Callahan,* and Daniel S. Lisciat ... Address reprint requests to Daniel S. Liscia, Pathology Section, S. .... antibodies (Becton & Dickinson, Mountain View, CA).
American Journal of Pathology, Vol. 140, No. 1, Januaiy 1992 Copyright © American Association of Pathologist
Loss of Heterozygosity on Chromosome 17p13 in Breast Carcinomas Identifies Tumors with High Proliferation Index Giorgio R. Merlo,* Tiziana Venesio,t Amelia Bernardi,t Lorena Canale,t Piero Gaglia,t Danilo Lauro,t Alberto P. M. Cappa,t Robert Callahan,* and Daniel S. Lisciat From the Oncogenetic Section,* Laboratory of Tumor Immunology and Biology, National Institutes of Health, Bethesda, Maryland; and the Pathology Section, t S. Giovanni Vecchio Hospital, Torino, Italy
The capacity of breast tumor cells to proliferate is considered a potential prognostic factor together with other histopathologic parameters. The authors determined the proliferation index on a large panel of human primary breast tumors by measuring the levels of incorporation of bromodeoxyuridine (BrdU) byfresh tumor specimens in culture. Previous analysis showed that the percentage of cells entering the S-phase of the cell cycle strongly correlates with tumor grade, tumor size, and estrogen and progesterone receptor status. The capacity of tumor cells to proliferate might be associated with specific genetic mutations in primary tumors. To test this hypothesis, a panel of 96 human breast carcinomas, for which the BrdU labeling index (LI) was known, were tested for loss of heterozygosity (LOH) or increased copy number (ICN) at chromosomes lq, 3p, 13q, 1 7p, and 18q. On chromosome 17p, LOH and ICN were observed in 27% and 12%, respectively, of the informative breast tumors. The LOH on chromosome 1 7p was significantly associated with tumors having an elevated BrdU proliferation index (P = 0.022). No association (P = 0. 45) was observed between BrdU LI and tumor size (T2 + T3 compared with T1), tumor grade, and lymph node status. Increased copy number on chromosome 17p, LOH orICN on 1q, and LOH on 13q14, 18q, and 3p also showed no significant correlation with cell kinetic parameters. These data are consistent with the presence of a gene or genes on chromosome 17p13 near the YNZ22.1 locus whose normal functioning is necessary for controlling breast tumor cells proliferation in vivo. (Am JPathol 1992, 140:215-223)
Proliferation of tumor cells within the tumor mass is in-
creasingly being recognized, together with other clinical and pathologic parameters, as an important biologic marker for the diagnosis1 2 and the management34 of breast cancer disease. Factors that result in higher mitotic activity, either by overstimulating a normal mitogenic pathway or by impairing critical internal components of the cell cycle regulatory machinery, may contribute a selective growth advantage to the affected cells during their progression toward a more malignant state56 ultimately leading to cancer formation.7 Genetic mutations affecting regulatory genes critical for controlling the cell cycle may be one of the mechanisms by which cells escape from a normal pattern of division.810 Multiple genetic alterations have been documented in breast cancer.1" This is reflected in the high variability of the breast neoplastic disease for either its pathologic and histologic appearance or the clinical outcome. Perhaps the most frequent type of mutation detected in breast tumor DNAs is the somatic loss on one allelic copy of a specific gene in the tumor cells, or loss of heterozygosity (LOH). Such mutations may indicate the presence of a tumor-suppressor gene within the affected chromosomal region.12 According to the two-hit hypothesis proposed by Knudson,13 LOH unmasks the presence of a first inactivating mutation, which is recessive in nature. In primary human breast tumors, several different chromosomal sites are frequently affected by LOH, including chromosomes 1p,14 1q,15 3p, 16 11p,17 13q 18,19 1 7p,2021 1 7q, and 1 8q.22 With the exception of the Rb-1 and the p53 genes, however, located on chromosomes 13q14 and 17p13, respectively, the target genes involved in the neoplastic transformation of the mammary gland are unknown. Of particular interest is the finding that both the p532324 and the Rb-125 tumor-suppressor genes also are involved in the regulation of the cell cycle, or their expression/post-translational modification is a cell-cycleSupported in part by a grant from the Italian Association for Cancer Research (AIRC). Accepted for publication August 20, 1991. Address reprint requests to Daniel S. Liscia, Pathology Section, S. Giovanni Vecchio Hospital, USL-1, Via Cavour 31, 10123 Tonno, Italy.
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regulated event. Although the precise molecular details of these effects have not been elucidated yet, it is reasonable to think that mutations affecting such genes might have an impact on the rate of tumor cell proliferation. In the present study, we measured the proliferation index by the incorporation of the thymidine analog Bromo-deoxyuridine (BrdU) on 406 fresh carcinoma samples, and on a subpanel of 96 tumors we correlated these values with specific genetic lesions detected on genomic DNA samples. In particular we have focused on LOH on chromosomes lq, 3p, 13q14 (Rb-1 gene), 1 7p1 3, and 1 8q. The 1 7p1 3 chromosomal region that we analyzed is near the p53 putative tumor-suppressor gene. Our findings are consistent with an association between LOH on 17p13 and high tumor proliferation in breast carcinomas.
Material and Methods
Collection and Storage of Tissue Samples Primary breast carcinoma samples were collected at the S. Giovanni Vecchio Hospital (Torino, Italy) from patients who had not received any treatment before surgery. Immediately after surgical removal of the tumor, cryostat sections were prepared for intraoperative histopathologic diagnosis, and to select the neoplastic tissue for further analysis. A portion of each specimen was frozen in liquid nitrogen and kept at - 70°C until the genomic DNA was extracted. The remaining fresh material was used for the evaluation of the labeling index by BrdU incorporation and for routine histology. Peripheral blood from the same patients also was obtained and was used as a source of patients' somatic DNA for comparison. All the breast tumors were classified according to the World Health Organization (WHO).26 Patients were graded histopathologically according to the Bloom and Richardson method27 and by the UICC28 TNM (tumor, nodes, metastases) staging system.
DNA Marker Probes Used For chromosome 1 7p1 3, the pYNZ22. 1/D1 7S30 marker probe (ATCC, Rockville, MD), located on 17p13.3, was used.29 This probe detects a fragment length polymorphism (RFLP) in either Pst-l and Bam HI-digested human genomic DNAs. In our study, Pst-l restriction enzyme was used. To check for LOH on chromosome 1 7p telomeric with respect to pYNZ22.1, the probe p144D6 was
used,30 which detects a VNTR polymorphism on Bam Hland Pst I-digested DNA. For chromosome 1 q, the probe pDF1.8 (Pst-l) was used on Pst-l restricted cellular DNA.15 This probe recognizes a VTR polymorphism on chromosome 1q21-24. For the Rb-1 locus, the probe p68RS2.0, which recognizes a VTR RFLP,31 was used. The OS-4 probe detects a VTR RFLP on chromosome 18q.32 33 Finally the Erb-A2 probe detects a two-allele Bam HI RFLP on chromosome 3p21 -pter.16
Southern Blotting and Genetic Analysis High-molecular-weight DNA was extracted as described.34 Routinely 10 pLg DNA was digested with the enzyme of choice (BRL, Gaithersburg, MD), and the DNA fragments were separated by electrophoresis in 1% agarose gels and transferred to Genatran-45 Nylon membranes (Plasco, Woburn, MA). The DNA was immobilized to the membrane by UV-crosslinking for 2 minutes followed by prehybridization and hybridization with a solution containing 3 x SSC (salt sodium citrate) (1 x SSC is 0.15 mol/l [molar] NaCI, 0.015 mol/l NaCitrate, pH 7.4), 5% dextran sulfate, 50% formamide, at 370C. After 16 hours of hybridization, the filters were washed to a final stringency of 0.1 x SSC, 0.1% sodium dodecyl sulfate (SDS), at 55°C, and exposed to x-ray films for 2 to 5 days at - 700C.
BrdU Incorporation Tissue samples were placed in a petri dish containing Hank's salt solution no later than 15 minutes after the operation, cut with razor blades into 1- to 2-mm fragments and incubated for 3 hours at 370C in RPMI-1640 with 10% fetal calf serum, 20 mmol/l (millimolar) Hepes buffer, and 10-4 mol/l Bromodeoxyuridine (Sigma, St. Louis, MO). Tissue fragments then were washed with phosphate-buffered saline (PBS), fixed with Carnoy's solution and paraffin embedded. Five-micron sections were mounted, deparaffinized in Xylene, rehydrated, denatured for 10 minutes in 4 N HCI, washed with PBS, incubated for 20 minutes in 10% nonimmune horse serum and for 1 hour with 1:160-diluted anti-BrdU monoclonal antibodies (Becton & Dickinson, Mountain View, CA). Slides then were washed in PBS and incubated for 30 minutes with a biotinylated secondary antibody (horse anti-mouse IgG). This was followed by reaction with the avidin-biotin-peroxidase complex (ABC) reagent according to the manufacturer's instructions (Vector Laboratories, Burlingame, CA). The ABC complex was finally detected with 3-amino-9-ethilcarbazole (Sigma) and 0.03%
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H202 according to standard procedures. Positive nuclei scored using a Leitz Dialux microscope (Leitz, Wetzlar, Federal Republic of Germany) equipped with a 10 x 10 grid on the eyepiece and a 40x objective. Over 500 morphologically neoplastic cells were counted for each tumor. Values were expressed as percentage of positively stained nuclei. A panel of 406 breast tumors was used to determine the median value cut-off point for BrdU incorporation. Using this value, tumors were classified in a high or a low proliferation index rank. A subpanel consisting of these tumors minus all the tumors informative (heterozygous) at the pYNZ22.1 locus was reanalyzed for the median value. This subpanel had the same median proliferation value as the original panel. The cases analyzed for genetic mutations were representative of the larger panel of tumors for their BrdU value distribution and the other pathologic parameters. were
Estrogen and Progesterone Receptor Assay Cytosolic estrogen (ER) and progesterone (PR) recepwere measured by the dextran-coated charcoal (DCC) method according to the EORTC standards.35 The percentage of ER-positive cells was assessed by an immunohistochemical method on frozen sections with an anti-estrogen receptor monoclonal antibody (ER-ICA, Abbott Laboratories, Chicago, IL). tors
Statistical Analysis The Mann-Whitney and Kruskal-Wallis tests were used to compare cell proliferation indexes in tumors with a normal set of alleles, LOH or increased copy number (ICN). The results were confirmed by classifying the tumors in one of two groups according to the median value. Statistical associations with genetic alterations at different loci were assigned by chi-square analysis with Yates' correction or
by Fisher's exact test. Similar methods also were used to test for possible associations with other clinicopathologic parameters such as histologic tumor type, tumor size, number of positive lymph nodes, tumor grade, menopausal status, and steroid receptors status. All statistical analysis were performed with the SPSS statistical package
(SPSS Inc., Chicago, IL).
Results Genetic Analysis on Breast Tumor DNAs Genomic DNAs from breast carcinoma samples and from the matching lymphocytes from the same patients were Southern blotted and analyzed for LOH or ICN using RFLP marker probes located on different chromosomes. The probe pYNZ22.1 (1 7p1 3.3) detected at least seven alleles in Pst-l-digested human DNA. A total of 96 breast tumors were analyzed. Seventy-seven percent of these were from patients that were constitutionally heterozygous (informative) at this locus (Table 1). Twenty-seven percent of the informative tumors demonstrated a loss of signal (LOH) relative to one of the two alleles, indicating a reduction to homozygosity (Table 1). For instance, tumor samples 31/88 and 1 1/88 displayed an almost complete loss of one allele (Figure 1). In several cases, however, the LOH was not complete. This is usually attributed to the presence of a variable amount of normal cells in the tumor specimen that contributes both alleles in equal copy number (tumor sample 14/88, Figure 1), or to genetic heterogeneity within the tumor specimen. No homozygous loss at the pYNZ22.1 locus was observed in any of the tumor samples tested. ICN was observed in 12% of the informative tumors (Table 1). Figure 1 also illustrates two tumor samples, 44/88 and 21/88, with an ICN of one of the two alleles. Increased copy number presumably results from either an amplification of genes in this region of the chromosome 17 or an aneuploidy of the entire chromosome.
Table 1. Frequency of Genetic Alterations at Different Loci in Prinary Breast Cancer
Informative
pDF1.8 (1 q21-24) pYNZ22.1 (17p13.3) p68RS20 Rb-1 (13q14) Erb-A2 (3p21-pter) OS-4 (18q) p144D6 (17p13.3) *
Total 99 96 49 70 87 104
Percent of cases that were heterozygous by RFLP analysis.
t Percent of informative cases with loss of heterozygosity. t Percent of informative cases with increased copy number.
75 77 41 44 49 79
LOH
ICN
(%)t
(%)t
21 27 15 13 14 37
18 12 2 4
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