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Association of allelic loss at 8p22 with poor prognosis among breast cancer cases treated with high-dose adjuvant chemotherapy
Cancer Letters 180 (2002) 75–82 www.elsevier.com/locate/canlet
Association of allelic loss at 8p22 with poor prognosis among breast cancer cases treated with high-dose adjuvant chemotherapy Michiko Tsuneizumi a,b, Mitsuru Emi a,*, Akira Hirano a, Yoshihito Utada a,c, Koji Tsumagari a,c, Kaoru Takahashi c, Fujio Kasumi c, Futoshi Akiyama c, Goi Sakamoto c, Teruhisa Kazui b, Yusuke Nakamura d a
Department of Molecular Biology, Institute of Gerontology, Nippon Medical School, 1-396, Kosugi-cho, Nakahara-ku, Kawasaki 211-8533, Japan b First Department of Surgery, Hamamatsu University School of Medicine, 1-20-1, Handayama, Hamamatsu 431-3192, Japan c Department of Surgery and Pathology, Cancer Institute, Toshima-ku 170-8455, Japan d Human Genome Center, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639, Japan Received 28 September 2001; received in revised form 18 December 2001; accepted 25 December 2001
Abstract To identify specific allelic losses that might correlate with postoperative mortality of breast cancer patients treated with highdose adjuvant chemotherapy consisting of cyclophosphamide, methotrexate and fluorouracil, we examined tumors from a cohort of 150 such patients, who were followed clinically for 5 years postoperatively, for allelic losses (loss of heterozygosity, LOH) among 18 microsatellite markers throughout the genome. Patients whose tumors had lost an allele at 8p22 had significantly higher risks of mortality than those whose tumors retained both alleles at those loci. At 8p22, the 5-year mortality rate was 31% among patients with losses vs. 8% with retention (P ¼ 0:0354). No other region showed correlation between LOH and prognosis. The data indicate that LOH at 8p22 is a significant predictor of postoperative mortality for breast cancer patients who received high-dose postoperative adjuvant chemotherapy. Thus, LOH at 8p22 can serve as a negative prognostic indicator to guide postoperative management of patients. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Loss of heterozygosity; Postoperative prognosis; Adjuvant chemotherapy; Breast cancer; Tumor suppressor gene
1. Introduction In an effort to identify chromosomal regions where allelic losses are frequent in breast cancers, we previously examined an average of 200 primary breast cancers for loss of heterozygosity (LOH), using more than 150 polymorphic microsatellite markers derived from throughout the human genome [1–14]. The clin* Corresponding author. Tel.: 181-44-733-5230; fax: 181-44733-5192. E-mail address: [email protected] (M. Emi).
ical course of breast cancer varies widely among patients, from modest, non-invasive lesions to aggressive, inflammatory carcinomas. These differences in biological characteristics may be explained by differences in the pattern of alterations among genes that play roles in breast carcinogenesis. Prediction of postoperative prognosis for patients with breast cancer has increased in importance in view of the variety of adjuvant therapies that are now available. However, at present such decisions for an individual patient are still based on conventional prognostic factors such
0304-3835/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(02)00010-1
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as tumor size, clinical stage, lymph node metastasis, and hormone-receptor status [15,16]. We previously described an association of LOH at some chromosomal loci with postoperative prognosis in breast cancers overall [13,14]. In the present study we looked instead for LOH that might associate with poor prognosis among aggressive breast cancers, i.e. specifically those that necessitated high-dose postoperative chemotherapy consisting of cyclophosphamide (CPA), methotrexate (MTX) and fluorouracil (5FU) (CMF). We examined such tumors from 150 breast cancer patients for LOH in 18 regions where frequent LOH had been observed in breast cancers in general, using a representative polymorphic marker for each region.
2. Materials and methods 2.1. Patients, samples and DNA preparation The study population consisted of 150 patients who underwent postoperative adjuvant chemotherapy (CMF) for breast cancer between 1989 and 1993 at the Cancer Institute Hospital, Tokyo. Informed consent in the formal style approved by the ethical committee of the hospital was obtained from each patient prior to surgery. The majority of the patients received standard or modified radical mastectomy at the Cancer Institute Hospital during the period from 1989 through 1993. All patients were followed clinically for at least 5 years or until death. A part of the cohort of patients analyzed in the present study overlapped with those analyzed in our previous study. Details of each patient and the clinical data can be obtained from the corresponding author, provided that patients agree to disclose additional clinical data to the public. All clinical and histopathological data as well as staging at surgery were obtained from an electronic database maintained by the Cancer Institute Hospital in a recording format established by the Japanese Breast Cancer Society [17]. Symbol ‘n2’ in this staging system includes supraclavicular or infraclavicular nodes, unlike ‘N2’ status in the UICC TNM classification. Estrogen receptor (ER) and progesterone receptor (PgR) were measured by radioreceptor assay according to a standard dextran-coated charcoal (DCC) method, using [ 125I]estradiol as labeled ligand
on homogenates of fresh-frozen tissue (Otsuka Pharmaceutical, Tokushima). All samples containing .5 fmol of ER or PgR per mg protein were considered receptor-positive. As regards postoperative adjuvant therapy, all patients were treated according to the ‘Postoperative Clinical Protocol for Breast Cancer’ of the Cancer Institute Hospital. In principle the choice of adjuvant therapy that consists of CMF was applied to those having lymph node metastasis ranging from one to ten nodes or no lymph node metastasis, which was strictly determined on the basis of type of surgery, lymph node involvement, and presence of local or distant metastasis. None of the patients had distant metastasis prior to adjuvant chemotherapy or had undergone radiotherapy or chemotherapy prior to surgery. The standard chemotherapy regimen consisted of 100 mg cyclophosphamide orally on days 1 through 14 for 4 weeks, 40 mg/m 2 MTX intravenously on days 1 and 8 for 4 weeks, and 600 mg/m 2 5-FU intravenously on days 1 and 8 for 4 weeks; patients received chemotherapy for 16–24 weeks. Those patients had a minimum of 5 years of follow-up, unless they had disease recurrence. Tumors and samples of non-cancerous breast tissue were excised from each patient, frozen immediately, and stored at 280 8C. Genomic DNAs were extracted from the frozen materials as previously described [18]. 2.2. LOH analysis Procedures for LOH analysis were described elsewhere [12]. In brief, DNAs from matched normal and cancerous tissues were examined for LOH with respect to the 18 microsatellite markers listed in Table 2 [19]. Microsatellite sequences were amplified by polymerase chain reaction (PCR) using 10 ng of genomic DNA, and PCR products were electrophoresed and autoradiographed as described previously. Definition of LOH and distinction from chromosome multiplication were judged according to procedures we have described previously [12]. 2.3. Statistical analysis Postoperative survival was measured from the date of surgery to the date of last follow-up or death. Survival curves were constructed using the Kaplan–Meier method, and the significance of differences in survival
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rates was tested using the log-rank test as a univariate analysis. Cox’s proportional-hazards model for the risk ratio was used to assess the simultaneous contribution of each covariate in the multivariate analysis. P values of ,0.05 were considered statistically significant. All calculations were performed using Stat View version 4.5 software (SAS Institute Inc., San Francisco, CA).
3. Results The categories of clinical data for our cohort of 150 breast cancer patients who underwent postoperative high-dose adjuvant chemotherapy (CMF) are presented in Table 1. The mean age was 47.5 years (range 29–70) and all patients were females. All Table 1 Clinical characteristics of breast cancer patients who underwent postoperative adjuvant chemotherapy (CMF) No. of patients 1. Median age (years) 2. t (tumor stage) a t1 t2 t3 3. n (lymph node metastasis) a Negative n0 Positive n1a n1b n2 4. Menopausal status Pre-menopause Post-menopause Unknown 5. Histological type a a1 (papillotubular) a2 (solid-tubular) a3 (scirrhous) b3 (invasive lobular) b5 (squamous cell) 6. Estrogen receptor ER (1) ER (2) 7. Progesterone receptor PgR (1) PgR (2)
n ¼ 150 47.5 (range 29–70) 26 107 17
23 51 34 42 90 59 1 27 41 72 9 1 72 78 89 61
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surviving patients were followed for at least 5 years. The average time of postoperative follow-up in survival analysis was 66.4 months. Of the 150 patients, the disease relapsed in 29 women within 5 years; the 5year disease-free survival rate was 81%. Table 2 shows the frequencies of allelic loss (LOH) at each of the 18 chromosomal regions previously chosen as loci that displayed frequent LOH in breast cancers [13]; LOH ranged from 29 to 58% among the 150 tumors examined here. D8S136 (at 8p22) detected the highest frequency of LOH (50 of the 86 informative tumors, 58%); a representative autoradiogram demonstrating LOH at D8S136 in breast cancer is displayed in Fig. 1. Kaplan–Meier analysis of overall survival revealed that the postoperative risk of mortality was greater for patients whose tumors showed LOH at 8p22 compared with patients whose tumors retained both alleles (Fig. 2). Among the 86 patients whose tumors were informative at 8p22, 31% of those with LOH died within 5 years after surgery, compared with an 8% mortality rate among patients whose tumors retained both alleles of the 8p22 marker (3.4 times increase in relative risk of mortality; P ¼ 0:0354 by log-rank test) (Fig. 2, Table 3). No markers from the other 17 frequently deleted regions showed any correlation of LOH with mortality. Table 4 shows clinical characteristics of patient groups classified according to LOH status at 8p22. No significant correlation between 8p22 LOH status and major clinical characteristics was observed among the patients. Log-rank tests of mortality including other clinical parameters, i.e. age of onset, menopausal status, tumor size, nodal status, histological types, and hormone-receptor status, showed that lymph node metastasis and 8p22 LOH status exclusively had a significant correlation with mortality (Table 3). Multivariate analysis among tumor size, lymph node metastasis, ER status, PgR status, menopausal status and 8p22 LOH confirmed that 8p22 LOH predicts the highest relative risk of 5-year mortality among the six variables at the modest significance level (P ¼ 0:12) (Table 5).
4. Discussion
a
Clinical recording scheme according to the Japanese Breast Cancer Society (1989) [17].
In the present study we looked for specific allelic
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Table 2 Chromosomal regions, polymorphic markers, and LOH frequencies at the 18 loci examined in breast cancer patients who underwent postoperative adjuvant chemotherapy (CMF) Chromosomal region
DNA marker
Informative cases/ 150 patients
LOH (1) cases/ informative cases
LOH frequency (%)
1p36 1p34 1p22 3p25.1 3p14.3 6q26-27 8p22 9p21-22 11p15 11q23-24 13q12 13q14 16q24.3 17p13.3 17p13.1 17q21.1 18q21.1 22q13
D1S1612 D1S552 D1S551 D3S1286 D3S1295 D6S503 D8S136 D9S157 D11S922 D11S1998 D13S171 D13S270 D16S413 D17S849 TP53 D17S934 D18S474 D22S272
98 92 98 104 66 73 86 94 96 90 82 91 114 105 109 102 88 109
33/98 27/92 33/98 41/104 28/66 32/73 50/86 30/94 32/96 52/90 32/82 30/91 66/114 54/105 60/109 35/102 26/88 42/109
33.7 29.3 33.7 39.4 42.4 43.8 58.1 31.9 33.3 57.8 39.0 33.0 57.9 51.4 55.0 34.2 29.5 38.5
losses that might correlate with poor prognosis among 150 breast cancer patients who underwent postoperative high-dose adjuvant CMF chemotherapy. We found that the postoperative risk of mortality was higher for patients whose tumors showed LOH at 8p22 compared with patients whose tumors retained
Fig. 1. Representative autoradiogram demonstrating five cases of LOH at locus on chromosomes 8p22. N and T, non-cancerous and tumor DNAs, respectively. Horizontal lines point to positions of allelic bands observed in normal tissue of the patient.
both alleles at these loci among these group of patients. LOH in the 8p22 region has been reported in various other types of tumor [20–27]. A candidate tumor suppressor gene, N33, is at 8p22 near the macrophage-scavenger-receptor (MSR) gene locus [28– 31]. The N33 gene was silenced in several cancer cells, although no point mutations were found. Another candidate gene, PRLTS, at 8p21.3-22 showed point
Fig. 2. Kaplan–Meier curves of postoperative overall survival for patients whose tumors retained both alleles (Retention) or had lost one allele (LOH) at 8p22.
M. Tsuneizumi et al. / Cancer Letters 180 (2002) 75–82 Table 3 Univariate analysis of clinical variables and 5-year mortality n
1. t (tumor stage) a t1 t2 t3 2. Lymph node metastasis Negative Positive 3. Estrogen receptor ER (1) ER (2) 4. Progesterone receptor PgR (1) PgR (2) 5. 8p22 LOH LOH (1) LOH (2)
Mortality Rate
P value
26 107 17
0.15 0.19 0.23
NS
23 127
0.13 0.20
, 0.001
72 78
0.19 0.19
NS
89 61
0.14 0.26
NS
50 36
0.31 0.08
0.035
a
Clinical recording scheme according to the Japanese Breast Cancer Society (1989) [17].
mutations in four cancer cases [32,33]. The frequency of alterations in this gene was, however, very low [34]. Alterations of N-acetyltransferase (NAT)1 and NAT2 genes at 8p22 have also been studied in cancer, because of their carcinogen-metabolizing action [35,36]. The results showed no abnormality in cancer cells [35,36]. FEZ1 is altered in many tumors, including esophageal, prostate, and breast cancer. The region of LOH at 8p22 overlaps with regions described as commonly deleted in colorectal, prostate, hepatocellular and non-small cell lung carcinomas [37,38]. This overlapping strongly suggests that inactivation of one or more tumor suppressor genes at 8p22 may play an important carcinogenic role in a variety of human tissues. Because LOH at 8p22 in particular was a significant prognostic factor in the group of breast cancer patients who underwent high-dose postoperative adjuvant CMF chemotherapy in the present study, the candidate genes mentioned above might reflect the mechanism of response or sensitivity against chemotherapeutic drugs due to characteristics of each neoplasm or host patient. In previous work [13] we examined the relationship between postoperative mortality and LOH at 18 chromosomal regions in a cohort of patients with breast cancer overall after surgery, and found significant
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correlation with LOH at 1p34, 13q12, 17p13.3 and 17q21.1. We previously reported that allelic loss in the 1p34-36 region correlated with postoperative recurrence among breast cancers without lymph node metastasis [39]. Among 15 cases of n0 breast cancers displayed in Table 4, five of them showed LOH at 1p34-36 as well as LOH at 8p22; four cases showed LOH at 8p22 but not at 1p34-36; and two cases showed LOH at 1p34-36 but not at 8p22; three cases retained both alleles at both 1p34-36 and 8p22; the remaining case retained both alleles at 8p22 but was homozygous and uninformative for allelic status at 1p34-36. We also found a significant prognostic association with LOH at 8p22, specifically in Table 4 Clinical characteristics of breast cancer patients who underwent postoperative adjuvant chemotherapy (CMF) classified by 8p22 LOH status Clinicopathological feature 1. t (tumor stage) a t1 t2 t3 2. n (lymph node metastasis) a Negative n0 Positive n1a n1b n2 3. Menopausal status Pre-menopause Post-menopause 4. Histological type a a1 (papillotubular) a2 (solid-tubular) a3 (scirrhous) b3 (invasive lobular) b5 (squamous cell) 5. Estrogen receptor ER (1) ER (2) 6. Progesterone receptor PgR (1) PgR (2) 7. Distant metastasis Negative Positive a
LOH (1) (n ¼ 50)
LOH (2) (n ¼ 36)
10 32 8
4 31 1
9
6
15 12 14
11 12 7
28 21
22 14
8 12 26 3 1
8 12 12 4 0
26 24
19 17
31 19
24 12
50 0
36 0
Clinical recording scheme according to the Japanese Breast Cancer Society (1989) [17].
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Table 5 Multivariate analyses of clinical variables and 5-year mortality
Tumour size n (lymph node metastasis) ER status PgR status Menopausal status 8p22 LOH
RR
95% CI
P value
1.0070 1.0044 0.7690 0.5950 0.6940 2.4650
0.985–1.030 1.012–1.077 0.262–2.262 0.180–1.961 0.243–1.979 0.779–7.795
NS 0.01 NS NS NS 0.12
large tumors and in ER-negative breast cancers [40]. In a larger cohort of 504 patients, we later noticed a significant association between poor postoperative prognosis and LOH at 3p25.1 [41]. Although we previously reported that allelic loss in the 1p34-36 region correlated with postoperative recurrence of node-negative breast cancers, in the present study allelic loss of 1p was not a significant prognostic factor. These data corroborate that LOH at 1p specifically influences node-negative cancer. In the study reported here, we measured postoperative mortality among patients who received postoperative high-dose adjuvant CMF chemotherapy right after curative mastectomy, and found that in this group of tumors, distinct LOH status correlated with disease prognosis. Thus, LOH at 8p22 is considered to give tumor cells resistant character among CMF-treated breast cancers, suggesting that candidate genes which locate in these regions may relate to mechanisms of response or sensitivity against chemotherapeutic drugs of neoplasms. Further studies to explain such genetic differences will be necessary before we can fully understand the pathophysiology of breast cancer progression and treatment. Acknowledgements This work was supported in part by a special grant for Strategic Advanced Research on ‘Cancer’ and ‘Genome Science’ from the Ministry of Education, Science, Sports and Culture of Japan, by a Research Grant for Research from the Ministry of Health and Welfare of Japan, and by a Research for the Future Program Grant of The Japan Society for the Promotion of Science. The authors wish to thank Drs Takaaki Sato, Takuji Iwase, Toyomasa Katagiri, Yousuke Harada, Isao Ito, Kenji Kobayashi, Mieko
Matsushima, Aritoshi Iida, Takashi Tada, Hiroko Saito, Yoshio Miki, Takashi Yokota, Kouichi Bando, Kouichi Fukino, Mitsuko Kajita, Kyoko Shimizu, Mayumi Tanaka and Yumiko Sakai for their contributions.
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Association of allelic loss at 8p22 with poor prognosis among breast cancer cases treated with high-dose adjuvant chemotherapy Michiko Tsuneizumi a,b, Mitsuru Emi a,*, Akira Hirano a, Yoshihito Utada a,c, Koji Tsumagari a,c, Kaoru Takahashi c, Fujio Kasumi c, Futoshi Akiyama c, Goi Sakamoto c, Teruhisa Kazui b, Yusuke Nakamura d a
Department of Molecular Biology, Institute of Gerontology, Nippon Medical School, 1-396, Kosugi-cho, Nakahara-ku, Kawasaki 211-8533, Japan b First Department of Surgery, Hamamatsu University School of Medicine, 1-20-1, Handayama, Hamamatsu 431-3192, Japan c Department of Surgery and Pathology, Cancer Institute, Toshima-ku 170-8455, Japan d Human Genome Center, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639, Japan Received 28 September 2001; received in revised form 18 December 2001; accepted 25 December 2001
Abstract To identify specific allelic losses that might correlate with postoperative mortality of breast cancer patients treated with highdose adjuvant chemotherapy consisting of cyclophosphamide, methotrexate and fluorouracil, we examined tumors from a cohort of 150 such patients, who were followed clinically for 5 years postoperatively, for allelic losses (loss of heterozygosity, LOH) among 18 microsatellite markers throughout the genome. Patients whose tumors had lost an allele at 8p22 had significantly higher risks of mortality than those whose tumors retained both alleles at those loci. At 8p22, the 5-year mortality rate was 31% among patients with losses vs. 8% with retention (P ¼ 0:0354). No other region showed correlation between LOH and prognosis. The data indicate that LOH at 8p22 is a significant predictor of postoperative mortality for breast cancer patients who received high-dose postoperative adjuvant chemotherapy. Thus, LOH at 8p22 can serve as a negative prognostic indicator to guide postoperative management of patients. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Loss of heterozygosity; Postoperative prognosis; Adjuvant chemotherapy; Breast cancer; Tumor suppressor gene
1. Introduction In an effort to identify chromosomal regions where allelic losses are frequent in breast cancers, we previously examined an average of 200 primary breast cancers for loss of heterozygosity (LOH), using more than 150 polymorphic microsatellite markers derived from throughout the human genome [1–14]. The clin* Corresponding author. Tel.: 181-44-733-5230; fax: 181-44733-5192. E-mail address: [email protected] (M. Emi).
ical course of breast cancer varies widely among patients, from modest, non-invasive lesions to aggressive, inflammatory carcinomas. These differences in biological characteristics may be explained by differences in the pattern of alterations among genes that play roles in breast carcinogenesis. Prediction of postoperative prognosis for patients with breast cancer has increased in importance in view of the variety of adjuvant therapies that are now available. However, at present such decisions for an individual patient are still based on conventional prognostic factors such
0304-3835/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(02)00010-1
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as tumor size, clinical stage, lymph node metastasis, and hormone-receptor status [15,16]. We previously described an association of LOH at some chromosomal loci with postoperative prognosis in breast cancers overall [13,14]. In the present study we looked instead for LOH that might associate with poor prognosis among aggressive breast cancers, i.e. specifically those that necessitated high-dose postoperative chemotherapy consisting of cyclophosphamide (CPA), methotrexate (MTX) and fluorouracil (5FU) (CMF). We examined such tumors from 150 breast cancer patients for LOH in 18 regions where frequent LOH had been observed in breast cancers in general, using a representative polymorphic marker for each region.
2. Materials and methods 2.1. Patients, samples and DNA preparation The study population consisted of 150 patients who underwent postoperative adjuvant chemotherapy (CMF) for breast cancer between 1989 and 1993 at the Cancer Institute Hospital, Tokyo. Informed consent in the formal style approved by the ethical committee of the hospital was obtained from each patient prior to surgery. The majority of the patients received standard or modified radical mastectomy at the Cancer Institute Hospital during the period from 1989 through 1993. All patients were followed clinically for at least 5 years or until death. A part of the cohort of patients analyzed in the present study overlapped with those analyzed in our previous study. Details of each patient and the clinical data can be obtained from the corresponding author, provided that patients agree to disclose additional clinical data to the public. All clinical and histopathological data as well as staging at surgery were obtained from an electronic database maintained by the Cancer Institute Hospital in a recording format established by the Japanese Breast Cancer Society [17]. Symbol ‘n2’ in this staging system includes supraclavicular or infraclavicular nodes, unlike ‘N2’ status in the UICC TNM classification. Estrogen receptor (ER) and progesterone receptor (PgR) were measured by radioreceptor assay according to a standard dextran-coated charcoal (DCC) method, using [ 125I]estradiol as labeled ligand
on homogenates of fresh-frozen tissue (Otsuka Pharmaceutical, Tokushima). All samples containing .5 fmol of ER or PgR per mg protein were considered receptor-positive. As regards postoperative adjuvant therapy, all patients were treated according to the ‘Postoperative Clinical Protocol for Breast Cancer’ of the Cancer Institute Hospital. In principle the choice of adjuvant therapy that consists of CMF was applied to those having lymph node metastasis ranging from one to ten nodes or no lymph node metastasis, which was strictly determined on the basis of type of surgery, lymph node involvement, and presence of local or distant metastasis. None of the patients had distant metastasis prior to adjuvant chemotherapy or had undergone radiotherapy or chemotherapy prior to surgery. The standard chemotherapy regimen consisted of 100 mg cyclophosphamide orally on days 1 through 14 for 4 weeks, 40 mg/m 2 MTX intravenously on days 1 and 8 for 4 weeks, and 600 mg/m 2 5-FU intravenously on days 1 and 8 for 4 weeks; patients received chemotherapy for 16–24 weeks. Those patients had a minimum of 5 years of follow-up, unless they had disease recurrence. Tumors and samples of non-cancerous breast tissue were excised from each patient, frozen immediately, and stored at 280 8C. Genomic DNAs were extracted from the frozen materials as previously described [18]. 2.2. LOH analysis Procedures for LOH analysis were described elsewhere [12]. In brief, DNAs from matched normal and cancerous tissues were examined for LOH with respect to the 18 microsatellite markers listed in Table 2 [19]. Microsatellite sequences were amplified by polymerase chain reaction (PCR) using 10 ng of genomic DNA, and PCR products were electrophoresed and autoradiographed as described previously. Definition of LOH and distinction from chromosome multiplication were judged according to procedures we have described previously [12]. 2.3. Statistical analysis Postoperative survival was measured from the date of surgery to the date of last follow-up or death. Survival curves were constructed using the Kaplan–Meier method, and the significance of differences in survival
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rates was tested using the log-rank test as a univariate analysis. Cox’s proportional-hazards model for the risk ratio was used to assess the simultaneous contribution of each covariate in the multivariate analysis. P values of ,0.05 were considered statistically significant. All calculations were performed using Stat View version 4.5 software (SAS Institute Inc., San Francisco, CA).
3. Results The categories of clinical data for our cohort of 150 breast cancer patients who underwent postoperative high-dose adjuvant chemotherapy (CMF) are presented in Table 1. The mean age was 47.5 years (range 29–70) and all patients were females. All Table 1 Clinical characteristics of breast cancer patients who underwent postoperative adjuvant chemotherapy (CMF) No. of patients 1. Median age (years) 2. t (tumor stage) a t1 t2 t3 3. n (lymph node metastasis) a Negative n0 Positive n1a n1b n2 4. Menopausal status Pre-menopause Post-menopause Unknown 5. Histological type a a1 (papillotubular) a2 (solid-tubular) a3 (scirrhous) b3 (invasive lobular) b5 (squamous cell) 6. Estrogen receptor ER (1) ER (2) 7. Progesterone receptor PgR (1) PgR (2)
n ¼ 150 47.5 (range 29–70) 26 107 17
23 51 34 42 90 59 1 27 41 72 9 1 72 78 89 61
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surviving patients were followed for at least 5 years. The average time of postoperative follow-up in survival analysis was 66.4 months. Of the 150 patients, the disease relapsed in 29 women within 5 years; the 5year disease-free survival rate was 81%. Table 2 shows the frequencies of allelic loss (LOH) at each of the 18 chromosomal regions previously chosen as loci that displayed frequent LOH in breast cancers [13]; LOH ranged from 29 to 58% among the 150 tumors examined here. D8S136 (at 8p22) detected the highest frequency of LOH (50 of the 86 informative tumors, 58%); a representative autoradiogram demonstrating LOH at D8S136 in breast cancer is displayed in Fig. 1. Kaplan–Meier analysis of overall survival revealed that the postoperative risk of mortality was greater for patients whose tumors showed LOH at 8p22 compared with patients whose tumors retained both alleles (Fig. 2). Among the 86 patients whose tumors were informative at 8p22, 31% of those with LOH died within 5 years after surgery, compared with an 8% mortality rate among patients whose tumors retained both alleles of the 8p22 marker (3.4 times increase in relative risk of mortality; P ¼ 0:0354 by log-rank test) (Fig. 2, Table 3). No markers from the other 17 frequently deleted regions showed any correlation of LOH with mortality. Table 4 shows clinical characteristics of patient groups classified according to LOH status at 8p22. No significant correlation between 8p22 LOH status and major clinical characteristics was observed among the patients. Log-rank tests of mortality including other clinical parameters, i.e. age of onset, menopausal status, tumor size, nodal status, histological types, and hormone-receptor status, showed that lymph node metastasis and 8p22 LOH status exclusively had a significant correlation with mortality (Table 3). Multivariate analysis among tumor size, lymph node metastasis, ER status, PgR status, menopausal status and 8p22 LOH confirmed that 8p22 LOH predicts the highest relative risk of 5-year mortality among the six variables at the modest significance level (P ¼ 0:12) (Table 5).
4. Discussion
a
Clinical recording scheme according to the Japanese Breast Cancer Society (1989) [17].
In the present study we looked for specific allelic
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Table 2 Chromosomal regions, polymorphic markers, and LOH frequencies at the 18 loci examined in breast cancer patients who underwent postoperative adjuvant chemotherapy (CMF) Chromosomal region
DNA marker
Informative cases/ 150 patients
LOH (1) cases/ informative cases
LOH frequency (%)
1p36 1p34 1p22 3p25.1 3p14.3 6q26-27 8p22 9p21-22 11p15 11q23-24 13q12 13q14 16q24.3 17p13.3 17p13.1 17q21.1 18q21.1 22q13
D1S1612 D1S552 D1S551 D3S1286 D3S1295 D6S503 D8S136 D9S157 D11S922 D11S1998 D13S171 D13S270 D16S413 D17S849 TP53 D17S934 D18S474 D22S272
98 92 98 104 66 73 86 94 96 90 82 91 114 105 109 102 88 109
33/98 27/92 33/98 41/104 28/66 32/73 50/86 30/94 32/96 52/90 32/82 30/91 66/114 54/105 60/109 35/102 26/88 42/109
33.7 29.3 33.7 39.4 42.4 43.8 58.1 31.9 33.3 57.8 39.0 33.0 57.9 51.4 55.0 34.2 29.5 38.5
losses that might correlate with poor prognosis among 150 breast cancer patients who underwent postoperative high-dose adjuvant CMF chemotherapy. We found that the postoperative risk of mortality was higher for patients whose tumors showed LOH at 8p22 compared with patients whose tumors retained
Fig. 1. Representative autoradiogram demonstrating five cases of LOH at locus on chromosomes 8p22. N and T, non-cancerous and tumor DNAs, respectively. Horizontal lines point to positions of allelic bands observed in normal tissue of the patient.
both alleles at these loci among these group of patients. LOH in the 8p22 region has been reported in various other types of tumor [20–27]. A candidate tumor suppressor gene, N33, is at 8p22 near the macrophage-scavenger-receptor (MSR) gene locus [28– 31]. The N33 gene was silenced in several cancer cells, although no point mutations were found. Another candidate gene, PRLTS, at 8p21.3-22 showed point
Fig. 2. Kaplan–Meier curves of postoperative overall survival for patients whose tumors retained both alleles (Retention) or had lost one allele (LOH) at 8p22.
M. Tsuneizumi et al. / Cancer Letters 180 (2002) 75–82 Table 3 Univariate analysis of clinical variables and 5-year mortality n
1. t (tumor stage) a t1 t2 t3 2. Lymph node metastasis Negative Positive 3. Estrogen receptor ER (1) ER (2) 4. Progesterone receptor PgR (1) PgR (2) 5. 8p22 LOH LOH (1) LOH (2)
Mortality Rate
P value
26 107 17
0.15 0.19 0.23
NS
23 127
0.13 0.20
, 0.001
72 78
0.19 0.19
NS
89 61
0.14 0.26
NS
50 36
0.31 0.08
0.035
a
Clinical recording scheme according to the Japanese Breast Cancer Society (1989) [17].
mutations in four cancer cases [32,33]. The frequency of alterations in this gene was, however, very low [34]. Alterations of N-acetyltransferase (NAT)1 and NAT2 genes at 8p22 have also been studied in cancer, because of their carcinogen-metabolizing action [35,36]. The results showed no abnormality in cancer cells [35,36]. FEZ1 is altered in many tumors, including esophageal, prostate, and breast cancer. The region of LOH at 8p22 overlaps with regions described as commonly deleted in colorectal, prostate, hepatocellular and non-small cell lung carcinomas [37,38]. This overlapping strongly suggests that inactivation of one or more tumor suppressor genes at 8p22 may play an important carcinogenic role in a variety of human tissues. Because LOH at 8p22 in particular was a significant prognostic factor in the group of breast cancer patients who underwent high-dose postoperative adjuvant CMF chemotherapy in the present study, the candidate genes mentioned above might reflect the mechanism of response or sensitivity against chemotherapeutic drugs due to characteristics of each neoplasm or host patient. In previous work [13] we examined the relationship between postoperative mortality and LOH at 18 chromosomal regions in a cohort of patients with breast cancer overall after surgery, and found significant
79
correlation with LOH at 1p34, 13q12, 17p13.3 and 17q21.1. We previously reported that allelic loss in the 1p34-36 region correlated with postoperative recurrence among breast cancers without lymph node metastasis [39]. Among 15 cases of n0 breast cancers displayed in Table 4, five of them showed LOH at 1p34-36 as well as LOH at 8p22; four cases showed LOH at 8p22 but not at 1p34-36; and two cases showed LOH at 1p34-36 but not at 8p22; three cases retained both alleles at both 1p34-36 and 8p22; the remaining case retained both alleles at 8p22 but was homozygous and uninformative for allelic status at 1p34-36. We also found a significant prognostic association with LOH at 8p22, specifically in Table 4 Clinical characteristics of breast cancer patients who underwent postoperative adjuvant chemotherapy (CMF) classified by 8p22 LOH status Clinicopathological feature 1. t (tumor stage) a t1 t2 t3 2. n (lymph node metastasis) a Negative n0 Positive n1a n1b n2 3. Menopausal status Pre-menopause Post-menopause 4. Histological type a a1 (papillotubular) a2 (solid-tubular) a3 (scirrhous) b3 (invasive lobular) b5 (squamous cell) 5. Estrogen receptor ER (1) ER (2) 6. Progesterone receptor PgR (1) PgR (2) 7. Distant metastasis Negative Positive a
LOH (1) (n ¼ 50)
LOH (2) (n ¼ 36)
10 32 8
4 31 1
9
6
15 12 14
11 12 7
28 21
22 14
8 12 26 3 1
8 12 12 4 0
26 24
19 17
31 19
24 12
50 0
36 0
Clinical recording scheme according to the Japanese Breast Cancer Society (1989) [17].
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Table 5 Multivariate analyses of clinical variables and 5-year mortality
Tumour size n (lymph node metastasis) ER status PgR status Menopausal status 8p22 LOH
RR
95% CI
P value
1.0070 1.0044 0.7690 0.5950 0.6940 2.4650
0.985–1.030 1.012–1.077 0.262–2.262 0.180–1.961 0.243–1.979 0.779–7.795
NS 0.01 NS NS NS 0.12
large tumors and in ER-negative breast cancers [40]. In a larger cohort of 504 patients, we later noticed a significant association between poor postoperative prognosis and LOH at 3p25.1 [41]. Although we previously reported that allelic loss in the 1p34-36 region correlated with postoperative recurrence of node-negative breast cancers, in the present study allelic loss of 1p was not a significant prognostic factor. These data corroborate that LOH at 1p specifically influences node-negative cancer. In the study reported here, we measured postoperative mortality among patients who received postoperative high-dose adjuvant CMF chemotherapy right after curative mastectomy, and found that in this group of tumors, distinct LOH status correlated with disease prognosis. Thus, LOH at 8p22 is considered to give tumor cells resistant character among CMF-treated breast cancers, suggesting that candidate genes which locate in these regions may relate to mechanisms of response or sensitivity against chemotherapeutic drugs of neoplasms. Further studies to explain such genetic differences will be necessary before we can fully understand the pathophysiology of breast cancer progression and treatment. Acknowledgements This work was supported in part by a special grant for Strategic Advanced Research on ‘Cancer’ and ‘Genome Science’ from the Ministry of Education, Science, Sports and Culture of Japan, by a Research Grant for Research from the Ministry of Health and Welfare of Japan, and by a Research for the Future Program Grant of The Japan Society for the Promotion of Science. The authors wish to thank Drs Takaaki Sato, Takuji Iwase, Toyomasa Katagiri, Yousuke Harada, Isao Ito, Kenji Kobayashi, Mieko
Matsushima, Aritoshi Iida, Takashi Tada, Hiroko Saito, Yoshio Miki, Takashi Yokota, Kouichi Bando, Kouichi Fukino, Mitsuko Kajita, Kyoko Shimizu, Mayumi Tanaka and Yumiko Sakai for their contributions.
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