Affiliations of authors: P. O'Connell, K. Fischbach, S. K. Mohsin, D. C. Allred (Department of Pathology), S. Hilsenbeck, S. A. W. Fuqua, G. M. Clark, C. K. Osborne (Division of Oncology, Department of Medicine), The University of Texas Health Science Center at San Antonio.
Correspondence to present address: Peter O'Connell, Ph.D., Baylor College of Medicine, Baylor Breast Center, One Baylor Plaza, MS600, Houston, TX 77030.
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ABSTRACT |
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INTRODUCTION |
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There is convincing evidence that a large number of genetic events play a role in the etiology and progression of individual breast cancers (4-8). An important goal in our efforts to understand and treat breast cancer is the identification of the genetic events that herald progression from local to metastatic disease. When used in polymerase chain reaction (PCR) assays (9), microsatellite markers amplify locus-specific genetic polymorphisms present at high frequencies on all human chromosomes (10-14). For individuals who are genetically informative for a marker (distinguishable maternally and paternally derived alleles), loss of heterozygosity (LOH, or allelic imbalance) assays can compare normal and tumor DNA, and de novo genetic changes in the tumor DNA can be detected (15-18). LOH events arise during the evolution of tumorsloss (or amplification) of a chromosome segment confers a growth advantage to the mutated tumor cell relative to its neighbors and the expanding subclones of tumor cells all inherit the LOH event. Therefore, the presence of LOH indicates that the tumor is a clonal neoplasm, and high-frequency sites of deletion detected by LOH have been associated with sites of tumor suppressor genes.
Previous reports (15-18) document a complex relationship between LOH, tumor suppressor genes, and breast cancer, and many LOH loci have been identified. However, with the exception of the recently identified hereditary breast cancer susceptibility genes BRCA1 and BRCA2 (19,20), which may account for up to 5%-10% of the cases, no single genetic event is known to be necessary and sufficient to lead to breast cancer. Some reports (21,22) demonstrate a link between LOH and metastatic potential. High levels of expression of tumor suppressor genes, such as nonmetastatic 23 (NM23, the human homologue of the Drosophila abnormal wing development gene), may interfere with the development of lymph node metastases (21). A statistically significant increase in LOH events involving an unknown gene on chromosome 16q is associated with distant metastases in breast cancers (22).
In previous studies (23,24), we have detected increasing rates of LOH in progressively higher stages of breast cancer evolution, with the lowest rates being seen in hyperplasias and the highest rates being seen in primary invasive breast cancer and metastases. This proved to be the case for all of the 15 markers that we tested, with the single exception of D14S62 at 14q31.2 where rates were much higher in primary tumors than in metastases. To further investigate this unexpected result, we tested additional markers in this region of chromosome 14q (D14S302, D14S265, and D14S51) in an expanded set of primary breast cancers from patients with lymph node-negative and lymph node-positive disease.
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MATERIALS AND METHODS |
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Histologic slides from routine, archival, clinical specimens were screened microscopically for adequate amounts of control (normal) tissue and invasive or metastatic breast cancers. For all of the patients with lymph node-negative disease, normal tissue was recovered from normal lymph nodes (75% of the specimens) or from benign breast tissue (25% of the specimens). For patients with lymph node-positive disease, normal tissue was harvested from lymph nodes (90% of the specimens) or benign breast tissue (10% of the specimens). On the basis of data (25) suggesting that morphologically normal breast tissue (terminal duct lobular units) closely adjacent to breast cancer may occasionally show LOH for some loci, we may be slightly underestimating LOH for some markers when adjacent terminal duct lobular units were the only normal tissues available. Samples of normal/tumor tissue pairs were microdissected from a total of 42 specimens from women with lymph node-negative breast cancer and from a total of 34 specimens from women with lymph node-positive breast cancer. The overall study and the protocol used to select archival specimens were reviewed and approved by The University of Texas Health Science Center at San Antonio Institutional Review Board.
Microdissection of specimens was performed on serial sections from formalin-fixed, paraffin-embedded tissue blocks. Alternating 3- and 10-µm-thick sections were cut from the formalin-fixed, paraffin-embedded tissue blocks and float mounted on glass slides. The 3-µm-thick slides were stained with hematoxylin-eosin and examined under the light microscope to locate regions of normal and cancer tissues; these regions were outlined with a felt-tipped pen. The marked slide was used as a template to guide microdissection from corresponding regions of the unstained 10-µm-thick sections. Samples were screened microscopically to ensure that at least 75% or more of the cellularity of each sample was derived from the targeted tissue. Microdissection was performed on a light box under a dissecting microscope.
DNA was liberated from the microdissected specimens by the use of a modification of the procedure developed by O'Connell et al. (23,24) and by Wright and Manos (26). Briefly, paraffin and lipids were first extracted in 0.4 mL of octane, and the lysate was digested for 3-12 hours at 55 °C in 50 µL of 0.01 M Tris-HCl (pH 8.5), 0.001 M EDTA, 0.045% Nonidet P-40 (Sigma Chemical Co., St. Louis, MO), and 0.045% Tween 20 (Sigma Chemical Co.) containing 1.0 mg/mL proteinase K (Life Technologies, Inc. [GIBCO BRL], Gaithersburg, MD). The lysate was heated to 95 °C for 15 minutes to inactivate the protease and stored at -20 °C. Samples were diluted between 1 : 5 and 1 : 15 in lysis buffer without protease, and 5 µL of diluted lysate was used in PCR assays.
PCR-Based LOH Assays
Samples were independently evaluated for LOH at each of the four polymorphic 14q loci (see Table 1). The antisense primer was 5'-radiolabeled in
a standard polynucleotide kinase reaction by 3000 Ci/mmol[
-32P]adenosine triphosphate (ATP) (Du Pont NEN, Boston, MA) at a molar ratio of 18
[
-32P]ATP : 1 primer, as previously described (23,24). Thirty cycles of PCR (denaturing at 94 °C for 30 seconds, annealing at 55
°C for 1 minute, and extension at 72 °C for 1 minute) were carried out in a 15-µL
assay containing 5 µL of diluted lysate, 10 mM Tris-HCl (pH 8.3), 50 mM
KCl, 1.5 mM MgCl2, 0.1 M spermidine (Sigma Chemical Co.), 0.1
µM of each PCR primer, 0.1 µM deoxynucleoside triphosphate (Life
Technologies, Inc.), and 0.5 U of Taq polymerase (The Perkin-Elmer Corp.,
Branchburg, NJ). Amplified DNA was diluted 1 : 1 with stop solution (97% formamide,
1% EDTA, 0.1% bromophenol blue, and 0.1% xylene cyanol) and denatured
at 85 °C for 2 minutes. Three microliters of denatured DNA from each sample was loaded
onto a 7% denaturing polyacrylamide gel (19 : 1 acrylamide/bis-acrylamide) containing
32% formamide and 34% urea and was fractionated by gel electrophoresis for 2.5
hours at 60 W. Gels were transferred to filter paper (Whatman 3MM; WR Balston, Ltd.,
Maidstone, U.K.), covered with plastic wrap, equilibrated with 20% methanol and
20% acetic acid solution, and dried. Dried gels were exposed to x-ray film (Fuji Photo Film
Company, Tokyo, Japan) without an intensifying screen (usually 16 hours).
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Human YACs were isolated from the Human Polymorphism Study Center (CEPH, Paris, France) recombinant DNA library and were propagated in minimal medium, as previously described by Albertsen et al. (28). YACs were tested with the markers described above and with a nonpolymorphic marker, D14S1246 (14). PCR products were amplified as above but with oligonucleotide primers that were not isotopically labeled. PCR products were visualized on 2% agarose gels run in 1x Tris-borate-EDTA buffer and stained with 0.5 µg/mL of ethidium bromide. YACs were sized by pulsed-field gel electrophoresis (28). YACs 755c2, 772e4, and 859d4 were selected for our mapping studies on the basis of previously published data [(14); see also online data at http://www-genome.wi.mit.edu/].
Statistical Methods
LOH frequency was compared by Fisher's exact test between primary tumors from patients with axillary lymph node-negative and lymph node-positive primary breast cancer patients to identify loci that might be important in progression to metastatic disease. Metastatic lesions were not included in these calculations. In addition, information on histologic subtype, tumor grade, and tumor mitotic index was likewise compared with LOH status and with nodal status by use of Fisher's exact test (subtype and grade) or two-sample Student's t tests (mitotic index) to determine whether the observed differences in LOH frequency were possibly due to imbalances in the distribution of these factors. All statistical tests were two-sided at the P = .05 level of significance. Analyses were performed by use of SAS (Version 6.11; SAS Institute, Inc., Cary, NC).
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RESULTS |
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Specimens of breast cancer were microdissected as described in the "Materials and Methods" section to obtain normal tissue, primary tumors, and, for patients with lymph node-positive disease, matched nodal metastases (if available). Of the 76 subjects included in this study, 42 were negative for metastases in the axillary lymph nodes and 34 were positive for the presence of metastases in the axillary lymph nodes. Only the primary breast cancers were included in our analysis of LOH. Histologic subtypes included invasive ductal breast cancers of no special type (81%), invasive lobular breast cancers (11%), and other types, such as mucinous carcinoma and medullary variants (8%). LOH studies were carried out on both lymph node-negative and lymph node-positive primary breast cancers with four 14q markers. The sex-averaged recombination frequencies and map order for these markers were centromere-//-D14S302-10 cMD14S265-3 cMD14S62-2 cMD14S51-//-telomere.
Fig. 1, A, shows representative LOH data for each of the four
markers. Table 1
summarizes the tumor type, tumor grade, mitotic index,
and LOH status of these specimens. Table 2
shows the breakdown of
LOH frequency by marker and by nodal status. LOH at D14S62 was observed in 68% of
the lymph node-negative primary cancers but in only 24% of the lymph node-positive
primary tumors (two-sided Fisher's exact test; P = .001). The pattern of
D14S62-region marker homozygosity was identical in metastatic tissue samples matched to the
lymph node-positive primaries, with the exception of case 92-1509, which exhibited identical
LOH patterns for D14S302 and D14S51 in both the primary and the metastatic lesions but
exhibited LOH for D14S265 only in the primary breast tumor. As shown in Table 2
, high rates of LOH were observed for marker D14S302 in all primary breast cancers
regardless of nodal status. However, the more distal markers (D14S62 and D14S51) showed high
rates of LOH in the lymph node-negative primary breast cancers, but substantially decreased rates
of LOH in the lymph node-positive primary breast cancers. Thus, the presence of D14S62 LOH in
a primary breast tumor has a strong negative association with metastatic disease.
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Although most of the lymph node-negative tumors that showed LOH
exhibited allelic losses at sites distributed throughout the 14q
chromosomal region, a subset of six lymph node-negative samples had
patterns of LOH that imply the location of the metastasis-related
portion of chromosome 14q, as shown in Table 1 and Fig. 1
, B. Tumor
specimens from case subjects numbered 88-10429 and 88-2459 were
heterozygous for D14S265 but showed LOH for D14S62, indicating that the
metastasis-related gene is distal to D14S265. Tumor specimens from case
subjects numbered 87-5002, 90-2980, and 91-7552 were heterozygous for
D14S51 but showed LOH for D14S62, indicating that the
metastasis-related gene is proximal of D14S51. The tumor specimen from
the case subject numbered 87-0253 showed LOH for D14S265 but was
heterozygous for D14S62.
Together, these primary breast tumors from lymph node-negative patients indicate that the
metastasis-related gene is in the 5-cM region between the D14S265 and D14S51 loci. A previous
study (29) has indicated expanded genetic recombination distances for this
part of chromosome 14q. The physical distances between the markers were estimated by testing a
series of YAC clones that have been previously placed in this region (14).
Fig. 2 shows that D14S265 and D14S62 were found to reside on a
620-kilobase (kb) YAC, 755c2. Markers D14S1067, D14S979, and D14S51 reside on two
contiguous YACs, 772e4 (1170 kb) and 859d4 (870 kb). Marker D14S1246 connects these three
YAC clones into a contig. Therefore, the metastasis-related region defined in the high resolution
LOH studies cannot exceed the 1490-kb maximum distance between D14S265 and D14S51 (the
combined lengths of YACs 755c2 and 859d4). The six LOH breakpoints between D14S265 and
D14S51 (flanking D14S62, see Table 1
and Fig. 2
, B) indicate that the metastasis-related region is likely to be considerably smaller, on
the order of 1000 kb or less.
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DISCUSSION |
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To assess the impact of LOH at the D14S62 locus on metastasis, we measured LOH at the D14S62 locus in a set of primary cancers for which nodal status was known. A total of 68% of the lymph node-negative cancers showed LOH at D14S62 compared with only 24% of the lymph node-positive primary cancers. These differences were statistically significant. These data suggest that LOH at D14S62 could be a predictive marker for improved outcome. In addition, the number of patients with lymph node-negative disease who do not show D14S62 LOH (32%) approximates the recurrence rate (30%) in this otherwise low-risk group of patients (1,2). We are currently carrying out a large prognostic study of D14S62 LOH in primary breast cancer specimens with known clinical follow-up to confirm this hypothesis.
With one exception, the patterns of LOH exhibited by the lymph node-positive primary breast cancers in this study were also detected in matched metastatic lesions, indicating a clonal relationship between primary and metastatic disease. In case 92-1509, the LOH patterns for D14S302 and D14S62 were identical in both the primary and metastatic lesions, indicating a high probability that these lesions are clonally related in this patient. However, the LOH pattern for D14S265 between the primary and metastatic lesion in this case was not concordantthe primary tumor exhibited LOH for this marker and the metastasis did not. This represents a possible example of tumor heterogeneity.
The high rate of LOH at the D14S302 locus in all primary breast cancers (58%) suggests that a tumor suppressor gene is near D14S302 whose loss is associated with the development of breast cancer. LOH was seen frequently at the D14S51 locus in lymph node-negative primary breast cancers. Chang et al. (30) reported high levels of LOH in bladder cancer, including D14S51 and more telomeric markers, suggesting that D14S51 defines the location of a second tumor suppressor gene in this region. Of the 25 primary tumors informative for both D14S302 and D14S51, nine lost both markers, five lost D14S302 exclusively, and three lost D14S51 exclusively. The nearly threefold difference in the rates of LOH at D14S62 in tumor specimens from patients with lymph node-negative versus lymph node-positive primary breast cancers leads us to hypothesize that this region contains an additional gene(s) near D14S62, for which reduced gene dosage impedes metastasis. Given that a subset of our tumors loses either D14S302 or D14S51, the critical D14S62 region may lie between two regions of breast cancer-specific LOH.
It is possible that imbalance in the distribution of tumor characteristics could influence the association between LOH and nodal status. While our sample selection was biased toward larger tumors by the need to choose tumors that were large enough to microdissect manually, statistical analysis revealed no association between nodal status and pathologic grade (P = .53) or mitotic index (P = .32). Invasive lobular carcinoma (11% of the cases) was more often lymph node positive than the other subtypes (P = .05) but had a lower grade and mitotic index (P = .01 and P = .03, respectively). As expected, a highly statistically significant association was observed between histologic grade and mitotic index (P<.0001) for all cancers. There was an association between increased grade and mitotic index with LOH at D14S302 (P = .05) but not at the other markers. This is consistent with the presence of a tumor suppressor gene near D14S302.
Informative LOH events in lymph node-negative breast cancers indicate that the metastasis-related gene resides between D14S265 and D14S51. This region genetically spans 5 cM, but a previous mapping of chromosome 14q (29) indicates a fivefold expansion of the genetic map relative to the physical map in this region, an observation supported by the YAC mapping experiments. Therefore, the D14S265-D14S51 region may encompass as little as one megabase of genomic DNA, making gene isolation efforts feasible. In tumors with LOH, we do not know if a mutation is present in the remaining gene copy. It is possible that a 50% reduction in gene dosage at this locus produces the inhibitory effect on metastasis. The metastasis-related gene, irrespective of whether it encodes a protease, cell adhesion molecule, immunologic factor, or a circulatory survival factor or is a gene for some unknown function, may be at a rate-limiting control point in the metastatic process. Isolation and characterization of this gene will lead to new insights into the metastatic process and may lead to new therapies for patients with metastatic disease. Even without cloning of the proposed metastasis-related gene, these studies have the potential to provide a genetic test that could aid in diagnosing metastasis.
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NOTES |
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We thank Michelle Schmitz, J. Douglas Rains, and Oredius Presley for their excellent technical assistance.
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Manuscript received December 16, 1997; revised June 11, 1999; accepted June 21, 1999.
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