Early DNA damage response in residual carcinoma in situ at ductal stumps and local recurrence in...

ORIGINAL ARTICLE

Early DNA damage response in residual carcinoma in situat ductal stumps and local recurrence in patients undergoingresection for extrahepatic cholangiocarcinoma

Toshifumi Wakai • Yoshio Shirai •

Jun Sakata • Pavel V. Korita •

Yoichi Ajioka • Katsuyoshi Hatakeyama

Published online: 11 August 2012

� Japanese Society of Hepato-Biliary-Pancreatic Surgery and Springer 2012

Abstract

Background/purpose The aim of this study was to clarify

the association between the DNA damage response mediated

by p53-binding protein 1 (53BP1) in residual carcinoma in

situ at ductal stumps and local recurrence in patients under-

going resection for extrahepatic cholangiocarcinoma.

Methods A retrospective analysis was conducted of 11

patients with positive ductal margins with carcinoma in

situ. To evaluate the early DNA damage response, the

nuclear staining pattern of 53BP1 was examined by

immunofluorescence. TUNEL analysis was used to calcu-

late the apoptotic index.

Results Of the 11 tumor specimens of carcinoma in situ,

seven showed diffuse localization of 53BP1 in nuclei

(53BP1 inactivation) and four showed discrete nuclear foci

of 53BP1 (53BP1 activation); the apoptotic index was

significantly decreased in the seven tumor specimens with

53BP1 inactivation compared to the four with 53BP1 acti-

vation (median apoptotic index, 1 vs. 22 %; p = 0.003). The

cumulative probability of local recurrence was significantly

higher in patients with 53BP1 inactivation than in patients

with 53BP1 activation (cumulative 5-year local recurrence

rate, 60 vs. 0 %; p = 0.019).

Conclusions Clinically evident local recurrence of resid-

ual carcinoma in situ at ductal stumps is closely associated

with 53BP1 inactivation and decreased apoptosis.

Keywords Cholangiocarcinoma � Ductal resection

margin � Carcinoma in situ � P53-binding protein 1 �Apoptosis

Introduction

Ductal resection margin status is an established prog-

nostic factor in patients with extrahepatic cholangiocar-

cinoma [1–6], therefore, surgeons should carefully pursue

negative resection margins for extrahepatic cholangio-

carcinoma. In 2005, we reported that invasive carcinoma

at the ductal resection margins has a strong adverse

effect on patient survival. This was based on the survival

analysis between patients with a positive ductal margin

with invasive carcinoma compared with patients with a

positive ductal margin with carcinoma in situ [1]. In

contrast, patients with a positive ductal margin with

carcinoma in situ show a favorable survival time (med-

ian survival time of 99 months; cumulative 10-year

survival rate of 23 %), while residual carcinoma in situ

at the ductal resection margins may cause late local

disease recurrence [1]. Although a small proportion of

patients with positive ductal margins with carcinoma in

situ survive in the long term, some patients with residual

carcinoma in situ at ductal stumps die of local recurrence

within 5 years after resection [1]. These findings suggest

that a closer investigation of the molecular mechanisms

This article is a secondary publication based on a study first reported

in the JBA (Journal of Japan Biliary Association) 2010;24:667–674.

This study was presented at the 45th Japan Biliary Association,

Chiba, Japan, September 18, 2009 (oral presentation).

T. Wakai (&) � Y. Shirai � J. Sakata � K. Hatakeyama

Division of Digestive and General Surgery, Niigata University

Graduate School of Medical and Dental Sciences,

1-757 Asahimachi-dori, Chuo-ku, Niigata 951-8510, Japan

e-mail: [email protected]

P. V. Korita � Y. Ajioka

Division of Molecular and Diagnostic Pathology, Niigata

University Graduate School of Medical and Dental Sciences,

1-757 Asahimachi-dori, Chuo-ku, Niigata 951-8510, Japan

123

J Hepatobiliary Pancreat Sci (2013) 20:362–369

DOI 10.1007/s00534-012-0539-1

involved in these residual tumors at ductal stumps is

warranted.

The current study focuses on the early DNA damage

response mediated by p53-binding protein 1 (53BP1)

[7–10]. This study aimed to clarify the early DNA damage

response mediated by 53BP1 in tumor specimens of posi-

tive ductal resection margins with carcinoma in situ. In

addition, we sought to elucidate the association of clini-

cally evident local recurrence at ductal stumps in patients

undergoing resection for extrahepatic cholangiocarcinoma.

Methods

Of 84 patients with extrahepatic cholangiocarcinoma who

underwent surgical resection with curative intent from

January 1988 to December 2002, 11 had residual carci-

noma in situ at the ductal resection margins. These 11

patients formed the basis of the current retrospective study,

which included six men and five women with a median age

of 66 years (range, 53–80 years). The location of the pri-

mary tumor was hilar (n = 7), upper (n = 2), and middle

bile duct (n = 2). Surgical resection procedures depended on

the location of the primary tumor. Four patients underwent a

right hemihepatectomy extended to an inferior part of Cou-

inaud segment IV with extrahepatic bile duct resection, three

underwent an extrahepatic bile duct resection, one under-

went a combined extended right hemihepatectomy and

Whipple pancreaticoduodenectomy, one underwent a

Whipple procedure, one underwent a pylorus-preserving

pancreaticoduodenectomy, and a wedge resection of the

gallbladder bed with extrahepatic bile duct resection was

performed in one patient.

In the 11 patients with positive ductal margins with

carcinoma in situ, the continuity of carcinoma in situ at

ductal resection margins from the main tumor was con-

firmed histologically in multiple tissue sections. To evaluate

the early DNA damage response in tumor specimens of

main invasive tumors and positive ductal margins with

carcinoma in situ, nine serial 3-lm tissue sections were

re-cut and prepared from each block as follows: one for

hematoxylin–eosin staining, five for immunohistochemical

staining including a negative control, two for immunoflu-

orescence, and one for terminal deoxynucleotidyl transfer-

ase-mediated dUTP nick-end labeling (TUNEL) analysis.

To detect DNA double-strand breaks (DSBs), immuno-

histochemical staining with cH2AX antibody (Bethyl

Laboratories Inc., Montgomery, TX, USA) was performed.

To detect the DNA damage response mediated by 53BP1,

immunohistochemistry with 53BP1 antibody (Bethyl Lab-

oratories Inc.) and immunofluorescence with 53BP1 anti-

body were performed. To evaluate the DNA damage

response mediated by 53BP1, the nuclear staining pattern

of 53BP1 was examined by immunofluorescence, which

was captured with a confocal laser scanning microscope

(LSM510META; Carl Zeiss, Jena, Germany). Double-

labeled immunofluorescence with cH2AX and 53BP1

antibodies was performed to evaluate co-localization of

53BP1 with cH2AX at sites of DSBs. To evaluate the DNA

damage response, immunohistochemistry with p53 monoclo-

nal antibody (Leica Microsystems, Newcastle-upon-Tyne,

UK) and immunohistochemistry with Ki-67 monoclonal

antibody (Dako, Glostrup, Denmark) were performed.

Apoptotic cells were identified based on TUNEL analysis

using an Apop Tag� Peroxidase In Situ Apoptosis Detec-

tion Kit (Millipore Co., Billerica, MA, USA).

All statistical analyses were performed using PASW

Statistics 17 software (SPSS Japan, Tokyo, Japan). Con-

tinuous variables between two groups were compared by

the Mann–Whitney U test. The Kaplan–Meier method was

used to estimate the cumulative incidences of cancer-spe-

cific survival and local recurrence, and differences in these

incidences were evaluated by the log-rank test. All tests

were two-sided and p \ 0.05 was considered significant.

The median follow-up time was 98 months.

Results

Early DNA damage response in tumor specimens

of positive ductal resection margins with carcinoma

in situ

Immunohistochemical analysis of tissue sections from a

patient with positive ductal margins with carcinoma in situ

who died of local recurrence at ductal stumps are shown in

Fig. 1. Immunohistochemical staining for cH2AX shows

diffuse localization in the nuclei, which indicates DNA DSBs

in the tumor specimen of carcinoma in situ. Immunohisto-

chemical staining shows diffuse localization of 53BP1 in the

nuclei, and overexpression of p53. Ki-67 positive cells are

observed in the full thickness of carcinoma in situ, whereas no

apoptotic cells are observed by TUNEL analysis. In contrast,

immunohistochemical analyses of tissue sections from a

patient with positive ductal margins with carcinoma in situ,

who survived in the long term, are shown in Fig. 2. Immu-

nohistochemical staining for cH2AX shows diffuse locali-

zation in the nuclei, indicating the presence of DNA DSBs in

the tumor specimen of carcinoma in situ. Immunohisto-

chemical staining shows a dot-like pattern of discrete nuclear

foci of 53BP1, whereas there is overexpression of p53. Ki-67

positive cells are observed in the basement membrane of

carcinoma in situ, and apoptotic cells are observed in the

apical membrane of carcinoma in situ.

Of the 11 patients with positive ductal margins with

carcinoma in situ, seven were classified as having tumors

J Hepatobiliary Pancreat Sci (2013) 20:362–369 363

123

with diffuse localization of 53BP1 in the nuclei (Fig. 3a, b)

whereas four had the dot-like pattern of discrete nuclear

foci of 53BP1 (Fig. 3c, d). All of the main invasive tumor

from the 11 patients with positive ductal margins showed

diffuse nuclear localization of 53BP1. In four cases

showing the dot-like pattern of discrete nuclear foci of

53BP1, nuclear staining pattern of 53BP1 at the boundary

between carcinoma in situ and invasive carcinoma was also

examined. 53BP1 in the part of carcinoma in situ showed

the dot-like pattern of discrete nuclear foci, whereas

53BP1 in the part of invasive carcinoma showed dif-

fuse localization in the nuclei (Fig. 4). Although discrim-

ination between dot-like pattern and diffuse pattern by

using immunohistochemistry was sometimes difficult, the

Fig. 1 Immunohistochemical analysis of a patient with positive ductal margins with carcinoma in situ who died of local recurrence at ductal

stumps 9 years after surgical resection

Fig. 2 Immunohistochemical analysis of a patient with positive ductal margins with carcinoma in situ who survived with no evidence of

recurrence 19 years after surgical resection

364 J Hepatobiliary Pancreat Sci (2013) 20:362–369

123

nuclear staining pattern of 53BP1 was more clearly

visualized by immunofluorescence analysis than immuno-

histochemistry (Figs. 3, 4). The apoptotic index was

significantly lower in the seven tumors with diffuse local-

ization of 53BP1 (median index, 0 %) than in the four

tumors with discrete nuclear foci of 53BP1 (median index,

Fig. 3 Immunocytochemical

staining of p53-binding protein

1 (53BP1) in tumor specimens

of carcinoma in situ. a and

b correspond to the case in

Fig. 1. 53BP1 shows diffuse

localization in the nuclei

(a immunohistochemistry,

b immunofluorescence).

c and d correspond to the case in

Fig. 2. 53BP1 shows a dot-like

pattern of discrete nuclear foci

(c immunohistochemistry,

d immunofluorescence)

Fig. 4 Nuclear staining pattern of p53-binding protein 1 (53BP1) at

the boundary between carcinoma in situ and invasive carcinoma.

53BP1 in the part of carcinoma in situ shows dot-like pattern discrete

nuclear foci, whereas 53BP1 in the part of invasive carcinoma shows

diffuse localization in the nuclei. The nuclear staining pattern of

53BP1 is more clearly visualized by immunofluorescence analysis

than immunohistochemistry


123

22 %; p = 0.003; Fig. 5). Double-labeled immunofluores-

cence with cH2AX and 53BP1 antibodies in the four

tumors with discrete nuclear foci of 53BP1 (Fig. 6)

revealed a similar dot-like pattern of discrete nuclear foci

for cH2AX (Fig. 6b) and 53BP1 (Fig. 6c). A merged image

(Fig. 6d) shows that the 53BP1 foci predominantly co-

localize with cH2AX foci in the nuclei. The intensity

curves are visualized through the length of the red line in a

high magnification of Fig. 6d (inner box of Fig. 6e). The

two peaks (arrows) of the green intensity curve of 53BP1

co-localize with the two peaks of the red intensity curve of

cH2AX (Fig. 6e). In the four tumor specimens with dis-

crete nuclear foci of 53BP1, co-localization of 53BP1 with

cH2AX at sites of DNA DSBs was confirmed by double-

labeled immunofluorescence. This suggests that the early

DNA damage response mediated by 53BP1 activation is

associated with apoptosis (Fig. 5).

Long-term outcome after resection

The overall cumulative survival rates after resection were

56 % at 5 years and 19 % at 10 years (median survival

time, 99 months). Tumor recurrence had occurred in six

patients. The sites of initial disease recurrence were local at

ductal stumps (n = 2), liver (n = 1), local at ductal stumps

plus liver plus distant lymph nodes (n = 1), local at ductal

stumps plus distant lymph nodes (n = 1), local at ductal

stumps plus distant lymph nodes plus peritoneum (n = 1).

In total, five patients had clinically evident local recurrence

at ductal stumps, which were verified by biopsy and/or bile

cytology after percutaneous transhepatic biliary drainage

(n = 4) or a biopsy taken on explorative laparotomy

(n = 1). The cumulative probabilities of local recurrence

after resection were 38 % at 5 years and 67 % at 10 years

(median length of local recurrence, 89 months).

Among the 11 patients with positive ductal margins with

carcinoma in situ, cumulative survival after resection was

significantly worse in patients with diffuse localization

of 53BP1 (cumulative 5-year survival rate, 33 %) than in

patients with the dot-like pattern of discrete nuclear foci

of 53BP1 (cumulative 5-year survival rate, 100 %;

p = 0.038; Fig. 7). The cumulative probability of local

recurrence was significantly higher in patients with diffuse

localization of 53BP1 (cumulative 5-year local recurrence

rate, 60 %) than in patients with the dot-like pattern of

discrete nuclear foci of 53BP1 (cumulative 5-year local

recurrence rate, 0 %; p = 0.019; Fig. 8).

Discussion

The concepts of the DNA damage response as a final anti-

cancer barrier and an inefficient DNA damage response

leading to tumor initiation and progression have been pro-

posed. In addition, increased levels of DNA DSBs in pre-

cancerous lesions have been demonstrated [8, 11–14]. 53BP1

functioning as a DNA damage response-mediator upstream of

the tumor suppressor gene, p53, and Chk2 is rapidly recruited

to sites of DNA DSBs (53BP1 activation). The 53BP1-med-

iated DNA repair process has an important role in the early

DNA damage response [14–16]. The current study focused on

the evaluation of the early 53BP1-mediated DNA damage

response in residual carcinoma in situ at ductal stumps and

surgical outcomes. The clinically-evident local recurrence of

residual carcinoma in situ at ductal stumps is closely associ-

ated with 53BP1 inactivation and decreased apoptosis.

The nuclear staining pattern of 53BP1 in tumor speci-

mens of carcinoma in situ can be classified into two cate-

gories: diffuse localization or dot-like pattern of discrete

nuclear foci. The diffuse localization of 53BP1 in nuclei

implies that it is inactivated [14–16]. In the current study,

carcinoma in situ with diffuse localization of 53BP1 in

nuclei showed decreased apoptosis (Fig. 5) but tumor

specimens with discrete nuclear foci showed co-localiza-

tion with cH2AX at sites of DNA DSBs. This suggests that

the early DNA damage response mediated by 53BP1

activation is associated with apoptosis.

The DNA damage response at sites of DNA DSBs

includes recombination as well as apoptosis. The major

pathways of DNA repair include homologous recombina-

tion during the S/G2 phases of cell cycle and non-homol-

ogous end joining during the G1 phase [17]. However, the

precise molecular strategies that determine the choice of

DNA repair or apoptosis remain incompletely understood.

Cook et al. [18] reported that a protein tyrosine phospha-

tase, EYA, is involved in the promotion of efficient DNA

Fig. 5 Apoptotic index in tumor specimens of carcinoma in situ

according to the nuclear staining pattern of 53BP1


123

repair as opposed to apoptosis in response to genotoxic

stress. EYA mediates the damage-signal-dependent dep-

hosphorylation of an H2AX carboxy-terminal tyrosine

phosphate (Y142) after ionizing radiation in mammalian

embryonic kidney cells [18]. It is likely that additional

regulatory events govern this dual phosphorylation. Further

investigation will be required to elucidate the effect of this

post-translational modification on the pathways of DNA

repair and apoptotic cell death under physiological conditions

and in precancerous lesions, dysplasia, and cancer cells.

Fig. 6 Double-labeled immunofluorescence with cH2AX and 53BP1 antibodies in a tumor specimen of carcinoma in situ a dot-like pattern of

discrete nuclear foci for 53BP1. a DAPI (blue), b cH2AX (red), c 53BP1 (green), d merge, e high magnification of d and intensity curves

Fig. 7 Kaplan–Meier survival estimates according to the nuclear

staining pattern of 53BP1Fig. 8 Kaplan–Meier estimates of local recurrence at ductal stumps

according to the nuclear staining pattern of 53BP1


123

In histological studies of surgically resected specimens

of bile duct carcinoma, the longitudinal length of intra-

mural extension of invasive carcinoma is limited to less

than 10 mm beyond the macroscopic tumor border. How-

ever, the longitudinal length of superficial spread of non-

invasive carcinoma (carcinoma in situ) sometimes reaches

more than 20 mm [3, 19]. This superficial spread of non-

invasive carcinoma frequently observed in macroscopically

papillary or expansive tumor, which histologically corre-

sponds to papillary or well differentiated adenocarcinoma

[3, 20, 21]. This supports the hypothesis that superficial

spread results from intraepithelial ductal spread from the

main tumor, whereby tumor cells contiguously spreading

along the intact basement membranes of bile duct replace

the non-neoplastic biliary epithelium. An alternative

hypothesis is that superficial spread occurs by the carci-

noma in situ contiguously spreading along the intact

basement membranes of the bile duct before becoming

invasive. At some point the tumor cells invade beyond the

wall of the bile duct, and thus the remnant carcinoma in

situ results in superficial spread around the main invasive

tumor. Igami et al. [3] proposed that ‘‘field cancerization

(carcinogenesis)’’ participates in the formation of the

superficial spread. In the present study, the main invasive

tumors of the four patients with positive ductal margins

with carcinoma in situ showing 53BP1 activation (discrete

nuclear foci of 53BP1) showed 53BP1 inactivation (diffuse

localization of 53BP1 in nuclei). Thus, there is a distinct

difference in the presence of 53BP1 activation between the

carcinoma in situ at ductal stumps and the main invasive

carcinoma. This provides an interpretation of the superfi-

cial spread with respect to the early DNA damage response.

In these four patients, it is difficult to interpret the forma-

tion of superficial spread that results from intraepithelial

ductal spread from the main tumor when tumor cells spread

contiguously along the intact basement membranes of the

bile duct and replace non-neoplastic biliary epithelium. As

it is likely that an inefficient 53BP1-mediated early DNA

damage response participates in the formation of invasive

carcinoma, this fact is compatible with the phenomenon of

‘‘field carcinogenesis’’. However, both the main invasive

tumors and carcinoma in situ at ductal stumps of the

remaining seven patients showed 53BP1 inactivation, and

thus it is difficult to explain the mechanism of the forma-

tion of superficial spread from a viewpoint of early DNA

damage response. Further molecular biological investiga-

tion is warranted to elucidate the precise mechanism of the

formation of superficial spread.

The main limitation of the current study is the retro-

spective analysis of a small number of patients. We could

not find any differences regarding pathological character-

istics (histologic type, histologic grade, tumor size, pT

classification, pN classification, lymphatic vessel invasion,

blood vessel invasion, and perineural invasion) according

to the nuclear staining pattern of 53BP1 (data not shown).

The small number of patients (n = 11) analyzed retro-

spectively was a limiting factor in the current study and

limits firm conclusions being drawn. The current study

regarding early DNA damage response has potential clin-

ical implications. Firstly, immunofluorescence analysis of

53BP1 nuclear expression in tumor specimens of ductal

resection margins may be useful to estimate the risk for

local recurrence at ductal stumps. Secondly, evaluation

of the nuclear staining pattern of 53BP1 as an adjunct to

histologic diagnosis may be conducted on preoperative

endoscopic transpapillary biopsy samples, and even on

small biopsy samples from peroral cholangioscopy.

Thirdly, in ulcerative colitis [22] and esophagus [23],

where the histologic concept of dysplasia is used for pre-

cancerous lesions, it has been elucidated that accumulation

of DNA damage and an inefficient early DNA damage

response lead to cancer development [7]. The methodology

focused on early DNA damage response and evaluating

H2AX, cH2AX, and 53BP1 nuclear expression levels may

be a useful diagnostic tool for precancerous lesions.

In conclusion, after resection for extrahepatic cholan-

giocarcinoma, clinically-evident local recurrence of resid-

ual carcinoma in situ at ductal stumps is closely associated

with 53BP1 inactivation and decreased apoptosis. In con-

trast, apoptosis associated with early 53BP1-mediated

DNA damage response is one of the molecular biological

mechanisms that permit a small proportion of patients with

residual carcinoma in situ at ductal stumps to survive in the

long term with no evidence of local recurrence.

Acknowledgments The authors would like to thank Professor

Kouhei Akazawa, Department of Medical Informatics, Niigata Uni-

versity Medical and Dental Hospital, Niigata, Japan, for suggestions

concerning interpretation of statistical analysis in the present

manuscript.

Conflict of interest None.

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