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Cyclin A expression in superficial squamous cell carcinoma of the esophagus and coexisting infiltrated lymphocyte follicle
Cancer Letters 188 (2002) 221–229 www.elsevier.com/locate/canlet
Cyclin A expression in superficial squamous cell carcinoma of the esophagus and coexisting infiltrated lymphocyte follicle Tadahiro Nozoe a,*, Daisuke Korenaga a, Motonori Futatsugi b, Hiroshi Saeki b, Takefumi Ohga b, Keizo Sugimachi b b
a Department of General Surgery, Fukuoka Dental College Hospital, Fukuoka, 814-0193, Japan Department of Surgery and Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka, 812-8582, Japan
Received 22 April 2002; received in revised form 4 July 2002; accepted 8 July 2002
Abstract Cyclin A is a protein kinase to act a pivotal role in the mitotic phase of the cell cycle. The purpose of the current study was to elucidate the biological significance of immunohistochemical expression of cyclin A in superficial squamous cell carcinoma (SCC) of the esophagus. Immunohistochemical staining of cyclin A was performed for 45 samples of esophageal superficial SCCs. Clinicopathological features were compared between SCCs with and without cyclin A expression. Twenty-five superficial SCCs (55.6%) had positive expression of cyclin A and the other 20 (44.4%) did not. No significant difference regarding clinicopathological characteristics between esophageal SCCs with and without cyclin A expression. Infiltration of lymphocytes with germinal center cells was observed beneath 17 (68.0%) out of 25 superficial SCCs with cyclin A expression and 15 (75.0%) out of 20 superficial SCCs without cyclin A expression. Although 16 (94.1%) out of 17 superficial SCCs with cyclin A expression were associated with cyclin A expression in germinal center cells in infiltrated lymphoid follicles beneath the tumors, only 2 (13.3%) out of 15 superficial SCCs without cyclin A expression coexisted with cyclin A expression in lymphoid follicles beneath the tumors (P , 0:0001). Cyclin A expression in the germinal center cells of the lymphoid follicles beneath the superficial SCCs of the esophagus might be an immunological signal toward the proliferation and progression of the tumors. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Cyclin A; Immunohistochemical expression; Esophagus; Squamous cell carcinoma; Superficial carcinoma; Lymphocyte follicle; Immunological surveillance
1. Introduction Although squamous cell carcinoma (SCC) of the esophagus accounts for one of the human neoplasms associated with biologically most malignant behavior [1], recent advance in the diagnostic technique has been contributing to the increase of superficial SCCs * Corresponding author. Tel.: 1 81-92-801-0526; fax: 1 81-92801-0735. E-mail address: [email protected] (T. Nozoe).
[2,3], which has steadily brought about the improvement in the prognosis of the patients with esophageal SCC. Hence, the clinical focus of the physicians has been directed to superficial SCC of the esophagus [4–6]. Molecular and biological methodologies could be used to investigate the clinicopathological features in order to acquire the clinical hint directly correlated with better outcome of the patients with esophageal SCCs. Such bio-markers as expressions of cyclindependent kinase inhibitor p27 [7] and appoptosis-
0304-3835/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(02)00434-2
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related protein Bcl-X [8], amplification of int-2 [9], and serum detection of anti-p53 antibody [10] have been suggested to indicate the more malignant or lessinvasive potential in superficial SCC of the esophagus. Cyclins are the essential protein which have an important role to regulate the cell cycle [11], and proliferation of cell is controlled by protein complexes composed of cyclins associated with cyclin dependent kinase (Cdk) [12,13]. Cyclin A is a protein kinase to modulate the cell cycle and cell proliferation and has been known to act a pivotal role both in S-phase [14] and in the G2/M phase transition as a mitotic cyclin during the cell cycle [15]. Cyclin A binds to both cdk2 and cdc2 and modulated the DNA replication in S-phase [16] and the initiation of mitosis [14,17]. These studies could suggest that cyclin A expression might be correlated with the progression of the tumors. Moreover, there have been also some investigations suggesting that cyclin A can be considered to be possibly associated with the malignant transformation of the cells [18–20]. Some clinical investigations demonstrated the significance of cyclin A expression as an indicator of malignant potential of tumors and/or a prognosticator of the patients with some human SCCs [21,22]. In the current study, we investigated an immunohistochemical expression of cyclin A to find out its clinicopathological significance in superficial esophageal SCC, which has been left to be discussed.
2. Patients and methods 2.1. Patients The tissue samples were collected from 45 patients with superficial squamous cell carcinoma (SCC) and 14 with SCCs invading muscularis propria of the esophagus, who had been treated with resection of the esophagus and reconstruction in the Department of Surgery and Science, Graduate School of Medical Sciences, Kyushu University Hospital. The pathological depth of the superficial tumors, determined according to TNM classification of malignant tumors decided by International Union Against Cancer [23], were Tis in three, T1a in 14 and T1b in 28, respectively. The study population associated with superfi-
cial tumors consisted of 42 men and three women, and the age range from 40 to 77 years with a mean of 64 years. The population associated with SCCs invading muscularis propria (T2) was consisted of 13 men and one woman, and the age range from 44 to 81 years with a mean of 68 years. The clinicopathological investigation was made on the basis of guidelines for clinical and pathologic studies on carcinoma of the esophagus established by the Japanese Society for Esophageal Diseases [24]. Follow-up for the patients was continuously performed until death and only patients who died of esophageal carcinoma were included in the tumorrelated deaths. The interval of the follow-up after surgical treatment ranged from 5 months to 9 years and 6 months with a mean of 4 years and 7 months. 2.2. Methods Immunostaining of cyclin A was performed using a mouse monoclonal anti-human cyclin A antibody (6E6, Novocastra Laboratory, UK). According to the manufacturer, this antibody has no cross-reactive response to other cyclins. Four micrometer thick sections sliced from paraffin-embedded specimen were prepared on the slideglasses pre-coated with silane. After removing paraffin in xylene and washing in graded series of ethanol, the sections were placed in Tris-buffered saline (TBS) for 10 min. The sections were incubated with a blocking solution for 5 min to block an endogenous peroxidase activity, and were placed in TBS. Then the sections were placed in the 0.01 mM Tris buffer (pH 6.0) and heated at 1218C for 20 min in an autoclave oven. Next the sections were incubated with TBS including 1% concentration of bovine serum albumin for 20 min to block non-specific binding of the immunoreagents. After washing in TBS, the sections were incubated with 1:50 diluted primary antibody overnight at 48C. After washing in TBS, an immunoperoxidase staining was performed by an EnVision antibody complex method [25,26] using ENVISION kit (DAKO. Ltd., Tokyo, Japan). Finally, the localization of cyclin A was visualized with diaminobenzidine tetrahydrochloride. All of the samples were analyzed by two observers (T.N., D.K.) who were unaware of the clinical information. Positive response to an immunohistochemical expression of cyclin A was determined according to
T. Nozoe et al. / Cancer Letters 188 (2002) 221–229
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Fig. 1. Cyclin A was expressed only in the nuclei of the esophageal carcinoma cells. (A) Lower magnification of £ 100. (B) Higher magnification of £ 200. Cyclin A expression in basal cells of normal epithelium concomitant with superficial SCCs, £ 100 (C). Simultaneous expression of cyclin A both in superficial SCC and germinal center cells of the lymphoid follicle beneath the carcinoma, £ 100 (D). Cyclin A expression in germinal center cells of the lymphoid follicle, higher magnification of £ 200 (E). Negative expression of cyclin A both in superficial SCC and lymphoid follicles, £ 100 (F).
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the criteria presented by Volm et al. [27]: Category (A) is defined as the percentage of the carcinoma cells with cyclin A expression; 0 ¼ no cells expressing cyclin A, 1 ¼ less than 25% of carcinoma cells, 2 ¼ 25–50% of carcinoma cell cells, 3 ¼ more than 50% of carcinoma cell cells. Category (B) is defined by the intensity of cyclin A staining; 0 ¼ negative, 1 ¼ weak, 2 ¼ moderate, 3 ¼ strong. The carcinomas were regarded to have a positive response to cyclin A when the total scores of (A) 1 (B) were more than 3. The chi-square test and Student’s t test were used to
compare the clinicopathological data. The cumulative survival rates were calculated by the Kaplan–Meier method and the survival curves were tested by the Mantel–Cox method. A P value of less than 0.05 was regarded as significant.
3. Results Cyclin A was expressed only in the nuclei of the esophageal carcinoma cells (Fig. 1A, B). Twenty-five
Table 1 Relationship between cyclin A expression and clinicopathological factors in superficial squamous cell carcinoma a Variable
Cyclin A positive (n ¼ 25)
Cyclin A negative (n ¼ 20)
P-value
Sex Male Female
23 (92.0) 2 (8.0)
19 (95.0) 1 (5.0)
0.685
Age
62.1 ^ 9.3
65.5 ^ 8.4
0.213
Location of tumor Upper Middle Lower
3 (12.0) 13 (52.0) 9 (36.0)
3 (15.0) 12 (60.0) 5 (25.0)
0.843
Tumor size (cm)
3.6 ^ 2.2
3.5 ^ 2.3
0.851
Histology Well Moderate Poor
3 (12.0) 16 (64.0) 6 (24.0)
2 (10.0) 15 (75.0) 3 (15.0)
0.710
Depth of the tumor Tis T1a T1b
1 (4.0) 9 (36.0) 15 (60.0)
2 (10.0) 5 (25.0) 13 (65.0)
0.584
Lymph nodes metastasis Positive Negative
4 (16.0) 21 (84.0)
3 (15.0) 17 (85.0)
0.581
Lymphatic invasion Positive Negative
2 (8.0) 23 (92.0)
4 (20.0) 16 (80.0)
0.239
Venous invasion Positive Negative
0 25 (100)
1 (5.0) 19 (95.0)
0.258
TNM stage 0 I IIB
1 (4.0) 20 (80.0) 4 (16.0)
2 (10.0) 15 (75.0) 3 (15.0)
0.725
a Values in the parentheses are the percentages. All data are expressed as mean ^ SD Well, well differentiated squamous cell carcinoma; Moderate, moderately differentiated squamous cell carcinoma; Poor, poorly differentiated squamous cell carcinoma.
T. Nozoe et al. / Cancer Letters 188 (2002) 221–229
Fig. 2. There was no significant difference between the survival in SCCs with (thick line) and without (thin line) cyclin A expression (P ¼ 0:317).
superficial SCCs (55.6%) had positive expression of cyclin A and the other 20 (44.4%) did not. Cyclin A expressed in basal cells of normal epithelium concomitant with some of superficial SCCs (Fig. 1C). The clinicopathological characteristics in esophageal superficial SCCs with cyclin A expression did not differ from those of SCCs without cyclin A expression (Table 1). One-, 3-, and 5-year survival rates of patients with superficial SCCs with cyclin A expression were 100, 94.7, and 86.8%, respectively and they did not significantly differ from those of patients with superficial SCCs without cyclin A expression (100, 94.4, and 80.4%, respectively, P ¼ 0:317; Fig. 2). Infiltration of lymphocytes with germinal center cells was observed beneath 17 (68.0%) out of 25 superficial SCCs with cyclin A expression and 15 (75.0%) out of 20 superficial SCCs without cyclin A expression. No significant difference was observed between these percentages (P ¼ 0:605). Among these 32 SCCs with germinal center cells beneath superficial carcinoma, no significant difference regarding clinicopathological features was observed between carcinomas with and without cyclin A expression in germinal center cells (Table 2). One-, 3-, and 5-year survival rates of patients with superficial SCCs with cyclin A expression in germinal center cells were 100, 92.9, and 92.9%, respectively and they were comparatively better than those in
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patients with carcinomas without cyclin A expression in germinal center cells (100, 92.3, and 62.3%, respectively; P ¼ 0:132) (Fig. 3). The proportion of cyclin A expression in SCCs invading to muscularis propria (T2) was 85.7% (12 out of 14), which was significantly higher than that in superficial carcinomas (55.6%, 25 out of 45; P ¼ 0:032). However only three among 14 carcinomas invading to muscularis propria had germinal center cells beneath the concomitant carcinomas. Sixteen (94.1%) out of 17 superficial SCCs with cyclin A expression were associated with cyclin A expression in germinal center cells in infiltrated lymphoid follicles beneath the carcinomas. On the other hand, only two (13.3%) out of 15 superficial SCCs without cyclin A expression coexisted with cyclin A expression in lymphoid follicles beneath the carcinomas and the other 13 did not have cyclin A expression in lymphoid follicles (Fig. 1D–F). There was a significance difference between the proportions of cyclin A expression in lymphoid follicles coexisted beneath the superficial SCCs with and without cyclin A expression (P , 0:0001; Fig. 4).
4. Discussion Cyclins now have come to be defined as protooncogenes and have been suggested to be concerned with progression of tumors [13,28]. From the point of view in cyclin A, which has been well known to act an important role to modulate the mitosis of the cells, there have been many clinical investigations suggesting the correlation of cyclin A expression with the more malignant potential of the tumors and/or the more unfavorable prognosis of the patients [21,22,27,29–32]. To the best of our knowledge, only two studies investigating the cyclin A expression in SCC of the esophagus [22,33], and both results have demonstrated that cyclin A expression was found to be an indicator of more malignant potential of esophageal SCCs. Our motivated purpose of the current study was to investigate the expression of cyclin A in superficial SCCs of the esophagus, which still have some desirable promise in the prognosis of the patients. The study population of the investigation by Furihata et al.[22] included patients who had been treated with
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preoperative irradiation. The influence of irradiation induced to carcinoma cells to the over-expression of such protein kinase as cyclins has not been elucidated, only tumors treated without any preoperative therapies were studied in the current study. In malignant tumor cells, the localization of cellcycle related cyclins and kinases, namely nuclear and cytoplasmic expression has been suggested to have some biological significance. We recently reported that the prognosis of patients with carcinomas with
nuclear expression of cyclin B1 was found to be significantly poorer than that in patients with carcinomas with cytoplasmic expression [34]. However, localization of cyclin A in esophageal SCC cells was not observed, and the nuclear expression was observed in all tumors with positive response to cyclin A. Some normal epithelial cells had weak expression of cyclin A in the basal cells in the current study, which was consistent with a previous report [22]. The most prominent finding in the current study is
Table 2 Relationship between cyclin A expression in germinal center cells and clinicopathological factors in superficial squamous cell carcinoma a Variable
Cyclin A expression in GCCs positive (n ¼ 18)
Cyclin A expression in GCCs negative (n ¼ 14)
P-value
Sex Male Female
17 (94.4) 1 (5.6)
14 (100) 0
0.370
Age
61.4 ^ 10.9
63.6 ^ 8.5
0.530
Location of tumor Upper Middle Lower
2 (11.1) 9 (50.0) 7 (38.9)
3 (21.4) 8 (57.2) 3 (21.4)
0.618
Tumor size (cm)
3.9 ^ 2.2
3.2 ^ 1.6
0.366
Histology Well Moderate Poor
2 (11.1) 12 (66.7) 4 (22.2)
0 13 (92.9) 1 (7.1)
0.183
Depth of the tumor Tis T1a T1b
1 (5.6) 6 (33.3) 11 (61.1)
2 (14.3) 4 (28.5) 8 (57.2)
0.699
3 (16.7) 15 (83.3)
2 (14.3) 12 (85.7)
0.581
2 (11.1) 16 (88.9)
3 (21.4) 11 (78.6)
0.425
Venous invasion Positive Negative
0 18 (100)
1 (7.1) 13 (92.9)
0.249
TNM stage 0 I IIB
1 (5.6) 14 (77.7) 3 (16.7)
2 (14.3) 10 (71.4) 2 (14.3)
0.701
Lymph nodes metastasis Positive Negative Lymphatic invasion Positive Negative
a
Values in the parentheses are the percentages. All data are expressed as mean ^ SD GCC, germinal center cell; Well, well differentiated squamous cell carcinoma; Moderate, moderately differentiated squamous cell carcinoma; Poor, poorly differentiated squamous cell carcinoma.
T. Nozoe et al. / Cancer Letters 188 (2002) 221–229
Fig. 3. Although there was no significant difference, prognosis of patients with superficial SCCs with cyclin A expression in germinal center cells (thick line) was comparatively better than that in patients with carcinomas without cyclin A expression in germinal center cells (thin line; P ¼ 0:132).
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that cyclin A expression was observed in the germinal center cells of lymphocyte follicles beneath the SCCs. Although the proportion of lymphocyte infiltration concomitant with SCCs with cyclin A expression did not differ from that in SCCs without cyclin A expression, the proportion of cyclin A expression in germinal center cells of lymphocyte follicles beneath the SCCs expressing cyclin A was significantly higther than that in lymphocytes beneath the SCCs without cyclin A expression. The significance of the existence of lymphocyte follicles beneath SCC of the esophagus has been studied in some investigations [35–37]. Lymphocytes in follicles were found to infiltrate more frequently beneath superficial SCC than normal esophageal epithelium [35]. Furthermore lymphocytes forming follicles were found to infiltrate more frequently beneath superficial SCC expressing HLA-DR antigen [36]. Baba et al.[37] reported that lymphocyte infiltration appeared significantly more frequently beneath epithelial carcinoma where the
Fig. 4. Infiltration of lymphocytes and cyclin A expression in superficial SCC and coexisted lymphocyte follicles. Lymphocytes infiltration was observed beneath 17 (68.0%) out of 25 superficial SCCs with cyclin A expression and 15 (75.0%) out of 20 superficial SCCs without cyclin A expression. Although 16 (94.1%) out of 17 superficial SCCs with cyclin A expression were associated with cyclin A expression in germinal center cells in infiltrated lymphoid follicles beneath the carcinomas, only two (13.3%) out of 15 superficial SCCs without cyclin A expression coexisted with cyclin A expression in lymphoid follicles beneath the carcinomas (P , 0:0001).
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defect of the basement membrane exists and carcinoma cells are progressing, which could demonstrate the biological significance of lymphocyte infiltration as an immunological defense to the invasive carcinoma cells. The biological significance of cyclin A expression in non-tumorous lymphocytes has not been previously elucidated. However, our results suggest that cyclin A expression in the lymphocytes might reflect the immunologically defensive function of the lymphocytes and that cyclin A expression in germinal center cells of the lymphoid follicles beneath the superficial SCCs might be an immunological signal of precaution to the carcinoma cells expressing cyclin A, which are likely to indicate to have more aggressive ability to proliferate and progress [22,33]. Although we additionally performed immunohistochemical staining of lymphocytes T and B in serial sections in which germinal center cells expressed cyclin A, no special relationship could be found between expressions of cyclin A and lymphocytes T and B in germinal center cells beneath the superficial SCCs of the esophagus. The proportion of cyclin A expression in esophageal SCCs invading to muscularis propria was found to be significantly higher than that in superficial SCCs, which was consistent with a previous report presenting that cyclin A might have a pivotal role in progression in SCC of the esophagus [22]. Although only three among 14 carcinomas invading to muscularis propria with cyclin A expression had germinal center cells beneath the concomitant superficial carcinomas, in these three carcinomas, expression of cyclin A in germinal center cells was observed beneath concomitant intraepithelial carcinomatous regions, associated with cyclin A expression. The function of immunological supervision of germinal center cells which presents cyclin A expression associated with superficial carcinomas might be incompetent, as esophageal SCCs advanced more invasively. Although the clinicopathological features did not differ between patients with esophageal superficial SCCs with and without immunohistochemical expression of cyclin A, cyclin A expression in the germinal center cells of the lymphoid follicles beneath the superficial SCCs of the esophagus might demonstrate the biological significance of supervising the proliferation and progression of superficial SCCs.
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Cyclin A expression in superficial squamous cell carcinoma of the esophagus and coexisting infiltrated lymphocyte follicle Tadahiro Nozoe a,*, Daisuke Korenaga a, Motonori Futatsugi b, Hiroshi Saeki b, Takefumi Ohga b, Keizo Sugimachi b b
a Department of General Surgery, Fukuoka Dental College Hospital, Fukuoka, 814-0193, Japan Department of Surgery and Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka, 812-8582, Japan
Received 22 April 2002; received in revised form 4 July 2002; accepted 8 July 2002
Abstract Cyclin A is a protein kinase to act a pivotal role in the mitotic phase of the cell cycle. The purpose of the current study was to elucidate the biological significance of immunohistochemical expression of cyclin A in superficial squamous cell carcinoma (SCC) of the esophagus. Immunohistochemical staining of cyclin A was performed for 45 samples of esophageal superficial SCCs. Clinicopathological features were compared between SCCs with and without cyclin A expression. Twenty-five superficial SCCs (55.6%) had positive expression of cyclin A and the other 20 (44.4%) did not. No significant difference regarding clinicopathological characteristics between esophageal SCCs with and without cyclin A expression. Infiltration of lymphocytes with germinal center cells was observed beneath 17 (68.0%) out of 25 superficial SCCs with cyclin A expression and 15 (75.0%) out of 20 superficial SCCs without cyclin A expression. Although 16 (94.1%) out of 17 superficial SCCs with cyclin A expression were associated with cyclin A expression in germinal center cells in infiltrated lymphoid follicles beneath the tumors, only 2 (13.3%) out of 15 superficial SCCs without cyclin A expression coexisted with cyclin A expression in lymphoid follicles beneath the tumors (P , 0:0001). Cyclin A expression in the germinal center cells of the lymphoid follicles beneath the superficial SCCs of the esophagus might be an immunological signal toward the proliferation and progression of the tumors. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Cyclin A; Immunohistochemical expression; Esophagus; Squamous cell carcinoma; Superficial carcinoma; Lymphocyte follicle; Immunological surveillance
1. Introduction Although squamous cell carcinoma (SCC) of the esophagus accounts for one of the human neoplasms associated with biologically most malignant behavior [1], recent advance in the diagnostic technique has been contributing to the increase of superficial SCCs * Corresponding author. Tel.: 1 81-92-801-0526; fax: 1 81-92801-0735. E-mail address: [email protected] (T. Nozoe).
[2,3], which has steadily brought about the improvement in the prognosis of the patients with esophageal SCC. Hence, the clinical focus of the physicians has been directed to superficial SCC of the esophagus [4–6]. Molecular and biological methodologies could be used to investigate the clinicopathological features in order to acquire the clinical hint directly correlated with better outcome of the patients with esophageal SCCs. Such bio-markers as expressions of cyclindependent kinase inhibitor p27 [7] and appoptosis-
0304-3835/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(02)00434-2
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related protein Bcl-X [8], amplification of int-2 [9], and serum detection of anti-p53 antibody [10] have been suggested to indicate the more malignant or lessinvasive potential in superficial SCC of the esophagus. Cyclins are the essential protein which have an important role to regulate the cell cycle [11], and proliferation of cell is controlled by protein complexes composed of cyclins associated with cyclin dependent kinase (Cdk) [12,13]. Cyclin A is a protein kinase to modulate the cell cycle and cell proliferation and has been known to act a pivotal role both in S-phase [14] and in the G2/M phase transition as a mitotic cyclin during the cell cycle [15]. Cyclin A binds to both cdk2 and cdc2 and modulated the DNA replication in S-phase [16] and the initiation of mitosis [14,17]. These studies could suggest that cyclin A expression might be correlated with the progression of the tumors. Moreover, there have been also some investigations suggesting that cyclin A can be considered to be possibly associated with the malignant transformation of the cells [18–20]. Some clinical investigations demonstrated the significance of cyclin A expression as an indicator of malignant potential of tumors and/or a prognosticator of the patients with some human SCCs [21,22]. In the current study, we investigated an immunohistochemical expression of cyclin A to find out its clinicopathological significance in superficial esophageal SCC, which has been left to be discussed.
2. Patients and methods 2.1. Patients The tissue samples were collected from 45 patients with superficial squamous cell carcinoma (SCC) and 14 with SCCs invading muscularis propria of the esophagus, who had been treated with resection of the esophagus and reconstruction in the Department of Surgery and Science, Graduate School of Medical Sciences, Kyushu University Hospital. The pathological depth of the superficial tumors, determined according to TNM classification of malignant tumors decided by International Union Against Cancer [23], were Tis in three, T1a in 14 and T1b in 28, respectively. The study population associated with superfi-
cial tumors consisted of 42 men and three women, and the age range from 40 to 77 years with a mean of 64 years. The population associated with SCCs invading muscularis propria (T2) was consisted of 13 men and one woman, and the age range from 44 to 81 years with a mean of 68 years. The clinicopathological investigation was made on the basis of guidelines for clinical and pathologic studies on carcinoma of the esophagus established by the Japanese Society for Esophageal Diseases [24]. Follow-up for the patients was continuously performed until death and only patients who died of esophageal carcinoma were included in the tumorrelated deaths. The interval of the follow-up after surgical treatment ranged from 5 months to 9 years and 6 months with a mean of 4 years and 7 months. 2.2. Methods Immunostaining of cyclin A was performed using a mouse monoclonal anti-human cyclin A antibody (6E6, Novocastra Laboratory, UK). According to the manufacturer, this antibody has no cross-reactive response to other cyclins. Four micrometer thick sections sliced from paraffin-embedded specimen were prepared on the slideglasses pre-coated with silane. After removing paraffin in xylene and washing in graded series of ethanol, the sections were placed in Tris-buffered saline (TBS) for 10 min. The sections were incubated with a blocking solution for 5 min to block an endogenous peroxidase activity, and were placed in TBS. Then the sections were placed in the 0.01 mM Tris buffer (pH 6.0) and heated at 1218C for 20 min in an autoclave oven. Next the sections were incubated with TBS including 1% concentration of bovine serum albumin for 20 min to block non-specific binding of the immunoreagents. After washing in TBS, the sections were incubated with 1:50 diluted primary antibody overnight at 48C. After washing in TBS, an immunoperoxidase staining was performed by an EnVision antibody complex method [25,26] using ENVISION kit (DAKO. Ltd., Tokyo, Japan). Finally, the localization of cyclin A was visualized with diaminobenzidine tetrahydrochloride. All of the samples were analyzed by two observers (T.N., D.K.) who were unaware of the clinical information. Positive response to an immunohistochemical expression of cyclin A was determined according to
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Fig. 1. Cyclin A was expressed only in the nuclei of the esophageal carcinoma cells. (A) Lower magnification of £ 100. (B) Higher magnification of £ 200. Cyclin A expression in basal cells of normal epithelium concomitant with superficial SCCs, £ 100 (C). Simultaneous expression of cyclin A both in superficial SCC and germinal center cells of the lymphoid follicle beneath the carcinoma, £ 100 (D). Cyclin A expression in germinal center cells of the lymphoid follicle, higher magnification of £ 200 (E). Negative expression of cyclin A both in superficial SCC and lymphoid follicles, £ 100 (F).
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the criteria presented by Volm et al. [27]: Category (A) is defined as the percentage of the carcinoma cells with cyclin A expression; 0 ¼ no cells expressing cyclin A, 1 ¼ less than 25% of carcinoma cells, 2 ¼ 25–50% of carcinoma cell cells, 3 ¼ more than 50% of carcinoma cell cells. Category (B) is defined by the intensity of cyclin A staining; 0 ¼ negative, 1 ¼ weak, 2 ¼ moderate, 3 ¼ strong. The carcinomas were regarded to have a positive response to cyclin A when the total scores of (A) 1 (B) were more than 3. The chi-square test and Student’s t test were used to
compare the clinicopathological data. The cumulative survival rates were calculated by the Kaplan–Meier method and the survival curves were tested by the Mantel–Cox method. A P value of less than 0.05 was regarded as significant.
3. Results Cyclin A was expressed only in the nuclei of the esophageal carcinoma cells (Fig. 1A, B). Twenty-five
Table 1 Relationship between cyclin A expression and clinicopathological factors in superficial squamous cell carcinoma a Variable
Cyclin A positive (n ¼ 25)
Cyclin A negative (n ¼ 20)
P-value
Sex Male Female
23 (92.0) 2 (8.0)
19 (95.0) 1 (5.0)
0.685
Age
62.1 ^ 9.3
65.5 ^ 8.4
0.213
Location of tumor Upper Middle Lower
3 (12.0) 13 (52.0) 9 (36.0)
3 (15.0) 12 (60.0) 5 (25.0)
0.843
Tumor size (cm)
3.6 ^ 2.2
3.5 ^ 2.3
0.851
Histology Well Moderate Poor
3 (12.0) 16 (64.0) 6 (24.0)
2 (10.0) 15 (75.0) 3 (15.0)
0.710
Depth of the tumor Tis T1a T1b
1 (4.0) 9 (36.0) 15 (60.0)
2 (10.0) 5 (25.0) 13 (65.0)
0.584
Lymph nodes metastasis Positive Negative
4 (16.0) 21 (84.0)
3 (15.0) 17 (85.0)
0.581
Lymphatic invasion Positive Negative
2 (8.0) 23 (92.0)
4 (20.0) 16 (80.0)
0.239
Venous invasion Positive Negative
0 25 (100)
1 (5.0) 19 (95.0)
0.258
TNM stage 0 I IIB
1 (4.0) 20 (80.0) 4 (16.0)
2 (10.0) 15 (75.0) 3 (15.0)
0.725
a Values in the parentheses are the percentages. All data are expressed as mean ^ SD Well, well differentiated squamous cell carcinoma; Moderate, moderately differentiated squamous cell carcinoma; Poor, poorly differentiated squamous cell carcinoma.
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Fig. 2. There was no significant difference between the survival in SCCs with (thick line) and without (thin line) cyclin A expression (P ¼ 0:317).
superficial SCCs (55.6%) had positive expression of cyclin A and the other 20 (44.4%) did not. Cyclin A expressed in basal cells of normal epithelium concomitant with some of superficial SCCs (Fig. 1C). The clinicopathological characteristics in esophageal superficial SCCs with cyclin A expression did not differ from those of SCCs without cyclin A expression (Table 1). One-, 3-, and 5-year survival rates of patients with superficial SCCs with cyclin A expression were 100, 94.7, and 86.8%, respectively and they did not significantly differ from those of patients with superficial SCCs without cyclin A expression (100, 94.4, and 80.4%, respectively, P ¼ 0:317; Fig. 2). Infiltration of lymphocytes with germinal center cells was observed beneath 17 (68.0%) out of 25 superficial SCCs with cyclin A expression and 15 (75.0%) out of 20 superficial SCCs without cyclin A expression. No significant difference was observed between these percentages (P ¼ 0:605). Among these 32 SCCs with germinal center cells beneath superficial carcinoma, no significant difference regarding clinicopathological features was observed between carcinomas with and without cyclin A expression in germinal center cells (Table 2). One-, 3-, and 5-year survival rates of patients with superficial SCCs with cyclin A expression in germinal center cells were 100, 92.9, and 92.9%, respectively and they were comparatively better than those in
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patients with carcinomas without cyclin A expression in germinal center cells (100, 92.3, and 62.3%, respectively; P ¼ 0:132) (Fig. 3). The proportion of cyclin A expression in SCCs invading to muscularis propria (T2) was 85.7% (12 out of 14), which was significantly higher than that in superficial carcinomas (55.6%, 25 out of 45; P ¼ 0:032). However only three among 14 carcinomas invading to muscularis propria had germinal center cells beneath the concomitant carcinomas. Sixteen (94.1%) out of 17 superficial SCCs with cyclin A expression were associated with cyclin A expression in germinal center cells in infiltrated lymphoid follicles beneath the carcinomas. On the other hand, only two (13.3%) out of 15 superficial SCCs without cyclin A expression coexisted with cyclin A expression in lymphoid follicles beneath the carcinomas and the other 13 did not have cyclin A expression in lymphoid follicles (Fig. 1D–F). There was a significance difference between the proportions of cyclin A expression in lymphoid follicles coexisted beneath the superficial SCCs with and without cyclin A expression (P , 0:0001; Fig. 4).
4. Discussion Cyclins now have come to be defined as protooncogenes and have been suggested to be concerned with progression of tumors [13,28]. From the point of view in cyclin A, which has been well known to act an important role to modulate the mitosis of the cells, there have been many clinical investigations suggesting the correlation of cyclin A expression with the more malignant potential of the tumors and/or the more unfavorable prognosis of the patients [21,22,27,29–32]. To the best of our knowledge, only two studies investigating the cyclin A expression in SCC of the esophagus [22,33], and both results have demonstrated that cyclin A expression was found to be an indicator of more malignant potential of esophageal SCCs. Our motivated purpose of the current study was to investigate the expression of cyclin A in superficial SCCs of the esophagus, which still have some desirable promise in the prognosis of the patients. The study population of the investigation by Furihata et al.[22] included patients who had been treated with
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preoperative irradiation. The influence of irradiation induced to carcinoma cells to the over-expression of such protein kinase as cyclins has not been elucidated, only tumors treated without any preoperative therapies were studied in the current study. In malignant tumor cells, the localization of cellcycle related cyclins and kinases, namely nuclear and cytoplasmic expression has been suggested to have some biological significance. We recently reported that the prognosis of patients with carcinomas with
nuclear expression of cyclin B1 was found to be significantly poorer than that in patients with carcinomas with cytoplasmic expression [34]. However, localization of cyclin A in esophageal SCC cells was not observed, and the nuclear expression was observed in all tumors with positive response to cyclin A. Some normal epithelial cells had weak expression of cyclin A in the basal cells in the current study, which was consistent with a previous report [22]. The most prominent finding in the current study is
Table 2 Relationship between cyclin A expression in germinal center cells and clinicopathological factors in superficial squamous cell carcinoma a Variable
Cyclin A expression in GCCs positive (n ¼ 18)
Cyclin A expression in GCCs negative (n ¼ 14)
P-value
Sex Male Female
17 (94.4) 1 (5.6)
14 (100) 0
0.370
Age
61.4 ^ 10.9
63.6 ^ 8.5
0.530
Location of tumor Upper Middle Lower
2 (11.1) 9 (50.0) 7 (38.9)
3 (21.4) 8 (57.2) 3 (21.4)
0.618
Tumor size (cm)
3.9 ^ 2.2
3.2 ^ 1.6
0.366
Histology Well Moderate Poor
2 (11.1) 12 (66.7) 4 (22.2)
0 13 (92.9) 1 (7.1)
0.183
Depth of the tumor Tis T1a T1b
1 (5.6) 6 (33.3) 11 (61.1)
2 (14.3) 4 (28.5) 8 (57.2)
0.699
3 (16.7) 15 (83.3)
2 (14.3) 12 (85.7)
0.581
2 (11.1) 16 (88.9)
3 (21.4) 11 (78.6)
0.425
Venous invasion Positive Negative
0 18 (100)
1 (7.1) 13 (92.9)
0.249
TNM stage 0 I IIB
1 (5.6) 14 (77.7) 3 (16.7)
2 (14.3) 10 (71.4) 2 (14.3)
0.701
Lymph nodes metastasis Positive Negative Lymphatic invasion Positive Negative
a
Values in the parentheses are the percentages. All data are expressed as mean ^ SD GCC, germinal center cell; Well, well differentiated squamous cell carcinoma; Moderate, moderately differentiated squamous cell carcinoma; Poor, poorly differentiated squamous cell carcinoma.
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Fig. 3. Although there was no significant difference, prognosis of patients with superficial SCCs with cyclin A expression in germinal center cells (thick line) was comparatively better than that in patients with carcinomas without cyclin A expression in germinal center cells (thin line; P ¼ 0:132).
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that cyclin A expression was observed in the germinal center cells of lymphocyte follicles beneath the SCCs. Although the proportion of lymphocyte infiltration concomitant with SCCs with cyclin A expression did not differ from that in SCCs without cyclin A expression, the proportion of cyclin A expression in germinal center cells of lymphocyte follicles beneath the SCCs expressing cyclin A was significantly higther than that in lymphocytes beneath the SCCs without cyclin A expression. The significance of the existence of lymphocyte follicles beneath SCC of the esophagus has been studied in some investigations [35–37]. Lymphocytes in follicles were found to infiltrate more frequently beneath superficial SCC than normal esophageal epithelium [35]. Furthermore lymphocytes forming follicles were found to infiltrate more frequently beneath superficial SCC expressing HLA-DR antigen [36]. Baba et al.[37] reported that lymphocyte infiltration appeared significantly more frequently beneath epithelial carcinoma where the
Fig. 4. Infiltration of lymphocytes and cyclin A expression in superficial SCC and coexisted lymphocyte follicles. Lymphocytes infiltration was observed beneath 17 (68.0%) out of 25 superficial SCCs with cyclin A expression and 15 (75.0%) out of 20 superficial SCCs without cyclin A expression. Although 16 (94.1%) out of 17 superficial SCCs with cyclin A expression were associated with cyclin A expression in germinal center cells in infiltrated lymphoid follicles beneath the carcinomas, only two (13.3%) out of 15 superficial SCCs without cyclin A expression coexisted with cyclin A expression in lymphoid follicles beneath the carcinomas (P , 0:0001).
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defect of the basement membrane exists and carcinoma cells are progressing, which could demonstrate the biological significance of lymphocyte infiltration as an immunological defense to the invasive carcinoma cells. The biological significance of cyclin A expression in non-tumorous lymphocytes has not been previously elucidated. However, our results suggest that cyclin A expression in the lymphocytes might reflect the immunologically defensive function of the lymphocytes and that cyclin A expression in germinal center cells of the lymphoid follicles beneath the superficial SCCs might be an immunological signal of precaution to the carcinoma cells expressing cyclin A, which are likely to indicate to have more aggressive ability to proliferate and progress [22,33]. Although we additionally performed immunohistochemical staining of lymphocytes T and B in serial sections in which germinal center cells expressed cyclin A, no special relationship could be found between expressions of cyclin A and lymphocytes T and B in germinal center cells beneath the superficial SCCs of the esophagus. The proportion of cyclin A expression in esophageal SCCs invading to muscularis propria was found to be significantly higher than that in superficial SCCs, which was consistent with a previous report presenting that cyclin A might have a pivotal role in progression in SCC of the esophagus [22]. Although only three among 14 carcinomas invading to muscularis propria with cyclin A expression had germinal center cells beneath the concomitant superficial carcinomas, in these three carcinomas, expression of cyclin A in germinal center cells was observed beneath concomitant intraepithelial carcinomatous regions, associated with cyclin A expression. The function of immunological supervision of germinal center cells which presents cyclin A expression associated with superficial carcinomas might be incompetent, as esophageal SCCs advanced more invasively. Although the clinicopathological features did not differ between patients with esophageal superficial SCCs with and without immunohistochemical expression of cyclin A, cyclin A expression in the germinal center cells of the lymphoid follicles beneath the superficial SCCs of the esophagus might demonstrate the biological significance of supervising the proliferation and progression of superficial SCCs.
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