Affiliations of authors: F. M. Waldman, S. DeVries, K. L. Chew, D. H. Moore (Cancer Center), K. Kerlikowske (Department of Epidemiology and Biostatistics and General Internal Medicine Section, Department of Veterans Affairs), B.-M. Ljung (Department of Pathology), University of California, San Francisco.
Correspondence to: Frederic Waldman, M.D., Ph.D., Cancer Genetics Program, UCSF Cancer Center, Rm. S436, University of California, San Francisco, San Francisco, CA 94143 (e-mail: waldman{at}cc.ucsf.edu).
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ABSTRACT |
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INTRODUCTION |
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Breast-conserving surgery raises the possibility that DCIS may recur in the remaining breast tissue. The incidence of local recurrences following breast-conserving surgery for DCIS ranges from 5% to 25%, depending on the follow-up period and postoperative treatment (3,7-12). The mechanism of recurrence is uncertain. Several investigators (5,13,14) have pointed out the importance of wide surgical excisions in preventing recurrent disease after lumpectomy. The recurrence of DCIS following mastectomy is only 1%-2% (3,4,9), which suggests that many recurrent lesions are due to residual tumor cells rather than to growth of a different neoplasm.
If recurrent DCIS lesions reflect residual disease, we would expect to see a clonal relationship between the initial DCIS and the recurrent in situ tumor. One approach to assessing clonality is comparative genomic hybridization (CGH). CGH screens for losses and gains of DNA sequences along the entire genome by comparing the competitive hybridization of tumor and normal DNAs that are differentially labeled to normal metaphase chromosomes (15-17). This approach allows the entire genome to be characterized in one analysis because tumor DNA is analyzed directly rather than with individual probes. To establish the extent to which DCIS recurrences arise from residual tumor cells as opposed to being new lesions, we have used CGH to compare genetic changes in 18 primary DCIS lesions and their ipsilateral recurrences.
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MATERIALS AND METHODS |
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Eighteen female patients from California Pacific Medical Center were identified who presented initially with DCIS and returned more than a year after the initial diagnosis with DCIS in the same breast. This study was approved by the Institutional Review Board of the University of California at San Francisco. All surgical slides were reviewed to confirm the DCIS diagnosis and to exclude the presence of microinvasion or more extensive invasive tumor. Initial DCIS cases were reviewed independently of the corresponding recurrence. Nuclear grade of the DCIS was recorded as low, intermediate, or high, and the histologic pattern was classified as comedo, solid, cribriform, or micropapillary type. Comedo-type DCIS was defined as solid-type DCIS with high nuclear grade and moderate or extensive necrosis. Tumors exhibiting a mixture of histologic types were classified by the predominant population. Tumor involvement of the surgical margin was also noted. Clear margins were defined if there was no tumor involvement within 1 mm of the surgical margin.
Tissue Dissection and DNA Extraction
Sections (5 µm) containing the DCIS were placed on slides for microdissection as previously described (18). With the use of an adjacent hematoxylin-eosin-stained slide for orientation, one or two 5-µm deparaffinized, methyl green (0.1%)-stained sections were microdissected. Previously selected areas of DCIS were separated from surrounding lymphocytes and stroma. DNA was isolated with the use of a 3-day Proteinase K digestion (18).
Polymerase Chain Reaction Amplification
Amplification of the microdissected DNA was by degenerate oligonucleotide primer polymerase chain reaction (PCR) (18). Samples were amplified in duplicate but in separate PCR reactions, each containing a 1- to 2-µL aliquot of microdissected DNA. Each PCR run included samples of female genomic DNA from healthy donors (considered the reference and isolated from peripheral blood), MPE600 (breast cancer cell line with known CGH aberrations), and a PCR blank. Fifty nanograms of reference and MPE600 cell line DNA resulted in approximately 2-3 µg of amplified DNA, ranging in size from 200 base pairs (bp) to 6 kilobase pairs (kbp). Microdissected DNA yielded up to 1 µg of PCR product, averaging around 600 bp (range, 100 bp to 2 kbp).
Probe Labeling and CGH
PCR-amplified DNA from the initial DCIS as well as from the recurrent lesion was labeled in duplicate by nick translation. PCR-amplified normal reference DNA (25 µL) was labeled with fluorescein-12-deoxyuridine triphosphate (dUTP) (Du Pont NEN, Boston, MA) or indirectly with biotin-deoxyadenosine triphosphate (dATP) (Life Technologies, Inc. [GIBCO BRL], Gaithersburg, MD). The MPE600 cell line and PCR-amplified test DNA were labeled with digoxigenin-11-dUTP (Boehringer Mannheim Biochemicals, Indianapolis, IN). Nick-translation PCR products were close to the original product size, 100-1000 bp. Smaller probes tended to yield less than optimum CGH results and appeared to be granular or dim.
CGH was performed as previously described (18,19). Samples were hybridized onto normal male metaphase spreads. Digoxigenin-labeled test DNA samples were hybridized in duplicate against reference DNA labeled either with fluorescein-12-dUTP or with biotin-dATP. Digoxigenin-labeled samples were stained with anti-digoxigenin rhodamine (Boehringer Mannheim Biochemicals). Biotin-labeled samples were stained with fluorescein isothiocyanate-labeled avidin (Vector Laboratories, Inc., Burlingame, CA).
Successful hybridizations showed good intensity signals with smooth, homogeneous staining over the entire metaphases. At least five metaphase spreads were acquired for each case. Acquisition was performed with the use of our Quantitative Image Processing System [QUIPS (20)]. Two to three metaphases per sample were analyzed in each color. Tumor-to-reference fluorescence intensity ratios were calculated along chromosomal arms, and gains and losses were defined if the mean and standard deviation were above 1.2 or below 0.85. Inverse CGH pairs were examined together, and all changes must have been seen in both hybridizations. Interpretations of changes at 1pter, 19, and 22 (and 4 and 13 in the opposite direction) were interpreted with caution. Definition of changes at these loci required the cut point to be exceeded in both hybridizations.
Statistical Analysis
For scoring of genetic alterations, whole chromosome changes were scored as one event. All other changes were scored by arm. A loss and a gain on one arm were scored as two changes, whereas two separate losses (or gains) on the same arm were scored as one change.
To compare frequencies of alterations in different groups of tumors, we calculated a chi-squared statistic for each 2 x 2 contingency table. All statistical tests were two-sided and were considered to be statistically significant at P<.05.
The following three methods were used to measure concordance between the initial and recurrent lesions for the CGH alterations: 1) percent concordance, 2) similarity score, and 3) hierarchical clustering.
The percent concordance was calculated in the following manner:
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The similarity score was calculated for each pair as a weighted sum of the alterations for each chromosome arm. The weights were based on the overall probabilities of gains and losses for each chromosome arm, with greater weight being given to agreement when a gain or loss was rare than when it was common. The weights were proportional to the log of the probability for the observed CGH alterations in observed pairs.
The similarity score can be defined mathematically as follows: Let Xij be an indicator variable equal to 1 if there is a gain (or loss) at the jth chromosome arm in the ith member of the pair (i = 1, 2; j = 1, . . ., n) and define pj to be the overall probability of gain or loss at the jth chromosome arm (pj are estimated from the 18 tumors in this study). The similarity score is defined by
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The individual terms of the similarity score will be negative when alterations are discordanti.e., when there is an alteration on a chromosome arm of one pair member but not on the corresponding arm of the other member of the pair. The similarity score will be positive when the pair members are alikei.e., each has alterations of the same type on the same chromosome arms, or neither pair member has an alteration. The probability weighting ensures that agreements at rare alteration sites get more weight than alterations at common alteration sites. We calculated a similarity score for every possible pairing of initial-recurrent tumors. We then compared the distribution of similarity scores for initial-recurrent pairs from the same patient with that for initial-recurrent pairs where the initial lesion and the recurrence were from different patients. This latter distribution was used to define nonclonality.
For hierarchical clustering, the program "Agnes" from the S-PLUS statistical package was used (21). Agnes is based on pairwise similarities of the CGH data; similarities are not weighted by their likelihoods. Results from clustering are best displayed graphically as trees; interpretation tends to be subjective rather than to be based on objective measures. Nonparametric confidence intervals (CIs) for the median concordance and median similarity scores were obtained from the order statistics (22).
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RESULTS |
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The mean age of the 18 patients at the time of diagnosis for the
initial DCIS was 52.6 years (Table 1).
Breast-conserving surgery was the only treatment for 15 of the cases,
with two cases having both surgery and radiation therapy. (Radiation
treatment was unknown for one case.) The mean time to recurrence for
all 18 patients was 4.2 years (range, 16 months to 9.3 years).
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Microscopic review of the initial and recurrent in situ tumor pairs revealed a
striking similarity in histopathologic
features (Fig. 1). Thirteen (72%) of the recurrent
tumors had the same histologic type as their corresponding initial
tumor (Table 1
). Eight of the initial lesions were high grade, and 10
were low to intermediate grade. When low and intermediate grades were
combined, 15 (83%) of the recurrent tumors had the same nuclear grade
as their paired initial lesions, and the remaining three changed from
intermediate to high grade.
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Chromosomal Alterations by CGH
All of the DCIS lesions showed at least one genetic aberration by
CGH (Table 2). The total number of aberrations was
higher in the recurrences (mean number = 10.7; 95% CI = 7.8-13.7)
than in the initial lesions (mean number = 8.8; 95% CI = 6.0-11.7)
(P = .019, paired two-sided t test). The most common
changes in the initial DCIS were gains involving 17q (61%) and losses
involving 8p (61%) and 17p (50%). There were no statistically
significant differences in the prevalence of individual CGH alterations
between the initial and recurrent lesions (Table 3
).
However, there was an increased prevalence of a small number of
alterations (gains involving 3q and 17q and losses involving 8p and
14q) in these DCIS lesions compared with our previously reported set of
invasive ductal cancers (15,17).
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Concordance. The median percent concordance for the tumor pairs was 81% (range, 0%-100%; 95% CI = 77%-90%). One pair (F22) had a concordance of 0% (having two and 20 alterations in the initial and recurrent tumors), whereas the other 17 cases had a median concordance of 82% (range, 65%-100%). The concordance was similar whether the initial tumor showed clear surgical margins or margins that were involved by tumor (73% versus 85%).
Similarity score. When the initial lesion and the recurrence from the same subject
were paired, the median similarity score was 24.3 (95% CI = 21.4-28.1) (Fig. 2). If the initial lesions were paired with recurrences from different
subjects (306 possible pairs), the median similarity score was -11.9 (95% CI
= -14.2 to -10.3). The difference in the distribution of similarity scores
was statistically significant (P<.0001). Fig. 2
shows similarity
scores plotted separately for each subject. In only two cases did initial lesions have a better match
with recurrent tumors from other patientsone subject (F4) had a better match with a
different recurrent tumor from another patient (F1) than with its initial lesion, whereas the other
subject (F22) had better matches for six other recurrences than with its own recurrence. In the
remaining cases, the initial tumor and the recurrent tumor from the same subject were more alike
than matches with any other recurrences.
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Losses involving 16q occurred more frequently in low/intermediate-grade DCIS lesions than in high-grade lesions, both for the initial (P = .094) and the recurrent (P = .024) lesions, although the difference was statistically significant only for the recurrent lesions. Conversely, losses involving 8p were statistically significantly associated with high grade in the recurrences (91% high grade versus 43% low/intermediate grade; P = .026) but not in the initial lesions (75% high grade versus 50% low/intermediate grade; P = .28).
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DISCUSSION |
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Whether an initial DCIS lesion is related to its local recurrence may be difficult to determine. CGH is a powerful molecular tool that yields a genetic profile for each tumor, thus allowing a comparison of each lesion to its recurrence. Our analyses confirmed that DCIS recurrences are predominantly clonally related to their initial DCIS lesions, suggesting that the subsequent lesions are due to persistence of neoplastic cells rather than to newly arising lesions. These analyses used alterations involving chromosome arms as the unit for statistical analysis of genetic similarities. It is possible that differences between paired samples existed at the resolution of individual genes, since the resolution of CGH is limited in metaphase chromosomes and cannot routinely define alterations less than 10 megabase pairs in size. Our previous studies (17,23) support the use of CGH to define clonal relationships in other paired sets, including primary tumors and metastases from breast and bladder cancers. In the future, array-based CGH analyses will allow copy number alterations at gene resolution to be determined (24).
In this study, time to tumor recurrence was unrelated to both the number of genetic aberrations present in the initial lesion and the degree of concordance between the tumor pairs. In one case (F25), a single loss of chromosome 12p was seen in both the initial DCIS and the recurrent tumor after 5.9 years. In another case (F10), 20 genetic aberrations were seen both in the initial lesion and in the recurrence that was detected after 4.8 years. The one case (F22) without concordance by all three statistical methods showed two genetic changes in the initial DCIS lesion and 20 different changes in the recurrence. These data are most consistent with the second lesion being a new neoplasia rather than being a recurrence of the original lesion. The time to recurrence for this case was 9 years, one of the longer time intervals in our study.
Few studies have reported comparisons of genetic alterations in initial and recurrent breast lesions. Lininger et al. (25) showed a high concordance of loss of heterozygosity (LOH) in three ipsilateral DCIS primary/recurrence pairs. Similarly, a number of reports (26-32) have shown genetic similarities between DCIS lesions and their concurrently associated invasive tumors.
DCIS, especially of high grade, is a genetically advanced lesion despite the absence of invasion through the basement membrane. A mean of 8.8 chromosomal changes was seen in the 18 primary DCIS cases studied, similar to the 8.7 changes per tumor found in the combined set of 94 invasive breast carcinomas previously analyzed in our laboratory (15,17). These results confirm the presence of multiple genetic alterations in DCIS, previously shown by LOH (30,33,34), CGH (35,36), and other approaches (37-39).
The specific genetic changes seen in our set of DCIS lesions are similar to those seen by
others and, for the most part, are present in invasive cancers as well (Table 3) (26,30-32). Our finding of a small, yet statistically
significant, overall increase in the number of genetic alterations in the recurrences (8.8-10.7)
suggests that genetic progression occurred, although no specific alterations appeared more likely
than others to be increased. This observation is consistent with reports of clonal evolution
detected by LOH in synchronous pairs of DCIS and invasive cancer (27,30,31).
In addition to genetic similarities, a striking similarity in histologic appearance was seen
between the initial lesions and the recurrences (Fig. 1). This overall
histologic similarity was associated with an agreement in grade, with only three cases showing a
change from intermediate to high grade, as well as an agreement in architectural pattern, with 13
cases showing the same classification. A similarity in histologic type between DCIS lesions and
associated invasive cancers has been described previously (40). These
observations reinforce the conclusion that genetic alterations are the determinant of morphologic
appearance for breast neoplasia.
The clonal relationship of initial DCIS lesions and recurrences suggests that DCIS recurrences arise from residual tumor cells that are not removed at the time of surgery. The importance of wide excision margins in the treatment of DCIS has been described in multiple studies (5,9,13,14). Silverstein et al. (5) recently reported that clear surgical margins of 10 mm or more lead to a very small recurrence rate that is unaffected by radiation treatment. In our study, nine cases with margins of 1 mm or more still recurred; eight of these cases were clonally related to the initial lesion. This result supports the conclusion that residual DCIS may be left behind when surgical margins are less than 10 mm, as previously suggested (5,13).
We conclude that most DCIS recurrences result from growth of persistent neoplastic cells, which may remain indolent for long periods. These data explain the importance of wide surgical margins and/or radiation therapy during treatment of these noninvasive neoplasias. Further insights into the genetic determination of preinvasive histology and biology will allow treatment tailored to the likelihood of clinically aggressive tumors.
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NOTES |
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