Int J Colorectal Dis (2014) 29:1349–1354 DOI 10.1007/s00384-014-1996-6
ORIGINAL ARTICLE
DNA damage response and its clinicopathological relationship in appendiceal tumors Nobuhisa Yajima & Ryuichi Wada & Yutaka Matsuzaki & Soroku Yagihashi
Accepted: 10 August 2014 / Published online: 26 August 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Background Appendiceal tumors are rare, and their pathogenesis is not well known. DNA damage response (DDR) is a sequence from the detection of damaged DNA to the repair, and its impairment is implicated in the progression of cancers. The aim of the current study is to explore the expression and phosphorylation of checkpoint kinase 2 (Chk2) and TP53, which are key molecules in DDR, and their clinicopathological correlation in the appendiceal tumors. Methods Chk2, phosphorylated Chk2 (pChk2), and TP53 were immunostained in 4 cases of adenoma (AD), 5 nonmucinous adenocarcinomas (AC), 29 low-grade appendiceal mucinous neoplasms (LAMN), and 7 mucinous adenocarcinomas (MAC). Ki-67 labeling index was also evaluated by immunostaining. Results Chk2 was highly expressed in the nuclei of all the appendiceal tumors. While pChk2 was high in AD, LAMN, and MAC, it was reduced in AC. Nuclear positive reaction of TP53 was lower in LAMN compared with those of other tumors. The Ki-67 labeling index was slightly lower in LAMN than those in other tumors. The recurrence and death in LAMN is infrequent compared with those in AC and MAC. Conclusions The current study suggested the impairment of DDR in AC and MAC. DDR appeared to be preserved in LAMN, and it may account for low proliferating activity and a favorable clinical course in LAMN.
N. Yajima : R. Wada : Y. Matsuzaki : S. Yagihashi Department of Pathology and Molecular Medicine, Graduate School of Medicine, Hirosaki University, Hirosaki, Japan R. Wada (*) Department of Integrated Diagnostic Pathology, Nippon Medical School, 1-1-5 Sendagi Bunkyo-ku, 113-8602 Tokyo, Japan e-mail: [email protected]
Keywords Low-grade appendiceal mucinous neoplasm . DNA damage response . Chk2 . Ki-67 . Prognosis
Introduction Appendiceal tumors are uncommon, and they constitute less than 1 % among the tumors arising from the lower gastrointestinal tract [1]. They may present as a huge abdominal mass, whereas in some cases, the tumor is so small as to be incidentally found in the resected appendix. High-grade adenocarcinomas are characterized by the invasive proliferation of neoplastic cells with severe atypia and occasional production of mucin. Lowgrade mucinous neoplasms (LAMN) is characterized by villous or serrated proliferation of cells with the production of large amount of mucin, and it usually forms cystic lesion known as “mucocele” of the appendix. The rupture of the tumor causes pseudomyxoma peritonei (PMP). The progression of the disease is slow, and the prognosis is usually favorable [2]. DNA damage response (DDR) is a sequence of DNA damage detection, cell cycle arrest, and subsequent repair of damaged DNA [3, 4]. When DNA is injured, ataxia telangiectasia mutated (ATM) and ataxia telangiectasia related (ATR) are recruited to the damaged lesion. They activate key regulators of DDR, such as checkpoint kinase 1 (Chk1) and checkpoint kinase 2 (Chk2). The cell cycle is arrested by TP53, and damaged DNA is repaired. The impairment of DDR is implicated in the progression of colorectal carcinomas [5, 6] and other human carcinomas [7–9]. Under rapid proliferation, genomic DNA of neoplastic cells can be frequently damaged. While DDR is sufficient, the neoplastic cells are genetically stable. When the molecules involved in DDR are affected, the cells become genetically instable, and the progression of neoplastic cells to highly malignant cells may occur. It is thus speculated that the DDR serve as a barrier to prevent the progression to highly malignant
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Int J Colorectal Dis (2014) 29:1349–1354
Table 1 The list of antibodies used in the current study Antibody (clone)
Species
Pretreatment
Dilution
Source
Chk2 (DCS-270) Phosphorylated Chk2 TP53 (DO-1) Ki-67 (MIB-1)
M R M M
CB CB TE TE
1:5,000 1:1,000 1:200 1:200
Thermo Scientific, CA, USA Cell Signaling Technology, Tokyo, Japan EMD Chemicals, Inc, Darmstadt, Germany Dako Japan, Tokyo, Japan
M mouse, R rabbit, CB citrate buffer (pH 6.0), TE Tris-EDTA buffer (pH 9.0)
tumor [7]. In the appendiceal tumors, there is no information on the expression and functional state of the molecules in DDR. The aim of the current study is to elucidate the expression and phosphorylation of the molecules in DDR and their correlation with clinicopathological features in the appendiceal tumors.
Materials and methods Cases of appendiceal tumors The cases of appendiceal tumors were retrieved from the archives of pathological files of the Department of Pathology and Molecular Medicine, Hirosaki University Graduate School of Medicine. From more than 30,000 pathology reports for 20 years, cases were extracted with key words of “appendix,” “adenoma,” “carcinoma,” “tumor,” “mucocele,” “mucinous,” and “cyst.” Cases of metastatic tumors to the appendix were excluded. Finally, 45 cases in total of primary appendiceal tumors were procured. Clinical records and follow-up information of the patients were obtained as much as possible. Informed consents were obtained from the living patients. Histological examinations Four-micrometer-thick paraffin sections of tumors fixed in 10 % formalin were stained with hematoxylin and eosin (H
& E), Alcian Blue (ALB) (pH 2.5), and periodic acid-Schiff (PAS). The mucin production was defined as positive when more than 50 % of the tumor areas showed mucin production by ALB or PAS staining, based on the criteria proposed by WHO [1]. Tumors without mucin production were classified as adenoma (AD) and adenocarcinoma (AC). Mucinproducing tumors were classified into two categories of LAMN and mucinous adenocarcinoma (MAC). The histology of the tumors was reviewed by three pathologists (NY, RW, SY). Immunostaining and quantitation of positive cells Immunostaining was performed on the paraffin sections. The antibodies used in the current study and their pretreatments were shown in Table 1. Briefly, the paraffin sections were deparaffinized and pretreated with buffers listed in Table 1. After blocking with 1 % bovine serum albumin for 10 min, the primary antibodies were diluted with Renoir Red Antibody Diluent (Biocare Medical, Concord, CA, USA) and applied to the sections. Then, endogenous peroxidase was blocked in 10 % hydrogen peroxide for 5 min and the sections were processed with MACH3 HRP Polymer Kit (Biocare Medical, Concord, CA, USA), and peroxidase activity was visualized by Metal Enhanced DAB (ThermoFischer Scientific, K.K., Tokyo, Japan). The sections for TP53 and Ki-67 were immunostained by Ventana GX System (Roche
Table 2 The clinicopathological features of appendiceal tumors
Number of cases Age Sex (M/F) Nodal status (+/−) TNM stage (0/I/II/II/IV) Number of follow-up patients Observation period (months) PMP/recurrence Death of disease Time to death (months) PMP pseudomyxoma peritonei
AD
AC
LAMN
MAC
4 71.8±7.9 2/2 – – 3 1–27 0 (0 %) 0 (0 %) –
5 50.4±19.3 5/0 2/3 0/1/2/0/2 5 13–146 1 (20 %) 1 (20 %) 19
29 65.0±15.3 10/19 0/29 15/4/9/0/2 23 1–187 2 (9 %) 1 (4 %) 32
7 60.0±12.6 3/4 1/6 0/1/5/0/1 5 20–95 4 (80 %) 2 (40 %) 32, 35
Int J Colorectal Dis (2014) 29:1349–1354
Diagnostics, Co., Tokyo, Japan). The primary antibodies were diluted with Antibody Diluent (Roche Diagnostics, Co.). The stained sections were observed under ×40 objective, and nuclear brown staining was considered as positive. The number of cells with nuclear positivity was counted in 1,500 neoplastic cells. Ki-67 labeling index was calculated by dividing the number of positive cells by 1,500. Statistical analysis Statistical analysis was performed using JMP10 (SAS Institutes, Inc., CA, USA). Comparison of multiple groups was done with nonparametric Steel-Dwass test. The p value less than 0.05 was considered to be significant. Fig. 1 The histology of appendiceal tumors. a Macroscopic appearance of LAMN. Scale bar corresponds to 2 cm. b Loupe image of the neoplasm. Scale bar corresponds to 2 cm. c–f Hematoxylin and eosin staining of AD (c), LAMN (d), AC (e), and MAC (f). g–h Periodic acid-Schiff stain of AC (g) and LAMN (h). Mucin (red) was demonstrated in LAMN (h, arrow)
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Results Clinicopathological features and histology of appendiceal tumors The age of the patients ranged from 20 to 94 years of age, and male to female ratio was 20:25 (Table 2). Based on the classification of appendiceal tumors by WHO, 4 cases were classified as AD, 5 as AC, 29 as LAMN, and 7 as MAC (Table 2). The age distribution was similar among the four histological groups, but the patients of AC were slightly younger than other groups. All AC patients were male. In LAMN, female patients were twice of the male patients. Representative histology of
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appendiceal tumors is shown in Fig. 1. Mucin production was evident in LAMN and MAC (Fig. 1h). The tumor cells invaded into the submucosa in all five cases in AC and in 15 out of 29 in LAMN. The tumor cells exposed to the serosa in all seven cases of MAC. Nodal metastasis was not present in LAMN. Expression and phosphorylation of Chk2 The expressions of Chk2 and pChk2 were localized in the nuclei of the tumor cells (Fig. 2), and there was no cytoplasmic positive reaction. In most tumors, more than 80 % of tumor cells showed positive nuclear expression of Chk2 (Fig. 3a). In contrast, the percentage of pChk2-positive cells varied largely from 2.9 to 95.2 % among the appendiceal tumors (Figs. 2 and 3b). The percentage of positive cells was significantly lower in AC than those in other tumors, and it was higher in LAMN (Fig. 3b). Expression of TP53 The expression of TP53 was localized to the nuclei of tumor cells (Fig. 2). The percentage of TP53-positive cells varied widely in each histological group, and it is significantly lower in LAMN than those in AC and MAC (Fig. 3c). Proliferation activity The Ki-67-labeling index varied widely, and it was lower in LAMN compared with those in other tumors (Figs. 2 and 3d). Fig. 2 Immunostaining of molecules involved in DNA damage response. Representative immunostaining of Chk2, pChk2, TP53, and Ki-67 in AD, AC, LAMN, and MAC. Nuclear brown staining of the tumor cells is judged as positive reaction. Arrowheads indicate positive nuclear reactions, which are very sparsely distributed in the tumor. Magnification of the images is ×200
Int J Colorectal Dis (2014) 29:1349–1354
Clinicopathological features of appendiceal tumors Totally, 36 patients have been followed up (Table 2). Local recurrence was not present in the cases of AD. In AC, one out of five patients (20 %) died of the disease. In LAMN, 2 out of 23 cases (9 %) presented with PMP, and one (4 %) died of the extensive disease, while 21 out of 23 cases (91 %) were free of disease. Among the cases of MAC, recurrence occurred in four out of five cases (80 %). Two cases (40 %) died of the disease at 32 and 35 months after operation, and other two cases are alive with PMP and local recurrence.
Discussion The current study is the first that showed distinct patterns of expression and phosphorylation of molecules involved in DDR in the appendiceal tumors. Chk2 was ubiquitously expressed in the nuclei of the appendiceal tumor cells. However, the frequency of pChk2-positive cells was reduced in AC, whereas it was high in LAMN and MAC (Fig. 3b). On the other hand, the frequency of TP53-positive cells was high in AC and MAC, whereas it was low in LAMN (Fig. 3c). We have previously reported the high frequency of nuclear positivity of TP53 in MAC, which was associated with mutation of TP53 gene [10]. The reduction of pChk2-positive cells and mutated TP53 suggests that DDR is impaired in AC and MAC (Fig. 4).
Int J Colorectal Dis (2014) 29:1349–1354
1353
Fig. 3 Quantitation of expression and phosphorylation of the molecules in appendiceal tumors. a Chk2. b pChk2. *p
ORIGINAL ARTICLE
DNA damage response and its clinicopathological relationship in appendiceal tumors Nobuhisa Yajima & Ryuichi Wada & Yutaka Matsuzaki & Soroku Yagihashi
Accepted: 10 August 2014 / Published online: 26 August 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Background Appendiceal tumors are rare, and their pathogenesis is not well known. DNA damage response (DDR) is a sequence from the detection of damaged DNA to the repair, and its impairment is implicated in the progression of cancers. The aim of the current study is to explore the expression and phosphorylation of checkpoint kinase 2 (Chk2) and TP53, which are key molecules in DDR, and their clinicopathological correlation in the appendiceal tumors. Methods Chk2, phosphorylated Chk2 (pChk2), and TP53 were immunostained in 4 cases of adenoma (AD), 5 nonmucinous adenocarcinomas (AC), 29 low-grade appendiceal mucinous neoplasms (LAMN), and 7 mucinous adenocarcinomas (MAC). Ki-67 labeling index was also evaluated by immunostaining. Results Chk2 was highly expressed in the nuclei of all the appendiceal tumors. While pChk2 was high in AD, LAMN, and MAC, it was reduced in AC. Nuclear positive reaction of TP53 was lower in LAMN compared with those of other tumors. The Ki-67 labeling index was slightly lower in LAMN than those in other tumors. The recurrence and death in LAMN is infrequent compared with those in AC and MAC. Conclusions The current study suggested the impairment of DDR in AC and MAC. DDR appeared to be preserved in LAMN, and it may account for low proliferating activity and a favorable clinical course in LAMN.
N. Yajima : R. Wada : Y. Matsuzaki : S. Yagihashi Department of Pathology and Molecular Medicine, Graduate School of Medicine, Hirosaki University, Hirosaki, Japan R. Wada (*) Department of Integrated Diagnostic Pathology, Nippon Medical School, 1-1-5 Sendagi Bunkyo-ku, 113-8602 Tokyo, Japan e-mail: [email protected]
Keywords Low-grade appendiceal mucinous neoplasm . DNA damage response . Chk2 . Ki-67 . Prognosis
Introduction Appendiceal tumors are uncommon, and they constitute less than 1 % among the tumors arising from the lower gastrointestinal tract [1]. They may present as a huge abdominal mass, whereas in some cases, the tumor is so small as to be incidentally found in the resected appendix. High-grade adenocarcinomas are characterized by the invasive proliferation of neoplastic cells with severe atypia and occasional production of mucin. Lowgrade mucinous neoplasms (LAMN) is characterized by villous or serrated proliferation of cells with the production of large amount of mucin, and it usually forms cystic lesion known as “mucocele” of the appendix. The rupture of the tumor causes pseudomyxoma peritonei (PMP). The progression of the disease is slow, and the prognosis is usually favorable [2]. DNA damage response (DDR) is a sequence of DNA damage detection, cell cycle arrest, and subsequent repair of damaged DNA [3, 4]. When DNA is injured, ataxia telangiectasia mutated (ATM) and ataxia telangiectasia related (ATR) are recruited to the damaged lesion. They activate key regulators of DDR, such as checkpoint kinase 1 (Chk1) and checkpoint kinase 2 (Chk2). The cell cycle is arrested by TP53, and damaged DNA is repaired. The impairment of DDR is implicated in the progression of colorectal carcinomas [5, 6] and other human carcinomas [7–9]. Under rapid proliferation, genomic DNA of neoplastic cells can be frequently damaged. While DDR is sufficient, the neoplastic cells are genetically stable. When the molecules involved in DDR are affected, the cells become genetically instable, and the progression of neoplastic cells to highly malignant cells may occur. It is thus speculated that the DDR serve as a barrier to prevent the progression to highly malignant
1350
Int J Colorectal Dis (2014) 29:1349–1354
Table 1 The list of antibodies used in the current study Antibody (clone)
Species
Pretreatment
Dilution
Source
Chk2 (DCS-270) Phosphorylated Chk2 TP53 (DO-1) Ki-67 (MIB-1)
M R M M
CB CB TE TE
1:5,000 1:1,000 1:200 1:200
Thermo Scientific, CA, USA Cell Signaling Technology, Tokyo, Japan EMD Chemicals, Inc, Darmstadt, Germany Dako Japan, Tokyo, Japan
M mouse, R rabbit, CB citrate buffer (pH 6.0), TE Tris-EDTA buffer (pH 9.0)
tumor [7]. In the appendiceal tumors, there is no information on the expression and functional state of the molecules in DDR. The aim of the current study is to elucidate the expression and phosphorylation of the molecules in DDR and their correlation with clinicopathological features in the appendiceal tumors.
Materials and methods Cases of appendiceal tumors The cases of appendiceal tumors were retrieved from the archives of pathological files of the Department of Pathology and Molecular Medicine, Hirosaki University Graduate School of Medicine. From more than 30,000 pathology reports for 20 years, cases were extracted with key words of “appendix,” “adenoma,” “carcinoma,” “tumor,” “mucocele,” “mucinous,” and “cyst.” Cases of metastatic tumors to the appendix were excluded. Finally, 45 cases in total of primary appendiceal tumors were procured. Clinical records and follow-up information of the patients were obtained as much as possible. Informed consents were obtained from the living patients. Histological examinations Four-micrometer-thick paraffin sections of tumors fixed in 10 % formalin were stained with hematoxylin and eosin (H
& E), Alcian Blue (ALB) (pH 2.5), and periodic acid-Schiff (PAS). The mucin production was defined as positive when more than 50 % of the tumor areas showed mucin production by ALB or PAS staining, based on the criteria proposed by WHO [1]. Tumors without mucin production were classified as adenoma (AD) and adenocarcinoma (AC). Mucinproducing tumors were classified into two categories of LAMN and mucinous adenocarcinoma (MAC). The histology of the tumors was reviewed by three pathologists (NY, RW, SY). Immunostaining and quantitation of positive cells Immunostaining was performed on the paraffin sections. The antibodies used in the current study and their pretreatments were shown in Table 1. Briefly, the paraffin sections were deparaffinized and pretreated with buffers listed in Table 1. After blocking with 1 % bovine serum albumin for 10 min, the primary antibodies were diluted with Renoir Red Antibody Diluent (Biocare Medical, Concord, CA, USA) and applied to the sections. Then, endogenous peroxidase was blocked in 10 % hydrogen peroxide for 5 min and the sections were processed with MACH3 HRP Polymer Kit (Biocare Medical, Concord, CA, USA), and peroxidase activity was visualized by Metal Enhanced DAB (ThermoFischer Scientific, K.K., Tokyo, Japan). The sections for TP53 and Ki-67 were immunostained by Ventana GX System (Roche
Table 2 The clinicopathological features of appendiceal tumors
Number of cases Age Sex (M/F) Nodal status (+/−) TNM stage (0/I/II/II/IV) Number of follow-up patients Observation period (months) PMP/recurrence Death of disease Time to death (months) PMP pseudomyxoma peritonei
AD
AC
LAMN
MAC
4 71.8±7.9 2/2 – – 3 1–27 0 (0 %) 0 (0 %) –
5 50.4±19.3 5/0 2/3 0/1/2/0/2 5 13–146 1 (20 %) 1 (20 %) 19
29 65.0±15.3 10/19 0/29 15/4/9/0/2 23 1–187 2 (9 %) 1 (4 %) 32
7 60.0±12.6 3/4 1/6 0/1/5/0/1 5 20–95 4 (80 %) 2 (40 %) 32, 35
Int J Colorectal Dis (2014) 29:1349–1354
Diagnostics, Co., Tokyo, Japan). The primary antibodies were diluted with Antibody Diluent (Roche Diagnostics, Co.). The stained sections were observed under ×40 objective, and nuclear brown staining was considered as positive. The number of cells with nuclear positivity was counted in 1,500 neoplastic cells. Ki-67 labeling index was calculated by dividing the number of positive cells by 1,500. Statistical analysis Statistical analysis was performed using JMP10 (SAS Institutes, Inc., CA, USA). Comparison of multiple groups was done with nonparametric Steel-Dwass test. The p value less than 0.05 was considered to be significant. Fig. 1 The histology of appendiceal tumors. a Macroscopic appearance of LAMN. Scale bar corresponds to 2 cm. b Loupe image of the neoplasm. Scale bar corresponds to 2 cm. c–f Hematoxylin and eosin staining of AD (c), LAMN (d), AC (e), and MAC (f). g–h Periodic acid-Schiff stain of AC (g) and LAMN (h). Mucin (red) was demonstrated in LAMN (h, arrow)
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Results Clinicopathological features and histology of appendiceal tumors The age of the patients ranged from 20 to 94 years of age, and male to female ratio was 20:25 (Table 2). Based on the classification of appendiceal tumors by WHO, 4 cases were classified as AD, 5 as AC, 29 as LAMN, and 7 as MAC (Table 2). The age distribution was similar among the four histological groups, but the patients of AC were slightly younger than other groups. All AC patients were male. In LAMN, female patients were twice of the male patients. Representative histology of
1352
appendiceal tumors is shown in Fig. 1. Mucin production was evident in LAMN and MAC (Fig. 1h). The tumor cells invaded into the submucosa in all five cases in AC and in 15 out of 29 in LAMN. The tumor cells exposed to the serosa in all seven cases of MAC. Nodal metastasis was not present in LAMN. Expression and phosphorylation of Chk2 The expressions of Chk2 and pChk2 were localized in the nuclei of the tumor cells (Fig. 2), and there was no cytoplasmic positive reaction. In most tumors, more than 80 % of tumor cells showed positive nuclear expression of Chk2 (Fig. 3a). In contrast, the percentage of pChk2-positive cells varied largely from 2.9 to 95.2 % among the appendiceal tumors (Figs. 2 and 3b). The percentage of positive cells was significantly lower in AC than those in other tumors, and it was higher in LAMN (Fig. 3b). Expression of TP53 The expression of TP53 was localized to the nuclei of tumor cells (Fig. 2). The percentage of TP53-positive cells varied widely in each histological group, and it is significantly lower in LAMN than those in AC and MAC (Fig. 3c). Proliferation activity The Ki-67-labeling index varied widely, and it was lower in LAMN compared with those in other tumors (Figs. 2 and 3d). Fig. 2 Immunostaining of molecules involved in DNA damage response. Representative immunostaining of Chk2, pChk2, TP53, and Ki-67 in AD, AC, LAMN, and MAC. Nuclear brown staining of the tumor cells is judged as positive reaction. Arrowheads indicate positive nuclear reactions, which are very sparsely distributed in the tumor. Magnification of the images is ×200
Int J Colorectal Dis (2014) 29:1349–1354
Clinicopathological features of appendiceal tumors Totally, 36 patients have been followed up (Table 2). Local recurrence was not present in the cases of AD. In AC, one out of five patients (20 %) died of the disease. In LAMN, 2 out of 23 cases (9 %) presented with PMP, and one (4 %) died of the extensive disease, while 21 out of 23 cases (91 %) were free of disease. Among the cases of MAC, recurrence occurred in four out of five cases (80 %). Two cases (40 %) died of the disease at 32 and 35 months after operation, and other two cases are alive with PMP and local recurrence.
Discussion The current study is the first that showed distinct patterns of expression and phosphorylation of molecules involved in DDR in the appendiceal tumors. Chk2 was ubiquitously expressed in the nuclei of the appendiceal tumor cells. However, the frequency of pChk2-positive cells was reduced in AC, whereas it was high in LAMN and MAC (Fig. 3b). On the other hand, the frequency of TP53-positive cells was high in AC and MAC, whereas it was low in LAMN (Fig. 3c). We have previously reported the high frequency of nuclear positivity of TP53 in MAC, which was associated with mutation of TP53 gene [10]. The reduction of pChk2-positive cells and mutated TP53 suggests that DDR is impaired in AC and MAC (Fig. 4).
Int J Colorectal Dis (2014) 29:1349–1354
1353
Fig. 3 Quantitation of expression and phosphorylation of the molecules in appendiceal tumors. a Chk2. b pChk2. *p
