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Analysis of serum ErbB-2 protein and HLA-DRB1 in Japanese patients with lung cancer
Cancer Letters 152 (2000) 87±95 www.elsevier.com/locate/canlet
Analysis of serum ErbB-2 protein and HLA-DRB1 in Japanese patients with lung cancer Chie Yoshimura a, Shosaku Nomura a,*, Manabu Yamaoka b, Tetsuji Ohtani b, Tatsunori Matsuzakiz b, Kazuyuki Yamaguchi a, Shirou Fukuharal a,b a b
The First Department of Internal Medicine, Kansai Medical University, 10-15 Fumizonocho, Moriguchi, Osaka 570-8507, Japan The First Department of Blood Transfusion, Kansai Medical University, 10-15 Fumizonocho, Moriguchi, Osaka 570-8507, Japan Received 6 September 1999; received in revised form 5 December 1999; accepted 5 December 1999
Abstract We investigated the relationship between ErbB-2 and HLA in order to clarify the clinical and genetic factors related to Japanese patients with lung cancer. Thirty-nine of the 73 lung cancer patients (53.4%) had elevated levels of ErbB-2. Only seven of 23 (30.4%) patients with small cell carcinoma had elevated ErbB-2 levels. The prevalence of ErbB-2 positivity was highest (23 of 32; 71.8%) in patients with adenocarcinoma, while that in patients with squamous cell carcinoma was 50% (9 of 18). The frequencies of HLA A33, B44, B62, and B75 were lower in the lung cancer patients than in the control group. HLADR9 was higher in frequency in lung cancer patients than in the healthy controls (P , 0:05), but HLA-DR6 was lower in frequency in lung cancer patients than in controls (P , 0:01). DRB1*0901 was signi®cantly higher in frequency in lung cancer patients than in controls (P , 0:05). On the other hand, DRB1*0802, DRB1*1302 and the DRB1*14 group (*1401, *1403, *1405, *1406, and *1407) were completely absent in lung cancer patients. The frequencies of HLA B35, B52, B62, DRB1*0404, and DRB1*0406 were higher in the ErbB-2-positive lung cancer patients than in the ErbB-2-negative lung cancer patients. However, these types of HLA were not included in signi®cant frequencies in our group of lung cancers. Our results suggest that some HLA-antigens/alleles participate in the pathogenesis of lung cancer in Japanese patients. In addition, the relationship between HLA-associated genetic factors and ErbB-2 seems to be weak. These ®ndings suggest that ErbB-2 is correlated with prognostic factors for lung cancer independently of HLA-associated genetic factors. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Lung cancer; Prognostic factor; ErbB-2; HLA-DRB1*0901; HLA-DRB1*1302
1. Introduction Lung cancer is currently the leading cause of cancer-related death [1]. Most patients have inoperable tumors at the time of diagnosis, and metastatic relapse in patients with operable non-small cell lung * Corresponding author. Tel.: 181-66-992-1001; fax: 181-66994-8344. E-mail address: [email protected] (S. Nomura)
carcinoma remains a frequent event [2]. Therefore, major research efforts are being directed at identifying the relationship between speci®c gene alterations and the clinical behavior of lung tumors. Genetic abnormalities in lung cancer frequently include the mutation, rearrangement, or overexpression of several genes and their protein products, such as c-myc, K-ras, p53 and cerbB-2 [3±6]. The c-erbB-2 oncoprotein is a 1 85-kd transmembrane protein kinase receptor with homol-
0304-3835/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(99)00437-1
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ogy to the epidermal growth factor receptor, and has been shown to contribute to malignant transformation and progression in a number of epithelial cell carcinomas [7±9]. The c-erbB-2 protein is strongly stained immunohistochemically on the cell membrane of adenocarcinoma, and erbB-2 overexpression relative to that of normal alveolar lung tissue has been found to be associated with a worse prognosis [5]. In addition, recent studies have shown that a soluble form of the cerbB-2 protein can be found in serum [10±12]. In recent years, there have been many reports of genetic predispositions for particular malignancies and numerous studies of genetic effects, such as that of HLA, on lung cancer. Rogentine et al. [13] suggested that some HLA-class 1 antigens conferred resistance to the progression of bronchogenic carcinoma in Caucasians. More recently, Prazak et al. [14] reported a relationship between survival time and HLA for epidermoid lung carcinoma, and Romano et al. [15] reported an association between HLADR7 and resistance to lung cancer. Some of these divergent results may be attributable to the study of differing populations and the existence of numerous other factors related to the development of lung cancer. Thus, analysis of c-erbB-2 and HLA may be important for determining not only the etiology but also the appropriate therapy and prognosis of lung cancer. However, there are few reports studied for both relationship.
2. Materials and methods 2.1. Subjects We studied 73 unrelated Japanese patients (49 men and 24 women) with lung cancer, admitted to our hospital between June 1995 and September 1998. The diagnosis of lung cancer was made histologically. The subjects ranged in age from 27 to 81 years. Eighteen patients had squamous cell carcinoma, 32 had adenocarcinoma, and 23 had small cell carcinoma. One hundred and twenty-two controls were randomly selected from among healthy unrelated Japanese. All subjects gave informed consent for participation in this study. Clinical staging of carcinoma was performed using the TMN classi®cation.
2.2. Measurement of serum ErbB-2 Serum ErbB-2 was quanti®ed by EIA, using the ErbB-2 EIA `Nichirei' kit (Nichirei Inc., Tokyo) [10,16,17]. In brief, 50 ml of serum or standard was diluted 1:5 with buffer solution and incubated at 15± 258C for 2 h in anti-ErbB-2 antibody-coated beads. After washing with PBS, peroxidase-labeled antiErbB-2 antibody was added, and incubated for 2 h at room temperature. After washing, the beads were transferred to plastic tubes. The incubation was continued for 30 min in the dark using o-phenylenediamine with H2O2.The color development was terminated using 4 M H2S04, and absorbency at 492 nm was read within 3 h. 2.3. Serological HLA typing The patients and the healthy controls were subjected to serological typing for HLA class I and class II antigens using the standard complementdependent microcytotoxicity method [18]. Lymphocytes were isolated from heparinized peripheral blood by centrifugation over a Ficoll±Isopaque gradient, and HLA-A, -B, and -C typing was performed using a standard lymphocyte microcytotoxicity technique and Terasaki trays. 2.4. HLA DNA typing Genomic DNA from patients and controls was isolated by phenol extraction of sodium dodecyl sulphate-lysed and proteinase K-treated cells [19]. DNA was ampli®ed by the polymerase chain reaction (PCR) with Taq DNA polymerase and typed by the PCR-restriction fragment length polymorphism (RFLP) method. The reaction mixture was subjected to 30 cycles of 1 min at 96±978C for denaturation, 1 min at 55±628C for annealing, and 2 min at 728C for extension of the annealed primers in an automated PCR thermal sequencer (Iwaki Glass, Inc., Tokyo). After ampli®cation, aliquots of the reaction mixture were digested with allele-speci®c restriction endonucleases for 3 h after addition of the appropriate reaction buffer. Samples of the restriction enzymecleaved ampli®ed DNAs were subjected to electrophoresis on 12% polyacrylamide gel in a minigel apparatus (Mupid2, Cosmo Bio Co., Ltd., Tokyo). Cleavage or non-cleavage of ampli®ed fragments
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was detected by staining with ethidium bromide. Discrimination of genotypes was performed on the basis of RFLP band patterns thus generated. 2.5. Statistical analysis The frequency of each HLA antigen was tabulated for our control population. These frequencies were compared to those published for the Japanese population at the 11th Japan Workshop. The chi-square method with continuity correction and Fisher's exact test were used for data analysis. Relative risk was calculated according to Wolf's method with Holdane's correction. Brie¯y, relative risk was calculated as (a £ d)/(b £ c), where a, b, c, and d were the numbers of marker-positive patients, marker-negative patients, marker-positive controls, and markernegative controls, respectively.
Fig. 2. Positivities for ErbB-2 for three histological types of lung cancer: small, small cell carcinoma; squamous, squamous cell carcinoma; adeno, adenocarcinoma.
3. Results 3.1. Serum ErbB-2 and lung cancer Fig. 1 shows the distribution of serum ErbB-2 levels in normal controls and patients with lung cancer. The ErbB-2 levels obtained for the normal controls were 5.9 ^ 0.7 (ng/ml) (mean ^ SD). Values more than 2 SD above the mean for the normal controls (7.3 ng/ml) were considered positive. By this criterion, 39 of the 73 lung cancer patients (53.4%) had elevated levels of ErbB-2. The ErbB-2 level was a mean of 14.6 ^ 3.1 ng/ml in the 73 lung cancer patients. Fig. 2 shows positivities for ErbB-2 for the three histological types of lung cancer. Only seven of 23 (30.4%) patients with small cell carcinoma had elevated values. The prevalence of ErbB-2 positivity was highest (23 of 32; 71.8%) in patients with adenocarcinoma, while that for patients with squamous cell carcinoma was 50% (9 of 18). 3.2. HLA serotyping in lung cancer patients
Fig. 1. Distribution of serum ErbB-2 levels in normal controls and patients with lung cancer. Values more than 2 SD above the mean in normal controls (shown by the horizontal line) were considered positive. Normal, normal controls; LC, lung cancer.
Table 1 shows the results of serotyping of HLA-A, B and -C antigens. The frequencies of HLA A33, B44, B62, and B75 were lower in the lung cancer patients than in the control group. However, for HLA C antigens, frequencies did not differ between the patients and controls. Table 2 shows the frequencies of HLA-DR antigens in the patients with lung cancer and the healthy
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Table 1 Relative frequencies of HLA class 1 antigens in Japanese patients with lung cancer and healthy controls HLA antigens
Lung cancer (n 73)
Healthy controls (n 122)
11 JWS (%) a
A33 B44 B62 B75
3 (4.1%) 5 (6.8%) 4 (5.5%) 1 (1.4%)
20 (16.4%) b 17 (13.9%) b 27 (22.1%) b 6 (4.9%) b
7.48 7.43 7.06 0.80
a b
11 JWS: Japanese population at the 11th Japan workshop. P , 0:05:
controls. HLA-DR9 was higher in frequency in lung cancer patients than in controls (P , 0:05), but HLADR6 was lower in frequency in lung cancer patients than in the controls (P , 0:01). HLA-DR5 includes DR11 and DR12, while HLA-DR6 includes DR13 and DR14. 3.3. HLA DNA typing HLA-DRB1 genotyping was carried out by the PCR-RFLP method. The DRB1-related allele frequencies in lung cancer patients and healthy controls are shown in Table 3. DRB1 *0901 was signi®cantly higher in frequency in lung cancer patients than in controls (P , 0:05). On the other hand, DRB1*0802, DRB1*1302 and the DRB1*14 group (*1401, *1403, *1405, *1406, and *1407) were completely absent in lung cancer patients.
3.4. Relationships among ErbB-2, HLA, and clinical staging Table 4 shows the HLA class I antigens and HLADRB1 alleles for which signi®cant differences were found in frequency between ErbB-2-positive and negative patients with lung cancer. The frequencies of HLA B35, B52, B62, DRB1*0404, and DRB1*0406 were higher in the ErbB-2-positive lung cancer patients than in the ErbB-2-negative lung cancer patients. Table 5 shows differences in clinical stage between ErbB-2-positive and -negative patients with lung cancer; we found no signi®cant differences in the stage between the two groups.
4. Discussion Improvement of the survival of patients with small-
Table 2 Relative frequencies of HLA-DR antigens in lung cancer patients and healthy controls HLA-DR speci®ties
Lung cancer (n 73)
Healthy controls (n 122)
11 JWS (%) a
DR1 DR2 (DR1S/DR16) DR3 DR4 DRS (DR11/DR12) DR6 (DR13/DR14) DR7 DR8 DR9 DR10
5 (6.8%) 23 (31.5%) 1 (1.4%) 35 (47.9%) 8 (11.0%) 4 (5.5%) 2 (2.7%) 21 (28.8%) 43 (58.9%) 0 (0%)
23 (18.9%) 49 (40.2%) 1 (0.8%) 59 (48.4%) 9 (7.4%) 41 (33.6%) b 2 (1.6%) 41 (33.6%) 26 (21.3%) c 1 (0.8%)
5.81 18.33 0.12 22.05 9.07 17.04 0.25 12.68 14.08 0.70
a
11 JWS: Japanese population at the 11th Japan workshop. P , 0:01. c P , 0:05. b
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Table 3 Relative frequencies of HLA-DRB1 alleles in Japanese patients with lung cancer and healthy controls HLA-DRB1
Lung cancer (n 73)
Healthy controls (n 122)
0802 b 0901 b 1302 b 1401 b 1403 b 1405 b
2 (2.7%) 43 (58.9%) 1 (1.4%) 0 (0%) 0 (0%) 0 (0%)
18 26 13 7 5 6
(14.8%) c (21.3%) b (10.7%) c (5.7%) b (4.1%) b (4.9%) b
11 JWS a (%) 4.18 14.08 6.83 3.37 1.91 2.22
a
11 JWS: Japanese population at the 11th Japan workshop. P , 0:05. c P , 0:01. b
cell lung cancer (SCLC) has resulted from the use of increasingly aggrieve combinations of chemotherapeutic agents and radiation therapy. A number of strategies have been tested to improve response rates and duration in SCLC. For example, there is evidence that the addition of anticoagulants to chemotherapy and radiation therapy may be bene®cial in the treatment of SCLC [20±22]. However, the overall prognosis of patients suffering from non-small-cell lung cancer (NSCLC) is extremely poor, the 5-year survival rate being less than 10% [23±26]. Ampli®cation of certain oncogenes or an increase in their oncoproteins has been shown to be of prognostic signi®cance for several cancers [27,28]. It has been reported that overexpression of the c-erb-2 gene in ovarian carcinoma is correlated with poor prognosis [29±32], although this association has been controversial [33,34]. In lung
adenocarcinoma, erbB-2 overexpression relative to normal alveolar lung tissue has been found to correlate with a worse prognosis [5]. Recent studies have shown that a soluble form of the erbB-2 protein is present in the serum of carcinoma patients [10±12]. In the present study, we quanti®ed serum erbB-2 using level the Nichirei ErbB-2 EIA. This method appears to be useful for evaluating serum ErbB-2 levels in lung cancer patients since it has already been demonstrated in breast cancer patients [10,12,16,17]. Both in the literature and depending on the methods used for ErbB-2 detection, ErbB-2 expression varies in adenocarcinoma. In the present study, the prevalence for ErbB-2 positivity was the highest (23 of 32; 71.8%) in patients with adenocarcinoma. Thus, our results accorded with those of the report by Rachwall et al. [9].
Table 4 Relative frequencies of HLA class I antigens and HLA-DRB1 alleles in lung cancer patients with or without ErbB-2 HLA speci®ties
ErbB-2 positive (n 39)
ErbB-2 negative (n 34)
P-value
A3 B35 B39 B44 B48 B52 B54 B59 B62 DRB1 *0403 DRB1*0404 DRB1 *0406 DRB1*1201
3 5 2 4 1 8 3 1 4 0 4 5 2
0 (0%) 2 (5.9%) 5 (14.7%) 1 (2.9%) 3 (8.8%) 4 (11.8%) 6 (17.6%) 4 (11.8%) 0 (0%) 4 (11.8%) 0 (05) 1 (2.9%) 4 (11.8%)
,0.05 ,0.05 ,0.05 ,0.05 ,0.05 ,0.05 ,0.05 ,0.05 ,0.05 ,0.05 ,0.05 ,0.05 ,0.05
(7.7%) (12.8%) (5.1%) (10.3%) (2.6%) (20.5%) (7.7%) (2.6%) (10.3%) (0%) (10.3%) (12.8%) (5.1%)
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Table 5 Relative frequencies of HLA class I antigens and HLA-DRB1 alleles in lung cancer patients with or without ErbB-2 Stage
ErbB-2 negative (n 34)
ErbB-2 positive (n 39)
0 IA IB IIA IIB IIIA IIIB IV Unknown
0 1 0 0 0 7 9 9 8
0 3 0 2 0 7 12 6 9
Some investigators have reported associations between various HLA antigens and certain malignant tumors. For example, there have been reports of an association between HLA antigens and lung cancer [35]. However, a prospective study of 70 cases by Rogentine et al. [13] found no difference in HLA antigen incidence between lung cancer patients and controls. Furthermore, Markman et al. [36] reported an increased frequency of HLAB44 and Dellon et al. [37] reported that Al9 and B5 were associated with prolonged survival in bronchogenic carciboma patients. However, no clear trends have been con®rmed because most studies have assessed only HLA phenotyes in lung cancer. DNA typing of HLA class II antigens is available, making it possible to better assess the relationships between HLA class II and various diseases. The HLA-DRB1 region exhibits the most extensive polymorphism among HLA class II genes. The present study demonstrated a signi®cant increase in HLA-DRB1*0901 and a decrease in HLADRB1*1302 and DRB1*14-related alleles in Japanese patients with lung cancer. DRB1*0901 and DRB1*0405 are the most frequent alleles in the Japanese population. Futaki et al. [38] reported that elucidation of the characteristics of naturally processed peptides bound to DRB1*0901 molecules might increase our understanding of the pathogenesis of birch pollen allergy in Japanese subjects and myasthenia gravis of Japanese children. Recently, Fujisao et al. [39] determined binding peptide motifs for HLA-DR9 using a phage random peptide library, and reported that only two anchors of the NH2-terminal half of the binding peptides play an important role, and that a small neutral hydrophilic
serine is the second anchor for high-af®nity binding. Our results suggested that the carcinoma-related antigen in lung cancer was similar to these peptides. On the other hand, relationships between HLA-DR9 and clinical outcome or severity have been reported for other diseases. Zhou et al. [40] found that HLADR9 was associated with the frequency of relapse and, therefore, with the severity of nephrotic syndrome. DRB1 *0901 might thus also in¯uence the prognosis or severity of lung cancer. In the present study, DRB1*1302 and the DRB1*14 group (*1401, *1403, *1405, *1406, and *1407) were absent in lung cancer patients. Interestingly, these alleles are not decreased in hematological malignancies, so the decrease in lung cancer may be speci®c to the latter disease. It was previously reported that HLA-DR13 alleles protect against some diseases. DRB1*1302, but not *1301, was found to be associated with resistance to a severe form of malaria [41]. Both DRB1*1301 and *1302 are associated with resistance to chronic hepatitis B virus infection [42]. In another study, both alleles were associated with protection against cervical cancer [43]. Recently, Davenport et al. [44] identi®ed a peptide-binding motif for DRB1*1301 and *1302, and reported a disease-resistant effect of the dimorphism at position 86 of the HLA-DRb chain. They indicated that the effect of the glycine/valine dimorphism was to alter the preference for particular hydrophobic anchor residues in naturally processed and presented peptides. Interestingly, DRB1*1302, *1403, and *1407 all have glycine at position 86 of the HLA-DRb chain. In previous studies, it was found that HLA and HLArelated genes are among the most important genetic factors controlling susceptibility to lung cancer. For example, Shimura et al. [45] reported an association between tumor necrosis factor-a and gene polymorphism. Our ®ndings for DRB1*1302 and DRB1*14-related alleles appear to offer new insights into the role of HLA in lung cancer, such as resistance to this disease. Racial differences also appear to signi®cantly affect the association between HLA and malignancy. Sastre-Garau et al. [46] reported a decrease in the frequency of the DRB1*1301/1302DQA1*0103-DQB1*0603 haplotype in Caucasian patients with cervical cancer relative to controls. For this reason, there may be an association between the lung cancer patients characteristic Japanese haplotype
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and DRB1*0302 or DRB1*14-related alleles. Furthermore, it was reported that dysregulation of HLA-dependent genes could also occur at different stages of the process of oncogenesis [47] and lead to abnormal expression of HLA genes by neoplastic epithelial cells. This is likely to disorder the immune mechanisms involved in tumor rejection, and might be either a speci®c event or an indirect consequence of malignancy. Immunohistochemical staining for cerbB-2 protein is strong on the membranes of adenocarcinoma cells, and the soluble form of this protein can be observed in serum [5,10±12]. In the present study, we found no correlation between erbB-2 concentration in the serum of patients and expression of erbB-2 in tumor. Overexpression of the ErbB-2 gene was shown by Kern et al. [5] to be a negative prognostic indicator for ten of 29 patients with advanced adenocarcinoma of the lung. In contrast, Kerns et al. [48] found a close correlation between high level of immunohistochemical reactivity in paraf®n sections of breast tumors and the presence of ErbB-2 gene ampli®cation. Furthermore, they found a signi®cantly increased risk of recurrence and subsequent cancer death for the 58 patients (21%) who expressed ErbB-2. Thus, ErbB-2 status appears to be the second most signi®cant independent predictor of early recurrence and cancer death [49]. In the present study, the frequencies of HLA B35, B52, B62, DRB1*0404, and DRB1*0406 were higher in the ErbB-2-positive lung cancer patients than in the ErbB-2-negative lung cancer patients. However, the frequencies of these types of HLA were not signi®cantly high in our whole lung cancers. Thus, the ErbB-2 did not show the signi®cant relation to the DRB1*0901, DRB1*1302 and DRB1* 14-related alleles could be associated with genetic susceptibility and resistance to lung cancer. These results suggest that ErbB-2 is correlated with prognostic factors for lung cancer independent of HLAassociated genetic factors. In conclusion, we studied the relationship in lung cancer between ErbB-2 and HLA using ethnicallymatched patients and controls and a combination of molecular and serological techniques. DRB1*0901 was signi®cantly higher in frequency in lung cancer patients than in healthy controls, and DRB1*0802, DRB1*1302 and the DRB1*14 group (*1401, *1403,
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*1405, *1 406, and *1 407) were completely absent in lung cancer patients. These ®ndings suggest that ErbB2 is correlated with prognostic factors for lung cancer independent of HLA-associated genetic factors.
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Analysis of serum ErbB-2 protein and HLA-DRB1 in Japanese patients with lung cancer Chie Yoshimura a, Shosaku Nomura a,*, Manabu Yamaoka b, Tetsuji Ohtani b, Tatsunori Matsuzakiz b, Kazuyuki Yamaguchi a, Shirou Fukuharal a,b a b
The First Department of Internal Medicine, Kansai Medical University, 10-15 Fumizonocho, Moriguchi, Osaka 570-8507, Japan The First Department of Blood Transfusion, Kansai Medical University, 10-15 Fumizonocho, Moriguchi, Osaka 570-8507, Japan Received 6 September 1999; received in revised form 5 December 1999; accepted 5 December 1999
Abstract We investigated the relationship between ErbB-2 and HLA in order to clarify the clinical and genetic factors related to Japanese patients with lung cancer. Thirty-nine of the 73 lung cancer patients (53.4%) had elevated levels of ErbB-2. Only seven of 23 (30.4%) patients with small cell carcinoma had elevated ErbB-2 levels. The prevalence of ErbB-2 positivity was highest (23 of 32; 71.8%) in patients with adenocarcinoma, while that in patients with squamous cell carcinoma was 50% (9 of 18). The frequencies of HLA A33, B44, B62, and B75 were lower in the lung cancer patients than in the control group. HLADR9 was higher in frequency in lung cancer patients than in the healthy controls (P , 0:05), but HLA-DR6 was lower in frequency in lung cancer patients than in controls (P , 0:01). DRB1*0901 was signi®cantly higher in frequency in lung cancer patients than in controls (P , 0:05). On the other hand, DRB1*0802, DRB1*1302 and the DRB1*14 group (*1401, *1403, *1405, *1406, and *1407) were completely absent in lung cancer patients. The frequencies of HLA B35, B52, B62, DRB1*0404, and DRB1*0406 were higher in the ErbB-2-positive lung cancer patients than in the ErbB-2-negative lung cancer patients. However, these types of HLA were not included in signi®cant frequencies in our group of lung cancers. Our results suggest that some HLA-antigens/alleles participate in the pathogenesis of lung cancer in Japanese patients. In addition, the relationship between HLA-associated genetic factors and ErbB-2 seems to be weak. These ®ndings suggest that ErbB-2 is correlated with prognostic factors for lung cancer independently of HLA-associated genetic factors. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Lung cancer; Prognostic factor; ErbB-2; HLA-DRB1*0901; HLA-DRB1*1302
1. Introduction Lung cancer is currently the leading cause of cancer-related death [1]. Most patients have inoperable tumors at the time of diagnosis, and metastatic relapse in patients with operable non-small cell lung * Corresponding author. Tel.: 181-66-992-1001; fax: 181-66994-8344. E-mail address: [email protected] (S. Nomura)
carcinoma remains a frequent event [2]. Therefore, major research efforts are being directed at identifying the relationship between speci®c gene alterations and the clinical behavior of lung tumors. Genetic abnormalities in lung cancer frequently include the mutation, rearrangement, or overexpression of several genes and their protein products, such as c-myc, K-ras, p53 and cerbB-2 [3±6]. The c-erbB-2 oncoprotein is a 1 85-kd transmembrane protein kinase receptor with homol-
0304-3835/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(99)00437-1
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ogy to the epidermal growth factor receptor, and has been shown to contribute to malignant transformation and progression in a number of epithelial cell carcinomas [7±9]. The c-erbB-2 protein is strongly stained immunohistochemically on the cell membrane of adenocarcinoma, and erbB-2 overexpression relative to that of normal alveolar lung tissue has been found to be associated with a worse prognosis [5]. In addition, recent studies have shown that a soluble form of the cerbB-2 protein can be found in serum [10±12]. In recent years, there have been many reports of genetic predispositions for particular malignancies and numerous studies of genetic effects, such as that of HLA, on lung cancer. Rogentine et al. [13] suggested that some HLA-class 1 antigens conferred resistance to the progression of bronchogenic carcinoma in Caucasians. More recently, Prazak et al. [14] reported a relationship between survival time and HLA for epidermoid lung carcinoma, and Romano et al. [15] reported an association between HLADR7 and resistance to lung cancer. Some of these divergent results may be attributable to the study of differing populations and the existence of numerous other factors related to the development of lung cancer. Thus, analysis of c-erbB-2 and HLA may be important for determining not only the etiology but also the appropriate therapy and prognosis of lung cancer. However, there are few reports studied for both relationship.
2. Materials and methods 2.1. Subjects We studied 73 unrelated Japanese patients (49 men and 24 women) with lung cancer, admitted to our hospital between June 1995 and September 1998. The diagnosis of lung cancer was made histologically. The subjects ranged in age from 27 to 81 years. Eighteen patients had squamous cell carcinoma, 32 had adenocarcinoma, and 23 had small cell carcinoma. One hundred and twenty-two controls were randomly selected from among healthy unrelated Japanese. All subjects gave informed consent for participation in this study. Clinical staging of carcinoma was performed using the TMN classi®cation.
2.2. Measurement of serum ErbB-2 Serum ErbB-2 was quanti®ed by EIA, using the ErbB-2 EIA `Nichirei' kit (Nichirei Inc., Tokyo) [10,16,17]. In brief, 50 ml of serum or standard was diluted 1:5 with buffer solution and incubated at 15± 258C for 2 h in anti-ErbB-2 antibody-coated beads. After washing with PBS, peroxidase-labeled antiErbB-2 antibody was added, and incubated for 2 h at room temperature. After washing, the beads were transferred to plastic tubes. The incubation was continued for 30 min in the dark using o-phenylenediamine with H2O2.The color development was terminated using 4 M H2S04, and absorbency at 492 nm was read within 3 h. 2.3. Serological HLA typing The patients and the healthy controls were subjected to serological typing for HLA class I and class II antigens using the standard complementdependent microcytotoxicity method [18]. Lymphocytes were isolated from heparinized peripheral blood by centrifugation over a Ficoll±Isopaque gradient, and HLA-A, -B, and -C typing was performed using a standard lymphocyte microcytotoxicity technique and Terasaki trays. 2.4. HLA DNA typing Genomic DNA from patients and controls was isolated by phenol extraction of sodium dodecyl sulphate-lysed and proteinase K-treated cells [19]. DNA was ampli®ed by the polymerase chain reaction (PCR) with Taq DNA polymerase and typed by the PCR-restriction fragment length polymorphism (RFLP) method. The reaction mixture was subjected to 30 cycles of 1 min at 96±978C for denaturation, 1 min at 55±628C for annealing, and 2 min at 728C for extension of the annealed primers in an automated PCR thermal sequencer (Iwaki Glass, Inc., Tokyo). After ampli®cation, aliquots of the reaction mixture were digested with allele-speci®c restriction endonucleases for 3 h after addition of the appropriate reaction buffer. Samples of the restriction enzymecleaved ampli®ed DNAs were subjected to electrophoresis on 12% polyacrylamide gel in a minigel apparatus (Mupid2, Cosmo Bio Co., Ltd., Tokyo). Cleavage or non-cleavage of ampli®ed fragments
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was detected by staining with ethidium bromide. Discrimination of genotypes was performed on the basis of RFLP band patterns thus generated. 2.5. Statistical analysis The frequency of each HLA antigen was tabulated for our control population. These frequencies were compared to those published for the Japanese population at the 11th Japan Workshop. The chi-square method with continuity correction and Fisher's exact test were used for data analysis. Relative risk was calculated according to Wolf's method with Holdane's correction. Brie¯y, relative risk was calculated as (a £ d)/(b £ c), where a, b, c, and d were the numbers of marker-positive patients, marker-negative patients, marker-positive controls, and markernegative controls, respectively.
Fig. 2. Positivities for ErbB-2 for three histological types of lung cancer: small, small cell carcinoma; squamous, squamous cell carcinoma; adeno, adenocarcinoma.
3. Results 3.1. Serum ErbB-2 and lung cancer Fig. 1 shows the distribution of serum ErbB-2 levels in normal controls and patients with lung cancer. The ErbB-2 levels obtained for the normal controls were 5.9 ^ 0.7 (ng/ml) (mean ^ SD). Values more than 2 SD above the mean for the normal controls (7.3 ng/ml) were considered positive. By this criterion, 39 of the 73 lung cancer patients (53.4%) had elevated levels of ErbB-2. The ErbB-2 level was a mean of 14.6 ^ 3.1 ng/ml in the 73 lung cancer patients. Fig. 2 shows positivities for ErbB-2 for the three histological types of lung cancer. Only seven of 23 (30.4%) patients with small cell carcinoma had elevated values. The prevalence of ErbB-2 positivity was highest (23 of 32; 71.8%) in patients with adenocarcinoma, while that for patients with squamous cell carcinoma was 50% (9 of 18). 3.2. HLA serotyping in lung cancer patients
Fig. 1. Distribution of serum ErbB-2 levels in normal controls and patients with lung cancer. Values more than 2 SD above the mean in normal controls (shown by the horizontal line) were considered positive. Normal, normal controls; LC, lung cancer.
Table 1 shows the results of serotyping of HLA-A, B and -C antigens. The frequencies of HLA A33, B44, B62, and B75 were lower in the lung cancer patients than in the control group. However, for HLA C antigens, frequencies did not differ between the patients and controls. Table 2 shows the frequencies of HLA-DR antigens in the patients with lung cancer and the healthy
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Table 1 Relative frequencies of HLA class 1 antigens in Japanese patients with lung cancer and healthy controls HLA antigens
Lung cancer (n 73)
Healthy controls (n 122)
11 JWS (%) a
A33 B44 B62 B75
3 (4.1%) 5 (6.8%) 4 (5.5%) 1 (1.4%)
20 (16.4%) b 17 (13.9%) b 27 (22.1%) b 6 (4.9%) b
7.48 7.43 7.06 0.80
a b
11 JWS: Japanese population at the 11th Japan workshop. P , 0:05:
controls. HLA-DR9 was higher in frequency in lung cancer patients than in controls (P , 0:05), but HLADR6 was lower in frequency in lung cancer patients than in the controls (P , 0:01). HLA-DR5 includes DR11 and DR12, while HLA-DR6 includes DR13 and DR14. 3.3. HLA DNA typing HLA-DRB1 genotyping was carried out by the PCR-RFLP method. The DRB1-related allele frequencies in lung cancer patients and healthy controls are shown in Table 3. DRB1 *0901 was signi®cantly higher in frequency in lung cancer patients than in controls (P , 0:05). On the other hand, DRB1*0802, DRB1*1302 and the DRB1*14 group (*1401, *1403, *1405, *1406, and *1407) were completely absent in lung cancer patients.
3.4. Relationships among ErbB-2, HLA, and clinical staging Table 4 shows the HLA class I antigens and HLADRB1 alleles for which signi®cant differences were found in frequency between ErbB-2-positive and negative patients with lung cancer. The frequencies of HLA B35, B52, B62, DRB1*0404, and DRB1*0406 were higher in the ErbB-2-positive lung cancer patients than in the ErbB-2-negative lung cancer patients. Table 5 shows differences in clinical stage between ErbB-2-positive and -negative patients with lung cancer; we found no signi®cant differences in the stage between the two groups.
4. Discussion Improvement of the survival of patients with small-
Table 2 Relative frequencies of HLA-DR antigens in lung cancer patients and healthy controls HLA-DR speci®ties
Lung cancer (n 73)
Healthy controls (n 122)
11 JWS (%) a
DR1 DR2 (DR1S/DR16) DR3 DR4 DRS (DR11/DR12) DR6 (DR13/DR14) DR7 DR8 DR9 DR10
5 (6.8%) 23 (31.5%) 1 (1.4%) 35 (47.9%) 8 (11.0%) 4 (5.5%) 2 (2.7%) 21 (28.8%) 43 (58.9%) 0 (0%)
23 (18.9%) 49 (40.2%) 1 (0.8%) 59 (48.4%) 9 (7.4%) 41 (33.6%) b 2 (1.6%) 41 (33.6%) 26 (21.3%) c 1 (0.8%)
5.81 18.33 0.12 22.05 9.07 17.04 0.25 12.68 14.08 0.70
a
11 JWS: Japanese population at the 11th Japan workshop. P , 0:01. c P , 0:05. b
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Table 3 Relative frequencies of HLA-DRB1 alleles in Japanese patients with lung cancer and healthy controls HLA-DRB1
Lung cancer (n 73)
Healthy controls (n 122)
0802 b 0901 b 1302 b 1401 b 1403 b 1405 b
2 (2.7%) 43 (58.9%) 1 (1.4%) 0 (0%) 0 (0%) 0 (0%)
18 26 13 7 5 6
(14.8%) c (21.3%) b (10.7%) c (5.7%) b (4.1%) b (4.9%) b
11 JWS a (%) 4.18 14.08 6.83 3.37 1.91 2.22
a
11 JWS: Japanese population at the 11th Japan workshop. P , 0:05. c P , 0:01. b
cell lung cancer (SCLC) has resulted from the use of increasingly aggrieve combinations of chemotherapeutic agents and radiation therapy. A number of strategies have been tested to improve response rates and duration in SCLC. For example, there is evidence that the addition of anticoagulants to chemotherapy and radiation therapy may be bene®cial in the treatment of SCLC [20±22]. However, the overall prognosis of patients suffering from non-small-cell lung cancer (NSCLC) is extremely poor, the 5-year survival rate being less than 10% [23±26]. Ampli®cation of certain oncogenes or an increase in their oncoproteins has been shown to be of prognostic signi®cance for several cancers [27,28]. It has been reported that overexpression of the c-erb-2 gene in ovarian carcinoma is correlated with poor prognosis [29±32], although this association has been controversial [33,34]. In lung
adenocarcinoma, erbB-2 overexpression relative to normal alveolar lung tissue has been found to correlate with a worse prognosis [5]. Recent studies have shown that a soluble form of the erbB-2 protein is present in the serum of carcinoma patients [10±12]. In the present study, we quanti®ed serum erbB-2 using level the Nichirei ErbB-2 EIA. This method appears to be useful for evaluating serum ErbB-2 levels in lung cancer patients since it has already been demonstrated in breast cancer patients [10,12,16,17]. Both in the literature and depending on the methods used for ErbB-2 detection, ErbB-2 expression varies in adenocarcinoma. In the present study, the prevalence for ErbB-2 positivity was the highest (23 of 32; 71.8%) in patients with adenocarcinoma. Thus, our results accorded with those of the report by Rachwall et al. [9].
Table 4 Relative frequencies of HLA class I antigens and HLA-DRB1 alleles in lung cancer patients with or without ErbB-2 HLA speci®ties
ErbB-2 positive (n 39)
ErbB-2 negative (n 34)
P-value
A3 B35 B39 B44 B48 B52 B54 B59 B62 DRB1 *0403 DRB1*0404 DRB1 *0406 DRB1*1201
3 5 2 4 1 8 3 1 4 0 4 5 2
0 (0%) 2 (5.9%) 5 (14.7%) 1 (2.9%) 3 (8.8%) 4 (11.8%) 6 (17.6%) 4 (11.8%) 0 (0%) 4 (11.8%) 0 (05) 1 (2.9%) 4 (11.8%)
,0.05 ,0.05 ,0.05 ,0.05 ,0.05 ,0.05 ,0.05 ,0.05 ,0.05 ,0.05 ,0.05 ,0.05 ,0.05
(7.7%) (12.8%) (5.1%) (10.3%) (2.6%) (20.5%) (7.7%) (2.6%) (10.3%) (0%) (10.3%) (12.8%) (5.1%)
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Table 5 Relative frequencies of HLA class I antigens and HLA-DRB1 alleles in lung cancer patients with or without ErbB-2 Stage
ErbB-2 negative (n 34)
ErbB-2 positive (n 39)
0 IA IB IIA IIB IIIA IIIB IV Unknown
0 1 0 0 0 7 9 9 8
0 3 0 2 0 7 12 6 9
Some investigators have reported associations between various HLA antigens and certain malignant tumors. For example, there have been reports of an association between HLA antigens and lung cancer [35]. However, a prospective study of 70 cases by Rogentine et al. [13] found no difference in HLA antigen incidence between lung cancer patients and controls. Furthermore, Markman et al. [36] reported an increased frequency of HLAB44 and Dellon et al. [37] reported that Al9 and B5 were associated with prolonged survival in bronchogenic carciboma patients. However, no clear trends have been con®rmed because most studies have assessed only HLA phenotyes in lung cancer. DNA typing of HLA class II antigens is available, making it possible to better assess the relationships between HLA class II and various diseases. The HLA-DRB1 region exhibits the most extensive polymorphism among HLA class II genes. The present study demonstrated a signi®cant increase in HLA-DRB1*0901 and a decrease in HLADRB1*1302 and DRB1*14-related alleles in Japanese patients with lung cancer. DRB1*0901 and DRB1*0405 are the most frequent alleles in the Japanese population. Futaki et al. [38] reported that elucidation of the characteristics of naturally processed peptides bound to DRB1*0901 molecules might increase our understanding of the pathogenesis of birch pollen allergy in Japanese subjects and myasthenia gravis of Japanese children. Recently, Fujisao et al. [39] determined binding peptide motifs for HLA-DR9 using a phage random peptide library, and reported that only two anchors of the NH2-terminal half of the binding peptides play an important role, and that a small neutral hydrophilic
serine is the second anchor for high-af®nity binding. Our results suggested that the carcinoma-related antigen in lung cancer was similar to these peptides. On the other hand, relationships between HLA-DR9 and clinical outcome or severity have been reported for other diseases. Zhou et al. [40] found that HLADR9 was associated with the frequency of relapse and, therefore, with the severity of nephrotic syndrome. DRB1 *0901 might thus also in¯uence the prognosis or severity of lung cancer. In the present study, DRB1*1302 and the DRB1*14 group (*1401, *1403, *1405, *1406, and *1407) were absent in lung cancer patients. Interestingly, these alleles are not decreased in hematological malignancies, so the decrease in lung cancer may be speci®c to the latter disease. It was previously reported that HLA-DR13 alleles protect against some diseases. DRB1*1302, but not *1301, was found to be associated with resistance to a severe form of malaria [41]. Both DRB1*1301 and *1302 are associated with resistance to chronic hepatitis B virus infection [42]. In another study, both alleles were associated with protection against cervical cancer [43]. Recently, Davenport et al. [44] identi®ed a peptide-binding motif for DRB1*1301 and *1302, and reported a disease-resistant effect of the dimorphism at position 86 of the HLA-DRb chain. They indicated that the effect of the glycine/valine dimorphism was to alter the preference for particular hydrophobic anchor residues in naturally processed and presented peptides. Interestingly, DRB1*1302, *1403, and *1407 all have glycine at position 86 of the HLA-DRb chain. In previous studies, it was found that HLA and HLArelated genes are among the most important genetic factors controlling susceptibility to lung cancer. For example, Shimura et al. [45] reported an association between tumor necrosis factor-a and gene polymorphism. Our ®ndings for DRB1*1302 and DRB1*14-related alleles appear to offer new insights into the role of HLA in lung cancer, such as resistance to this disease. Racial differences also appear to signi®cantly affect the association between HLA and malignancy. Sastre-Garau et al. [46] reported a decrease in the frequency of the DRB1*1301/1302DQA1*0103-DQB1*0603 haplotype in Caucasian patients with cervical cancer relative to controls. For this reason, there may be an association between the lung cancer patients characteristic Japanese haplotype
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and DRB1*0302 or DRB1*14-related alleles. Furthermore, it was reported that dysregulation of HLA-dependent genes could also occur at different stages of the process of oncogenesis [47] and lead to abnormal expression of HLA genes by neoplastic epithelial cells. This is likely to disorder the immune mechanisms involved in tumor rejection, and might be either a speci®c event or an indirect consequence of malignancy. Immunohistochemical staining for cerbB-2 protein is strong on the membranes of adenocarcinoma cells, and the soluble form of this protein can be observed in serum [5,10±12]. In the present study, we found no correlation between erbB-2 concentration in the serum of patients and expression of erbB-2 in tumor. Overexpression of the ErbB-2 gene was shown by Kern et al. [5] to be a negative prognostic indicator for ten of 29 patients with advanced adenocarcinoma of the lung. In contrast, Kerns et al. [48] found a close correlation between high level of immunohistochemical reactivity in paraf®n sections of breast tumors and the presence of ErbB-2 gene ampli®cation. Furthermore, they found a signi®cantly increased risk of recurrence and subsequent cancer death for the 58 patients (21%) who expressed ErbB-2. Thus, ErbB-2 status appears to be the second most signi®cant independent predictor of early recurrence and cancer death [49]. In the present study, the frequencies of HLA B35, B52, B62, DRB1*0404, and DRB1*0406 were higher in the ErbB-2-positive lung cancer patients than in the ErbB-2-negative lung cancer patients. However, the frequencies of these types of HLA were not signi®cantly high in our whole lung cancers. Thus, the ErbB-2 did not show the signi®cant relation to the DRB1*0901, DRB1*1302 and DRB1* 14-related alleles could be associated with genetic susceptibility and resistance to lung cancer. These results suggest that ErbB-2 is correlated with prognostic factors for lung cancer independent of HLAassociated genetic factors. In conclusion, we studied the relationship in lung cancer between ErbB-2 and HLA using ethnicallymatched patients and controls and a combination of molecular and serological techniques. DRB1*0901 was signi®cantly higher in frequency in lung cancer patients than in healthy controls, and DRB1*0802, DRB1*1302 and the DRB1*14 group (*1401, *1403,
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*1405, *1 406, and *1 407) were completely absent in lung cancer patients. These ®ndings suggest that ErbB2 is correlated with prognostic factors for lung cancer independent of HLA-associated genetic factors.
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