| Japanese Journal of Clinical Oncology | Pages |
Introduction
Patients And Methods
Histological Typing and Grading of n0 Breast Cancer
Follow-up Data
Gene and Chromosome Alterations in n0 Breast Carcinomas
Statistical Analysis
Results
Discussion
Acknowledgments
References
Prognostic Significance of Accumulation of Gene and Chromosome Alterations and Histological Grade in Node-negative Breast Carcinoma
The histological grade of atypia is a known prognostic indicator for breast cancer patients and correlates with many gene and chromosome alterations. To investigate the independent prognostic significance of gene and chromosome alterations in axillary node-negative (n0) breast cancers of the invasive ductal and invasive lobular types, the prevalence of eight gene and chromosome alterations and their association with histological grade and recurrence was studied in 129 consecutive patients who had undergone resection over an average follow-up period of 43.4 months. Loss of heterozygosity on 16q, 17p, 16p, 17q and 18q, p53 gene mutation and c-erbB-2 and int-2 gene amplifications were detected in 55%, 37%, 25%, 24%, 22%, 23%, 15% and 11% respectively. Individual alterations in 17p, 17q, 18q, c-erbB-2 and p53 were detected most frequently and gene and chromosome alterations tended to be accumulated in Grade 3 n0 invasive ductal/lobular carcinomas. Histological grade, 18q, int-2 and the number of these gene and chromosome alterations were significant prognostic indicators on Cox's univariate proportional hazard model, and 18q and int-2 were still significant after adjustment for histological grade. Results suggested that examination of the presence of certain gene and chromosome alterations, as well as histological grade, were effective in identifying n0 breast cancer patients at high risk of early recurrence.
INTRODUCTION
The 10-year disease-free survival rate in patients with node-metastasis-negative (n0) primary breast carcinomas after curative surgical therapy has been reported as 80-90% in Japanese and 60-70% in Caucasian patients (1-5). Identification of a high-risk n0 breast carcinoma group would be helpful because it would allow adjuvant therapies to be targeted on this group, thus potentially resulting in an improved prognosis. A number of histological, biochemical and immunohistochemical factors have been reported as being effective prognostic indicators in n0 breast carcinoma. Histological type, histological grade, nuclear grade and the number of mitotic figures of cancer cells are pure morphological prognostic indicators in the n0 group (5-10). Ductal carcinoma in situ (DCIS), invasive ductal carcinoma with a predominantly intraductal component, or predominantly DCIS, medullary carcinoma and mucinous carcinoma have been shown to recur only rarely after curative surgery and are accepted as low-risk groups. In invasive ductal carcinoma (IDC), the highest histological grade (Grade 3), is associated with a higher recurrence rate than lower grades (6-10). A case-control study revealed that patients with Grade 3 n0 IDC have a relative risk of early recurrence and death nearly 42 times higher than that of Grade 1 patients (11). If the evaluation of these histological parameters is performed objectively and reproducibly, histological type and grade would provide critical information with a strong impact on the prognosis. In addition to these histological parameters, tumor size on palpation, estrogen-receptor (ER) and progesterone-receptor (PgR) status, DNA aneuploidy, the S-phase fraction, expression of the epidermal growth factor receptor, nuclear immunoreaction of the p53 protein, microvessel density and loss of expression of the bcl-2 oncoprotein, which are detected by biochemical or immunohistochemical assays, are also known to have prognostic significance in n0 breast cancer (1-3,9-15). Nonetheless, the consensus is that there is no single factor which can accurately identify the high-risk n0 group and a combination of several factors must be used in practice to select patients likely to fall into this group (16).
Table 1
| Atypia | Score | Criteria |
| I Structural atypia | ||
| 1 | Predominant papillary, tubular and/or cribriform pattern | |
| 2 | Between 1 and 3 | |
| 3 | Predominantly solid nest pattern | |
| II Nuclear atypia | ||
| 1 | Uniform nuclear size and shape. Chromatin does not appear coarse | |
| 2 | Between 1 and 3 | |
| 3 | Pleomorphic nuclei. Chromatin has a coarse or fine granular appearance | |
| III Number of mitotic figures | ||
| 1 | <5 per 10 high-power fields (* 400) | |
| 2 | 5-10 per 10 high-power fields | |
| 3 | >10 per 10 high-power fields | |
There are strong correlations between the histological grade of atypia in breast carcinoma (comprising I, structural atypia; II, nuclear atypia; III, the number of mitotic figures), and the number of gene and chromosome alterations at the DNA level, e.g. amplifications of oncogenes, chromosome alterations such as loss of heterozygosity (LOH) on chromosomes 7q, 11p, 17p and 17q and point mutation of the p53 tumor-suppressor gene (17-21). These gene and chromosome alterations are most commonly detected in breast cancers with the highest histological grade, although certain chromosome alterations, e.g. LOH on 16q, are frequently detected in cancers regardless of their histological grade but do not occur in benign neoplasms of the breast.
To determine the prognostic significance of histological grade and gene and chromosome alterations in n0 breast cancers, we prospectively examined the incidence of eight gene and chromosome alterations and their association with histological grade and with cancer recurrence in 129 patients with IDC/invasive lobular carcinoma (ILC). The prognostic significance of the number of gene and chromosome alterations was also analyzed.
Fresh breast cancer and non-cancerous tissue specimens were obtained from 167 Japanese patients with n0 breast carcinoma who underwent curative surgery at the hospital between June 1990 and December 1992. Except for one patient who underwent quadrantectomy and lymph node dissection with radiation therapy, in whom the surgical margins were free of cancer, radical mastectomies were performed on all patients. We confirmed that the tumor tissue specimens contained >50% of the cancer cell area by histological observation of the tissue sections adjacent to the specimens.
Histological typing of the primary tumor was performed according to World Health Organization (WHO) criteria (22). Thirty-two patients were found to have DCIS or predominantly DCIS, 125 IDC, four ILC, three mucinous carcinoma, two medullary carcinoma and one tubular carcinoma. The criteria for histological grading of IDC were based on a modification of those recommended by the WHO (Table 1) (23). These criteria were also applied to ILC, in which the score for structural atypia was always three. This grade was also allocated to the invasive components in carcinoma with an invasive component. When there was heterogeneity in the grade within a tumor specimen, the higher grade was adopted if the region with the higher grade comprised >= 10% of the primary tumor area while the lower grade was assigned if the area with the higher grade comprised <10% of the specimen. Twenty IDC specimens were classified as Grade 1, 46 as Grade 2 and 59 as Grade 3. One ILC was classified as Grade 2 and three as Grade 3.
Thirty-eight patients with low-risk histological types, i.e., DCIS or predominantly DCIS, mucinous carcinoma, medullary carcinoma or tubular carcinoma were excluded from the analysis, since only one patient with a predominantly DCIS type tumor suffered a lung metastasis 44 mo after surgery. In these 38 patients, the mean follow-up period after surgery was 42.6 mo. We examined the association between gene and chromosome alterations and prognosis in the remaining 129 patients with n0 IDC/ILC. These patients, aged from 29 to 83, comprised 60 pre/perimenopausal and 69 postmenopausal patients; 40 with tumors <= 2.0 cm in diameter, 81 with 2.1-5.0 cm diameter tumors and eight with tumors >= 5.1 cm in diameter on palpation; 95 positive for ER (>10 fmol/mg protein), 22 negative for ER ( <= 10 fmol/mg protein) and 12 with no data regarding ER status; 81 positive for PgR (>10 fmol/mg protein), 36 negative for PgR ( <= 10 fmol/mg protein) and 12 with no PgR status data. No patient received perioperative adjuvant chemotherapy or endocrine therapy. Among the 129 IDC/ILC patients, 20 (18 with IDC and two with ILC) suffered a recurrence within 49 months of surgery and six died from disseminated cancer. In the remaining 123 patients, the mean follow-up period was 43.4 months after surgery, ranging from 15 to 61 months.
LOH on 16p, LOH on 16q and mutation of the p53 gene had already been examined in 65, 65 and 37 patients respectively (21,24). DNA was isolated from cancerous and non-cancerous tissue specimens of 64 other carcinomas. Five µg of DNA isolated from each of the 129 carcinomas was digested with RsaI, TaqI (New England BioLab, Beverly, MA), BamHI or MspI (Takara, Kyoto, Japan), electrophoresed in 0.8% agarose gel, denatured in alkali, neutralized and blotted onto Nitroplus filters (MSI, Westboro, MA) (21). The filters were prehybridized then hybridized to the polymorphic genomic DNA markers or proto-oncogene cDNA probes. The probes used were D16S83 (pEKMDA2.1A, RsaI), HBA (JW101, RsaI) and D16S159 (CJ52.94, TaqI) for 16p (25,26); D16S4 (ACH207, TaqI), HP (hp2[alpha], BamHI), TAT (BB0.4, BamHI), D16S7 (79-2-23, TaqI, RsaI), and APRT (pAT2.3aprt, TaqI) for 16q (26); D17S34 (p144D6, TaqI), D17S30 (pYNZ22, TaqI, RsaI), D17S31 (pMCT35.1, MspI), D17S1 (pHF12-2, MspI), MYH2 (p10-5, MspI) and D17S71 (pA10-41, MspI) for 17p (27-29); D17S74 (pCMM86, TaqI, RsaI) for 17q (29); and D18S8 (OLVII E10, MspI), D18S7 (OLVII A8, MspI) and D18S5 (OS4, TaqI) for 18q (30-32). Gene amplification was examined using the c-erbB-2 (pCER204) and int-2 (SS6) probes (33,34). Mutations in exons 4-8 of the p53 gene were examined by polymerase chain reaction single-strand conformation polymorphism analysis (35).
The mean number of gene and chromosome alterations per tumor was modulated by the formula:
The correlation of each gene or chromosome alteration with the histological grade was examined using the [chi]2 test. Disease-free survival curves were plotted by the Kaplan-Meier method and differences between the three curves were investigated by the log-rank test (36,37). The risk estimation of each prognostic indicator by univariate and multivariate analyses were performed by Cox's proportional hazards general linear model using the PHREG procedure of the SAS program package (SAS Institute Inc., Cary, NC) (38).
The 20 IDC/ILC patients with recurrence comprised 17 with Grade 3 tumors (15 IDC and two ILC) and three with Grade 2 IDC. No patient with a Grade 1 tumor experienced recurrence. Disease-free survival curves differed significantly between the Grade 1, Grade 2 and Grade 3 groups (P = 0.004, Fig. 1). Tumor size, menopausal status, ER and PgR did not influence disease-free survival in n0 IDC/ILC.
In n0 breast IDC/ILC, LOH on 16q was confirmed as occurring frequently regardless of difference in histological grade, whereas LOH on 17p, 17q and 18q, p53 mutation and c-erbB-2 amplification were accumulated in the Grade 3 group. LOH on 18q and int-2 gene amplification were indicators of high recurrence rate in n0 IDC/ILC, and the impact of these two alterations was of independent significance on early recurrence in a multivariate analysis. Several studies have shown the importance of p53 gene mutations and nuclear immunoreaction of the p53 protein as a prognostic indicator in n0 breast cancer (12,13). In a case-control study, Iwaya, et al. showed that p53 immunoreaction in cancer cell nuclei was a significant indicator of recurrence within 2 yrs of curative surgical therapy (11). The results of the present study differ from those of previous studies in that mutations of the p53 gene did not correlate with a high recurrence rate. Longer follow-up and comparison of the p53 gene mutation status with nuclear p53 protein immunoreactivity for individual patients would be necessary to ascertain the role of p53 in n0 cancer recurrence. It is still unclear whether c-erbB-2 gene amplification and overexpression of its protein have a significant impact on the prognosis of n0 breast carcinomas (39,40). Amplification of c-erbB-2 alone was of no significance as a prognostic factor in the n0 cancer group even after excluding DCIS and predominantly DCIS.
The number of these gene and chromosome alterations was shown to correlate with a high recurrence rate in invasive n0 breast cancers. In the majority of cases, disease-free survival curves significantly differed between the group with the gene or chromosome alteration score of 3.0 or more and the group with a score of 1.0-2.9. However, among Grade 3 cases, the curves did not differ between the group with a score of 3.0 or more and the group with a score of 1.0-2.9. The Grade 3 case group with the alteration score of 0-0.9 tended to be of better prognosis. These data and the results of the multivariate analysis indicated that the histological grade was superior to the accumulation of the eight gene and chromosome alterations that were examined in the present study as a practical prognostic factor of n0 breast cancer.
The accumulation of alterations of tumor-associated genes or chromosome sites were not investigated in the present study. Multicolor fluorescence in situ hybridization and comparative genomic hybridization methods have recently allowed the accumulation of a large number of gene and chromosome alterations to be observed in breast cancers (41-45). Application of these novel methods, which can analyze a large number of chromosome alterations at the same time, would be useful in the detection of n0 breast cancer patients at high risk of early recurrence.
In the present study, histological type and the histological grade of atypia were confirmed as indicators of a high-risk group for early recurrence of n0 breast carcinomas. Grade 1 IDC as well as histologically low-risk types such as DCIS and predominantly DCIS, medullary carcinoma, mucinous carcinoma and tubular carcinoma, were confirmed as carrying a low risk of early recurrence, whereas Grade 3 IDC/ILC, which showed a 4-yr recurrence rate of 23%, was considered to carry a high risk factor for early recurrence. In the Grade 2 group, the 4-yr recurrence rate was 5%, but recurrence tended to occur slowly. According to the data of Yoshimoto,et al., nuclear grade 3 is a prognostic indicator of early recurrence but not of recurrence after 5 years. While the disease-free survival rate of their nuclear grade 3 group decreased abruptly within 5 years and reached a plateau thereafter, that of the nuclear grade 2 group decreased gradually and in a linear manner even after 5 years (5). Because the grade of nuclear atypia is one of the constituents of the histological Grade, and both are well correlated, histological grade 2 might be an indicator of late recurrence, but not of early recurrence.
To identify groups at high risk of recurrence in multi-institutional protocol studies for the efficacy of adjuvant therapies, variations in the criteria for histological or nuclear grading between institutions should be reduced to a minimum. Such variations may originate largely from interobserver deviations in diagnostic criteria and in part from variations in the procedures for specimen processing among institutes. It has been shown that, when a grading scheme with specified guidelines is used, good reproducibility of grading can be achieved among pathologists (46,47). When these difficulties are overcome, histological grading or nuclear grading will be a practical and more reliable prognostic indicator for n0 breast carcinomas. Active treatment for such poor prognostic cases would be recommended.
This work was supported in part by Grants-in-Aid for Cancer Research and for the Comprehensive 10-Year Strategy for Cancer Control from the Ministry of Health and Welfare of Japan, and a Grant-in-Aid from the Uehara Memorial Foundation for Bioscience. The authors are grateful to Drs H. Yamamoto and T. Nanasawa for providing the surgically resected specimens and to Drs J. Arrand, D. Drayna, Y. Nakamura, G. Scherer and G. Thomas for providing the DNA probes. DNA probes were also provided by the Japanese Cancer Research Resources Bank (Tokyo, Japan) and the American Type Culture Collection (Rockville, MD).
where N1 is the number of alterations, including LOH on 16p, 17p, 17q and 18q, c-erbB-2 and int-2 gene amplifications, and p53 gene mutations, in each tumor, and N2 is the number of gene or chromosome loci informative for gene alterations in each tumor. In this formula, n, the number of gene alteration types examined, was always 7. Because LOH on 16q occurs in breast cancer regardless of the grade of atypia (21), this alteration was excluded from the calculation.
References
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For reprints and all correspondence: Hitoshi Tsuda, Pathology Division, National Cancer Center Research Institute, 1-1, Tsukiji 5-chome, Chuo-ku, Tokyo 104, Japan
Abbreviations: LOH, loss of heterozygosity; IDC, invasive ductal carcinoma; ILC, invasive lobular carcinoma; DCIS, ductal carcinoma in situ; ER, estrogen-receptor; PgR, progesterone-receptor.
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