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Relationship of p53 oncoprotein and proliferating cell nuclear antigen expression between primary and relapsing non-small cell lung cancer
Lung Cancer 18 (1997) 253 – 257
Short communication
Relationship of p53 oncoprotein and proliferating cell nuclear antigen expression between primary and relapsing non-small cell lung cancer Atsuhisa Tamura a,b,*, Akira Hebisawa b, Hikotaro Komatsu 1,a, Hideki Yotsumoto a, Masashi Mori a a
Department of Respiratory Diseases, Tokyo National Chest Hospital, 311 Takeoka, Kiyose, Tokyo, 204, Japan b Depatment of Pathology, Tokyo National Chest Hospital, 311 Takeoka, Kiyose, Tokyo, 204, Japan Received 22 April 1997; received in revised form 30 June 1997; accepted 4 July 1997
Abstract To investigate whether p53 and proliferating cell nuclear antigen (PCNA) are maintained at relapse of non-small cell lung cancer (NSCLC), we examined tumor materials from nine patients with NSCLC who had undergone resection for primary cancer and also a second resection for its relapse to the lung. In each case, histological types of primary and relapsing tumor were identical (eight adenocarcinomas and one squamous cell carcinoma). Immunohistochemical staining analysis for p53 oncoprotein expression revealed that seven of the nine cases had identical p53 expression in primary and relapsing tumor (p53 positive in three cases and negative in four) and that in the remaining two cases, p53 positive conversion during relapse was found in one case and negative conversion in one. Immunostaining for PCNA expression revealed that PCNA expression was observed in five primary tumors, and at relapse these cases were also PCNA positive. Three of the remaining four cases showed PCNA positive conversion during relapse. This study of a small number of patients indicates that results of p53 and PCNA immunostaining of resected materials of NSCLC seem to be of little significance for predicting future relapse. © 1997 Elsevier Science Ireland Ltd. * Corresponding author. Tel.: +81 424 912111; fax: + 81 424 942168. 1 Present address: Department of Respiratory Diseases, Chushin Matsumoto National Chest Hospital. 0169-5002/97/$17.00 © 1997 Elsevier Science Ireland Ltd. All rights reserved. PII S 0 1 6 9 - 5 0 0 2 ( 9 7 ) 0 0 0 6 6 - 4
254
A. Tamura et al. / Lung Cancer 18 (1997) 253–257
Keywords: Non-small cell lung cancer; p53; Proliferating cell nuclear antigen
1. Introduction Alteration of the p53 gene has been shown to be one of the most common genetic changes in lung cancer [1]. Instead of demonstrating the gene mutation directly by DNA sequencing, immunohistochemical methods have been used to indirectly assess p53 mutations [2]. Since Quinlan’s first report [3], many others have been published on the relationship between p53 oncoprotein expression and prognosis of non-small cell lung cancer (NSCLC), with very conflicting results. Some showed that p53 expression is associated with a poor prognosis [3,4] while others showed an association with a favorable prognosis [5]. Proliferating cell nuclear antigen (PCNA) is associated with DNA replication [6]. PCNA expression is also associated with poor prognosis of NSCLC [7] and correlation was observed between expression of p53 and PCNA [8]. However, these studies evaluated p53 or PCNA expression only at one point, the time of resection of primary NSCLC, and it is uncertain whether p53 or PCNA expression changes or is maintained at relapse of NSCLC. Therefore, we examined the expression of p53 oncoprotein and PCNA in resected lung samples at two points, at presentation and at relapse of NSCLC.
2. Materials and methods
2.1. Patients We examined tumor materials from nine patients with NSCLC who had undergone completely curative resection for primary cancer and had also undergone a second resection for its relapse to the lung (lobectomy in five cases and wedge resection in four cases) within 5 years after the first resection. These resections were all done between 1985 and 1992 at Tokyo National Chest Hospital. Diagnosis of relapse in this study was based on radiographic findings (spherical or ovoid in shape) and similarity of histopathological findings of tumor materials obtained by the first and second resection. Patient data are listed in Table 1. Patients included six females and three males (average age 57 years). Of the primary tumors, eight were adenocarcinomas and one was squamous cell carcinoma; seven tumors were pathologic stage I and two were stage II. Histological studies at the second surgery revealed that the histological type of each relapsing tumor was identical to that of the primary tumor.
2.2. Immunohistochemistry Immunohistochemical staining analysis for p53 oncoprotein and PCNA expression was performed on 5 mm-thick, formalin-fixed paraffin-embedded sections, using an anti-p53 antibody (DAKO, Copenhagen, Denmark), anti-PCNA antibody (Novocastra Laboratories, Newcastle, UK), and labeled streptavidin-biotin (LSAB)
M/60 M/57 F/72 F/61 F/49 F/53 M/74 F/40 F/51
1 2 3 4 5 6 7 8 9
RLL/LLL RML/RLL LLL/RML RLL/LLL
RUL/RLL LUL/LLL LUL/LLL RUL/RLL RLL/RML
Site of lung cancer (primary/relapsing)
Ad, papillary Ad, acinar Ad, papillary Ad, papillary Ad, bronchioloalveolar Ad, papillary Sq Ad, acinar Ad, acinar
Histologic types of primary tumor
I I II II
I I I I I
Pathologic stage of primary tumor
26 6 58 25
16 20 30 53 60
Interval (months) between 1st and 2nd surgery
9 23 18 31
18 12 20 32 45
(wedge resection) (wedge resection) (lobectomy) (lobectomy)
(lobectomy) (wedge resection) (wedge resection) (lobectomy) (lobectomy)
Ad, papillary Ad, acinar Ad, solid Ad, papillary Ad, bronchioloalveolar Ad, papillary Sq Ad, acinar Ad, acinar
Diameter (mm) of re- Histologic types of lapsing tumor relapsing tumor (method of surgery)
RUL, right upper lobe; RLL, right lower lobe; LUL, left upper lobe; LLL, left lower lobe; RML, right middle lobe; Ad, adenocarcinoma; Sq, squamous cell carcinoma.
Sex/age
Case
Table 1 Clinicopathologic findings
A. Tamura et al. / Lung Cancer 18 (1997) 253–257 255
256
A. Tamura et al. / Lung Cancer 18 (1997) 253–257
kit (DAKO, Copenhagen, Denmark). Sections for p53 expression were pretreated with microwave antigen retrieval in citric acid buffer [9]. The antibodies were diluted to 1:30 for p53 and 1:10 for PCNA, and the incubation was performed at 4°C overnight. Peroxidase activity was visualized by 3-,3%-diaminobenzidine hydrochloride with 0.05% hydrogen peroxide and by counterstaining the sections with hematoxylin. The specificity of the immunostaining was confirmed by a negative control using nonimmune mouse IgG in place of the primary antibody. For both p53 and PCNA, only nuclear staining was considered. Nuclear staining was scored as follows: − ( B1% positive cells) and + (] 1% positive cells). 3. Results Details of p53 and PCNA expression are shown in Table 2. In three cases, both primary and relapsing tumors were p53 positive, while in four cases both tumors were p53 negative. Although a trend indicating an association between primary and relapsing tumors in p53 expression was found, data did not indicate significance (P= 0.167, by Fischer’s exact test). As for the remaining two cases, p53 positive conversion during relapse was found in one case, and p53 negative conversion in the second. In the latter case, the primary tumor consisted of p53 positive papillary adenocarcinoma with a focus of p53 negative solid carcinoma with mucin, whereas the relapsing tumor consisted of only p53 negative solid carcinoma with mucin. PCNA expression was observed in five cases of primary tumors, and at relapse these cases were also PCNA positive. Further, three of the remaining four cases showed PCNA positive conversion during relapse. There was no association with primary and relapsing tumors in PCNA expression and with p53 and PCNA expression. 4. Discussion There have been a few studies reporting that p53 expression was maintained Table 2 Immunohistochemical findings Case
1 2 3 4 5 6 7 8 9
Staining results (primary/relapsing) p53
PCNA
+/+ +/+ +/− −/+ −/− −/− +/+ −/− −/−
−/+ +/+ +/+ −/+ +/+ +/+ −/+ −/− +/+
−, B1% positive cells; +, ]1% positive cells.
A. Tamura et al. / Lung Cancer 18 (1997) 253–257
257
during metastatic progression to hilar or mediastinal lymph nodes [8,10]. Our findings also showed that p53 was apt to be maintained during relapse to the lung. However, p53 expression in our primary tumors varied, being negative in some and positive in others. These results tend to support previous controversial conclusions of p53 expression and prognosis of NSCLC [3–5]. As for the two cases in which p53 expression differed between primary and relapsing tumors, possibilities responsible for the p53 mismatching are: (1) tumors obtained by the second resection did not represent a relapse of the primary cancer but represented a second cancer; (2) there was a discrepancy between immunostaining of p53 oncoprotein and the actual p53 gene mutation [11]; (3) p53 expression occurred during relapse in the cases of p53 positive conversion; and (4) p53 negative cells metastasized in the case in which p53 negative conversion took place, reflecting the heterogeneity of adenocarcinoma cells. PCNA expression has been shown to increase from early to advanced phases of NSCLC [7,8]. Our study also showed that PCNA expression increased from primary to relapsing phases. It may reflect the highly proliferative activity of cancer cells in the relapsing phase, and such proliferative activity of relapsing cancer cells seems to be irrelevant to the activity of primary cancer cells. Through the results of the present study, we feel that in individual cases, p53 and PCNA immunostaining in the resected materials of NSCLC seems to be of little significance for predicting future relapse. Since our investigation involved a very small number of patients, further study is required to clarify the actual significance of p53 and PCNA expression for predicting prognosis of NSCLC.
References [1] Takahashi T, Nau MM, Chiba T, et al. p53: a frequent target for genetic abnormalities in lung cancer. Science 1989;246:491–4. [2] Vojte' s' ek B, Ba´rtek J, Midgley CA, Lane DP. An immunohistochemical analysis of the human nuclear phosphoprotein p53. J Immunol Methods 1992;151:237 – 44. [3] Quinlan DC, Davidson AG, Summers CL, Warden HE, Doshi HM. Accumulation of p53 protein correlates with a poor prognosis in human lung cancer. Cancer Res 1992;52:4828 – 31. [4] Mitsudomi T, Oyama T, Kusano T, et al. Mutations of p53 gene as a predictor of poor prognosis in patients with non-small cell lung cancer. J Natl Cancer Inst 1993;85:2018 – 23. [5] Lee JS, Yoon A, Kalapurakal SK, et al. Expression of p53 oncoprotein in non-small-cell lung cancer: a favorable prognostic factor. J Clin Oncol 1995;13:1893 – 903. [6] Laskey RA, Fairman MP, Blow JJ. S phase of the cell cycle. Science 1989;246:609 – 14. [7] Ishida T, Kaneko S, Akazawa K, et al. Proliferating cell nuclear antigen expression and argyrophilic nucleolar organizer regions as factors influencing prognosis of surgically treated lung cancer patients. Cancer Res 1993;53:5000–3. [8] Fontanini G, Vignati S, Bigini D, et al. Human non-small cell lung cancer: p53 protein accumulation is an early event and persists during metastatic progression. J Pathol 1994;174:23 – 31. [9] Lambkin HA, Mothersill CM, Kelehan P. Variations in immunohistochemical detection of p53 protein overexpression in cervical carcinomas with different antibodies and methods of detection. J Pathol 1994;172:13–8. [10] Marchetti A, Buttitta F, Merlo G, et al. p53 alterations in non-small cell lung cancers correlate with metastatic involvement of hilar and mediastinal lymph nodes. Cancer Res 1993;53:2846 – 51. [11] Battifora H. p53 immunohistochemistry: a word of caution. Hum Pathol 1994;25:435 – 7.
Short communication
Relationship of p53 oncoprotein and proliferating cell nuclear antigen expression between primary and relapsing non-small cell lung cancer Atsuhisa Tamura a,b,*, Akira Hebisawa b, Hikotaro Komatsu 1,a, Hideki Yotsumoto a, Masashi Mori a a
Department of Respiratory Diseases, Tokyo National Chest Hospital, 311 Takeoka, Kiyose, Tokyo, 204, Japan b Depatment of Pathology, Tokyo National Chest Hospital, 311 Takeoka, Kiyose, Tokyo, 204, Japan Received 22 April 1997; received in revised form 30 June 1997; accepted 4 July 1997
Abstract To investigate whether p53 and proliferating cell nuclear antigen (PCNA) are maintained at relapse of non-small cell lung cancer (NSCLC), we examined tumor materials from nine patients with NSCLC who had undergone resection for primary cancer and also a second resection for its relapse to the lung. In each case, histological types of primary and relapsing tumor were identical (eight adenocarcinomas and one squamous cell carcinoma). Immunohistochemical staining analysis for p53 oncoprotein expression revealed that seven of the nine cases had identical p53 expression in primary and relapsing tumor (p53 positive in three cases and negative in four) and that in the remaining two cases, p53 positive conversion during relapse was found in one case and negative conversion in one. Immunostaining for PCNA expression revealed that PCNA expression was observed in five primary tumors, and at relapse these cases were also PCNA positive. Three of the remaining four cases showed PCNA positive conversion during relapse. This study of a small number of patients indicates that results of p53 and PCNA immunostaining of resected materials of NSCLC seem to be of little significance for predicting future relapse. © 1997 Elsevier Science Ireland Ltd. * Corresponding author. Tel.: +81 424 912111; fax: + 81 424 942168. 1 Present address: Department of Respiratory Diseases, Chushin Matsumoto National Chest Hospital. 0169-5002/97/$17.00 © 1997 Elsevier Science Ireland Ltd. All rights reserved. PII S 0 1 6 9 - 5 0 0 2 ( 9 7 ) 0 0 0 6 6 - 4
254
A. Tamura et al. / Lung Cancer 18 (1997) 253–257
Keywords: Non-small cell lung cancer; p53; Proliferating cell nuclear antigen
1. Introduction Alteration of the p53 gene has been shown to be one of the most common genetic changes in lung cancer [1]. Instead of demonstrating the gene mutation directly by DNA sequencing, immunohistochemical methods have been used to indirectly assess p53 mutations [2]. Since Quinlan’s first report [3], many others have been published on the relationship between p53 oncoprotein expression and prognosis of non-small cell lung cancer (NSCLC), with very conflicting results. Some showed that p53 expression is associated with a poor prognosis [3,4] while others showed an association with a favorable prognosis [5]. Proliferating cell nuclear antigen (PCNA) is associated with DNA replication [6]. PCNA expression is also associated with poor prognosis of NSCLC [7] and correlation was observed between expression of p53 and PCNA [8]. However, these studies evaluated p53 or PCNA expression only at one point, the time of resection of primary NSCLC, and it is uncertain whether p53 or PCNA expression changes or is maintained at relapse of NSCLC. Therefore, we examined the expression of p53 oncoprotein and PCNA in resected lung samples at two points, at presentation and at relapse of NSCLC.
2. Materials and methods
2.1. Patients We examined tumor materials from nine patients with NSCLC who had undergone completely curative resection for primary cancer and had also undergone a second resection for its relapse to the lung (lobectomy in five cases and wedge resection in four cases) within 5 years after the first resection. These resections were all done between 1985 and 1992 at Tokyo National Chest Hospital. Diagnosis of relapse in this study was based on radiographic findings (spherical or ovoid in shape) and similarity of histopathological findings of tumor materials obtained by the first and second resection. Patient data are listed in Table 1. Patients included six females and three males (average age 57 years). Of the primary tumors, eight were adenocarcinomas and one was squamous cell carcinoma; seven tumors were pathologic stage I and two were stage II. Histological studies at the second surgery revealed that the histological type of each relapsing tumor was identical to that of the primary tumor.
2.2. Immunohistochemistry Immunohistochemical staining analysis for p53 oncoprotein and PCNA expression was performed on 5 mm-thick, formalin-fixed paraffin-embedded sections, using an anti-p53 antibody (DAKO, Copenhagen, Denmark), anti-PCNA antibody (Novocastra Laboratories, Newcastle, UK), and labeled streptavidin-biotin (LSAB)
M/60 M/57 F/72 F/61 F/49 F/53 M/74 F/40 F/51
1 2 3 4 5 6 7 8 9
RLL/LLL RML/RLL LLL/RML RLL/LLL
RUL/RLL LUL/LLL LUL/LLL RUL/RLL RLL/RML
Site of lung cancer (primary/relapsing)
Ad, papillary Ad, acinar Ad, papillary Ad, papillary Ad, bronchioloalveolar Ad, papillary Sq Ad, acinar Ad, acinar
Histologic types of primary tumor
I I II II
I I I I I
Pathologic stage of primary tumor
26 6 58 25
16 20 30 53 60
Interval (months) between 1st and 2nd surgery
9 23 18 31
18 12 20 32 45
(wedge resection) (wedge resection) (lobectomy) (lobectomy)
(lobectomy) (wedge resection) (wedge resection) (lobectomy) (lobectomy)
Ad, papillary Ad, acinar Ad, solid Ad, papillary Ad, bronchioloalveolar Ad, papillary Sq Ad, acinar Ad, acinar
Diameter (mm) of re- Histologic types of lapsing tumor relapsing tumor (method of surgery)
RUL, right upper lobe; RLL, right lower lobe; LUL, left upper lobe; LLL, left lower lobe; RML, right middle lobe; Ad, adenocarcinoma; Sq, squamous cell carcinoma.
Sex/age
Case
Table 1 Clinicopathologic findings
A. Tamura et al. / Lung Cancer 18 (1997) 253–257 255
256
A. Tamura et al. / Lung Cancer 18 (1997) 253–257
kit (DAKO, Copenhagen, Denmark). Sections for p53 expression were pretreated with microwave antigen retrieval in citric acid buffer [9]. The antibodies were diluted to 1:30 for p53 and 1:10 for PCNA, and the incubation was performed at 4°C overnight. Peroxidase activity was visualized by 3-,3%-diaminobenzidine hydrochloride with 0.05% hydrogen peroxide and by counterstaining the sections with hematoxylin. The specificity of the immunostaining was confirmed by a negative control using nonimmune mouse IgG in place of the primary antibody. For both p53 and PCNA, only nuclear staining was considered. Nuclear staining was scored as follows: − ( B1% positive cells) and + (] 1% positive cells). 3. Results Details of p53 and PCNA expression are shown in Table 2. In three cases, both primary and relapsing tumors were p53 positive, while in four cases both tumors were p53 negative. Although a trend indicating an association between primary and relapsing tumors in p53 expression was found, data did not indicate significance (P= 0.167, by Fischer’s exact test). As for the remaining two cases, p53 positive conversion during relapse was found in one case, and p53 negative conversion in the second. In the latter case, the primary tumor consisted of p53 positive papillary adenocarcinoma with a focus of p53 negative solid carcinoma with mucin, whereas the relapsing tumor consisted of only p53 negative solid carcinoma with mucin. PCNA expression was observed in five cases of primary tumors, and at relapse these cases were also PCNA positive. Further, three of the remaining four cases showed PCNA positive conversion during relapse. There was no association with primary and relapsing tumors in PCNA expression and with p53 and PCNA expression. 4. Discussion There have been a few studies reporting that p53 expression was maintained Table 2 Immunohistochemical findings Case
1 2 3 4 5 6 7 8 9
Staining results (primary/relapsing) p53
PCNA
+/+ +/+ +/− −/+ −/− −/− +/+ −/− −/−
−/+ +/+ +/+ −/+ +/+ +/+ −/+ −/− +/+
−, B1% positive cells; +, ]1% positive cells.
A. Tamura et al. / Lung Cancer 18 (1997) 253–257
257
during metastatic progression to hilar or mediastinal lymph nodes [8,10]. Our findings also showed that p53 was apt to be maintained during relapse to the lung. However, p53 expression in our primary tumors varied, being negative in some and positive in others. These results tend to support previous controversial conclusions of p53 expression and prognosis of NSCLC [3–5]. As for the two cases in which p53 expression differed between primary and relapsing tumors, possibilities responsible for the p53 mismatching are: (1) tumors obtained by the second resection did not represent a relapse of the primary cancer but represented a second cancer; (2) there was a discrepancy between immunostaining of p53 oncoprotein and the actual p53 gene mutation [11]; (3) p53 expression occurred during relapse in the cases of p53 positive conversion; and (4) p53 negative cells metastasized in the case in which p53 negative conversion took place, reflecting the heterogeneity of adenocarcinoma cells. PCNA expression has been shown to increase from early to advanced phases of NSCLC [7,8]. Our study also showed that PCNA expression increased from primary to relapsing phases. It may reflect the highly proliferative activity of cancer cells in the relapsing phase, and such proliferative activity of relapsing cancer cells seems to be irrelevant to the activity of primary cancer cells. Through the results of the present study, we feel that in individual cases, p53 and PCNA immunostaining in the resected materials of NSCLC seems to be of little significance for predicting future relapse. Since our investigation involved a very small number of patients, further study is required to clarify the actual significance of p53 and PCNA expression for predicting prognosis of NSCLC.
References [1] Takahashi T, Nau MM, Chiba T, et al. p53: a frequent target for genetic abnormalities in lung cancer. Science 1989;246:491–4. [2] Vojte' s' ek B, Ba´rtek J, Midgley CA, Lane DP. An immunohistochemical analysis of the human nuclear phosphoprotein p53. J Immunol Methods 1992;151:237 – 44. [3] Quinlan DC, Davidson AG, Summers CL, Warden HE, Doshi HM. Accumulation of p53 protein correlates with a poor prognosis in human lung cancer. Cancer Res 1992;52:4828 – 31. [4] Mitsudomi T, Oyama T, Kusano T, et al. Mutations of p53 gene as a predictor of poor prognosis in patients with non-small cell lung cancer. J Natl Cancer Inst 1993;85:2018 – 23. [5] Lee JS, Yoon A, Kalapurakal SK, et al. Expression of p53 oncoprotein in non-small-cell lung cancer: a favorable prognostic factor. J Clin Oncol 1995;13:1893 – 903. [6] Laskey RA, Fairman MP, Blow JJ. S phase of the cell cycle. Science 1989;246:609 – 14. [7] Ishida T, Kaneko S, Akazawa K, et al. Proliferating cell nuclear antigen expression and argyrophilic nucleolar organizer regions as factors influencing prognosis of surgically treated lung cancer patients. Cancer Res 1993;53:5000–3. [8] Fontanini G, Vignati S, Bigini D, et al. Human non-small cell lung cancer: p53 protein accumulation is an early event and persists during metastatic progression. J Pathol 1994;174:23 – 31. [9] Lambkin HA, Mothersill CM, Kelehan P. Variations in immunohistochemical detection of p53 protein overexpression in cervical carcinomas with different antibodies and methods of detection. J Pathol 1994;172:13–8. [10] Marchetti A, Buttitta F, Merlo G, et al. p53 alterations in non-small cell lung cancers correlate with metastatic involvement of hilar and mediastinal lymph nodes. Cancer Res 1993;53:2846 – 51. [11] Battifora H. p53 immunohistochemistry: a word of caution. Hum Pathol 1994;25:435 – 7.