all breast cancer patients and that an alternative source, such as skin fibroblasts, should be used in future studies.
Because blood was used as the source of normal DNA, breast cancer cells circulating in the blood may have contributed to what was interpreted as the normal fingerprint in some cases.
While this may not be a problem when tumor burden is low, it may, in some cases, diminish the ability of this testing method to identify the true breast cancer fingerprint, as was demonstrated in the patient with inflammatory disease. Given this study result, we suspect that our tally of band-change differences between the tumor DNA fingerprint and that of the normal DNA obtained from the blood is lower than what we would have observed if a different source of normal tissue had been used.
The discovery of conservation of the tumor DNA fingerprint in lymph nodes and distant metastases provides a method for determining the origin of secondary tumors. Our results suggest that with more experience analyzing DNA fingerprints from multicentric breast cancer, it will be possible to detect the presence of an occult secondary tumor by inspecting the patient’s lymph node DNA fingerprints. This has treatment implications with respect to deciding on lumpectomy or mastectomy procedures for local control of the breast. In addition, the two cases in this study in which DNA fingerprints were examined prior to, during, and following treatment demonstrated the possibility of this test to evaluate the efficacy of treatment regimens as well as to identify breast cancer cell populations that were refractory to treatment.
In this study, three normal control patients demonstrated a single band change. It is interesting that in all three cases, the change was in the mammary tissue, involving the increase of a single band that was approximately the same length. Only one breast cancer DNA fingerprint showed an abnormality of this band length, and in that case there was a substantial amplification of that band. The clinical implication of this band change among the normal patients is unknown. In choosing our normal patient population, we chose readily available tissue. However, it must be noted that these women were all patients receiving medical treatment for breast hypertrophy. It is unclear whether our findings represent a false-positive result for malignancy, a marker for hypertrophy, or a premalignant lesion.
As we follow the women in this study over time it may be possible to provide clinical outcome information on women for whom this test revealed an abnormal DNA fingerprint for lymph nodes that were normal by pathology. Although reports in the literature suggest that the malignancy status of lymph nodes based on PCR analysis shows no difference in survival, that analysis is based on gene expression assays.17 This study, by examining the malignancy status through direct examination of genomic changes, may provide different outcome results.
Our future work will include the further development of this assay so that it will be possible to detect the subclinical presence of breast cancer cells through a simple blood test by scanning for the presence of the patient’s breast cancer DNA fingerprint in their circulating blood. It is hoped that with the means for sensitive surveillance of small numbers of breast cancer cells circulating in the blood, before bones and vital organs are grossly affected, treatment regimens can be more effective with less total body toxicity.
The genetic material from this study has provided a repository of the DNA fingerprint changes detected by Alu-PCR. Currently, the abnormal 1300-base-pair fragment detected in the three normal breast tissue samples is being cloned and sequenced. Similarly, we have initiated the cloning of DNA fragments from abnormal breast cancer fingerprints. They will be sequenced and mapped to the human genome in order to identify genes involved in breast cancer development and dissemination. This information will outline the scope of genetic abnormalities that can lead to breast cancer and promote refractory disease. This valuable information will provide invaluable direction for developing therapeutic protocols that target or perhaps multitarget breast cancer with greater tumor specificity and cytotoxic efficacy.
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ACKNOWLEDGMENTS
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The acknowledgments are available online in the fulltext version at www.annalssurgicaloncology.org. They are not available in the PDF version.
The authors thank Dr. Julianna Hansen for contributing normal control patients to the study; John Waskey for technical assistance with illustrations; Barbara and Anne for their courage; and women’s golf clubs of the Portland area and the Oregon Chapter of the Order of the Eastern Star for their funding support.
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FOOTNOTES
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This study provides a method of using the unique genetic changes of each patient’s breast cancer for producing a patient-specific marker. The marker is conserved in lymph node and distant metastases. Preliminary evidence indicates that responses to treatment and disease progression can be detected in the blood with the use of such markers.
Received for publication March 8, 2003.
Accepted for publication February 23, 2004.
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ABSTRACT
INTRODUCTION
